CN1616485A - SARS coronavirus structural protein ORF3 and its application - Google Patents
SARS coronavirus structural protein ORF3 and its application Download PDFInfo
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- CN1616485A CN1616485A CN 200310108604 CN200310108604A CN1616485A CN 1616485 A CN1616485 A CN 1616485A CN 200310108604 CN200310108604 CN 200310108604 CN 200310108604 A CN200310108604 A CN 200310108604A CN 1616485 A CN1616485 A CN 1616485A
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Abstract
本发明涉及一种新的SARS冠状病毒结构蛋白及其应用。这个蛋白质属于ORF3(位于SARS冠状病毒基因组S基因和E基因之间)的基因产物。ORF3蛋白质被定位在病毒颗粒上，与识别宿主细胞和感染有关。ORF3蛋白或其免疫原性片段可作为抗原，用于生产抗体以及开发检测和治疗SARS的制剂。The present invention relates to a kind of novel SARS coronavirus structural protein and application thereof. This protein belongs to the gene product of ORF3 (located between the S and E genes of the SARS coronavirus genome). The ORF3 protein is localized on virus particles and is involved in host cell recognition and infection. ORF3 protein or its immunogenic fragments can be used as antigens for producing antibodies and developing agents for detecting and treating SARS.
Description
Technical field
The present invention relates to the structural protein and the application thereof of sars coronavirus (SARS-CoV).Particularly, the present invention relates to a kind of new SARS virus structural protein ORF3 and fragment thereof, and anti-ORF3 albumen or its segmental antibody.
Background technology
WHO has sent the early warning of transmissible disease first over nearly 10 years on March 12nd, 2003 to the whole world, this transmissible disease that once once had been called as atypical pneumonia (atypical pneumonia) was found in Chinese Guangdong first within the border from mid-November, 2002, its onset is anxious, infectivity was strong, and antibiotic therapy is invalid, by 18 o'clock on the 10th May, this disease is found in existing 33 countries and regions, the whole world, accumulative total number of patients 7296 people, death toll 526 people, mortality ratio reaches 3-6%.
On April 16th, 2003, World Health Organization's official confirmation caused that the pathogenic agent of SARS (Severe Acute Respiratory Syndrome) (Severe Acute Respiratory Syndrome is hereinafter to be referred as SARS) is a kind of novel coronavirus-sars coronavirus (hereinafter to be referred as SARS-CoV).It is a kind of normal chain, single strand RNA virus (ssRNA positive-strand viruses, no DNA stage), belong to cover virales (orderNidovirales), coronaviridae (family Coronaviridae), coronavirus genus (genusCoronavirus), sars coronavirus (SARS coronavirus).
Under the Electronic Speculum, the coronavirus particle is irregular shape, and diameter is about 60-220nm, and coating is arranged, and has glycoprotein on the coating, and wherein a kind of envelope glycoprotein projection length of length is about 20nm, and coating contains nucleocapsid.The expressing viral albumen that will be packaged into usually in the virion is called structural protein, but some Nonstructural Protein also can be inserted in the peplos.It is generally acknowledged the formation of structural protein and virion and the identification of release and host cell, host's infection and cause a disease relevant (S.G..Siddell, Ed., TheCoronaviridae Plenum Press New York, 1995).Sequential analysis shows the same structural protein that exist equally with known coronavirus of SARS-CoV: replicative enzyme (replicase), S albumen (spikeprotein), E albumen (envelope protein), M albumen (membrane protein), N albumen (nucleocapsid protein), the aminoacid sequence of these structural protein is similar to known coronavirus on 26S Proteasome Structure and Function.Wherein the conservative gene sequence that comprised of the E of SARS-CoV, M, N albumen is present in other coronavirus equally; S albumen is the glycoprotein of coronavirus, is positioned at virus surface, is responsible for participating in the receptors bind and the film fusion of recipient cell, and the proteic C-terminal of S is made up of membrane-spanning domain and the cytoplasmic tail that is rich in cysteine residues.Also comprised important viral neutralizing epitope on the S albumen, but, because the S albumen of SARS-CoY and the aminoacid sequence difference between other coronavirus S albumen can't therefrom be known the proteic receptors bind specificity of S of SARS-CoV or the constitutional features of epitope (Paul A.Rota et al ' Characterization of a Novel CoronavirusAssociated with Severe Acute Respeiratory Syndrome ' Sciencexpress/1May 2003) at present by inference.Because the variation of S gene, its host range, tissue trend and virulence all may change, and therefore, how will help to understand S albumen for the research of S albumen and covalent complex thereof influences the pathogenesis of SARS and the development of related drugs.
The research group of Toronto genome scientific center has issued viral complete sequence on the net April 12, the SARS-CoV whole genome sequence is about 30kb, have 5 ' cap structure and 3 ' polyA tail (Marco A.Marra et al ' The Genome Sequence of the SARS-AssociatedCoronavirus ' Sciencexpress/1May 2003), from Canadian SARS-CoV strain isolated prediction 14 open reading frame (Rota PA are arranged, Oberste MS, The Genome sequence of theSARS-associated coronavirus.Science.2003 May 30; 300 (5624): 1399-404.).Known structural protein replicative enzyme, S albumen, E albumen, M albumen, the proteic encoding sequence of N lay respectively at ORF1a and ORF1b, ORF2, ORF5, ORF6, ORF12 (Rota PA, Oberste MS, The Genomesequence of the SARS-associated coronavirus.Science.2003 May30; 300 (5624): 1399-404.).In general, RNA viruses has the feature of high genetic mutation rate, and these needs that are evolution also are the actual mechanisms (Yijun Ruan etal) that RNA viruses is used to escape host defense.Gene sequencing shows that the genome sequence of SARS-CoV and known three kinds of coronavirus subgroup sequences are completely different, very likely is independent the evolution, so it is classified as the new branch of coronavirus.The 14 strain SARS-CoV genome sequence comparative results demonstration of taking from different sources always has 127 single nucleotide variations, wherein 94 have changed amino-acid residue (Yi jun Ruan et al), but whether these differences play critical effect for the meaning of SARS-CoV evolution itself and for different plant types on each species diversity that exists between infected host's individuality, also unknowable at present.
Existing SARS detection means has 3 kinds, comprises that the method that adopts antibody test: ELISA (IGM/IGA) method can detect reliably clinical symptom antiviral antibody among the SARS patients serum after 20 days to occur.Some patient can detect antibody in the time of 14-21 days; Adopt immunofluorescence technique to detect and to detect the M immunoglobulin (Ig) that virus infection VERO cell produced after 10 days; Adopt molecular detecting method: developed 7 pairs of primers and be used in the testing process, the positive control RNA in the detection can be from Bernhard-NochtInstitute in Hamburg, and Germany freely obtains.Test sample comprises blood, ight soil, respiratory secretions.PCR result can assist clinical diagnosis evaluation, but can not affirm or get rid of the possibility of disease; Cell culture processes: utilize the VERO cell to detect patient's SARS respiratory secretions and blood sample, positive findings has been represented SARS patient infection coronavirus, negative findings does not show that patient is not SARS.But these detection meanss can't generally all also need comprehensively to judge in conjunction with supplementary meanss such as clinical symptom timely and effectively as the effective ways that detect diagnosis SARS.
At present, effectively control and even eliminate SARS and become the important topic of pendulum in face of global scientist for human life's harm.People are from finding confirm the time that gene order-checking, sequential analysis also have only short some months of pathogeny to pathogenic agent, although obtained some progress, people know little about it to sars coronavirus at present.In order to obtain how valuable clue, people invest more sight in the research of the functional analysis of separation to viral protein, viral protein and application thereof gradually, people press for by more approach and search out more, valuable SARS-CoV expressing protein, further understand their effects aspect virus replication, pathogeny, the detection method and the reagent of effective sars coronavirus are provided, promote the development of SARS vaccine and the exploitation of SARS new drug.At present, except structural protein S, E, M, N are identified, at present, except above-mentioned open reading frame is identified encoding SARS-CoV structure gene, also clearly do not confirm other SARS-CoV structure genes and confirm the function of this structure gene and the report of living features, especially be positioned at other albumen of virus surface.
Because the clinical medicine that lacks effective SARS, be effectively preventing means so present stage isolates for treatment infected patient rapidly.In order to develop SARS detection method and methods of treatment, this area presses for new SARS structural protein of exploitation and relevant diagnostic method and test kit.
Summary of the invention
The object of the invention just provides a kind of new SARS structural protein and application thereof.
In a first aspect of the present invention, a kind of structural protein are provided, this albumen is selected from down group:
(a) has the polypeptide of SEQ ID NO:1 aminoacid sequence;
(b) replacement, disappearance or the interpolation of SEQ ID NO:1 aminoacid sequence through one or more amino-acid residues formed, and be positioned on the SARS virus particle and have immunogenicity, can with S albumen formed the interchain disulfide bond covalent complex by (a) polypeptides derived.
In another preference, this proteic molecular weight is 31KD-34KD, and it can form the interchain disulfide bond covalent complex with S albumen on non-reduced glue.
Preferably, described albumen contains the aminoacid sequence of SEQ ID NO:1.More preferably described proteic aminoacid sequence is shown in SEQ ID NO:1.
In a second aspect of the present invention, a kind of antibody is provided, the above-mentioned structural protein of described antibodies specific ground and the present invention combine.More preferably, described antibody is monoclonal antibody.
In a third aspect of the present invention, a kind of antigenic polypeptide is provided, it has successive 25-100 amino acid among the SEQ ID NO:1, and combines with the above-mentioned specific antibody specificity of the present invention.In another preference, described polypeptide has the aminoacid sequence of SEQ ID NO:9.The conjugate of described antigenic polypeptide also is provided.
In a fourth aspect of the present invention, a kind of detection kit is provided, it contains the above-mentioned structural protein of the present invention, above-mentioned antibody, above-mentioned polypeptide or its combination.
In a fifth aspect of the present invention, the method that whether has SARS virus or anti-SARS virus antibody in a kind of vitro detection sample is provided, comprise step:
(a) sample is contacted with the material that is selected from down group: the above-mentioned structural protein of the present invention, above-mentioned antibody, above-mentioned polypeptide or its combination;
(b) detect whether form antigen-antibody complex, wherein form mixture and just represent to exist in the sample SARS virus or anti-SARS virus antibody.
In another preference, described sample is serum or sputum.
In another preference, described material is that aminoacid sequence is the albumen of SEQ ID NO:1.
In another preference, described material is the described antibody of claim 2, and described antibody is monoclonal antibody.
In a sixth aspect of the present invention, a kind of composition is provided, contain above-mentioned structural protein of the present invention and pharmaceutically acceptable carrier.
Description of drawings
Fig. 1: the protein electrophorese collection of illustrative plates in 24 hours the tenuigenin of infective virus, the 1st swimming lane are molecular weight standard, are followed successively by 175kD from top to bottom, 83kD, 66kD, 43kD, 33kD, 25kD, 14kD and 7kD.The 2nd swimming lane is the protein in 24 hours the tenuigenin of infective virus.
Fig. 2: the mass fragmentograph of ORF3 protein N terminal 7-19 position peptide section FFTLGSITAQPVK (SEQ ID NO:3).
Fig. 3: the mass fragmentograph of ORF3 protein 180-193 position peptide section LKEDYQIGGYSEDR (SEQ ID NO:4).
Fig. 4: ORF3 protein C end 236-274 position peptide section
The mass fragmentograph of LVKDPPNVQIHTIDGSSGVANPAMDPIYDEPTTTTSVPL (SEQ ID NO:5).
Fig. 5: protein C end 236-274 position peptide section
The mass fragmentograph of LVKDPPNVQIHTIDGSSGVANPAM#DPIYDEPTTTTSVPL (SEQ ID NO:6).
Wherein M# is the methionine(Met) of oxidation.
Fig. 6: A is the ORF3 protein immunoblot experiment, and at the cell (the 2nd road) of virus infection be secreted in the extracellular virus (the 3rd road) and all detect ORF3 albumen, the 1st road with the cell of uninfecting virus in contrast.B is S albumen and the proteic immunoblot experiment of ORF3.Left side figure is the proteic immunoblot experiment of S, going back (the 1st road) on the virgin rubber, can detect S albumen at 180KD, and on non-reduced glue (the 2nd road), can detect S albumen simultaneously at 180KD and 210KD place.Right figure is the proteic immunoblot experiment of ORF3, has also detected ORF3 albumen (the 2nd road) at non-reduced Jiao210KDChu, illustrates that ORF3 albumen and S albumen have formed the interchain disulfide bond covalent complex really.
Fig. 7: ORF3 protein gold-marking immunity electron microscopic observation and agglutination reaction, ORF3 protein is positive on the coronavirus surface, thereby confirms that the ORF3 protein positioning at virus surface, is a structural protein.
Fig. 8: the detection of ORF3 protein antibody in the patients serum also detects the ORF3 protein antibody by the ELISA experiment and is positive in the patients serum.Adopting the SBP1 polypeptide among the A figure is detection probes; Adopting the SBP2 polypeptide among the B figure is detection probes; Adopting the S1 polypeptide among the C figure is detection probes; D figure expression SBP2 polypeptide and S1 polypeptide are to the dependency of patients serum's detection reaction.
Embodiment
The inventor is by the natural protein of mass spectroscopy SARS virus, finds first and separated a kind of new structural protein.This protein belongs to the gene product of ORF3 (between sars coronavirus genome S gene and E gene).ORF3 protein also is positioned on the virion, belongs to a kind of structural protein, and it may and infect relevant with the recognition of host cell.Finished the present invention on this basis.
The invention provides a kind of new sars coronavirus structural protein, this proteinic molecular weight is 31KD-34KD, and it can form the interchain disulfide bond covalent complex with S albumen on non-reduced glue.Described structural protein is meant, be packaged into the expressing viral albumen in the virion, be meant the gene product of ORF3 especially, it has aminoacid sequence shown in the SEQ ID NO:1, and this sequence can be that to come by genetic engineering technique also can be that synthetic comes.The molecular weight of ORF3 gene product is 31KD-34KD, and hint ORF3 gene product has modification after the translation or multi-form hypotype (seeing embodiment 3 for details).
Identify by liquid chromatography-electron spray(ES) ion trap mass spectrometry by the ORF3 gene product that the present invention obtained, and confirmed this protein amino acid sequence (shown in SEQ ID NO:1) by the mass spectrum order-checking.ORF3 is between S and E gene, and structural analysis shows that ORF3 albumen has three and strides the film district, and proteic N end 1-22 position is outside film, and 160 amino acid of C end are in film.Also there is a potential atpase activity site in C end 209-264 position.ORF3 albumen contains 8 halfcystines, all is positioned at the intracellular region of C end.This 22 at proteic N end has a potential glycosylation site.11 at N end is not having sudden change in the homophyletic in this albumen, and this experiment proves that also the 11st amino acids becomes G (seeing embodiment 2 for details) by R.
Confirm that by further Antibody Preparation and immunoblot experiment, immuno-electron microscope experiment, clinical serum ELISA experiment (seeing embodiment 3,4,5 for details) ORF gene product of the present invention is positioned the virion surface, be structural protein, it can form the disulfide linkage mixture with S albumen, and ORF3 gene product and S albumen are closely related on function, thereby can mediate virus and the combining of host cell jointly with S albumen, and then finish attack the host.
As used herein, " structural protein ORF3 " or " ORF3 albumen " refers to have the polypeptide of SEQ ID NO:1 aminoacid sequence.This term comprises that also replacement, disappearance or the interpolation with the one or more amino-acid residues of SEQ ID NO:1 aminoacid sequence process forms, and be positioned at equally on the SARS virus particle and have immunogenicity, or can with S albumen form the interchain disulfide bond covalent complex by (a) polypeptides derived.
The present invention also comprise have with 75%, 85%, 90%, 95% homology of aminoacid sequence shown in the SEQ ID NO:1 and have the active sequence of identical function, can use the Clustal computer program that European information biology institute (EBI) provides to measure sequence homology.
The present invention also comprises the proteic fragment of the ORF3 of SARS, derivative and analogue.As used herein, term " fragment ", " derivative " are meant identical biological function or the active polypeptide of ORF3 albumen that keeps natural SARS of the present invention basically with " analogue ".Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merges formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or with the fusion rotein of the segmental formation of antigen I gG).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
The present invention further provides the antigenic portions or the section of sars coronavirus structural protein.Described " antigenic portions or section " is meant the fragment of the ORF3 gene product with certain molecular structure, particularly have antigenic portions or the section shown in the SEQ ID NO:9, when this molecular structure existed with appropriate form, it can bring out immunne response to affiliated ORF3 gene product in ill organism.The whole bag of tricks that is used at this useful epitope of known amino acid order test is known, according to these sequences, can use these epi-positions of determination of experimental method, for example by means of the triage techniques among the WO8606487.These antigenic portions or section can be induced the provide protection to sars coronavirus equally.Therefore, these antigenic parts or section are included in the scope of the present invention too.
In the present invention, term " the ORF3 polypeptide of SARS " refers to have the SEQ ID NO.:1 polypeptide of sequence of the ORF3 protein-active of SARS.This term also comprises variant form ORF3 albumen identical function, SEQ ID NO:1 sequence that has with SARS.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises proteic active fragments of the ORF3 of SARS and reactive derivative.
In the present invention, " ORF3 albumen conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:1, has 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The invention still further relates to the polynucleotide of coding ORF3.Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:1, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:2.
ORF3 Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.When sequence is longer, usually needs to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The present invention also relates to comprise the carrier of polynucleotide of the present invention and the host cell that produces through genetically engineered with carrier of the present invention.Described carrier can be plasmid, virion and other carrier formats.Polynucleotide sequence in the expression vector can operationally be connected with expression control sequenc, also comprises the ribosome bind site that is used for translation initiation and Transcription Termination, preferably comprises one or more selected markers.Described host cell can be bacterium such as intestinal bacteria, fungi such as yeast, insect cell such as Sf9, zooblast such as CHO, COS etc.By the elaboration of this paper, the selection of appropriate carriers, host cell is all in those skilled in the art's ken.
The present invention also relates to structural protein of the present invention, the particularly method of ORF3 gene product through the recombinant technology generation.Can polynucleotide sequence be inserted in the carrier with method well known in the art, carrier can enter host cell by modes such as conversion, transduction, transfections.Host cell is cultivated for some time through method known in the art, with ordinary method such as physics or the broken host cell of chemical process, reclaims and the purifying culture.
In addition, the present invention also provides another kind of separation and purifying structural protein of the present invention, the particularly method of ORF3 gene product.This method comprises: cultivate sars coronavirus, isolated viral albumen also adopts gradient separations glue isolated viral albumen, downcuts the band of 31KD-34KD, carries out enzymolysis and mass spectrum evaluation in the glue.
Structural protein ORF3 of the present invention or its fragment can be directly used in the antibody that detects anti-SARS, also can be used for the albumen coupling with the BSA equimolecular quantity, thereby form the polypeptide conjugate.Usually, described conjugate is made of orf protein or fragment, linking agent and BSA, the preferred glutaraldehyde of wherein said linking agent (when the N-with ORF3 holds coupling), EDAC (when the C-with ORF3 holds coupling).
ORF3 albumen of the present invention or its conjugate can be used for animals such as immunize rabbit, mouse, thereby obtain the antibody (" antibody of the present invention ") of anti-structural protein ORF3 of the present invention.Antibody of the present invention comprises specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into SARS virus, gene product or fragment.The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises and have immunocompetent antibody fragment (as Fab ' or (Fab)
2Fragment), heavy chain of antibody, light chain of antibody, chimeric antibody, humanized antibody etc.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the structural protein ORF3 of the present invention or the conjugate of purifying can be applied to animal to induce the generation of polyclonal antibody.For monoclonal antibody, can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerl ing, In MonoclonalAntibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).
Whether there are SARS virus or anti-SARS virus antibody in ORF3 albumen of the present invention, immunogenic fragments (small peptide), conjugate and all available test sample of antibody, thereby as one of level of signification of early detection SARS.
The present invention also provides a kind of detection kit, it contains ORF3 gene product of the present invention, immunogenic fragments (small peptide), conjugate and antibody, this detection kit can be used for whether existing in the test sample SARS virus or anti-SARS virus antibody, thereby as one of level of signification of early detection SARS.
The present invention also provides the method that whether has SARS virus or anti-SARS virus antibody in a kind of vitro detection sample, comprise (a) with sample and structural protein of the present invention particularly the ORF3 gene product and with antigenic portions of the present invention or section, corresponding antibodies, comprise that polyclonal antibody, monoclonal antibody and polypeptide contact; (b) detect whether form antigen-antibody complex, wherein form mixture and just represent to exist in the sample SARS virus or anti-SARS virus antibody, described sample can be serum or sputum.
ORF3 albumen of the present invention, immunogenic fragments and conjugate also can be used for preparing curative pharmaceutical composition or preventative vaccine composition.
Therefore, the present invention also provides a kind of composition, and it contains immunogenic fragments, conjugate or its combination of structural protein ORF3 of the present invention, the ORF3 of (a) of the present invention safe and effective amount; And (b) pharmaceutically acceptable carrier or vehicle.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol, adjuvant and combination thereof.Composition of the present invention can be prepared by ordinary method.In structural protein ORF3, its consumption for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.In addition, the example of carrier and compound method can be referring to " Remington pharmaceutical science " (Remington ' sPhamaceutical Science).
In addition, ORF3 gene product of the present invention can be used to develop inhibitor and the promotor and the inhibition of ORF3 gene product or promote the inhibitor and the promotor of the effect aspect the receptors bind of mediation and recipient cell and film fusion of S albumen.Can stop virus to penetrate host cell such as membrane fusion inhibitor.
The present invention confirms first and obtains ORF3 gene product and the protein bound interchain disulfide bond covalent complex of S; In the patients serum, obtain the antibody of ORF3 first, and be strong correlation with the S protein antibody; Obtain large-scale ORF3 gene expression product by genetic engineering technique first; Obtain the ORF3 gene product by the separation and purification of protein method first.Structural protein ORF3 determines, and the evaluation of function, the improvement that function and antigenic determinant be proteic to be determined to help further to understand the difference of genome sequence and the relation between the viral pathogeny and help the SARS detection means, the exploitation of new generation vaccine, medicine.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (NewYork:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
1, cell cultures and proteins extraction
African green monkey kidney cell strain Vero-E6 (available from ATCC) is adding 10% foetal calf serum (FBS), 100U/ml penicillin (penicillin), the DMEM nutrient solution of 50 μ g/ml Streptomycin sulphates (streptomycin) (Dulbecco ' s Modified Eagle ' s Medium) in, in 37 degrees centigrade and 5%CO
2Cultivate under the condition.All cells is cultivated with reagent available from GIBCO BRL company.To contain sars coronavirus strain BJ-01 (available from Military Medical Science Institute) stoste (virus titre be TCID50 10
6) diluting 10 times with DMEM nutrient solution (containing 2% foetal calf serum), every 1ml diluent infects 107 Vero-E6 cells; Infect and remove viral dilution liquid after 1 hour, add DMEM nutrient solution (containing 2% foetal calf serum) then, under 37 degrees centigrade and 5%CO2 condition, cultivate.
Behind the cell harvesting with viral liquid or infective virus, use 100mM Tris, 0.5%NP-40 dissolving collecting cell matter is boiled after 10 minutes and is preserved.(above work is all carried out in 3 grades of laboratories of Biosafety)
2, SDS-PAGE running gel internal protein enzymolysis:
Adopt the gradient separations glue isolated viral albumen of 7.5-17%.Fig. 1 is the protein electrophorese collection of illustrative plates in 24 hours the tenuigenin of infective virus.The 1st swimming lane is a molecular weight standard, is followed successively by 175kD from top to bottom, 83kD, 66kD, 43kD, 33kD, 25kD, 14kD and 7kD.The protein of second swimming lane for from 24 hours cell of infective virus, extracting.Protein band in the 2nd swimming lane finds to have the N albumen of SARS virus to exist after mass spectrum is identified, simultaneously, in 1,2 and 3 bands, all finds to have ORF3 protein (seeing embodiment 2).
Band among Fig. 11 and band 2 are downcut, place 50%H
2After rinsing is decoloured among the O-40%MeOH-10%HAc, freeze-drying.Add 5mM dithiothreitol (DTT) and some 20mM iodo-acetamide reduction and sealing halfcystine successively.Adhesive tape is smashed to pieces and is placed 100mM NH
4HCO3 (pH 8.3) adds TPCK-trypsinase, and 37 ℃ are incubated 24 hours.Behind the enzymolysis, the gel internal protein is after 0.1%TFA-60%ACN extracting 2-3 time, and freeze-drying concentrates.
Be determined on the Finnigan MAT LCQ ion trap mass spectrometer and carry out capillary temperature, 200 ℃; Spray voltage, 4.25kV; The sheath gas velocity, 50; The HPLC of coupling is the Survey type, anti-phase C18 post (Keystone company product), 50 * 1.0.18I.D.mm, flow velocity: 2ul/min; Elutriant, A liquid, 0.1%FA-H2O; B liquid, 0.1%FA-ACN.The MS/MS energy is 35%.Database retrieval adopts the automatic retrieval software of Sequest to carry out searching database SARS virus genome database.With band 1,2,3 carry out enzymolysis peptide spectrum analysis with mass spectrum respectively.Fig. 2,3,4,5 is the mass spectrum of peptide section.
According to detected result, and through database retrieval, find to occur in the band 1,2,3 gene product of ORF3, the proteinic aminoacid sequence of ORF3 is listed in SEQ ID NO:1.The peptide section that mass spectrum identifies is: FFTLGSITAQPVK (SEQ ID NO:3), L KEDYQIGGYSEDR (SEQ ID NO:4), LVKDPPNVQIHTIDGSSGVANPAMD PIYDEPTTTT SVPL (SEQ ID NO:5), the amino acid fraction of coverage of these peptide sections accounts for 24.08% of gross protein, and to have proved conclusively this proteinic sequence (SEQ ID NO:1) by mass spectrum order-checking be reliable.
The proteinic aminoacid sequence of ORF3 is listed in SEQ ID NO:1.
MDLFMR
FFTL?
GSITAQPVKI??DNASPASTVH????ATATIPLQAS????LPFGWLVIGV????50
AFLAVFQSAT????KIIALNKRWQ???LALYKGFQFI????CNLLLLFVTI????YSHLLLVAAG????100
MEAQFLYLYA????LIYFLQCINA???CRIIMRCWLC????WKCKSKNPLL????YDANYFVCWH????150
THNYDYCIPY????NSVTDTIVVT???EGDGISTPK
L?
KEDYQIGGYS??
EDRHSGVKDY???200
VVVHGYFTEV????YYQLESTQIT???TDTGIENATF????FIFNK
LVKDP?
PNVQIHTIDG??
250
SSGVANPAMD??
PIYDEPTTTT?
SVPL????????????????????????????????????
274
(SEQ?ID?NO：1)
The ORF3 encoding sequence is referring to SEQ ID NO:2.
Embodiment 3 Antibody Preparation and immunoblot experiment
On Peptide synthesizer, by synthetic the proteinic antigen peptide S1 of S (332-351 position, TKFPSVYAWERKKISNCVAD, SEQ ID NO:7) and S2 (758-780 position, RNTREVFAQVKQMYKTPTLKYFG, SEQ ID NO:8) and the proteinic antigen peptide SBP1 of ORF3 (176-199 position, STPKLKEDYQIFFYSEDRHSGVKD, SEQ ID NO:9), to preparing polyclonal antibody after the rabbit immunity.
The N protein antibodies adopts total length N albumen (embodiment 1) immunity to obtain.
Proteins react after adopting S2 and SBP1 antibody respectively and changeing film develops the color with ECL.Use the proteinic antibody of ORF3 (anti-SBP1) to carry out immunoblot experiment respectively, at the cell of virus infection be secreted into and all detect ORF3 protein (Fig. 6 A) in the extracellular virus.The protein molecular weight that detects conforms to the result of SDS-PAGE about 31KD-34KD, and hint ORF3 protein has posttranslational modification or multi-form hypotype.
(anti-S2) carries out immunoblot experiment with the proteic antibody of S, going back on the virgin rubber, can detect S albumen at the 180KD place, and on non-reduced glue, can detect S albumen simultaneously at 180KD and 210KD place, illustrate that S albumen and other protein have formed the interchain disulfide bond mixture, the molecular weight of mixture is at 210KD.Use the proteic antibody of ORF3 (anti-SBP1) to carry out immunoblot experiment again, also found ORF3 albumen at non-reduced Jiao210KDChu.This explanation ORF3 albumen and S albumen have formed the interchain disulfide bond covalent complex really.The results are shown in Figure 6B.
The experiment of embodiment 4 immuno-electron microscopes
At first be ORF3 protein antibodies (anti-SBP1) to be mixed back temperature bathe with the sars coronavirus particle, mark (Protein A-Gold) with gold then and mark mark and under transmission electron microscope, observe.The result shows that ORF3 albumen is present in (Fig. 7 A) on the virion.
Carried out the immune agglutination experiment subsequently again.ORF3 protein antibodies (anti-SBP1) is mixed the back temperature bathe with the sars coronavirus particle, under transmission electron microscope, observe then, use anti-S2 and the proteic antibody of anti-N simultaneously as positive control.The result shows, has occurred the aggegation (Fig. 7 B) of virion under the situation that anti-SBP1 antibody exists.Experimental verification ORF3 albumen is positioned at virion, is structural protein.
ORF3 protein antibodies (anti-SBP1) gold-marking immunity electron microscopic observation and agglutination reaction result are as shown in Figure 7.Fig. 7 A is the sars coronavirus particulate gold-marking immunity Electronic Speculum figure under ORF3 protein antibodies (anti-SBP1) exists.Fig. 7 B is the sars coronavirus particulate immune agglutination Electronic Speculum figure under the proteic antibody of ORF3 protein antibodies (anti-SBP1), anti-S2 and anti-N exists.
Selected 56 patients SARS and 36 normal peoples' serum for use, carried out the ELISA experiment with the antigen peptide SBP1 of ORF3 and SBP2 respectively, with the S1 antigen peptide as positive control.
The results are shown in Figure 8.The serum of some people all has remarkable reaction with SBP1 and SBP2 are arranged among patient SARS, and another part then is lower than normal people's reaction.In addition, there is the patients serum of remarkable reaction similarly reaction when detecting the reaction of S1 antigen peptide, to occur with SBP2.They have significant dependency (Fig. 8 D) statistical results show, and this shows that ORF3 albumen and S albumen are closely related on function.
Embodiment 6 antigenic peptides and be connected (preparation of conjugate) carrier
For the proteinic antigen peptide SBP1 of embodiment 3 synthetic ORF3 (SEQ ID NO:9), dissolve (to some insoluble polypeptide with PBS earlier, can be earlier with partly measuring the PBS dissolving, then make it molten entirely as if insoluble) with 1M NaOH adjusting PH, get 0.5ml polypeptide (concentration is respectively 0.5mg/ml, 1mg/ml) and 15mgBSA (1.5ml) mixing, add linking agent then (at the difference of crosslinked direction, select different linking agents, N-holds crosslinked adding glutaraldehyde, the C-end adds EDAC), mix the back in room temperature reaction 5 hours, lucifuge is constantly rocked; After the crosslinked end, put into dialysis tubing, to 4 ℃ of dialysed overnight of PBS; The amount volume, with the BSA calculating concentration, standby in-20 ℃ of preservations.
Get the about 0.5ml of conjugate (conjugate content is respectively 0.1mg, 0.5mg) of embodiment 6 preparations, mix with CFA (complete Freund's adjuvant) after adding PBS 1.5ml, in Y-tube, push away repeatedly and beat, until the sensation effort, place after 3-5 minute, nothing two is separated and gets final product.
Will be through the antigen immune rabbit and the mouse of adjuvant emulsion processing; About every rabbit injections of antigens 3ml, mouse is only then injected 0.8ml/.Difference subcutaneous injection animal two sufficient pads, neck, back multi-point injection (every some 0.2ml).
Initial immunity is got same dosage polypeptide antigen, PBS after three weeks, mixes 2ml IFA (incomplete Freund's adjuvant), after the emulsification animal is carried out booster immunization, and injected dose is constant.
After three weeks animal is got blood, collect antiserum(antisera).Use the chromatography column of BSA bag quilt, remove antibody at BSA.Select antibody titers antigens with higher polypeptide, make monoclonal antibody.
Embodiment 8 detects and uses
(1) antigenic peptide detects patients serum's sample:
Use antigenic peptide (SEQ ID NO:9) the bag quilt of treated embodiment 3 preparations, with patients serum's hybrid reaction, result's antigen peptide section of the present invention can be discerned early stage specific antibody at SARS among the patients serum, and combination with it, and the washing back adds the HRP enzyme and connects two anti-reactions; Add chromogenic substrate at last, detect.
The result shows, ORF immunogenic peptide of the present invention can be specifically and the antibodies of anti-SARS virus.
(2) antibody (monoclonal antibody is with how anti-) detects pathogenic agent:
Antibody (resisting and monoclonal antibody) for embodiment 7 obtains with ELISA method and patients serum's reaction, detects the virus combination in tissue of patient liquid, the blood more, and pathogenic agent is detected.
The result shows that resist with monoclonal antibody of the present invention can combine with SARS virus specifically.
Embodiment 9 sars coronavirus ORF3 albumen pronucleus expression and purifying:
In the present embodiment, use primer corresponding to the proteic full-length gene clone of sars coronavirus ORF3 (SEQID NO:2) two terminal sequence 20bp, obtain the full length sequence of ORF3 by reverse transcription PCR amplification, insert the multiple clone site of (Qiagen company) among the prokaryotic expression carrier pQE30 then.With recombinant plasmid transformed intestinal bacteria M15 abduction delivering albumen.Choose mono-clonal in corresponding resistance nutrient solution after the overnight incubation, be seeded to 1: 10 ratio in the nutrient solution of identical resistance shaking culture to OD600 be 0.6～0.8, add IPTG to final concentration be 0.5mmol/L abduction delivering albumen.Behind the Ni-NTA affinitive layer purification, SDS-PAGE detects the ORF3 albumen of reorganization according to operational manual.Molecular weight is about 34KD, conforms to predictor.
The Western trace:
ORF3 albumen behind the purifying behind the 12%SDS-PAGE electrophoresis electrotransfer to pvdf membrane.After confining liquid (3%Gelatin) sealing, add immunity back rabbit anti-serum (1/1000 dilution), to hatch 1 hour for 37 ℃, the washing back adds the IgG (H+L) of the goat-anti rabbit of HRP mark, hatches 1 hour ECL colour developing behind the thorough washing for 37 ℃.
As a result, recombinant expressed ORF3 has a colour developing band at about 34kDa place, conform to prediction.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Inst. of Life Science, CAS
＜120〉sars coronavirus structural protein ORF3 and application thereof
<130>035182
<160>9
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＜213〉SARS virus (SARS virus)
<400>1
Met?Asp?Leu?Phe?Met?Arg?Phe?Phe?Thr?Leu?Gly?Ser?Ile?Thr?Ala?Gln
1???????????????5???????????????????10??????????????????15
Pro?Val?Lys?Ile?Asp?Asn?Ala?Ser?Pro?Ala?Ser?Thr?Val?His?Ala?Thr
20??????????????????25??????????????????30
Ala?Thr?Ile?Pro?Leu?Gln?Ala?Ser?Leu?Pro?Phe?Gly?Trp?Leu?Val?Ile
35??????????????????40??????????????????45
Gly?Val?Ala?Phe?Leu?Ala?Val?Phe?Gln?Ser?Ala?Thr?Lys?Ile?Ile?Ala
50??????????????????55??????????????????60
Leu?Asn?Lys?Arg?Trp?Gln?Leu?Ala?Leu?Tyr?Lys?Gly?Phe?Gln?Phe?Ile
65??????????????????70??????????????????75??????????????????80
Cys?Asn?Leu?Leu?Leu?Leu?Phe?Val?Thr?Ile?Tyr?Ser?His?Leu?Leu?Leu
85??????????????????90??????????????????95
Val?Ala?Ala?Gly?Met?Glu?Ala?Gln?Phe?Leu?Tyr?Leu?Tyr?Ala?Leu?Ile
100?????????????????105?????????????????110
Tyr?Phe?Leu?Gln?Cys?Ils?Asn?Ala?Cys?Arg?Ile?Ile?Met?Arg?Cys?Trp
115?????????????????120?????????????????125
Leu?Cys?Trp?Lys?Cys?Lys?Ser?Lys?Asn?Pro?Leu?Leu?Tyr?Asp?Ala?Asn
130?????????????????135?????????????????140
Tyr?Phe?Val?Cys?Trp?His?Thr?His?Asn?Tyr?Asp?Tyr?Cys?Ile?Pro?Tyr
145?????????????????150?????????????????155?????????????????160
Asn?Ser?Val?Thr?Asp?Thr?Ile?Val?Val?Thr?Glu?Gly?Asp?Gly?Ile?Ser
165?????????????????170?????????????????175
Thr?Pro?Lys?Leu?Lys?Glu?Asp?Tyr?Gln?Ile?Gly?Gly?Tyr?Ser?Glu?Asp
180?????????????????185?????????????????190
Arg?His?Ser?Gly?Val?Lys?Asp?Tyr?Val?Val?Val?His?Gly?Tyr?Phe?Thr
195?????????????????200?????????????????205
Glu?Val?Tyr?Tyr?Gln?Leu?Glu?Ser?Thr?Gln?Ile?Thr?Thr?Asp?Thr?Gly
210?????????????????215?????????????????220
Ile?Glu?Ash?Ala?Thr?Phe?Phe?Ile?Phe?Asn?Lys?Leu?Val?Lys?Asp?Pro
225?????????????????230?????????????????235?????????????????240
Pro?Asn?Val?Gln?Ile?His?Thr?Ile?Asp?Gly?Ser?Ser?Gly?Val?Ala?Asn
245?????????????????250?????????????????255
Pro?Ala?Met?Asp?Pro?Ile?Tyr?Asp?Glu?Pro?Thr?Thr?Thr?Thr?Ser?Val
260?????????????????265?????????????????270
Pro?Leu
<210>2
<211>825
<212>DNA
＜213〉SARS virus (SARS virus)
<400>2
atggatttgt?ttatgagatt?ttttactctt?ggatcaatta?ctgcacagcc?agtaaaaatt??60
gacaatgctt?ctcctgcaag?tactgttcat?gctacagcaa?cgataccgct?acaagcctca??120
ctccctttcg?gatggcttgt?tattggcgtt?gcatttcttg?ctgtttttca?gagcgctacc??180
aaaataattg?cgctcaataa?aagatggcag?ctagcccttt?ataagggctt?ccagttcatt??240
tgcaatttac?tgctgctatt?tgttaccatc?tattcacatc?ttttgcttgt?cgctgcaggt??300
atggaggcgc?aatttttgta?cctctatgcc?ttgatatatt?ttctacaatg?catcaacgca??360
tgtagaatta?ttatgagatg?ttggctttgt?tggaagtgca?aatcccagaa?cccattactt??420
tatgatgcca?actactttgt?ttgctggcac?acacataact?atgactactg?tataccatat??480
aacagtgtca?cagatacaat?tgtcgttact?gaaggtgacg?gcatttcaac?accaaaactc??540
aaagaagact?accaaattgg?tggttattct?gaggataggc?actcaggtgt?taaagactat??600
gtcgttgtac?atggctattt?caccgaagtt?tactaccagc?ttgagtctac?acaaattact??660
acagacactg?gtattgaaaa?tgctacattc?ttcatcttta?acaagcttgt?taaagaccca??720
ccgaatgtgc?aaatacacac?aatcgacggc?tcttcaggag?ttgctaatcc?agcaatggat??780
cccatttatg?atgagccgac?gacgactact?agcgtgcctt?tgtaa??????????????????825
<210>3
<211>13
<212>PRT
＜213〉SARS virus (SARS virus)
<400>3
Phe?Phe?Thr?Leu?Gly?Ser?Ile?Thr?Ala?Gln?Pro?Val?Lys
1???????????????5???????????????????10
<210>4
<211>14
<212>PRT
＜213〉SARS virus (SARS virus)
<400>4
Leu?Lys?Glu?Asp?Tyr?Gln?Ile?Gly?Gly?Tyr?Ser?Glu?Asp?Arg
1???????????????5???????????????????10
<210>5
<211>39
<212>PRT
＜213〉SARS virus (SARS virus)
<400>5
Leu?Val?Lys?Asp?Pro?Pro?Asn?Val?Gln?Ile?His?Thr?Ile?Asp?Gly?Ser
1???????????????5???????????????????10??????????????????15
Ser?Gly?Val?Ala?Asn?Pro?Ala?Met?Asp?Pro?Ile?Tyr?Asp?Glu?Pro?Thr
20??????????????????25??????????????????30
Thr?Thr?Thr?Ser?Val?Pro?Leu
35
<210>6
<211>39
<212>PRT
＜213〉SARS virus (SARS virus)
<220>
<221>misc?feature
<222>(23)..(23)
＜223〉Met is the methionine(Met) of oxidation
<400>6
Leu?Val?Lys?Asp?Pro?Pro?Asn?Val?Gln?Ile?His?Thr?Ile?Asp?Gly?Ser???1????????????????5???????????????????10??????????????????15
Ser?Gly?Val?Ala?Asn?Pro?Ala?Met?Asp?Pro?Ile?Tyr?Asp?Glu?Pro?Thr
20??????????????????25??????????????????30
Thr?Thr?Thr?Ser?Val?Pro?Leu
35
<210>7
<211>20
<212>PRT
＜213〉SARS virus (SARS virus)
<400>7
Thr?Lys?Phe?Pro?Ser?Val?Tyr?Ala?Trp?Glu?Arg?Lys?Lys?Ile?Ser?Asn
1???????????????5???????????????????10??????????????????15
Cys?Val?Ala?Asp
20
<210>8
<211>23
<212>PRT
＜213〉SARS virus (SARS virus)
<400>8
Arg?Asn?Thr?Arg?Glu?Val?Phe?Ala?Gln?Val?Lys?Gln?Met?Tyr?Lys?Thr
1???????????????5???????????????10???????????????15
Pro?Thr?Leu?Lys?Tyr?Phe?Gly
20
<210>9
<211>24
<212>PRT
＜213〉SARS virus (SARS virus)
<400>9
Ser?Thr?Pro?Lys?Leu?Lys?Glu?Asp?Tyr?Gln?Ile?Phe?Phe?Tyr?Ser?Glu
1???????????????5???????????????????10??????????????????15
Asp?Arg?His?Ser?Gly?Val?Lys?Asp
20
Claims (10)
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| CNB200310108604XA CN100497377C (en) | 2003-11-14 | 2003-11-14 | SARS coronavirus structure protein ORF3 and its use |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085812B (en) * | 2006-06-08 | 2010-12-01 | 中国科学院上海生命科学研究院 | A kind of SARS coronavirus polypeptide antigen and application thereof |
| CN111398597A (en) * | 2020-03-12 | 2020-07-10 | 中科欧蒙未一(北京)医学技术有限公司 | Kit for detecting IgM antibody against novel coronavirus SARS-CoV-2 in sample |
| CN111458504A (en) * | 2020-03-12 | 2020-07-28 | 中科欧蒙未一(北京)医学技术有限公司 | Kit for detecting IgG antibody against novel coronavirus SARS-COV-2 in sample |
| CN112321686A (en) * | 2020-11-16 | 2021-02-05 | 北京大学深圳研究生院 | A kind of stable polypeptide targeting new coronary pneumonia virus spike protein and its use |
-
2003
- 2003-11-14 CN CNB200310108604XA patent/CN100497377C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101085812B (en) * | 2006-06-08 | 2010-12-01 | 中国科学院上海生命科学研究院 | A kind of SARS coronavirus polypeptide antigen and application thereof |
| CN111398597A (en) * | 2020-03-12 | 2020-07-10 | 中科欧蒙未一(北京)医学技术有限公司 | Kit for detecting IgM antibody against novel coronavirus SARS-CoV-2 in sample |
| CN111458504A (en) * | 2020-03-12 | 2020-07-28 | 中科欧蒙未一(北京)医学技术有限公司 | Kit for detecting IgG antibody against novel coronavirus SARS-COV-2 in sample |
| CN112321686A (en) * | 2020-11-16 | 2021-02-05 | 北京大学深圳研究生院 | A kind of stable polypeptide targeting new coronary pneumonia virus spike protein and its use |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100497377C (en) | 2009-06-10 |
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