JPH07504800A - Molecular cloning of astroviral human gastroenteritis agents and corresponding cDNAs - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Molecular cloning of astroviral human gastroenteritis agents and corresponding cDNAs This study , NIHGrants DKO1811and Merit Review, Supported in part by Veterans Administration. subordinate Therefore, the United States Government has certain rights in this invention. This application was filed in 1991. Co-pending, jointly filed U.S. Patent Application No. 07/ Part of No. 702゜731 is pending.

Field of invention・ The present invention provides a method for identifying the nucleic acid sequence of the astrovirus genome. The polypeptides encoding these polypeptide antigens are added to the polypeptide viral antigens. nucleotide sequence, an expression system capable of producing the above polypeptide antigen, human For detecting Astrovirus infection in serum Methods of using the polypeptide antigens described above include astroviruses in stool or other body secretions. and how to use astrovirus nucleic acid sequences to detect Antibodies directed against polypeptide antigens.

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Background of the invention Viruses of different serotypes have been shown to be associated with common sources of gastroenteritis. (Kapikian et al; Greenberg et al. al. (1989); Dolin et al.). These viruses are Astrovirus (Astrovirus), Calicivirus (calici) virus), Norwalk and Norwalk uniform virus (small ring structure Virus small round-structured virus (SRS ■), parvovirus, coronavirus coronavirus, torovirus, Contains pestivirus. All cases of acute gastroenteritis in the United States It is estimated that up to 65% of cases can be attributed to viral agents. (Blacklow et al. (1982)). Virus-induced gastroenteritis is Typically lasts only 1 to 2 days, but is worth the number of days lost from work or school. The overall effect is substantial (National center for he alth 5 statistics.

5eries 10. No, 85. (1972): D) IEW Publ. cation (HRA) 74-1512゜Washington DC, Government printing Office (1973)).

Human Nastrovirus was detected in newborns with diarrhea in the delivery room in 1975. It was first identified in 2013 (Appleton et al., Madeley et al.). The virus has since spread to worldwide epidemics and endemic gastroenteritis. It was found that there is a relationship between the Na Stroviruses often cause watery stools that may last several days. 1 -Children in the 3-year age group tend to show the most obvious signs of infection (Kurtz et al., (1977)), the children surveyed in the study 87% had developed antibodies against Nastrovirus by age 10 (Kurtz et al., (1978)). Nastrovirus is found in the following environments: It has also been shown to be related to the occurrence of gastroenteritis, including in adults: l) In local medical facilities. Elderly people (Gray et al, Shiro et al,), 2) Pollution One third of adults consumed oysters (Kurtz et al. (1989)) and 3) groups of teachers and students who drank water from contaminated sources (Kurtz et al. l, (1989) Is it distinguishable as an antigen from human Nastrovirus? Animal Nastroviruses, which cannot be morphologically distinguished from each other, are derived from various species. It has also been identified in diarrheal stool samples from Kjeldsberg et al. l,).

Two previous studies investigated Nastrovirus gastroenteritis in adult human volunteers. Attempting a systemic experiment. In one study in the UK, Nastrooil Fecal filtrate from one strain (JW) of J. spp. was administered to volunteers (Kurtz et al (1979)). Only one out of eight volunteers (DA+ ) developed into severe diarrhea on the 6th day, and on the 5th and 6th day after ingestion, nast was present in the stool. Excreted lovirus. Subsequently, nine volunteers were administered the fecal filtrate of the above DM. Two people in the tier excreted Nastrovirus in their stools but did not develop clinical disease. There wasn't. A second study in the United States isolated an outbreak in a nursing home. The infectivity of the Marin County agent (astrovirus type 5) was investigated (H.

Greenberg and K., Midthun, unpublished data (1987 )), only 1 out of 19 volunteers developed symptoms. This means that This suggests a relatively low pathogenicity of Nastrovirus in human volunteers.

Serological responses to Nastrovirus infection did not develop in both studies. It was found in 35-50% of volunteers.

So far, five serotypes of human nastroviruses have been identified (K urtz et al. (1984)), a study from Oxford, UK, Serotype 1 Nastrovirus explains 72% of community-acquired infections On the other hand, serotypes 2.3.4 and 5 were released in 1975 and 1986, respectively. revealed that it is found at a frequency of 6-7% in this region during the year. (Kurtz et al, (1989)), in the United States, the above CD C is an antibody against all 5 types of Nastrovirus. Determined to exist in the globulin pool (LeBaron etal, ). Each of these Nastrovirus serotypes is Populations in tissue culture (LLCλ (2 rhesus kidney cells) supplemented with has been adapted, but at relatively low titers (Lee et al.).

Hyperimmune rabbit serum (group- and serotype-specific) and group 1-responsive mono A clonal antibody (8E7) was recently generated (Herrmann.

et al. (1989)). Rabbit blood against specific Nastrovirus serotypes has been used in neutralization assays (Hudson et al.), However, Nastrovirus ELISA (He rrmann, et al. (1990)). treatment ahead A recent pediatric study found that 3-5% of hospitalizations due to diarrhea were caused by Nastrovirus. (Ellis et al.). However, Nastrowill The true incidence of gastroenteritis remains to be conclusively established.

The Nastrovirus genome described above is a plus- (Herringet al., 8 Ionr. oe et al.

Willcock et al.) This genome is -7.2 Kb in length. riQlonr.

e et al.) and three structural lines aligned in masses from 20-35 kDa. It encodes a protein (Willcock et al., & Ionro e et at,). So far, the nucleic acids and antibodies of the Nastrovirus genome have been The amino acid composition has not been determined.

This specification describes effective methods for the isolation and expression of Nastrovirus genomic sequences. Disclose the method.

Summary of the invention One general object of the invention is to provide the following groups: SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. No. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, and SEQ ID NO. 13. a purified Nastrovirus polynucleotide comprising at least one sequence It is to be.

Another object of the invention is to immunize rabbits with an infectious inoculum of Nastrovirus. encodes a Nastrovirus polypeptide that is immunoreactive with serum from We provide recombinantly produced Nastrovirus polynucleotides. There are many things. Specific embodiments of these polynucleotides have the following sequences: Number 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 1O1 SEQ ID NO: 12, and encoding an immunoreactive portion of any one of SEQ ID NO: 14. It is. Specific polynucleotides encoding these polypeptides are Column number 1, Sequence number 3, Sequence number 5, Sequence number 7, Sequence number 9, Sequence number 11, and SEQ ID NO: 13.

The present invention provides Lambda-A2, Lambda-All, Lambda-Al1 and Lambda-A33. (These are lambda gtl+ vectors containing designed inserts.

See Example 3. ) containing a vector selected from the group consisting of Also included are Nastrovirus-produced polypeptides produced by cells.

The present invention uses blood from rabbits immunized with an infectious inoculum of Nastrovirus. Also provided are recombinant Nastrovirus polypeptides that are immunoreactive with the virus.

Such recombinant polypeptides include those with matching and the following polypeptides: Petide: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and a sequence substantially the same as one of SEQ ID NO: 14. This group Replacement polypeptides may be encoded by other protein moieties, e.g. It also includes in-frame fusions to the β-galactosidase protein. like this Recombinant polypeptides include, for example, lambda-A2, lambda-All, lambda- A lambda gtll vector with any of the above inserts containing A13 and lambda-A33. can be obtained from the cells of bacteria, including bacteria.

In one aspect, the present invention provides for Includes methods for detecting toroviruses. This method works by Partial purification of polynucleotides or enrichment of polynucleotides from environmental samples , and its successor, an oligonucleotide specific to the above Nastrovirus polynucleotide. Including hybridization to otide probes. Also, this method is similar to the above Detecting the binding of the probe to the sample present in the stool or to the environmental sample. It also includes means for Such a detection means biotinylates the probe. standard assays, such as labeling with radioactive isotopes (tinylation) or Including how to exit. This method uses RNA templates present in the partially purified samples described above. and the sequence-independent amplification of the resulting cDNA molecules. But that's fine. Oligonucleospecific to the above astrovirus polynucleotide Tido probes and oligonucleotide primers are also defined by the present invention. Ru. Both the above probes and primers were used with previously described astroviruses. ・Can be obtained from the code sequence.

Another aspect of the invention provides human blood containing antibodies specific for astrovirus antigens. A diagnostic kit and method of use in screening for. This kit is Serum and immune reactivity from rabbits immunized with strovirus infectious inoculum. Certain recombinant astrovirus polypeptide antigens and and means for detecting binding of said antibody. The previously described astrovirus epitope Polypeptide antigens containing any of the immunoreactive portions of can be done. This kit consists of a solid support to which the polypeptide antigen described above is attached. The antibodies may also include ribo-labeled anti-human antibodies. In this case the smell In other words, the binding of anti-astrovirus serum antibodies to the above-mentioned antigens is - can be detected by binding of the labeled antibody to the solid surface.

Furthermore, another aspect of the invention provides that the tester can be immunized with a testrovirus infectious inoculum. Development of immunoreactive astrovirus polypeptides with serum from rabbits Including current system and manufacturing method. This method involves oven-reading in a suitable host. ・A recombinant expression system containing frame (012F) whose ORF is Astro Sera from rabbits immunized with virus-infected inoculum and immunoreactive antibodies It has a polynucleotide sequence encoding a strovirus polypeptide. This includes introducing an expression system that In this approach, this vector is , designed to express the ORF in the selected host. Then this The host is cultured under conditions that result in expression of the ORF sequence. Escherichia coli (Escher) A large number of expression systems, including the above lambda gtl1 expression system in Ichia coli, can be used in this regard.

Other expression systems are available for use in yeast, bacteria, insect, and salivary mammalian cells. Contains an expression vector.

Embodiments of the invention also provide methods for immunizing patients against astrovirus infection. vaccine. This vaccine is immunized with an ametrovirus infectious inoculum. Sera from cultured rabbits and immunoreactive recombinant astrovirus poly Contains peptide antigens. Such polypeptide antigens include the previously described Astro can include immunoreactive portions of virus coding sequences. This polypeptide anti The drug substance is typically prepared with a pharmacologically acceptable adjuvant.

The present invention further provides the following groups: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, A sequence selected from sequence number 8, sequence number 10, sequence number 12, and sequence number 14 including antibodies specific for polypeptides having the sequence. In addition, the antibody can be used with the following vectors. ter, Lambda-A2, Lambda-A11, Lambda-A13 and Lambda-A33. Astroviruses produced by bacterial cells transformed by one of the can be produced against Rousseau-specific antigens. These antibodies are polyclonal It may be monoclonal or monoclonal. This antibody is produced by administering the antibody parenterally to the patient. creating passive immunity within the patient against astroviruses, including can be used in a method.

These and other objects and features of the invention will be apparent from the following detailed description together with the accompanying drawings. It will become more fully clear when reading the description.

Brief description of the drawing Figure 1 shows that only the A43 clone-specific probe was detected from astrovirus-infected cells. This example shows hybridization to a nucleic acid.

Figure 2 shows that the A43 clone-specific probe was tested by other astrovirus serotypes. Hybridization to nucleic acids from infected cells is exemplified.

Figure 3. Astrovirus with corresponding antigen and hybridization analysis. Figure 3 shows a CsC1 density gradient separation of a sample preparation.

Figure 4A illustrates that nucleic acids from infected cells are RNase sensitive.

Figure 4B shows that the above astrovirus RNA is plus-sense RNA. Which indicates that.

FIG. 5 shows the following sequences: A35, SEQ ID NO: I; A43, SEQ ID NO: 3. and AI It shows a row in which SEQ ID NO. 7 is lined up.

Figure 6 shows that the new astrovirus sequence was how it is identified by tope spanning method) It shows.

Figure 7 shows the presence of two populations of astrovirus-specific poly(A) + RNA. The results of the Northern plot analysis are shown. Astrovirus genome R NA (7,2 Kb) clone A43- and clone A39-specific probe On the other hand, the genomic RNA (2°8 Kb) is recognized by two A 39 - recognized by only one of the specific probes.

The terms defined below have the following meanings herein: Astrovirus - The polynucleotide may contain variants of the disclosed sequence, e.g. degenerate codons, or presence in isolates or strains of astrovirus that are immunologically cross-reactive with type I isolates. refers to a variant in an existing sequence.

2. Below (7) Washing conditions: 2 x SCC, 0.1%; SDS, room temperature twice; 30 minutes each; then 2X SCC, 0,1% 5l)S, 5o''C-times, 3 0 min M secondary, 2 x 5C01 room temperature twice, 1o min each, was used, & 1aniatis et at.

Hybridization described in 肚, ■t,, pp. 320-323 When two complementary strands are able to hybridize to each other under the conditions Nucleic acid fragments are "homologous" and have up to about 25-30% base pair unpairing. Homologous sequences containing combinations can be identified.

More preferably, the homologous nucleic acid strands have 15-25% base pairs. It contains a mismatch, more preferably a base pair mismatch of 5-15χ. These homologous The extent to which a genetic library (or genetic material more stringent washes or halves to identify clones from other sources) can be selected by using hybridization conditions.

3. The protein is an astrovirus polypeptide, or an astrovirus ・The polypeptide is a cDNA or RNA fragment representing the above-mentioned astrovirus agent. coded by the oven reading frame of the fragment. is obtained from the astrovirus polypeptide.

4. One of the sequences determined for the disclosed astrovirus epitope and Proteins with substantially the same sequence can be combined without removing the antigenic properties of the protein. Defined as a protein with an amino acid substitution (i.e., a neutral substitution) within the white coding sequence do. Neutral substitutions between different amino acids that do not adversely affect the overall antigenic action A matrix that defines the relevance of (Taylor et al., 1989+) Recently available in combination with Dayhoff; 5chulz et al) Primary and secondary structure analysis (Needleman et al; Doolitt le: Taylor et al, 1989: Hopp et al,) can be logically predicted by using . Proteins with sequence substitutions The quality of serum (e.g., Examples 3 and 6), polyclonal tested for immunoreactivity with specific monoclonal or specific monoclonal antibodies. be able to.

5. LLCMK2 cells infected with serotype I astrovirus were treated with “AI This is referred to as ``LL (JIK2''). This means that this is the astrovirus. meant to be one line of more continuously infected LLCMK2 cells. isn't it.

■ Molecular clone selection by immune screening of the five identified serotypes. Some human astroviruses (Kurtz et al. (1984)) . The following method can be used to isolate and differentiate any astrovirus, regardless of serotype. It can be used for analysis. Astrovirus from the above AI LLCMK2 Partial purification of serotype I is described in Example I. These virus particles are removed by centrifugation. and the presence of viral antigens was determined at each stage by ELISA assay. (Example 1). One-stage guanidium/phenol extraction of RNA from a partially purified virus preparation.

Two groups of cDNA molecules from a partially purified nucleic acid mixture.

The first one using oligo d(T) as a primer and the first strand cDNA A second one using random briming for A synthesis was created (Example 2 A). The resulting mixture of cDNA molecules, regardless of the specific sequence of the DNA molecules, is Sequence-independent single-primer amplification (Sequen ce-Independent Single-Primer Amplifi cation (S[5PA)) (Example 2B). Simply put , this sequence-independent amplification typically uses blunt-end ligation to link known sequences. By attaching a car to the ends of double-stranded DNA molecules present in the mixture, achieved. These linkers are then linked with primers that are complementary to this linker sequence. primers to provide primer-initiated amplification to the terminal sequences. typically , the above-mentioned 51SPA method (Reyeset al.) is a continuous modification of second-strand DNA. using temperature cycles to achieve primer-initiated polymerization. &lullis performed during 30 cycles of amplification al, ) o This S[SPA amplification is performed using the astrovirus present in the starting material. Provides the advantage of enrichment by amplifying genomic polynucleotides.

Two sets of amplified cDNA molecules from above were then added to the lambda gtll vector (e.g. 2C), made as an in-frame fusion to β-galactosidase. allows the expression of the protein encoded by the newly introduced cDNA sequence. (Young et al.

). Plaques created by blating this recombinant phage were then Rabbit blood before immunization reacts with hyperimmune rabbit serum made against serotype A2. Screened for products of polypeptide antigens that did not react with serum (Example 3) ). Candidates initially identified by this immunoscreen were then plaque purified. , and rescreened. 10 clones were finalized for further study. (A35, A43, A39, AI, A2, All, 813 , A14, A21, and A33) (Example 3). These clones are specifically reacts with hyperimmune serum from non-immunized rabbits and with serum from non-immunized rabbits. (No reaction occurred. The sizes of the cDNA inserts are given in Table 1.

Il, Detailed characterization of the lambda gt11 clone A, Immunological characterization is The above clones were paired with hyperimmune rabbit serum (old) by preimmune rabbit serum. , and an astrovirus-specific monoclonal paired with preimmune mouse serum. 8E7 ()lermann et al. (1988)) , further immunologically characterized by extended immune screening. Table 2 As can be seen from the data, none of the above cloned inserts The previously identified 8E7 astrovirus was shown to be a virus group-specific antigen. does not encode an epitope of rus (Herrmann et al., ( 1988)).

B. Characterization by hybridization of the 10 cloned inserts described above. , characterized by no-bridization analysis as follows. first, top The cDNA insert described above was isolated and compared with human lymphocyte genomic DNA and E. coli. (E. coli) strain 1088 for hybridization reaction with genomic DNA. (Example 5A). None of the above inserts This indicates that the above clone was a non-viral DNA. These two possible sources of viral DNA (i.e., of the human genome and of E. coli) (E. coli)).

Further hybridization studies (Example 5B) revealed the following between the above clones: A cross-hybridization relationship was shown (Table 3).

When clone A43 was used as a probe, clone A35, AI, A2 , and AI3 were cross-hybridized. These results indicate that the above clone is indicating that they arose from adjacent segments of the astrovirus genome.

Clones A39, All, A14, A21, and A33 are Does not cross-hybridize with any of the clones and represents an independent epitope Therefore, there is.

The above cloned inserts were labeled and astrovirus-infected and unsusceptible. was hybridized to cDNA made from the mRNA of the cells of the dye (Example 5C). 5 The ISPA amplified cDNA was obtained from the above AI LLCAIK2 (Astrovirus infection). ) and LLCNIK2 (uninfected) cell lines (Example 2B). Up All probes listed above were added to the 5ISPA amplified cDNA from AI LLCAIK2. 5IS from a cell line that specifically hybridized but was uninfected with LLCIIK2. It did not hybridize to PA amplified cDNA. The above A14 insert is weakly hardened. hybridized and the A21 insert was inserted into the AI LLCMK2 cDNA. Hybridized to a certain extent. These results indicate that the cloned inserts described above are The results support the finding that it originates from the astrovirus coding sequence.

The cloned gtll insert described above represents part of the astrovirus genome. To further support that Astrovirus-infected) cells and LLCIIK2 (uninfected) cell lines (Example 1) Extracted from. The hybridization probe used was lambda gtll Clones A43, A35, A39, All, A14, A21, and It was a radiolabeled cDNA of A33. Hybridization of the above The analysis results (Example 5D, Table 4, and Figure 1) show that the above insertion intensity is Specific for RNA extracted from K2 cells (i.e., astrovirus-infected cells) It was proved that it hybridized with

There was no hybridization with RNA isolated from uninfected cell lines as described above. It was. This data further indicates that the cloned sequences described above are astrovirus code. It supports matching the code sequence.

In addition to astrovirus serotype 1, the A43-specific probe also was shown to hybridize to serotypes 2.4 and 5 (Example 5F and Figure 2 ).

When astrovirus particles are separated on a CsC1 gradient, the astrovirus It was also demonstrated that the activity co-migrated with the A43-specific sequence (Example 5E and Figure 3). .

The properties of the extracted nucleic acid from which the above-mentioned astrovirus cDNA was obtained were further determined. investigated (Example 5G). As shown in Figure 4A, from AI LLC & IK2 cells. RNase A treatment (column 1) of the nucleic acid extract was performed using the A43 clone-specific protein described above. hybridization with the lobes was removed, while DNase treatment (color Mu29 had no effect at all. Nucleic acids from untreated samples can also be tested using the above probe. The test was positive by hybridization with 100% of the protein (column 3). death Therefore, this result indicates that the astrovirus genome consists of RNA. Consistent with the previous results shown (Herring et al., Klonr oe et al, and Willcocksetal). Furthermore, shade Only single-stranded probes of polarity (determined by oven reading frame analysis) ) is hybridized to the above AI LLCλlK2 cell-produced nucleic acid. Figure 4B, spot l), which indicates that the above extracted astrovirus RN It shows that A was a pulse chain.

In addition, poly(A) + RNA was added to AI LLCItlK2 (astrovirus-infected ) cells and LLCλIK2 (uninfected) cell line (Example 1). This isolation PolyA+ RNA was gel fractionated and transferred to nitrocellulose filters.

The RNA bound to this filter was then injected into a clone A43-specific probe. , and hybridized to two different clone A39-specific probes ( Example 9). Clone A39 has an internally mutated 5 ISPA linker sequence (sequence number: 15, nucleotides 192-223), so one A39- The specific probe contains the insertion sequence (sequence) from its 5' region to the above mutant linker. Column number I5, nucleotides +8-191) and the others are that Insert sequence from the 3゛ region to the above mutant linker (SEQ ID NO: 15, nucleo 24+-417). The results of this hybridization analysis (Example 9 , Figure 7) shows poly(A) + RNA in astrovirus-infected cell lysate, The existence of two populations of astrovirus-specific RNA was demonstrated. A43 and A 39 (3' of the mutant linker) clone-specific probe or both of the above 7 ．． 2 was hybridized with the genomic RNA of b (Fig. 7, lanes 1 and 3). Sara Next, the 2.8 Kb genomic RNA was applied to the clone A39 probe described above. (Figure 7, lane 3). This hybridization analysis further The internal 5ISPA sequence in clone A39 is not specific for astrovirus. This suggests that something is wrong.

The above results are consistent with the lambda gtll clone previously identified by immunoscreening. This suggests that the sequence corresponds to the antigen-coding sequence.

111, Sequence analysis of astrovirus cDNA clones Lambda gtll clone cDNA insertion of A43, A35, A39, AI, A14, and A21 subcloning the product into a vector useful for DNA sequence analysis and its insertion. The sequence data for the two clones sequenced above (Example 6) are represented in the sequence listing. .

Compile the cloned insert sequence above into the “GENBANK” data set. homologous sequences at both the nucleic acid and amino acid sequence levels. We investigated gender. This search indicates that this cloned insert sequence has not been previously characterized. It was shown that the sequence does not represent a nucleic acid or amino acid sequence.

The six inserts described above were aligned for sequence comparison. those with substantial sequence overlap; The only cloned sequences identified were A43, A35, and AI. (Figure 5). In Figure 5, the bold range indicates the overlapping range between the above designed clones. represents. As seen in this figure, clones A43 and AI No overlapping range: clone A35 sequence bridges the two.

This data showed cross-hybridization between AI and A43. This seems consistent with cross-hybridization. Check this array data Since there is no contradiction, we continued the above cross-hybridization experiment. I didn't go. This sequencing data indicates that at least two epitopes are present in this region. one of which matches the A43 sequence, and the other matches the Al sequence. I supported that.

The above identified immunoreactive clones were synthesized from a complete set of overlapping genomic cDNA clones. Can be used to obtain a set. For example, the lambda gill clone above The A43 insert was isolated and transduced into lambda gtl.

can be used as a probe against cDNA libraries established within Wear. This lambda gtlO library was used for immunoscreening. Essentially as described in Example 2C for the library in the ram ygtll made in

Inserts from clones identified in this manner are then added to the lambda gtlo EcoRI treatment of loans, electrophoretic fractionation of the treatment products, and the inserted DNA. can be isolated by electroelution of the band corresponding to (Maniatis et al, : Au5ubel et al, ) o other virus epi To identify the tope, this isolated insert was then transformed into a random fragment. To make, it can be treated with DNase l (Maniatis et al.), and the resulting treated fragments were used for immunoscreening. For this purpose, it was inserted into a lambda gt11 phage vector. Alternatively, sequencing After that, oligonucleotide primers are added to either selected expression vectors. used to isolate specific overlapping segments for 1n-phase insertion into You may.

Inserts from the immunocompetent clones identified above were made in lambda gt10. in a similar manner to probe the original cDNA library. can be done. A specific subfragment of this insert was isolated in a polymerase chain reaction. It can also be isolated by restriction endonucleases, either by reaction or after cleavage. this radiolabeled a subfragment of It can be used as a probe for NA libraries. In particular, The 5′ and 3′ end sequences of the source were analyzed to identify clones that overlap within this region. useful as a probe.

Furthermore, the sequences provided by the terminal sequences of the clone inserts are, for example, AI Second-strand DNA synthesis using LLC^lK2 cell RNA and A43 terminal sequence Reaction (&Ianiatis et al, ; 5charf et al,) (Figure 6). Or partially Purified astrovirus nucleic acid was added to fecal samples obtained from infected volunteers. (see Example 8) or from cell culture produced material. can. The second strand of cDNA is randomly initiated by CBoehringer. Klannheim, Indianapolis INX Figure 6, El). child The procedure is to generate a cDNA corresponding to the nucleic acid region flanking the known clone A43 insert sequence. A molecule is identified or produced. These newly isolated sequences, in turn, can be used to identify further flanking sequences etc., and there Overlapping cDNA clones from which the complete astrovirus genome can be determined Can be used to identify loans. As mentioned earlier, the new After isolating the strovirus genome sequence, this polynucleotide was cloned. and immunization to identify specific sequences encoding astrovirus antigens. Can be screened.

Alternatively, polymerases can be used to clone gears between known epitopes. A chain reaction (Qlullis) can be used. For example, the above Astroui The end sequences from either of the S. rus clones were then placed in this polymerase chain reaction. (e.g., Fig. 6, A43); The end sequences from unrelated clones (A39, AI4, and A21) were It can be used as a double primer (Fig. 6, El). These two sets Since the genomic relationships between the epitope-coding sequences of roach (i) e.g. both strands of one end of A43 and A1 a mixture of primers from both strands of one end of A43; (ii) one end of A43; Primers from one strand at the end (Fig. 6, A43), A39, A14, and a primer from one strand of one end of A21 (Fig. 6, El). reactions and the same primers as above from A43 and A39, AI4, and and primer (E2) from the opposite strand of the same end of A2+ as above. Another reaction creates an epitope spanning polymerase chain reaction amplification product. (Figure 6). Figure 6 uses, for example, A43 and El. We also describe the productive polymerase chain reaction that creates epitope-spanning regions. ing.

The above knowledge of the astrovirus genome sequence provides: (i) access to other viruses; in the study and establishment of relationships between astroviruses; and (ii) astroviruses. A single sequence from a virus related to the virus that has not yet been cloned. It will help you when you are away.

]V Anti-astrovirus antigen antibody In another aspect, the invention provides antibodies directed specifically against the recombinant antibodies of the invention. Contains antibodies that have been tested. Typically, a host animal, e.g. a rabbit, is used to prepare antibodies. Rabbits are immunized with the above-described generated antigen or fusion protein antigen. C hybrid or fused proteins to other proteins, e.g. Various coding sequences derived from glutathione-8-transferase or glutathione-8-transferase It can be created using columns. Serum or plasma of the above host is collected after an appropriate time interval. and this serum is tested for antibodies specifically directed against the antigens mentioned above. Testing. Example 7 is specific for the A43 antigen in the 5j26/A43 fusion protein. describes the production of different rabbit serum antibodies. These techniques are based on the present invention. can be equally utilized for other antigens.

For example, use of saturated ammonium sulfate, um or DEAE 5ephadex or by using other techniques known to those skilled in the art to make polyclonal antibodies. gamma globulin fraction or 1gG antibody of immunized animals can be obtained from Ru.

Alternatively, purified or fused antigen proteins can be used to make monoclonal antibodies. May be used. Here, the spleen or lymphocytes are removed from the immunized animal, and to prepare hybridomas by methods known to those skilled in the art. use. °Human lymphocyte donor to create human-to-human hybridomas Select.

Patients found to be infected with astrovirus are suitable lymphocyte donors. It can be helpful. Lymphocytes can be isolated from peripheral blood samples. Ru. Epstein-Barr virus (EBV) immortalized in human lymphocytes or human fusion partners can be used for human-to-human hybridization. Can be used to make bridomas. Virus specific polypeptide Initial in vitro immunization or immunization with human monoclonal anti- Can be used in body manufacturing.

The antibodies secreted by the above-mentioned immortalized cells can be detected by, for example, ELISA or Westerly Determine the desired specificity using the plot method (Ausubel et al.). screen to determine clones that secrete antibodies with

■、Usefulness The routes of transmission described for astroviruses and other gastroenteritis viruses are water, Food (especially shellfish, crustaceans and salads), aerosols, fomites , and includes person-to-person contact. Infectivity may continue for 2 days after resolution of symptoms (Gr. Eenberg et al, (+989)).

In cases of gastroenteritis, the investigator must identify the etiologic agent responsible for the development. We often encounter problems that need to be addressed before they can be done. caused by a virus Because of the difficulties associated with diagnosing gastroenteritis, this issue is particularly important for viral outbreaks. Related. Differentiating bacterial or protozoan viral gastroenteritis is sometimes difficult due to its overlapping clinical symptoms. Virus-specific antigens and Adequate supply of virus and antibodies is essential for any virus test system. Special Identification of distinct astrovirus antigens implicated in gastroenteritis outbreaks This would facilitate direct diagnosis of the disease.

A1 diagnostic method and diagnostic kit (i) Immunological approach Antigens obtained by the method of the present invention, especially clones A43, A35, A39 , AI, A2, All, AI3, AI4, A21, and A33. The antigen expressed by astrovirus is the astrovirus present in the serum of patients infected with astrovirus. It is advantageous for use as a diagnostic agent for viral antibodies.

In one preferred form of diagnosis, the test serum is obtained by the method of the invention. surface-bound astrovirus antigens, such as the A43 insert-encoded antigen described above. React with two solid phase reagents. The anti-astrovirus antibody is conjugated to the above reagent and the After washing to remove unbound serum components, the reagents are transferred onto the solid support. Bind the reporter to the above reagent in proportion to the amount of bound anti-astrovirus antibody. The reporter label is then reacted with anti-human antibody. Unbind this reagent again Amount of reporter washed to remove labeled antibody and with the above reagents Measure. Typically, the reporter is a suitable fluorometric or colorimetric detected by incubating the solid phase in the presence of a quantifiable substrate. It is an enzyme.

The solid surface reagent in the assays described above transfers the protein material to a solid support material, e.g. Rimmer beads, dip stick (dip 5tick), 96-+v prepared by known techniques for attachment to ELL plates or filter materials. It will be done. These attachment methods generally involve non-specific absorption of the protein onto the support. , or to chemically reactive groups on the solid support, e.g. activated carbonyl, hydrogen. to the droxyl or aldehyde group, typically via the free amine group, Including covalent attachment of the above proteins.

In a second form of diagnosis, antibodies bound to a solid support, known as homology assays. There are some changes in the reaction medium that can be directly detected in that medium. create. The known general types of homology assays proposed above are: (a) The reported mobility (spin splitting peak) spin label reporter when detected by a change in -1(b) a fluorescent reporter when detected by a change in the efficiency of fluorescence; 1(C) Enzyme liposomes when affecting antibody binding or enzyme/substrate interactions Tar-1 and (d) binding leads to liposome lysis and encapsulated reporter release. Contains liposome-bound reporter-1. Proteins of the present invention in these methods Application to antigens follows conventional manufacturing methods for homology assay reagents.

In each of the assays previously described, the assay method involves generating antibodies from the test patient. reacting with the above protein and testing the antigen for the presence of bound antibodies; Including. This test uses labeled anti-human antibodies against the antibodies being tested, i.e. , rg (low acute phase/-secondary response) or IgG (convalescent phase or chronic phase/secondary response). and bond to the solid support as in the first method above. or as in the second method above. , may include observing the effect of antibodies binding on homology assay reagents. .

Further, the aspect of the present invention also includes an assay system or a test system for performing the assay method described immediately above. It's a kit. The kit generally uses surface-bound recombinant astroviruses. a support with an antigen (e.g., A43 insert-encoded antigen) and a surface-bound antibody. -Reporter-labeled antibody for detecting astrovirus-antigen antibodies - Contains human antibodies. In particular, those produced by clone A43, AI, or A35 A39, A in the Astrovirus Kh kit. 2. Manufactured by All, A13, A14, A21, and A33 Can be combined with antigens. In addition, a single antigen containing multiple antigens as shown in Figure 5 can be used. recombinant polypeptides can be included in such kits.

In addition, these antigens can be expressed in rotaviruses, pestiviruses, coronaviruses, and toxins. virus, adenovirus, Norwalk virus, and Norwalk-like virus. Other gastroenteritis-inducing viruses, including Virus (LeBaron et al.) can be combined with several antigens. N above.

Several members of the rwalk-like virus family have the following variants: Hawa i i, Snow Mountain SM Montgomery County 5 Taunton SAmulree 1Sapporo and 0tofuke including. These antigens can be obtained in a number of ways, e.g. rus can be cultured (LeBaron et al.) and will The viral antigen protein can be isolated from the capsid of the virus. Karushi Viral polypeptides (Terashima et al.), and Norv The alk virus (Greenberg et al. (1981)) is similar to can be isolated. Kits containing selected gastroenteritis virus antigens are A diagnostic set capable of immunoreacting with a broad spectrum of gastroenteritis serum samples. provide products.

A third form of diagnosis is astrovirus, as described above in Section IV. Includes the use of anti-astrovirus antibodies that can detect specific antigens.

The above-mentioned astrovirus antigens are tested, e.g., in an antigen capture assay, in the serum of the candidate. , fecal material or environmentally generated samples (e.g. sewage and food sources, e.g. shellfish, crustaceans) ) is an astrovirus-specific monocloner. It can be detected using a molecule that reacts with a molecule. This anti-astrophyte rus monoclonal antibody is attached to a solid substrate, and then the antigen is attached to a second Detected by different labeled anti-astrovirus antibodies / Astrovirus specific Monoclonal antibodies of the invention directed against foreign antigens are particularly useful in this diagnostic method. It suits. Other variations of this antigen capture assay are known to those skilled in the art ()la rlow et al, ). The diagnostic form of this antigen capture assay is rotavirus, stivirus, cultivirus, astrovirus, parvovirus, coronau viruses, toroviruses, adenoviruses, and Norwalk-like viruses. Expanded to include antibodies directed against other gastroenteritis virus families. You can also get lost. For example, such an antigen-detection system is (Greenberg et al. (1978)), Cultivirus ( Nakata et al.), pestivirus (Yolken et al. ), and rotavirus (Dennehy et al.). It may also contain antibodies.

The present invention provides an easily renewable source of astrovirus antigens and for use in diagnosis. provides antibody reagents for For example, these reagents can be used for astrovirus antigens. in a solid-phase radioimmunoassay for astroviruses and against astroviruses. Can be used in radioimmunoassays for antibody pools (Gree Nberg et al. (1989)). The monoclonal and poly Clonal's astrovirus antibodies are also used in immunoelectron microscopy techniques. (Kapikian et al.).

(2) Nucleic acid approach The astrovirus nucleic acid sequences identified by the method of the present invention are Hybridization protocols to identify the presence of torovirus coding sequences Provides an array that can be used as a lobe. Polymerase chain reaction (PCR) ( Primers useful for S. klullis: Klullis et al. is shown in this sequence listing, or clone A2, All, A13, or Any of the nucleic acid sequences obtained from the insert of A33 can be obtained. difference Additionally, astrovirus sequences identified as described above in Section 111 can be used in this capacity. Typically these primers - are two nucleic acid sequences consisting of 8 or more co-linear nucleotides with a given separated by a distance, e.g. 500 bases, and homologous to opposite strands It is something.

Astrovirus-specific nucleic acid sequences can be used, for example, as done in Example 5C or 5D. Hybridization to detect the presence of astrovirus in a sample It can also be used as a probe.

A kit containing such PCR primers and hybridization probes may also contain similar primers useful for the identification of other gastroenteritis viruses. can.

Kits based on any of the above diagnostic methods are used to detect gastroenteritis viruses that cause human gastroenteritis. is a useful tool for aid in the rapid determination of whether the agent responsible for be. Furthermore, although person-to-person transmission is an important aspect of viral gastroenteritis, The initiating event for most cases of viral gastroenteritis is common source contamination (Lebaron et al.). Enteric viruses reproduce outside their host. Contrary to the bacterial pathogens present in the above common sources, The original inoculum determines the infectivity of its source. Therefore, the kit described above , also useful for identifying and proving contaminated sources (e.g. shellfish, crustaceans or water sources) It is.

A43 can be formed for use in astrovirus vaccines. Wear. The vaccine may be prepared by standard methods such as 1 in a suitable diluent, e.g. , in water, saline, buffered saline, complete or incomplete adjuvant, etc. can be accomplished. This immunogen was prepared using standard techniques for antibody induction. , for example, physiological substances containing inactivated or attenuated viral particles or antigens. Administered by subcutaneous administration of a sterile solution compatible with Immune response to viral antigens The reaction dose is typically a vaccination injection (vaccinizing injection). Typically, a volume of 1 milliliter or less is administered per ction).

A specific example of a vaccine composition is the combination in a pharmacologically acceptable adjuvant. A43 polypeptide produced by Alternative.

This vaccine was tested for significant titers of anti-astrovirus antibodies in its serum. Administer at regular intervals until release.

Such a vaccine can (1) cause the onset of gastroenteritis to be the same as that caused by astrovirus; short-term effects in community members who are uninfected with astrovirus. and (11) long-term prevention of astroviral infection. can be useful for active immunoprophylaxis.

C1 passive immunoprophylaxis The effect of viral gastroenteritis agents on patients with immune deficiencies is of particular interest (L ebaron et al.). Chronic astrovirus infection in the immunocompromised state Interventions, such as anti-astronomical Administration of viral immunoglobulin may be used in other situations, e.g. chronic diarrhea associated with malnutrition. It is also useful in protecting uninfected community members ( Lebaron et al.).

Antibody-virus complexes are recognized by macrophages and other effector cells. Therefore, the anti-astrovirus antibody of the present invention has anti-astrovirus immunity. It can be used as a means to strengthen responses. This antibody is Administered in the same amounts used for other therapeutic administrations. For example, the pool gamma globulin, which is used to block viruses from entering cells. initial incubation of viral diseases such as rabies, measles and hepatitis B. During treatment, 0.02-0.1 ml/Ib body weight is administered. Therefore, for example, Antibodies reactive with A43 antigens may enhance the immune response and/or the effectiveness of antiviral drugs. alone, in a 'cocktail' with other anti-viral antibodies, or with other anti-viral antibodies. passively administered together with a viral agent to patients infected with astrovirus can be done.

The following examples illustrate various aspects of the invention, but in no way do they illustrate the invention. It is not intended to limit the scope of the disclosure.

ringer & lannheim Biochemicals (Indian OT4 DNA ligase and T4 DNA ligase obtained from Apolis, IN) Limerase was purchased from New England Biolabs (Beverly, IA) and a dinitrocellulose filter from Cheicher. d5chuell (Keene, NH).

Synthetic oligonucleotide linkers and primers were prepared using commercially available oligonucleotides. Prepared using a nucleotide synthesizer. Or, it is designed as a standard product. For example, 5ynthetic Genetics (San Diego, CA). cDNA synthesis kit and and Random Briming Labeling Kit from BOehringer-& Iannheim Biochemical (BMB, Indianapo Obtained from lis', IN).

Cell lines LLCMK2 and AILL (JIK2 (infected by astrovirus type 1) stained monkey kidney cell line) was prepared by John E., Herrmann, Ph.D. ,, Division of Infectious Disease, Uni versity of Massachusetts &1dical Ce Obtained from nter.

Anti-astrovirus type I rabbit high virus serum H1 was prepared by John E, Herr. Obtained from rma'nn. A mouse model that recognizes all five types of astrovirus. Noclonal antibody 8E7 (Herrmann et at. (1988)) , John [:, l (obtained from 6rrmann. Astrovirus serotype Cells infected by John E. 24 and 5 were also infected by John E. Obtained from.

The above AI LLCMK2 and LL (JIK2 cell lines were used as previously described). cultured (Lee et al.: Kurtz et al. (1979 , 1984)) o The cultured cells were harvested and incubated at 3.000 x g for 30 min. Pelletized at °C. The resulting pellet was placed on ice while the supernatant was , 100.000 x g for 2 hours at 4°C. slow and fast speed Combine the tris and add 20ml of TN fart buffer-(50mM Tris -HCI, pH 8, 0, 100mM NaCl, 25m6 IgC12, 25 resuspended in mM CaC12) and placed on ice.

The above pellet suspension was mixed with “BLACO-TRON” (trichlorotrifluorofluoride). Ethane: Baron-Blakeslee, San Francisco, CA) and the aqueous phase was pooled from each extraction. child The extracted aqueous phase was pelleted at 100.000 x g for 2 hours at 4°C. Obtained The pellet was resuspended in 2 ml of TNIIC. This partially refined Half of the material was used for RNA extraction and half was run in a CsC1 gradient. It was further purified.

The presence of viral antigens was determined at each stage by ELISA (Rabbit H1 Detection- Lee et al., Kurtz et al. (1984); 8E7 capture The acquisition was monitored by Herrmann et al. Cell line LL (JIK2? Negative control samples were treated in tandem with AILLC & IK2.

Nucleic acids were extracted using a one-step guanidium/-phenol extraction procedure (Chomczynsk i et al.), the above partially purified astroviral material Extracted from RNA was precipitated using 2 volumes of ethanol and incubated at -70° It was stored overnight in C.

A, approximately IO prepared in cDNA fragment library preparation example 1 μg of nucleic acid was subjected to the method of Gubler et al. using oligo-(dT) primers. Therefore, it was transcribed into cDNA. Separately, to create a second cDNA library Approximately IO μg of the nucleic acid prepared in Example 1 was purified by the method of Persons et al. using Mannheim Primer (Boehringer Mannheim). transcribed into cDNA.

To ensure that the resulting cDNA molecule has blunt ends, a portion of the above preparation was cDNA in each in the presence of all four nucleic acids using a T4 DNA polymerizer. Qlaniatis et al.).

B. Amplification of the above cDNA fragment The resulting cDNA molecules were amplified using the sequence-independent single primer amplification (SISPA) method. and amplified it. This 5ISPA technology was applied for on July 26, 1988. NA and DNA Amplification Technique,” Co-owned U.S. Patent Application No. 224.961 (herein incorporated by reference) (incorporated into).

The blunt-ended cDNA molecule from above has the following sequence: 5-GGAATTCGCGG CCGCTCG-3'3-TTCCTTAAGCGCCGGCGAGC-5゜ (^1aniatis et al.).

The above cDNA and linker were combined into 74 DNA with 0.3 to 0.6 WeiSS units. Mixed in the presence of ligase at a molar ratio of 1:100. 1.5nt mouth 1g CI2 and 10m&I Tris-)1cI buffer containing 50rnM KCI, pH 8 , 3 (buffer y A)] with about 1 x 10" μg of the above linker. The finished cDNA, the above sequence d (5-GGAATTCGCGGCCGCTCG- 2 μ^1 primers with 3’”), 200 μM dATP each, d CTP, dGTP, and dTTP, and 2.5 units of subanal aquat icus DNA polymerase (Tag polymerase) (Perkin-Elm er Cetus) was added. The reaction mixture was incubated at 94 °C for 3 Heat for 0 seconds and cool to 50°C for 30 seconds for primer annealing. and then 72° for primer extension with Tag polymerase. Heat to C for 0.5-3 minutes. Continuous heating, cooling, and polymerase reactions The above replication reaction containing Perkin-Elmer Cetus DNA Th Repeated another 25 times with the help of an ermal cycler.

The amplified cDNA fragment was treated with EcoRI. excess linker -, "5EPHACRYL 300" (Pharmacia, Piscat away (NJ).

RNA obtained from the above cell line LLC&lK2 was treated as above, with the exception that , the LLCλlK2 amplified cDNA molecule described above was treated with EcoRI. There wasn't.

C0 Transfer of the above amplified AI, LLCMK2 cDNA to lambda gt11 cloning Phosphatase-treated lambda gtll phage vector arm Obtained from Promega Biotec (Madison, Wl.). child The lambda gtll (Huynh) vector of Contains a unique EcoR1-cloning site 53 base pairs upstream from the ing. Amplified AI LLCI+lK2 cDNA from Bart B was added to 0. 5-1.0 μg of the above cDNA molecules with an EcoRI cleavage gt ratio of 0.3-3 μm, 0 ．． 5 μm IOX ligation buffer (above), 0.5 μl DNA ligase (20 0 units) and add up to 5 μl of distilled water to the above costal gland light site. It was introduced in This mixture was incubated at 14°C for -night, then a standard according to the method (Ianiatis et al., pp. 256-268). and packaged in vitro.

The above packaged phage was transferred to Kano, Kevin & 1oore, DNAX ( Escherichia coli obtained from Palo Afto, CA) coli strain KIl1392. (Alternatively, Arn erican Type Culture Co11ection (ATCC# Escherichia coli strain Y available from 1090 could also be used. ) Continuation of the above packaged phages Lawns of &l392 infected by dilution, the titer of the phage used to determine. For immune screening, above approximately 10’ pfu The recombinant phages described above were plated on 8Man plates for each 150 mm plate. iatis et al.).

Random briming (rp) or oligo-d(T) briming (dT) The cDNA library in lambda gtll (Example 2) obtained by High titer antibody against (ELISA titer ≧l: 1000: Lee et al.

; Kurtz et al. (1984)); Virus-immunized rabbit serum was used for immunoscreening. This serum Large cells infected with the lambda gtll vector described above that do not contain any cDNA inserts. It was pre-absorbed with a lysate of E. coli. Each serum was For epidemic screening, AIB (TBS buffer, 10 ntl Tris , pH 8, 0, 150mM NaCl: 400x (contains 1% gelatin) (Young et al, + Au5ubel et al, ).

In the first immunoscreen with H1 serum, lambda gtll (dt) The 10 putative reactive plaques from Thirty-three putative antimicrobials identified from replacement phages and from lambda gel (rp). Reactive plaques were identified from a total of 0.8 x 10' recombinant phages. Ta. After a third screening for plaque purification, oligo-d(T) Three clones (lambda-A35, lambda-A 43, and lambda-A39) and randomly brimmed 7 clones from the library (Lambda-AI, Lambda-A2, Lambda -All, Lambda-AI3, Lambda-A14, Lambda-A21, and Lambda-A 33) or remained reactive.

The size of the above cDNA insert (Table 1) was determined from the εc of the above lambda gtll clone. oRI treatment, followed by agarose gel run in parallel with DNA size-standards. Determined by electrophoretic separation of the treated fragments above.

Table 1 Clone number (approximate size of insert (base pairs)) A35 230 A43 190 A39 400 Al 430 A2 410 All 160 A13 480 AI4 410 A21 410 A33 410 Example 4 Further immunological characterization of the lambda gtll clones described above Immunological screening of tll clones (Example 3: Table 1) was performed using the following pairing (pa ired) serum 81, and pre-immune rabbit serum: and 8E7, and pre-immune rabbit serum. Expanded to include mouse serum from P. aeruginosa. Immunization of the above lambda gtll clone The responsivity was measured between plaque-purified phage and non-recombinant lambda gtll in a ratio of l, l. were mixed and assayed by plaque-blating. reactivity Plaque (Young et al.: Au5ubel et al.) , after incubation with the above test serum, alkaline phosphatase-binding Pressure was detected by using an anti-1gG second antibody (Pierce, Roc kford IL).

The responsivity of pre- and post-infection sera against the above lambda clones is given in Table 2. Ru.

Table 2 Old number: 8E7 (pre-immunized rabbit) Previously-immunized mouse (as seen in Table 2) However, the protein products of all IO clones reacted only with rabbit serum post-infection; It did not react with the monoclonal 8E7 antibody mentioned above.

A, Ibridizane with human and E. coli genomic DNA The above cDNA clones of A35, A43, and A39 were Hybridization (Southern: 1. Ianiatis e These DNA fragments in the circle were placed in parallel lanes by and electrophoretic separation on a 1% agarose gel. This DNA fragment is Transferred to trocellulose.

Radiation of the lambda gtll cDNA inserts of A35, A43, and A39 above A sex-labeled probe was made as follows. Adjacent to the above cDNA insert Primer of known lambda gtll sequence (5'-GGCAGACATGGC CTGCCCGG-3° and 5-TCGACGGTTTCCATATGGGGG −3°) and the above cD by &Iullis polymerase chain reaction (PCR). Used for amplification of NA inserts.

A typical PCR cycle has the following steps: lysis at 94°C for 30 seconds, followed by 5 This included annealing at 0°C for 1 minute, followed by extension at 72°C for 30 seconds.

The above reaction was repeated for 30 cycles. This PCR product was decomposed by Ta. The clone-specific amplified fragments are identified by size; Then, electrophoresis was performed to completely remove the DNA band using an NA45 membrane (Schleiche membrane). r & 5chue11. Keene, NH).

This DNA was transferred from the membrane to high salt buffer - (Schuell & 5chu eIf, Keene, NH) and phenol chloroform. and ethanol precipitation. After ethanol precipitation , this DNA was subjected to random-briming DNA labeling (8 oehring As a mold for Er8 Iannheim, Indianapolis, IN) I used it.

The above nitrocellulose filters are used for A35, A43, and A39 filters. Radiolabeled probes and hybrid dye B made from each of the above cDNAs Cross-hybridization test between clones The inserts of the 10 lambda gtll clones (Table 2) described above are described in Example 5A. Isolate as shown. These 10 inserts and negative control DNA were transferred to Nitrocell. Applied to loin filter.

The above inserts were subjected to random-briming DNA labeling (Boehringe As a template for R.Klannheim, [Indianapolis, IN) was used to create a 32P-labeled insert-probe. Then this filter - were hybridized separately with the labeled insert-probe.

The results of this hybridization test are based on the cross-hybridization shown in Table 3. ization pattern.

A35 A43 A39 All A14 A21 A33 A35 + + - ND" ND ND NDA43 + + -ND ND ND NDA39 − − + ND ND ND NDAIIND −−+ −−− A13ND+--W+-- °・Not measured These results showed that when clone A43 was used as a probe, clone A3 5, indicating that AI, A2, and A13 were cross-hybridized. . This result shows that all of these clones have the same genome as the astrovirus. This suggests that it originates from an adjacent region. Clone A39, All, A14, A2+, and A33 are cross-hybrid with any of the other clones. Did not breed.

C1 with the above lambda g [11 clone insert and astrovirus-infected and uninfected Hybridization of the above lambda with cDNA made from cellular mRNA gtll clone A43, A35, A39, All, A33, A14 , and A21 inserts were isolated and radiolabeled as before.

made from both the AI LLCMK2 and LLCλIK2 cell lines (Example 2B) above. The S[SPA amplified cDNA was loaded onto parallel lanes of a 1% agarose gel. , and electrophoretically separated. This cDNA was extracted using the standard procedure Qlaniatis etc.

) was transferred from the gel to nitrocellulose paper. This nitrocellulose Filters were hybridized to the radiolabeled probe described above. above All of the probes were specific for the 5tSPA cDNA from AI LL (JIK2). S[SPA] from the LLCMK2-uninfected cell line described above. It did not hybridize to cDNA. Clone A43, A35, A39, A ll, and A33 inserts are the amplified AILLfJIK2 cDNA described above. It strongly hybridized to A. The above A14 insert is the above AI LLCAI K2 cDNA, and the A21 insert described above hybridizes weakly to the K2 cDNA, and the A21 insert described above 2 cDNA was hybridized to AI LLCM (at medium strength).

D. Supports that it represents a part of the Dot-Blot test sum. In order to maintain the RNA, AI LLCMK2 (Astrovirus infection) and L Extracted from LIJIK2 (astrovirus uninfected) cell line (Example 1). This R The NA preparation was purchased from the manufacturer of the “MINIFOLD l” device (Schleicher and 5chue11. Hybridizers following the recommendations of Keene, N)I) prepared for the ization assay.

Hybridization was performed using 50% formamide and IX hybridization. Buffy - (5x Denhardts solution (8 Ianiatis et al. al,), 5x SSC (Maniatis et al,), 50mM NaH2PO4, Intl sodium pyrophosphate/Na2HPO4 , 100 μg/ml denatured salmon sperm DNA, 100 μg/ml ATP). went. The hybridization probes used were prepared as previously described. Lambda gtl + clone A43, A35, A39, All, A There were radiolabeled cDNA inserts of I4, A2+, and A33. C After hybridization, the filters were (i) incubated in 2x SCC for 15 min at room temperature. -30 min and (ii) 65° in 0.1x SCC containing 0.1% SDS. Washed with C for 1 hour. The filter is dried and then exposed to X-ray film. did.

The results of the above hybridization assay are shown in Table 4.

Table 4 AI LLC 8I to 2 RNA LLCλlK2 RNA above insert probe hybridized to nucleic acids from only the infected cell lines described above. Uninfected cell line as above No hybridization with nucleic acids isolated from was demonstrated. typical A typical dot plot test is shown in Figure 1. There, the A43 clone insert described above was used as a probe.

E, co-transfer of astrovirus antigenic activity with A43-specific sequences cells were prepared from AI LLCMK2 tissue culture cells as previously described in Example 1. Manufactured. This astrovirus preparation was further treated with 40-55% CsC1, Purified by centrifugation at 100,000 x g maintained at 4°C on a gradient. Ta. Fractions (0.25 ml each) were placed in a bottom puncture. I recovered it. Fractionation of IO over a density range of 1.27-1.46 g/ml was examined (Figure 3). Determined by εLISA (Herrman et at.) The peak of the determined astrovirus antigen was found near the fraction with a density of 1.37 g/ml. (Figure 3).

Nucleic acids were extracted from the 10 fractions described above using a one-step guanine rum/phenol extraction procedure. (Chomczynski et al.) and the above-M Manufacturer of the IN [FOLD I' device (Schleicher and 5chu) e11. RNA dot plot high following the recommendations of Keene, NH) prepared for hybridization assay.

Hybridization to an A43-specific, radiolabeled probe was performed using 0% formamide and IX hybridization buffer 7 (5X D enhardts solution (Maniatis et al.), 5X SSC ( Maniatis et al.), 50mM NaHtPO4, 1mM Na Thorium pyrophosphate/Nat HPO4, 50mg Denatured salmon sperm DN A1500rnl, 50mgATP1500ml). High used Hybridization probes were labeled as described in A4 above.

After hybridization, the filters were (i) in 2X SSC at room temperature. 15 min and (ii) 65° in O,IX SSC containing 0.1% SDS. Washed with C for 1 hour. The filter is dried and then exposed to X-ray film. did.

As can be seen from the results of the above hybridization assay (Figure 3), the above A4 3 hybridizes strongly to the nucleic acid from fraction 5 and hybridizes weakly to the next fraction. Redized. Hybridization with nucleic acids isolated from fractions 1-4 It wasn't shown at all.

The above astrovirus antigen peak was blocked by the above A43-specific probe. The hybridization peaks seen on the probed RNA plots and corresponding to.

F, Hypothesis of A43 clone-specific probe with other astrovirus serotypes. Bridification The A43-specific probe described above was made as described in Example 5E.

RNA was collected from cells infected with astrovirus serotypes 2.4 and 5 (as before). sea urchin) isolated. This RNA was analyzed using an RNA dot plot as described above. - Prepared for hybridization.

In FIG. 2, RNA from uninfected cells is indicated by “U” and as described above. RNA from strovirus serotypes [-1 (infected with serotype l) to I -5. The results shown in Figure 2 are based on the A43 clone-specific protein. hybridizes to nucleic acids from cells infected with other astrovirus serotypes. It has been proven that the

G. Nucleic acid properties of the above-mentioned astrovirus genome (i) The above-mentioned astrovirus The genome of is an RNA molecule.

The nucleic acid extracted from AI LLCMK2 cells (Example 1) was processed according to the manufacturer's instructions above. was treated with RHasaA and DNase (both enzymes were Obtained from inger & Iannheim, Indianapolis IN did. ). Next, convert this sample to a dot plot/ 1 hybridization. This probe was used in Example 5F. The above A43 clone-specific probe was used. Processing by RNaseA ( Figure 4A, columns I, R) show the interaction with the A43-specific, radiolabeled probe. completely devoid of hybridization, whereas DNasel treatment (Fig. 4A, Column 2) had no effect at all. RHaSaA or DNase [Samples that were not treated by any of the above are shown in Figure 4A, column 3 (U). show.

(ii) The genome of the above-mentioned astrovirus has positive polarity.

Both the A43 positive strand (i.e. coding) and the A43 negative strand above. Corresponding single-stranded oligonucleotide probes were synthesized by standard procedures.

This oligonucleotide is combined with the synthetic oligonucleotide primer γ-[ ``P] Radiolabel by phosphorylation by ATP (Richardson) I did it.

Nucleic acids from AI LLC & IK2 cells were subjected to hybridization as described above. Prepared for. Hybridization was carried out in 30% formamide and IX Hygiene. hybridization buffer (see above).

) at 42°C. This filter is 2X 5SC (&Ianiat is et al.) for 30 minutes at room temperature.

Only the negative sense, synthetic A43 oligonucleotide probe described above Figure 4B. (l) This indicates that the above RNA has positive polarity.

When using the positive sense synthetic A43 oligonucleotide probe described above. No hybridization with the above extracted RNA was detected. (Figure 4B, spot 2).

Lambda gti + clone A43, A35, A39, AI, A14, and The A21 cDNA insert was inserted into the “BLLIESCRIPT KS+” vector (S tratagene, Ia Jolla, CA).

Briefly, this insert was amplified as described above in Example 5A. Illis; Iullis et al.) a However, the following primer m; 11F-H3: TGGCTGAATATCGAAGCTTTCCATATGG GGllR-Xho: TGGATTTCCTTACTCGAGATACGGG CAGAC was used.

These primers contain a lambda sequence adjacent to the cDNA insertion site, as well as t(indllll restriction site and second primer cloak in one primer) It was cloned into the "LUESCRIPT KS+" vector.

The sequence of the above cDNA insert was sequenced using the dideoxy chain termination technique (Sanger, 197 9) was determined according to the manufacturer's instructions. Each of the above clones is into the β-galactosidase reading frame of the lambda gtll vector. It had one adjacent oven reading frame. This array data are represented in the sequence listing as follows: A35, SEQ ID NO. 1; A43, SEQ ID NO. 3: A39, SEQ ID NO: 15; AI, SEQ ID NO: 7; A14, SEQ ID NO: 9: and and A21, SEQ ID NO: 11゜SEQ ID NO: 15 are terminals used in the above amplification reaction. Contains the terminal S[SPA sequence (SEQ ID NO: 15 residues 1-17 and 428-444). Distribution sequence to the “GENBANK” sequence at both the nucleic acid and amino acid levels. did. This “GENBANK” search indicates that these sequences have been previously characterized. indicated that it did not represent a nucleic acid or amino acid sequence.

The DNA sequences of the above six clones were aligned for comparison. Expressed first in example 5 As expected from the cross-hybridization data, the above clone A43, A35, and AI have overlapping sequences (Fig. 5) and Loans A39, AI4, and A21 had independent sequences. In Figure 5 The sequence given in is also represented as SEQ ID NO: 13.

A. Production of antibodies Antibodies are raised against any of the cloned astrovirus antigen coding sequences described above. can be done. For example, the above insertion procedure from the above lambda gtH clone A43 The biological fragment is released by EcoRI-treatment of the phage and its The inserted region is purified by gel electrophoresis. This purified fragment was transferred to pGE Glutathione S-transferase protein in X expression vector (Smith) Introduce it into a frame with quality. Contains the above A43-encoded polypeptide antigen Expression of glutathione S-transferase fusion protein (Sj26 fusion protein) , can be achieved in E. coli strain KM392. This fusion Proteins were extracted from lytic bacteria according to published methods (Smith). Affinity chromatography on columns packed with tathion-conjugated beads Isolate by

The above purified 5j26/A43 fusion protein was incubated in rabbits with Freund's horsetail. It can be injected subcutaneously as a adjuvant. Typically about 1 mg of fusion protein was injected on days 08 and 21, and rabbit serum was injected on days 42 and 56. collected.

The above procedure was repeated for β-galactosidase/A43 polypeptide antigen fusion protein. It can also be used to make antibodies. Here, we will explain the fusion protein as an example. For example, use p-aminobenzyl l-thio β-D-galactosidase-agarose. Isolate by affinity chromatography.

B. Specificity test The specificity of the above antibodies was determined by Western blot screening (Ausubel et al.). western plot script For training, the lambda gtll/A43 phage from Example 3 was cultured in E. coli (E. , coli) BNN103 used to infect temperature-sensitive bacteria . These bacteria were transformed into American Type Culture Ca 1lection, Rockville AID.

This bacterial host is infected with the above vector under temperature induced conditions (Hunyh). Expresses the encoded β-galactosidase/-polypeptide antigen fusion protein I can do things.

Minilysate is prepared from the infected bacteria as follows. Prepare. The above-mentioned infectious bacteria were incubated at 32°C in a streak of L/L. Grow overnight or until colonies form, then replicate individual colonies. - Blated and tested for growth at 32°C and 42°C. Up Indicating the integration of the phage genome, it grows at 32°C but not at 42°C. NZYDT (Maniatis) blot of Irn1. was used to inoculate S.A. This overnight saturated bacterial culture was added to l It is used to inoculate 0 ml of culture solution, and this is inoculated with about 0.2 to 0 ．． to an absorbance of 4, typically requiring 1 hour incubation. Incubate. This culture medium is then used to induce lambda gtll polypeptide synthesis. To conduct the test, quickly (bring to 43°C and Shake for minutes and incubate for an additional hour at 37°C. this cell , pelleted by centrifugation, and 1 ml of this pelleted material was 1 of lysis buffer (5% mercaptoethanol, 2.4% SDS and 10% glycol) Resuspend in 62 mAl Tris containing recerol, pH 7,5).

The lysate was transformed into (a) pGEX, and (b) pG containing the A43 insert (above). From bacterial strains of &l392 containing EX, essentially as described immediately above, It is also possible to omit the initial temperature selection step described above.

The lysate is made of a To treat the terrier DNA, it was treated with DNase. This DNase treatment A portion of the bacterial lysate (typically about 15 μl) was transferred to “TRITON X-100” and diluted with sodium dodecyl sulfate (SDS) and 2% “TR[TON Final concentration of X-100- and 0.5% SDS. Centrifugation of undissolved substances and the supernatant was subjected to SDS polyacrylamide electrophoresis (SDS -PAGE). A portion of this gel was stained with the polypeptide antigen of interest. protein in the corresponding unstained portion of the gel, using a known method. Nitrocellulose filter according to the method (Ausubel et al.) Moved to.

Briefly, the above filter is blocked by AIB and then react with serum samples from rabbits immunized with sea urchins. Above nitrocellulose The presence of specific antibodies that bind to the filter is detected using alkaline phosphatase. It can also be assayed by immunobinding of Bell anti-rabbit IgG. The above cloth The results of the Tan blot assay showed that positive anti-astrovirus antibodies were present in pGEX-A 43 and lambda gill/A43, but the above pGEX and It is expected to show no reaction with lysates from uninfected control strains. lambda The reactivity with gtll/A43 is determined by the astrovirus coded portion of the above fusion protein. It shows the specificity to In addition, anti-β-galactosidase antibodies (commercially available (hand available) is a fusion protein obtained from the lambda gtll/A43 infected lysate described above. expected to respond only to

Anti-A43 antibodies present in serum from animals immunized with 5j26/A43 can be purified by affinity chromatography, where is derivatized to the above '5EPHARO5E' beads or Gant is derivatized to the above '5EPHARO5E' beads. This is a purified β-gal/A43 fusion protein. From astrovirus-infected human specimens The human anti-A43 antibody from the obtained serum was added to the above A43 antigen-polypeptide. by derivatizing the support beads and passing the serum over the support. can be obtained in essentially the same manner as described above.

Example 8 Preparation of astrovirus nucleic acid from infected stool samples A, Preparation of astrovirus nucleic acid Partially purified human volunteers are given orally an astrovirus-infected inoculum. (Kurtz et al. (1979)). Clinical stool specimens from the above patients Obtain. The presence of virus particles was detected using an immunoelectron microscope (IIJIXKaρ1kja). Detected by n etal, ). Previously infected stools from the volunteers mentioned above are: Serves as a negative control in the steps below.

Approximately 7.5 grams of the above astrovirus-positive stool specimen was transferred to a sufficient amount of TN^ (C bag). Fy-(50mM Tris-J (CI, pH 8, 0, 100mkl Na C1,25+r1 mouth 1gC12,25mM CaC12) and mix with 10( Make a fecal suspension of % w/v). This suspension was shaken vigorously and 3.000 Pelletize at 4°C for 30 minutes at Xg. Place the resulting beret on ice, Meanwhile, the supernatant is pelleted at 100.000 x g for 2 hours at 4°C. child Resuspend the fast pellet in 20n+I TNMC buffer.

The above pellet suspension was mixed with "BLACO-TRON" (trichlorofluoroether). Baron-Blakeslee, Inc., San Francisco o, CA) and pool the aqueous phase from each extraction. This extraction The aqueous phase was pelletized at +00.000 x g for 2 hours at 4°C and the pellets obtained Resuspend the sample in 2 ml TNMC. This suspension was mixed with a CsCl cushion ( 1,58/ml) on top of the 20% sucrose cushion. and rotated at 80,000 x g for 3 hours at 4°C using a Beckman SW28. make it turn The band at this interface is retrieved by lateral puncture. a The presence of strovirus antigens was determined by EL [SA (Herrmann et al. : Lee et al, : Kurtzet al,) Monitor on the floor.

Negative control stools as above and other IIJI Astroviruses from the same volunteers as above. rus-positive infected stool samples were processed in tandem.

A one-step guanidinium/phenol extraction procedure for nucleic acids from partially purified stool. (Chomczynski et al.).

B. Preparation of cDNA fragments Approximately 10 μg of the nucleic acid prepared in Example 8A was transformed into cDNA as described in Example 2A. can be transferred to

C, amplification of cDNA fragments The resulting cDNA molecule was conjugated to a sequence-independent single plate as described above in Example 2c. Amplify using immer amplification (SISPA).

Poly(A) from lysates of astrovirus-infected and control uninfected cells (Example 1) RNA was conjugated with oligo(dT) essentially as described by Sambrook et al. ) - 500 μg of total 1 on cellulose column? Isolated from NA.

The recovered poly(A) sufficient fraction was precipitated with ethanol and washed with 70% ethanol. , and the final pellet in preparation for electrophoresis with 6 μm diethyl Resuspend in pyrocarbonate (DEPC) treated water.

RNA samples were transferred to x, 2 (w/v')' Loaded into a well in lumaldehyde gel and incubated at 5 V/cm for 4 hours. Convert ([rminger, et al,). After electrophoresis is finished, remove the above gel. Rinse the tube with distilled water and place it in IOX SSC (Maniatis, et al.). The RNA was equilibrated in 2OX SSC by overnight capillary transfer in 2OX SSC. Nitrocellulose filter (Schleicher and 5chu ell). Next, this film was baked in a vacuum oven (80"C for 12 hours). ), and 50% Borumamide, 5X SSPε (^1anjaHs, etc. al,), 2X Denhardt's solution, 0.1% SO3, 1° Shiji, and 100 μg/ml of each modified fragment Iwake Test DN. A and wheat germ tRNA for at least 4 hours at 42°C. It was incubated.

After prehybridization as described above, radiolabeled and random-blended (“PRIME-IT” kit, Stragene.

La Jolla CA) Astrovirus clone-specific probe (one A43-specific probe: and one A39-specific probe, of which region 5 The internally mutated 5ISPA linker (SEQ ID NO: 15, nucleotide 18-191), and the above-mentioned projections from the region 3'. Insertion sequence up to the natural linker (SEQ ID NO: 15, nucleotides 241-417) separately) to the hybridization vessel containing the membrane described above. and incubated overnight at 42°C. Wash the above membrane 2X in order. SSC, 0.1% SDS, 65° Cl2O min: then 0.2X SSC, 0.1% SDS, 65° Cl2O min: and short rinse with 2X SSC ), and prepared for autoradiography.

The above plot between hybridizations with different probes stripped from the bush (Ianiatis, et al.).

The above membrane was exposed to X-M film. The obtained autoradiogram is shown in Figure 7. show. In Figure 7, lanes 1.3 and 5 are cells of the astrovirus-infected cell line. lanes 2.4 and 6 contain poly(A)+ RNA extracted from cells, and lanes 2.4 and 6 contain poly(A)+ RNA extracted from Contains poly(A) + RNA extracted from cells of the infected cell line (Figure 1). Re clones 1 and 2 were blown with astrovirus clone A43-specific probe. I nged. Lanes 3 and 5 were labeled with a probe specific for the clone A3903 half. Blobbing. Lanes 5 and 6 contain a protein specific for the 5' half of clone A39. Blobbing with a robe.

In Figure 7, the Astrovirus clone A43-specific probe 7.2 Kb poly(A)+RNA extracted from virus-infected cell line cells (Lane J). Furthermore, 3゛ half of clone A39 The specific probe (described above) probes the genome located within the lysate of astrovirus-infected cells. (7,2 Kb) and subgenomic (2,8 Kb) poly(A)+RNA (Lane 3).

By control, the probe specific for the 5' half of clone A39 (above) was Or, it does not recognize the viral poly(A) + RNA (lane 5), which means that The above 5' sequence of the internal 5 ISPA linker is not specific to Astrovirus. It suggests that. Astrovirus-specific 3° sequence of the above internal 5ISPA site is expressed as SEQ ID NO: 5. These astrovirus clone-specific proteins None of the probes hybridized to poly(A)+ RNA from uninfected cells. (lanes 2.4 and 6).

Although the invention has been described with reference to specific aspects, methods, constructions, and uses, It will be appreciated by those skilled in the art that various changes and modifications can be made without departing from the invention. That should be obvious.

Sequence list (1) General information.

(i) Applicant: Kim, Jungsuh P.

^1atsui, 5uzanne M.

Greenberg, Harry B.

Reyes, Gregory R.

(ii) Name of the invention: Niastrovirus human gastroenteritis agent and corresponding cDNA molecule cloning (iii) Number of arrays: 216 (iv) Contact information: (A) Address: Law Office of Peter J, Dehli nger (B) address: P, 0. Box 60850 (C) City: Pa1o Alt.

(D) State, CA (E) Country: USA (F) Postal code: 94306 (v) Computer readable format.

(A) Media format: Floppy disk (B) Computer: BMPC compatible (C) Operating system: P C-DOS/Nl5-DO3CD) Software: patenHn Re1e ase #1.0. Version ff1.25 (vii) Previous application data : (A) Application number: US 07/702.731 (B) Application date: May 1991 20th (C) Classification: (viii) Patent attorney/agent information: (A) Name: Fabian, Gary R.

(B) Registration number + 33.875 (C) Reference/Case Number: 4600-0079.41 (ix) Telecommunication Information: (A) Phone: (415) 324-0880 (B) Fax: (415) 324-0960 (2) Information regarding sequence number l: (i) Characteristics of array: (A) Length: 225 base pairs (B) Form - Nucleic acid (C) Number of strands - double strands (D) Topology bilinear (2) Type of sequence: cDNA-mRNA (1st item) hypothetical sequence 2N0 (iv) Antisense No. (vi) Origin: (C) Isolated clone name Astrovirus serotype AI (vii) Direct origin: (B) Clone salt: A35 (ix) Array characteristics: (A) Symbol representing characteristics: CD5 (B) Existence location 11.225 (xi) Sequence: Sequence number 1: 'jcA GAA Ding AT AA CAA CAA GT στ AAG τCτ AτCA＾GCCτ CAG Enter AG AGτ S8 s@r Glu Tyr GLu CLr+ Gin Val Val Lye Ser engineering 1ea Lys Pt mouth Gin Lye Ser Yu 5 10 Yu 5 GAcc: lj,% CCA TAC: TCλ AA 807 TAC (:CC entered AG CCA CCλ entered TC CCCAAA 96Glu Pro G in Pro Tyr 5er Gin Thr Tyr Gly Lye A la pro engineering 1e ding rP ni 1uTl:? 'r, CG,! ,? ττT G into GACT Enter AT into GA GAT GAT into GCCAA Trr A ττ　CτGCCA@144 Ser Tyr Agp Phe AI! p　Trp　Asn　Glu　A! I P Asp Ala LyIs Phe ko541) 45 GCG CCA nc CG ττOACτ AAG GCA AT GA A　A’a　G’rCr　GGA　r　AAA　192 people 1a　X? to Ty x　ju″g　Lau Thr　Ly　^yo Todoroki Asp　Glu　es　V al Lau Gly 5ar Lx torture so　ss　s.

ATCGTCλ=a Cττ AGA ACO8ff1Aττ GAA AGA ace 225 Engineering La VaL Lys Lau Arg Tehr Engineering 1c Engineering Lm Glu Thr ALa65 Fu O75 (2) Information regarding sequence number 2: (1) Array characteristics: (A) Length near 5 amino acids (B) Form, amino acid (ii) Type of sequence: protein (xi) Sequence: Sequence number 2: (2) Information regarding sequence number 3.

(i) Characteristics of array: (A) Length = 184 base pairs (B) Form: Nucleic acid (C) Number of chains: double strands (D) Topology, linear (ii) Type of sequence: cDNA-mRNA (iii) Hybothetical sequence 2N0 (1v) antisense=NO (vl) Origin: (C) Isolated clone name Niastrovirus serotype AI (vii) Direct origin: CB) Clone name: A43 (1x) Array characteristics: (A) Symbol representing characteristics: CD5 (B) Location: 2. ．． 184 (xi) Sequence: Sequence number 3゜ Glu pro Glu VLIGlu 5er Gin pro Lau A jp Leu Set Gin Lyll Lyli G attack ■ m cy rcx cλA’n ding Gλ^ CAA CAA GTG GTCA AG TCT ATCAAG 00 cac 142 (2) Regarding sequence number 4 Information: (1) Characteristics of array.

(A) Length: 61 amino acids (B) Form diamino acid (D) Topology, linear (11) Sequence type/protein (xi) Sequence: Sequence number 4: Pro Glu Val (:lu Ser Gin Pro Lau Jui p　Lau　5ar　(:ln　Lye　Lys　Glu　kY@ 20　25　koO G Yun Sex Glu Tyr Glu G engineering n G engineering n Va engineering Val Ly s 5ar Any Lys Pro Glx Lyeko 5 40 45 5sir Glu Pro Gin Pro’ir Set Ginτhr Information regarding Tyr Lys Jua (2) SEQ ID NO: 5: (i) Characteristics of array・ (A) Length: 202 base pairs (B) Form Nucleic acid (C) Number of strands: 2 strands (D) Topology, linear (11) Type of sequence: cDNA-mRNA (iii) Hybothetical sequence 2 N0 (iv) Antisense No. (vi) Origin: (C) Isolated clone name Niastrovirus serotype A1 (vii) Direct origin: (B) Clone name/3″ part of clone A39 (SEQ ID NO: 15) (C) Sequence characteristics Symptoms: (A) Symbol representing characteristics CD5 (B) Location: 1. ．． 201 (xi) Sequence: Sequence number 5: GTA CCT cTG ACA TTTGAA GGA ACTCCT GG A? CA CCA TTA AτAAτG product τ 48 Val Pro Val Thr Pha Glu Gly Sar Ala (:ly Ser Pr Mouth Lau Engineering 1 Mat As■ (:TC"rcx cλG to 5 JlkGC(mA ding ττT CCA C GA ACA (:Tr C7 CCA ccc τ(, Tang`eA 96 Van Sar Glu Gly 5ar Hls Phe Ala Argτ hr ValLeu Ala Arg 5erτ ACA CCA Ace A CT CrA GCG CGT CCA GGA GAG AGA ACCAC CτC＾　GλCACA　14S Tksx Pro Thx Thx Lsu Ala Arg AλaG yG Engineering U Arg Thr Thx 5 Asp Thx 5 40 45 G’TA TGG WAG G’rCGTCMτ ACA GCT (iTA T(T GCT GCT GAG CrA GτCACG P92 VLITrp Glxr Val Lau Agn Thr 1a Val 'S Nijiiri 1a Ala Glu Lsu Val hr5p 55 60 CCT　CCT　CCG　C2゜2 Pro　Pro　Pr.

(2) Information regarding sequence number 6: (i) Characteristics of array: (A) Length 267 amino acids (B) Form diamino acid (D) Topology: linear (ii) Sequence type/protein (xi) Sequence: Sequence number 6゜ Val Pro Val Ding’hr Ph・(lu Gly Sar Ala (: 工 y Sex Pro Lau 工 IS) let `sn 1 S 10 15 Val Sex Glu Gly 5er 1lis i’h@Ala JLr g Thr Val Lsu Ala JLrg Byτ Thr pro T hx Thr Lsu Ala Arg Alm (:ly Glu Argτ hr Thr Set AQ ThrVa Engineering Trp Gin Val Lsu jLgn 7hx Ala Val Ser Ala Ala (:lu L eu Val 〒■■ 5Q 55 60 Pro　Pro　Pr.

(2) Information regarding SEQ ID NO: 7: (1) Characteristics of array・ (A) Length, 136 base pairs (B) Form: Nucleic acid (C) Number of strands: 2 strands (D) Topology: linear (11) Type of sequence: cDNA-mRNA (iii) Hypothetical sequence 2 N.

(1v) Antisense 2N0 (vi) Origin: (C) Isolated clone name Niastrovirus serotype AI (viD direct origin: (B) Clone name: Al (ix) Array characteristics: (A) Symbol representing characteristics: CD5 (B) Location: 1. ．． 135 (xl) Sequence: Sequence number 7゜ JLTT AπG product ^CA GCCAτA JLAG Aσ demand T Te A Ding ACT 48C Nikoichi-〇口GτA 'ffl' GAGσC IC Thoughts Gyu CστATG product GCh 96Jug Lliu Pro GLu Jua Va l　Phe　Glu　Leu　AIIP　Lyll　Ala　Ala　T’yr 　Glu@Ala ccT TTG GAA GG? TTCCXC (JA ACG (TT AA) AτCA AAA AM A 136Gly L, u C1u Gly Pha Lsu Gin krq Val LyllISw Lyll LY Nikko S 4 0 45 (2) Information regarding SEQ ID NO. 8.

(i) Array special ifi: (A) Length: 45 amino acids (B) Form, amino acid (D) Topology: linear (ii) Sequence type/protein (xi) Sequence/Sequence number 8: Ala L, u I’ro Glu Jua Val phe GLu Leu kBp LYII Ala Ala Tyr Glu A attack■ Gay lJu Glu Gly Pha Lau Gin A! ″g Val ! , Y# Bar Lyg 1-Y Massage (2) Information regarding sequence number 9: (i) Characteristics of array・ (A) Length: 289 base pairs (B) Shape, nucleic acid (C) Number of strands, double strands (D) Topology, linear (E) Type of sequence: cDNA-mRNA (iii) Hypothetical sequence, N .

(1v) Antisense 0 (vi) Origin (C) Isolated clone name ° Astrovirus serotype AI (vii) Direct origin CB) Clone name A14 (1x) Array characteristics: (A) Symbol representing characteristics CD5 (B) Existence position +2. ．． 289 (xi) Sequence: SEQ ID NO. 92 (2) Information regarding sequence number 10・, (i) Sequence characteristics.

(A) Length: 96 amino acids CB) form, amino acid (D) Topology: linear (11) Type of sequence: protein (xi) Array: Sequence number IO= Arg At5n Pro Val Thr Thr Thr Leu Gin Phe Thr CLn 14*t ABn Gin P pillar■ Engineering 5 10 15 Sex Lau Gly I (is Gly C1u entering 1a pro entering La Ala Engineering 1a Giy Arg sar Engineering Le PrB 20　25　ko0 Ala Pro to ly lJu to lu T’yr Lys Val Val Lau Thr Phe Gly Sar Pr mouth Hs■ ser Pro Aan Ala Asn Agn Lya Gin Thr Trp Val ABn Lys Pro Lau AspAla Pro 5 ar Gly HLg Tyr Asn VaL LyII Engineering la Ala Lye　λsp　Val　As+p　H繧W Tyr　Lau　Thr　Ksit　CLn　Gly　Pha　Tehr　Ser Engineering 1a kla Saw Van enter up Sar λ X 85 9Q 95 (2) Information regarding SEQ ID NO: 11: (i) Characteristics of array・ (A) Length = 278 base pairs (B) Shape, nucleic acid (C) Number of strands/double strands (D) Topology bilinear (11) Type of sequence: cDNA-mRNA (iii) Hybothetical sequence N.

(iv) Antisense, N.

(vi) Origin: (C) Isolated clone name Niastrovirus serotype AI (vii) Direct origin: CB) Clone salt: A21 (1x) Array characteristics: (A) Symbol representing characteristics: CD5 (B) Existence position rlt: 2. ．． 277 (xl) Sequence/Sequence number ll; GAA (, XT AAG ffA AGA ATCG’ff AAτGOT TC; to CAA AAA AAA A 27B lu Agp Lys La u arg 1e Val Agn Alaτrp Gin Lye LysB Oas 90 (2) Information regarding SEQ ID NO: 12; (i) Characteristics of array: (A) Length: 92 amino acids (B) Form diamino acid (D) Topology: linear (11) Type of sequence: protein (xi) Sequence: Sequence number 12: mt engineering 1m Asp Pro P! “Arq to lu to lu AIJL LY@JLLJI AIJI 1m Sex IIs@Cys Yu 5 Yu O15 JLrg Gm Ala Gly Engineering ill LYJI Pro Val Ma t Xis Thr Gly Asp Jun Lys button r Lau Agn 11m Tyx Amn Asp (Ay h Ispco 5 40 45 L@u Ala IIs 5er Gly Lsu ni lu Lau (:lu LyIIIla sar Mp Asp lu Lauτhr Agn A sn IIs Asp Ly self T'yr Ser Val Tyr Alai 1 for Arg Val Lys Pro 65　70　　s　g.

Asp Lye Lau Arg Eyus VaI Agn Ala Trp Gin Lye LY4859° (2) Information regarding SEQ ID NO: 13: (i) Characteristics of array: (A) Length: 434 base pairs (B) Shape, nucleic acid (C) Number of strands: 2 strands (D) Topology: linear (ii) Type of sequence: cDNA-mRNA (iii) Hypothetical sequence, N0 (1v) antisense: No (vi) Origin: (C) Isolated clone name, astrovirus serotype A2 (vii) Direct origin: (B) Clone salt: A43351 (ix) Array characteristics: (A) Symbol representing characteristics: CD5 (B) Location: 2. ．． 433 (xi) Sequence: SEQ ID NO: 13: GAA CCj* GAA to TC (:AA TCA CAA CCA CTT GkT TTG TCCCAA AX h) J+ Gkf g4 Glu　C1u　Val　Glu　57Gin　Pro　Leu　As p Leu Sar Gin Lys Lys Glu2° 25 30 60ゞτcAGAAτλτGAA CAA CAA to T to TOAAG τ CT ATCAA CCT GAG 14265 70 F5 C1KG! C, CQ CCA Tie CGG ττGACT All: (:CA　CAT　GAA　AGτccττ　GGA@286 L@u Pro AIJL P! :OTyr　Arg　Lau　Thr　Lys Enter la Asp Glu Engineering 1e VLL Lau Gl■ go as 90 95 'z('7 AAA λτCCτCλACCτT AGA ACG ATT A ? 'r　GAA　AcA　GCCATA　AAG　ACr　R34 ゜AG Enter Aτ Ding＾TAGτ GCA TTA CCT GM GCA GT A　TTT　GW,CTCGACAAA　l:icA　ko8Q Gin un Tyr Sar Jua Leu Pro Glu Ala V al　PbS　Glu　Lau　AsP　LYII　Ala 15 uO 25 1ko0 Yuko5 140 AAA A 434 yes (2) Information regarding sequence number 14: (1) Array characteristics: (A) Length: 144 amino acids CB) form diamino acid (D) Topology, linear (11) Type of sequence: protein (xi) Sequence: SEQ ID NO: 14: Lys Pro Xla Pro Enter 1a Leu Arg Thr Dinghr Lya Pro Lye Thr CYII P! :ro Gku Yu 5 engineering 015 Pro GLu Val Glu 5ar Gin Pro Lau Asp Lau　5ar　G3x　Lye　Lye　Giu　Lys2o　25　ko0 GLn 5M Glu TYr Glu Gm Gln Val Val Ly s 5ar Zl-Lym Pro ni ill Lyeko 5 40 45 5ar Glu Pro Gin Pre Tyr Bar Gln Thr Tyr Gly Lys Ala Pro engineering le τrpGlu 5ar T yr　Jup　Phe　As+pτrpNiln　Glu　Agp　Agp　 Jua AY @ Phe 1e Lsu65 70 75” Pro LLa Pro Tyr entering rg Lau Thr Lys Ala Asp Glu IIs VaI Lau Gly 5exLys 1@Va l LYll Lau krq Thr 6 engineering i@C1u Thr Ala Xis Lye” rhx G1n1oo zos xz.

Agn Tyx Sgax JuJI K, au Pro Glu AIJI VJL↓Ph* Glu Lau AIIP Lys Al<■H1 Uni 5 120 Engineering 25 ? yr　Glu　Ala　Gly　Lsu　Glu　Gly　Phe　Lau　 Gin Arg Van Lys 5ar Ly+ Lys1ko O Yuko5 14 0 (2) Information regarding sequence number 15 (1) Array characteristics: (A) Length: 444 base pairs (B) Shape, nucleic acid (C) Number of strands: 2 strands (D) Topology, vertical shape (ii) Type of sequence: cDNA = mRNA (iii) Hypothetical sequence N0 (1v) Antisense: N.

(vi) Origin/ (C) Isolated clone name Niastrovirus serotype AI (vii) Direct origin (B) Clone name/A39 (1x) Array characteristics: (A) Symbol representing characteristics: CD5 (B) Existence location・1. ,444 (xl) Sequence: Sequence number 15 Cab TTcGCI; Gee (:CT 01; 11; CCCACA T eA　(:AT　ACOAACCAA　ACT　CTCTC`　4B Glu llbm Alm Ala Ala Arg Proτhr Sar Mp Arg Asn Ginτhr Va Engineering Saryu 5IQ15 1:cA　CGT　rrrr　c;AG　CCCNl:CTCCCCCT　ACC( :CT　TA　ATCGll;CGAA　W“C9GArg　Arg　Phg a Glu Pgmouth Sar 5er hXq Thr Aia Lsu get Gly Glu Gin Pr.

AACccττGOAGCCCA CTCCAG CrCCAG GAT GC G ATG AGCC1l; A CAT CGA) 44JLsn Pz’t) Trp Ser Arg Leu in Leu ni Ala Mst 5er Arg E! Engineering 1qGGT GCCARA CCr τcccc ding CGA Tll; τ GAY CTC? 'TG G to A AAG ATA kG CCTC1X2 Gly Jua Ly@Pro 5@r Arg Arg Cys Amp L su　Lau　(：工 y　Lye 工is　Sar　LsuにA　oACCQ　 OVAλTTMτTCCCCG CCG CTC (:M GTA CCT Gπ ACATrT 240G1u Arg Pro JLrg Eyu@Mn Sa r Arg Pro Lau clu Val Pro VaL Thr Ph a65 70] 5 a.

AA GCA ACT (:CT GGA TCA CCA TTA ATA ATG AAT GTG TCA GAG GW AGOQ8!1 Glu　Gly　5@r　Ala　Gay　5er　Pro　Leu　Il@ Met　Mn　Val　5er　Glu　Gay　5erdT　TTτ　GCA C1:A ACA (: ττ CTT CCA CC (: TCA ACJi ACA CCA Ace entered C? C■`　336 H1s Pha Ala Arg Ding! u″ Val Leu Ala Arg 5er Thr Thr I’ro Thr τni-c keu loo 105 1yu0 GCG CGT GCA GGA GAG AGA ACCACCτeA to A CACA GτA Ding GCGAG GπCTC3B4Ala Arg Ala c ly clu Argτ bet τhr 5er Asp Thr Valτrp G in Val LauAAT ACA GCT GTA TCT GCT GO T (:AG CTT llTc ACCCOT CC’r CCG CCG A Gekigeki FC4ko2 AlIn Thr Ala Val 5er Ala Ala Glu Lau Val Thr pro Pr mouth Pro Pr mouth Be■ Eco mouth 135 140 GGCCX, CGAA TTC444 Gly Arg ni Glu l’ha Yu45 (2) Information regarding sequence number 16 (i) Characteristics of array: (A) Length: 148 amino acids (B) Form diamino acid (D) Topology: linear (ii) Type of sequence: protein (xi) Sequence Sequence number 16, Glu Phe Alm Ala Arg Pro D’hr 5er Asp Arg Mn Gin Thr Val 5er s xo 1s Arg Mg Phe Glu Pro 5er Ser Arg Thr A la Iau Met Gly Glu Gin Pr.

20　25　ko0 Asn X’ro Trp 5er Arg Lau Gin Lsu Gin Asp Ala Met Ser Arg Hls Ar■ Gly Jua Lys Pro Ser krq Arg Cys Asp Leu Lsu Gly Lys Engineering 1a 5er LeuGlu kxq Pr o Arg Engineering 1a) sn Ser Arg Pro Leu Glu Va l Pro Val Dinghr PheGlu Gly Ser Ala Gly 5er Pro Leu le Met Asn Val Sex: Glu Gly 5er8'i 9Q 95 HLs Pha entering 1a Arg Thx Val Lau entering 1 Todoro entering rg ser Thx Thr i’ro Thr Thr Le■ loo 105 1yu0 Aia Arg Ala Gly Glu Arg Thr Thr Ser Asp Dinghr Val Trp Gin Val LeuMn Thr Ju a Val Ser Ala Ala Glu Leu Val Thr Pr o Pr mouth pro pro 5erGly AT-q Glu Phe Yu45 IU U I-I I-2I-4I-5 process Infected cells Infected cells・ Fig, 4B -One cry--Astrovirus antigen activity (A490 unit) "0 Epitope Subbanning) 4.4- 1.4- Submission of translation of written amendment (Article 184-8 of the Patent Law) November 1993 II''1st

Claims (49)

[Claims] 1. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and a purified asteroid comprising a sequence selected from the group consisting of SEQ ID NO: 13. Lovirus polynucleotide. 2. Serum and immunization from rabbits immunized with astrovirus infectious inoculum A recombinantly produced Asto protein encoding a responsive polypeptide Lovirus polynucleotide. 3. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: Encoding a polypeptide selected from the group consisting of SEQ ID NO. 12 and SEQ ID NO. 14. 3. The polynucleotide of claim 2. 4. 3. The polynucleotide of claim 2, which is a CDNA. 5. SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, and a nucleic acid sequence selected from the group consisting of SEQ ID NO: 13. 4 polynucleotide. 6. Serum and immunization from rabbits immunized with astrovirus infectious inoculum Responsive, recombinant astrovirus polypeptide. 7. The above polypeptide is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 , SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14. 7. The polypeptide of claim 6, which has substantially the same sequence as the polypeptide. 8. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: No. 12 and SEQ ID No. 14. 7. The point of claim 6 comprising an immunoresponsive portion of an astrovirus polypeptide comprising: Ripeptide. 9. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: No. 12, and all polypeptides selected from the group consisting of SEQ ID No. 14. 7. The polypeptide of claim 6, comprising a peptide sequence. 10. 7. The polypeptide of claim 6, which is a hybrid protein. 11. The above hybrid protein has a polypeptide sequence of β-galactosidase. and SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, Sequence A polypeptide sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 14. 11. The polypeptide of claim 10, comprising: 12. From Lambda-A2, Lambda-A11, Lambda-A13 and Lambda-A-33 A vector produced by bacterial cells containing a vector selected from the group consisting of The polypeptide of claim 10. 13. A method for detecting astrovirus in a human stool sample, the method comprising: Partial purification of polynucleotides present in the astrovirus polynucleotide Hybridization of nucleotide-specific oligonucleotide probes and the binding of its probe to polynucleotides present in stool samples. means for detecting A method comprising: 14. The partial purification described above results in the generation of CDN from the RNA template present in the sample. A claim comprising making A molecules and sequence-independent amplification of the resulting CDNA molecules. 13 ways. 15. The above probes are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 Claim 13 obtained from a DNA sequence encoding a polypeptide having the sequence the method of. 16. The above method detects the binding of the above probe to the sample polynucleotide. 14. The method of claim 13, wherein the means comprises biotinylation of the probe. 17. The above method detects the binding of the above probe to the sample polynucleotide. 14. The method of claim 13, wherein the means includes radiolabeling the probe. 18. Oligonucleotide specific for the above astrovirus polynucleotide ·probe. 19. The above probes are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 Claim 18 obtained from a DNA sequence encoding a polypeptide having the sequence probe. 20. The above probes are SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, A point selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, and SEQ ID NO: 13. 19. The probe of claim 18, obtained from a polynucleotide sequence. 21. Oligonucleotide specific for the above astrovirus polynucleotide ·Primer. 22. The above oligonucleotide sequences are SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6. , SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14. obtained from a DNA sequence encoding a polypeptide having a sequence selected from 22. The pliers of claim 21. 23. DNA in which the above oligonucleotide sequence is complementary to the above DNA sequence 23. The primer of claim 22, obtained from the sequence. 24. Screening of human serum containing antibodies specific for astrovirus infection A diagnostic kit for use in astrovirus infectious inoculum Recombinant Astrovirus immunoreactive with serum from more immunized rabbits rus polypeptide antigen, and means for detecting the binding of the antibody to its antigen. 25. The above recombinant polypeptide antigens are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, a group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 Astrovirus polypeptides encoded by sequences selected from 25. The kit of claim 24, comprising an immunoreactive portion of. 26. The above recombinant polypeptide antigens are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, a group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 25. The kit of claim 24, comprising the entire polypeptide sequence of a polypeptide selected from to. 27. The above recombinant polypeptide antigens are lambda-A2, lambda-A11, lambda-A11, by a vector selected from the group consisting of Muda-A13 and Lambda A-33. Claim 2 comprising a polypeptide produced by a transformed bacterial cell. 4 kits. 28. The detection means includes a solid support and a reporter to which the polypeptide is attached. - Contains labeled anti-human antibodies, which inhibits the binding of said serum antibodies to said antigen. The reaction is detected by binding of the reporter-labeled antibody to the solid surface. 25. The kit of claim 24, such that the kit can be dispensed. 29. A method for detecting astrovirus infection in a patient, the method comprising: Sera from test patients with astrovirus infection were immunized with an astrovirus infectious inoculum. recombinant astrovirus polypep immunoreactive with serum from rabbits react with the titanium antigen, and testing the antigen for the presence of bound antibodies. 30. The above recombinant polypeptide antigens are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, a group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 Astrovirus polypeptides encoded by sequences selected from 30. The method of claim 29, comprising an immunoreactive portion of. 31. The above recombinant polypeptide antigens are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, a group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 30. The method of claim 29, comprising the entire polypeptide sequence of a polypeptide selected from . 32. The above recombinant polypeptide antigens are lambda-A2, lambda-A11, lambda-A11, by a vector selected from the group consisting of Muda-A13 and Lambda A-33. Claim 2 comprising a polypeptide produced by a transformed bacterial cell. 9 ways. 33. The polypeptide antigen described above is attached to a solid support, and the reaction described above is The reaction between this polypeptide antigen and this support, and the reaction between this support and reporter The test includes the following reaction with a labeled anti-human antibody, and the test described above Claims include detection of the presence of a reporter-lapeled antibody on a solid support. The method of Section 29. 34. Serum and immunity from rabbits immunized with astrovirus infectious inoculum. A method for producing an epidemic-responsive polypeptide, the method comprising: Sera from rabbits immunized with a lovirus infectious inoculum and immunoreactive It also contains polynucleotide sequences encoding astrovirus polypeptides. A recombinant expression system containing one oven reading frame (ORF), , the vector is designed to express the ORF in the host. introducing a system, and culturing the host cell under conditions that result in expression of the ORF sequence; A method comprising: 35. The above polypeptide is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14. Immune responsiveness of astrovirus polypeptides encoded by sequences 35. The method of claim 34, comprising: 36. The above polypeptide is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14. 35. The method of claim 34, comprising the entire polypeptide sequence of the polypeptide obtained. 37. The above expression vector is a lambda gtll phage vector, and and the host is Escherichia coli. 34 methods. 38. The above lambda gtll phage vectors include lambda-A2, lambda-A ll, lambda-A13 and lambda A-33, 38. The method of claim 37. 39. Serum and immunity from rabbits immunized with astrovirus infectious inoculum. Development of recombinant astrovirus polypeptides that are disease-responsive. current and open reading frame within the chosen expression vector. and a host capable of supporting the expression of a polypeptide encoding the above-mentioned polypeptide antigen. an open reading frame (ORF) containing a polynucleotide sequence a selected expression vector containing, An expression system comprising. 40. The above recombinant polypeptide antigens are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, a group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 astrovirus polypeptide containing an immunoresponsive portion of an astrovirus polypeptide selected from Expression system of claim 39. 41. The above recombinant polypeptide antigens are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, a group consisting of SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, and SEQ ID NO: 14 40. The expression of claim 39, comprising the entire polypeptide sequence of a polypeptide selected from system. 42. The expression vector selected above is the lambda gtll phage vector. and the above host is Escherichia coli. , the expression system of claim 39. 43. The above lambda gtll phage vectors include lambda-A2, lambda-A ll, lambda-A13 and lambda A-33, 43. The expression system of claim 42. 44. Serum and immunity from rabbits immunized with astrovirus infectious inoculum. and SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, A sequence selected from the group consisting of column number 10, sequence number 12, and sequence number 14. containing a recombinant astrovirus polypeptide antigen containing an immunoreactive portion of a sequence. Astrovirus infection as a pharmacologically acceptable adjuvant consisting of vaccines to immunize patients against. 45. SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, Sequence A polypeptide having a sequence selected from the group consisting of number 12 and sequence number 14 Antibodies specific for Tide. 46. 46. The antibody of claim 45, wherein said antibody is monoclonal. 47. 46. The antibody of claim 45, wherein said antibody is polyclonal. 48. It is a method of creating passive immunity in patients against astroviruses. 46. A method comprising parenteral administration of the antibody of claim 45 to said patient. 49. From Lambda-A2, Lambda-All, Lambda-A13 and Lambda-A-33 by bacterial cells transformed with a vector selected from the group consisting of An antibody against the produced astrovirus-specific polypeptide.