JPH061799A - Non-A non-B hepatitis virus antigen peptide, nucleic acid fragment encoding the peptide, and use thereof - Google Patents

Abstract

(57) [Summary] [Objective] To develop a peptide useful as a diagnostic agent for non-A non-B hepatitis virus infection. [Structure] A non-A non-B hepatitis virus antigen peptide having at least a part of the peptides shown below, or a peptide having an antigenicity equivalent thereto. (1) Val Ser Gln Leu Pro Gly Ser His Leu Pro Ser Trp Ile Trp Trp Arg (2) Ser Thr Thr Thr Thr Gly Gly Gln Val Gly Arg Gln Thr Ser Arg His Gly Ala Leu Phe Ser Gln Gly Ala Ser Gln Glu Val Gln Leu

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an antigen protein of a causative virus of serohepatitis virus (hereinafter referred to as non-A non-B hepatitis virus) which is neither type A nor type B, and a nucleic acid fragment encoding the same. The present invention relates to a diagnostic agent for non-A non-B hepatitis virus infection containing the peptide as an active ingredient.

[0002]

Viral hepatitis is a liver disease caused by infection with hepatitis virus. To date, hepatitis viruses of type A, type B, and type D (delta type) have been identified as hepatitis viruses. However, it has been reported that a large number of viral hepatitis that does not depend on infection with these hepatitis viruses exists. Among such hepatitis, those in which the causative virus is neither type A nor type B have been generically called non-A non-B hepatitis. Therefore, the causative virus of non-A non-B hepatitis is not one type, but is transmitted through, for example, an orally transmitted virus that infects via food or water, or mainly via blood,
Blood-borne viruses that frequently occur after blood transfusion are known.

The non-A non-B hepatitis virus was discovered from the cloning of viral genes, unlike the history of the discovery of other viruses (Science, 244, 359-).
362 (1989)). This non-A non-B hepatitis virus is currently considered to be an enveloped virus and taxonomically similar to flavivirus or pestivirus, but may be a completely new type of virus. There is also.

An antibody assay system in which a part of the viral genome is expressed in yeast as a fusion protein C100-3 polypeptide with human superoxide mutase (SOD) and used as a diagnostic agent has been established (Sc).
ience, 244, 362-364 (1989)).
However, the above-mentioned regions in which the viral gene was successfully cloned and the proteins which were successfully expressed as a fusion protein were genes and proteins corresponding to the non-structural protein region of flavivirus.

Since then, several research groups have succeeded in cloning the gene for the structural protein region of non-A non-B hepatitis virus. For example, chiron type (WO90 / 11089, WO90 / 14436, etc.)
And cancer center type (Proc. Natl. Acad. S
ci. USA, 87, 9524-9528 (199
0)), Osaka University Micro Lab. (J. Virol., 65, 110)
5-1113 (1991)), and based on these data, it has been proposed to further divide blood-borne non-A non-B hepatitis viruses into subclasses (Hepatolo).
gy, 14, 381 (1991)). However, analysis of virus properties, virus isolation, virus structural protein analysis, etc. have not progressed.

As the diagnostic agent, it is possible to use either viral structural proteins or non-structural proteins, but it is possible to detect the disease early by using structural proteins that produce antibodies immediately after infection. And is preferable. In general, different virus strains have different immunoserologic properties. Therefore, in the same diagnostic method, differences in detection sensitivity will occur due to differences in strains, and in vaccines, differences in immunogenicity and defense against infection will occur. . Furthermore, it is also possible that the sensitivity of the therapeutic agent differs depending on the strain.

[0007] For example, with regard to hepatitis B virus, it is known that major epidemic strains (subtypes) exist in each region such as Europe, America and Southeast Asia, and the non-A non-B hepatitis virus which is the subject of the present invention also has a region. It is conceivable that there is a virus strain peculiar to. Therefore, non-A which is prevalent in Japan
A non-A non-B hepatitis virus infection diagnostic method capable of finding an antigen protein unique to non-B hepatitis virus and identifying a strain is essential.

[0008]

Under the above-mentioned conventional techniques and knowledge, the present inventors have found that non-A non-B, which is superior to the antigens conventionally used as diagnostic agents, is highly reliable and effective.
As a result of intensive studies to obtain hepatitis B virus antigen,
A non-A non-B hepatitis virus antigen gene was cloned from a serum derived from a Japanese person containing a blood-transmitted non-A non-B hepatitis virus gene, and among these, the same virus does not exist in other viruses. The inventors have found a gene encoding a peptide and completed the present invention.

[0009]

According to the present invention, the first aspect of the present invention is as follows: First, the following (1) or (2), which is capable of reacting with an anti-non-A non-B hepatitis virus serum by an antigen-antibody reaction.
A peptide having at least a part of an amino acid sequence of Ala Leu Phe Ser Gln Gly Ala Ser Gln Glu Val Gln Leu or a peptide having an antigenicity equivalent to these peptides is provided.

Further, as a second invention, novel nucleic acid fragments encoding these peptides are provided.

As a third invention, there is provided a diagnostic agent for non-A non-B hepatitis virus infection comprising the above peptide as an active ingredient. The non-A non-B hepatitis virus antigen peptide of the present invention has at least a part of the amino acid sequence of (1) or (2) above, preferably 6 amino acids or more, and more preferably 10 amino acids or more.

Further, modification (addition of amino acid, insertion, substitution,
It may be a peptide that has been deleted or lost. Further, other amino acid sequences may be added to at least a part of the above-mentioned amino acid sequence, but when this peptide is used as a diagnostic agent for non-A non-B hepatitis virus infection, in order to avoid deterioration of detection accuracy. Peptides other than antigenic peptides derived from human pathogens are preferred. As such an example, mouse parasite-derived glutathione-S-transferase (hereinafter referred to as GST), mouse-derived dehydrofolate reductase, human serum albumin and the like can be mentioned.

Even if these peptides are chemically synthesized, they are suitable in the form of a gene encoding these peptides alone or in a form in which they are linked in-frame with genes encoding other peptides. After being incorporated into a host and expressed, the host is disrupted and purified by an appropriate combination of conventional methods such as salting out, gel filtration, separation by ion exchange and affinity chromatography, high performance liquid chromatography, and fractionation by electrophoresis. It may have been done.

When a peptide is produced by gene recombination, if the DNA fragment is too short, it will not be expressed, or even if it is expressed, purification is extremely difficult.
It is preferable to incorporate the gene into the host together with the above-mentioned GST-encoding gene and express it.

On the other hand, the nucleic acid fragment of the present invention encodes the above peptide (1) or (2), and specific examples thereof include the following (1) or (2). (1) GTATCGCAGT TACCCGGATC CCACAAGCCG TCGTGGATAT GGTGGGCGGG (2) TCTACCTACA CGACAGGGGG ACACAGTGGGTAG CGCCAGACTA GTCGTCCAGGG CGCTGGCCCGCTAG

Such a nucleic acid fragment may be obtained by any method, for example, it can be chemically synthesized by a conventional method, and GPT is 1
Viral RNA was isolated by a known technique from Japanese blood diagnosed with non-A non-B hepatitis of 00 or more, and cDNA was prepared for reverse transcriptase. Sequence (J. Virol., 65, 119
PCR method (Science, 23, 9) using primers designed based on 5-1213 (1991)).
487 (1988)) and inserted into a cloning vector (see Example 1 of the present specification). IK4
The 1000th to 1047th nucleotide sequences and the 1150th to 1239th nucleotide sequences of a total of 1260 bp cDNA derived from the non-A non-B hepatitis virus gene possessed by the CE clone (analyzed by dideoxy method, see SEQ ID NO: 1)
The second nucleotide sequence corresponds to the gene of the present invention.

The sequence of the peptide represented by the above (1) or (2) of the present invention is a non-A non-B known so far.
It does not show homology with the amino acid sequence of hepatitis B virus peptide. Therefore, when this peptide is used as a diagnostic agent, an antibody positive reaction with non-A non-B hepatitis patient serum is detected at a higher rate as compared with normal person serum, and therefore, for example, microplates, polystyrene beads, latex particles It can be provided as a diagnostic agent in a state where the peptide is adsorbed on the surface of a support such as. Such a diagnostic agent may be a commonly used assay method for antigen-antibody reaction, such as RIA or ELI.
It can be used according to a conventional method such as SA, fluorescent antibody method, passive agglutination method, reverse passive agglutination reaction and the like. The nucleic acid fragment of the present invention can also be used as a probe to detect a virus from blood of a patient infected with a non-A non-B hepatitis virus.

[0018]

Thus, according to the present invention, peptides specific to certain viruses of non-A, non-B hepatitis virus include:
Since it specifically reacts with the serum of a patient infected with a certain non-A non-B hepatitis virus, it can be used for the diagnosis of the viral infection, and a nucleic acid fragment encoding this peptide is also used as a probe for the virus. It can be used for diagnosis of infectious diseases.

[0019]

EXAMPLES The present invention will be described in more detail with reference to the following examples. (Example 1) Cloning and analysis of non-A non-B hepatitis virus-derived gene (A) Gene cloning of non-A non-B hepatitis virus-derived structural protein region Hepatitis B negative GPT value 100
A 5 times amount of guanidinium thiocyanate solution (4M guanidinium thiocyanate, 5
0 mM Tris-HCl pH 7.6, 10 mM EDTA,
0.1 M 2-mercaptomethanol, 2% sarcosyl) was added, phenol / chloroform extraction was performed, and total RNA in the serum was purified by ethanol precipitation using glycogen as a carrier. On the other hand, four synthetic DNA primers having the following sequences were prepared based on the results of Okamoto et al. (See above). (1) 5'CATATGAGCACGAATCCTAA, (2) 5'CCCAACGAGCAAGTCGACGT, (3) 5'TGATCATGCATACTCCCGGGT, (4) 5'GCTGCCGTTGGTATTCACAA c of the above RNA using the synthetic DNA of (4) as a primer
DNA was prepared using a cDNA synthesis system (Boehringer Mannheim). The synthesized cDNA (1.
26 kbp) as a template and the primer (1)
This cDNA fragment was amplified by performing the PCR method using (2) and (2). After phosphorylating the 5'end of the amplified cDNA fragment with T4 polynucleotide kinase, SmaI
About 3.41 kb obtained by ligation with pUC18 digested with
The plasmid of p was named pIKC. The same operation as described above was performed using the primers (3) and (4), and the obtained plasmid of about 3.45 kbp was named pIKE. The 3'part of the cDNA fragment of non-A non-B hepatitis virus inserted in pIKC and the 5'part of the cDNA of non-A non-B hepatitis virus inserted in pIKE partially overlap. Therefore, pIKC and pIKE are treated with the restriction enzyme Av.
Approximately 4.01 kbp obtained after digestion with aI and ligation
The plasmid was designated as pIK4CE.

(B) Determination of nucleic acid base sequence and amino acid sequence of pIK4CE In order to investigate the cDNA base sequence of non-A non-B hepatitis virus cloned in pIK4CE, the incorporated cD
NA (1.26 kbp) is recloned and Sequed
No. Sequence Kit (United States)
The base sequence was determined by Biochemical). The nucleotide sequence obtained as a result and the decoding result of the amino acid sequence predicted therefrom are shown in SEQ ID NO: 1.

[0021] When these base sequences and amino acid sequences were searched in a database (DNASIS), bases specific to the IK4CE clone showing no homology with the peptides of the previously known blood-transmitting non-A non-B hepatitis virus. The 1000th to the 104th in the sequence (SEQ ID NO: 1)
7th base sequence and 1150th to 1239th base sequence (in terms of amino acid sequence, 334th to 34th base sequences)
It was found that there are 9th and 384th to 413th).

(Example 2) Examination of specificity of peptide specific to IK4CE clone for non-A non-B hepatitis Peptide Val Ser Gln Leu Pro
Gly Ser His Leu Pro Ser T
rp Ile Trp Trp Arg was chemically synthesized, and the E buffer (composition: Na ₂ CO ₃ 1.6 g / l, NaHCO ₃₎ was added so that the peptide concentration was 0.1 μg / ml.
2.9 g / l, NaN ₃ 0.16 g / l), and 100 μl thereof was dissolved in EIA plate (Nunc).
And allowed to stand for 1 hour at room temperature. After that, the antigenic protein was discarded, washed twice with 0.05% Tween20-containing phosphate buffer (hereinafter referred to as PBS-Tw), and then 3
The plate was washed 5 times with PBS-Tw containing% bovine serum albumin. Thereafter, 100 μl of 100-fold diluted normal human serum or acute hepatitis, liver cirrhosis, liver cancer patient serum was added, and the reaction was carried out at room temperature for 1 hour.

This reacted EIA plate was added to PBS-
After washing 5 times with Tw, 100 μl of a substrate solution (1 mg / ml p-nitrophenyl phosphate) of an alkaline phosphatase-conjugated anti-human IgG antibody (TAGO) diluted 7,500 times was added, and the mixture was kept in the dark for 30 minutes. After leaving it
The absorbance at 405 nm was measured.

In patients diagnosed with acute hepatitis, 5
On the 7th day, the absorbance reached 10 times higher than that of normal human serum,
Even after 80 days, the value was 6 times or more the absorbance of normal human serum. Furthermore, an abnormal increase in GPT value was observed around 60 days, or the absorbance increased remarkably in synchronization with this, reaching 15 times the absorbance of normal human serum. This indicates that the antibody detection against the peptide of the present invention can be applied to grasp the pathological condition of non-A non-B hepatitis virus.

Then, the antibodies against this peptide in the sera of 12 non-A non-B acute hepatitis, liver cirrhosis and liver cancer patients were measured. As a result, in 5 out of 12 cases, the absorbance was 6 times or more that of a normal person, and it became clear that these 6 cases were infections of the IK4CE strain type having the peptide of the present invention.

[0026]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 1257 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: DNA Origin Organ Name: Bloodborne Non-A Non-B Hepatitis Virus Sequence ATG AGC ACG AAT CCT AAA CCT CAA AGA AAA ACC AAA CGT AAC ACC AAC 48 Met Ser Thr Asn Pro Lys Pro Gln Arg Lys Thr Lys Arg Asn Thr Asn 1 5 10 15 CGC CGC CCA CAG GAC GTC AAG TTC CCG GGC GGT GGT CAG ATC GTT GGT 96 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly 20 25 30 GGA GTT TAC CTG TTG CCG CGC AGG GGC CCC AGG TTG GGT GTG CGC GCA 144 Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala 35 40 45 ACT AGG AAG ACT TCC GAG CGG TCG CAG CCT CGT GGA AGG CGA CAA CCT 192 Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro 50 55 60 ATC CCC AAG GCT CGC CGG CCC GAG GGC AGG GCC TGG GCT CAG CCC CGG 240 Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Ala Trp Ala Gln Pro Arg 65 70 75 80 TAC CCT TGG CCC CTC TAT GGC AAT GAG GGT CTG GGG TGG GCA GGA TGG 288 Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp 85 90 95 CTC CTG TCA CCC CGA GGC TCT CGG CCT AGT TTG TCA CCC ACG GAC CCC 336 Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Leu Ser Pro Thr Asp Pro 100 105 110 CGG CGT AGG TCG CGT AAT TTG GGT AAG GTC ATC GAT ACC CTT ACA TGC 384 Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys 115 120 125 GGC TTC GGA GCC CTC ATG GGG TAC ATT CCG CTC GTC GGC GCC CCC CTA 432 Gly Phe Gly Ala Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu 130 135 140 GGC ATG GGC CCC AGG GCC CTG CGG CAT GGC GTC CGG CGT CTG GAG GAC 480 Gly Met Gly Pro Arg Ala Leu Arg His Gly Val Arg Arg Leu Glu Asp 145 150 155 160 GGC GTG AAC TAC GCA ACA GGG AAT CTG CCC GGT TGC TCT TTC TCT ATC 528 Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile 165 170 175 TTC CTT CTA GCT TTG CTG TCT TGT CTG ACC ATC CCA GCT TCC GCT TAC 576 Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro Ala Ser Ala Tyr 180 185 190 GAA GTG CGC AAC GAG TCC GGG GTG TAC CAT GTC ACG AAC GAC TGC TCC 624 Glu Val Arg Asn Glu Ser Gly Val Tyr His Val Thr Asn Asp Cys Ser 195 200 205 AAC TCA AGT ATT GTG TAT GAG GCA GCG GAC ATG ATC ATG CAA CCC CCC 672 Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile Met Gln Pro Pro 210 215 220 GGG TGC GTG CCC TGC ATC CGA GAG AAC AAT TCC TCC CGT TGC TGG GTG 720 Gly Cys Val Pro Cys Ile Arg Glu Asn Asn Ser Ser Arg Cys Trp Val 225 230 235 240 GCG CTC ACC CCC ACG CTC GCG GCC GCC GCC GCC
AAC AAC AGC ATC CCC ACC ACG 768 Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Asn Ser Ile Pro Thr Thr 245 250 255 ACA ATA CGA CGC CAC GGA TCT TTG CTT GTT GGG GCA GCT GCT CTC TGT 768 Thr Ile Arg Arg His Gly Ser Leu Leu Val Gly Ala Ala Ala Leu Cys 260 265 270 TCC GCT ATG TAC GTA GGG GAT CTC TGC GGA TCT GTC TTC CTC GTC TCC 864 Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val Phe Leu Val Ser 275 280 285 CAG CTG TTC ACC TTC TCA CCT CGC CGG TAT GAG ACG GTA CAA GAA TGC 912 Gln Leu Phe Thr Phe Ser Pro Arg Arg Tyr Glu Thr Val Gln Glu Cys 290 295 300 AAC TGC TCA CTC TAT CCC GGC CAC GTA TCA GGT CAC CGC ATG GCT TGG 960 Asn Cys Ser Leu Tyr Pro Gly His Val Ser Gly His Arg Met Ala Trp 305 310 315 320 GAT ATG ATG ATG AAC TGG TCA CCT ACA ACG GCC CTA GTG GTA TCG CAG 1008 Asp Met Met Met Asn Trp Ser Pro Thr Thr Ala Leu Val Val Ser Gln 325 330 335 TTA CCC GGA TCC CAC AAG CCG TCG TGG ATA TGG TGG CGG GGC CAC TGG 1056 Leu Pro Gly Ser His Lys Pro Ser Trp Ile Trp Trp Ar g Gly His Trp 340 345 350 GGA GTC CTA GCG GGC CTT GCC TAC TAT TCC ATG GTG GGG AAC TGG GCT 1104 Gly Val Leu Ala Gly Leu Ala Tyr Tyr Ser Met Val Gly Asn Trp Ala 355 360 365 AAA GTG TTG GTT GTG ATG CTA CTC TTT GCC GGT GTT AGC GGC TCG TCT 1152 Lys Val Leu Val Val Met Leu Leu Phe Ala Gly Val Ser Gly Ser Ser 370 375 380 ACC TAC ACG ACA GGG GGA CAA GTG GGT CGC CAG ACT AGT CGT CAC GGG 1200 Thr Tyr Thr Thr Gly Gly Gln Val Gly Arg Gln Thr Ser Arg His Gly 385 390 395 400 GCC CTC TTC AGC CAG GGG GCG TCT CAG GAA GTC CAA CTT GTG AAT ACC 1248 Ala Leu Phe Ser Gln Gly Ala Ser Gln Glu Val Gln Leu Val Asn Thr 405 410 415 AAC GGC AGC ATT 1260 Asn Gly Ser 419

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Claims (3)

[Claims] 1. A non-A non-B hepatitis virus antigen peptide having at least a part of the amino acid sequence represented by the following (1) or (2), or a peptide having an antigenicity equivalent thereto. (1) Val Ser Gln Leu Pro G
ly Ser HisLeu Pro Ser Trp
Ile Trp Trp Arg (2) Ser Thr Thr Thr Thr G
ly Gly GlnVal Gly Arg Gln
Thr Ser Arg His GlyAla L
eu Phe Ser Gln Gly Ala Se
r GlnGlu Val Gln Leu 2. A nucleic acid fragment encoding the peptide according to claim 1. 3. A diagnostic agent for non-A non-B hepatitis virus infection, which comprises the peptide according to claim 1 as an active ingredient.