JPH03266987A - Gene fraction of non-a, non-b type hepatitis virus and detection of virus - Google Patents

Abstract

PURPOSE:To detect non-A, non-B type hepatitis virus by synthesizing peptide of non-A, non-B type hepatitis virus to be coded by gene fraction containing a specific base sequence or part thereof. CONSTITUTION:Host cells are transformed with a vector containing DNA sequence to code a gene fraction containing base sequence shown by formula I or part thereof. The transformed host cells are cultured in a proper medium and peptide of non-A, non-B type hepatitis virus is separated from the culture mixture and recovered. since non-A, non-B type hepatitis virus in blood can be detected when the peptide is used, hepatitis causing virus which could not be removed by assay of A type, B type and C type hepatitis virus can be further eliminated.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a non-A, non-B hepatitis virus gene fragment and a method for detecting a non-A, non-B hepatitis virus using the same.

It has long been known that there are two types of viral hepatitis: hepatitis A (infectious hepatitis) and hepatitis B (serum hepatitis). This is mainly based on differences in infection routes;
It has been confirmed that hepatitis A causes epidemics through oral infection, and hepatitis B is primarily transmitted through blood. These two hepatitis-causing viruses have already been isolated and identified, and the hepatitis A virus is an RNA virus with a diameter of 27 nm that belongs to the picornavirus [Finest
on, S.

M, et al., 5science 182 p1026 (1
973)], on the other hand, hepatitis B virus is an enveloped DNA with a diameter of 42 nm that belongs to the hepadnavirus.
It was determined that it was a virus [Dane,
O, S, et al., Lancetp695 (1970)]
. Furthermore, immunoserological diagnostic methods for these hepatitis viruses have now been established.

As definitive diagnostic methods for these two hepatitis viruses have been established, the existence of non-A, non-B hepatitis, which does not belong to either of them, has become clear [Prince, A.

Q., et al., Lancet, I p241 (
1974) Ko.

Post-transfusion hepatitis is caused by hepatitis B virus surface antigen (HBsAg).
) has been significantly reduced with the introduction of screening methods, but it has not been reduced to zero, and furthermore, patients with hepatitis who have developed
No evidence of hepatitis B or hepatitis B infection was found. For this reason, this hepatitis is generally called non-A, non-B hepatitis.

This hepatitis accounts for about 50% of sporadic hepatitis and more than 90% of post-transfusion hepatitis in Japan, and it is estimated that more than 50% of chronic hepatitis, liver cirrhosis, and liver cancer are caused by non-A, non-B hepatitis. It has become a major social problem.

Recently, this non-A, non-B virus that is mainly transmitted through blood
Regarding the hepatitis virus, the name CV (hepatitis C virus) is becoming established, and screening methods for antibodies related to HC have already been developed, but its detailed virological properties are still unknown. is [
Choo et al., 5science.

244.359-362 (1989), Ku
o et al., 5science, 244362-364
(1989) Ko.

Apart from this, India, Myanmar, Afghanistan,
In addition, it has become clear that there is a second type of viral non-A, non-B hepatitis that is prevalent through oral infection in countries such as Africa [Khuroo, M, S, Am, J.

Med, 68 p818-824. (1980
)]. This is commonly referred to as waterborne, or epidemic non-A, non-B hepatitis. Although this hepatitis epidemic has not been seen in Japan, there seems to be some cases of hepatitis being imported from endemic areas by travelers [Fukuhara et al., Abstracts of the 25th Annual Meeting of the Japanese Society of Hepatology, p. 151 (1989) ].

The present invention relates to the latter mentioned above, an epidemic non-A, non-B hepatitis virus that is transmitted mainly through water. It is called non-A, non-B hepatitis virus.

The virus itself for this non-A, non-B hepatitis has not been isolated and identified, and therefore no diagnostic, therapeutic, or preventive methods for this hepatitis have been established.

Furthermore, the only way to diagnose this hepatitis was to make a diagnosis of exclusion.

That is, the patient's serum is tested for hepatitis A and B, for which diagnostic methods have been established, to deny these hepatitis cases, and to show hepatitis symptoms as part of systemic infection. The possibility of herpes, cytomegalo, or Epstein-Barr virus infection was ruled out, and drug-induced, alcohol-induced, or autoimmune hepatitis was ruled out, and the patient was diagnosed with non-A, non-B hepatitis.

Non-A, non-B hepatitis, which is the subject of the present invention, is mainly endemic in India, Nepal, Myanmar, and Africa. The main route of infection is through drinking water and vegetables, and it is known that the symptoms after infection are usually only a temporary infection, that is, acute hepatitis, and there is generally no persistent infection. ing. However, it is known that when pregnant women are infected with this virus, the mortality rate is as high as 20% [Tandon et al.
J, Med, Res.

75, p739-744 (1982)], outbreaks can often be caused by contamination of drinking water. A recent example is the reported outbreak of 120,000 patients in the southwestern part of Urumqi Autonomous Region in China from 1986 to 1988 [Gendai Kagaku, 19
July 1987 issue p 62-67, Journal of Infectious Diseases, 64.
p105-111 (1990)]. Therefore, for people traveling to such endemic areas, there is a desire for the development of effective vaccines as well as diagnostic reagents that can easily detect the presence of antibodies, antigens, or the virus itself.

According to previous reports, detection of virus-like particles by immunoelectron microscopy has been reported [5reen 1vasan
et al. J. Gen. Virol, 65. plo0
5-1007 (1984)] Furthermore, it has been reported that infection experiments were successful in an animal model using monkeys [Kane et al., JAMA 252゜p3140-
3145 (1984)].

The present inventors also inoculated the veins of cynomolgus monkeys with a virus solution prepared from the feces of acute-stage patients in endemic areas, resulting in infection and success in subculture.

We also detected virus-like particles in the feces and monkey liver cells of infected monkeys [Soe et al., Liver 9.
p135-145 (1989) Ko. Furthermore, it was confirmed that large amounts of virus-like particles were detected in monkey bile [Gendai Kagaku, July 1989 issue, p. 62-67].

However, the specific properties of the virus that caused the patient's non-B hepatitis and the base sequence of the virus' gene have not yet been reported, and no detection method has been established to directly detect the virus that causes the patient's non-B hepatitis. .

Under these circumstances, the present inventors conducted extensive research to isolate non-A, non-B hepatitis viruses.
We succeeded in identifying a nucleic acid sequence specific to the hepatitis virus. Furthermore, by using such gene fragments specific to non-A, non-B hepatitis viruses, the present inventors have discovered that it is possible to detect non-A, non-B hepatitis viruses, which have been undetectable so far. They have also found that such a detection method can be used to test human serum, leading to the completion of the present invention.

That is, the present invention provides a gene fragment of a non-A, non-B hepatitis virus that is effective for detecting non-A, non-B hepatitis viruses, which has been considered impossible until now.

The non-A, non-B hepatitis virus gene fragment of the Japanese invention is extracted and extracted from the bile of cynomolgus monkeys infected with the non-A, non-B hepatitis virus.
It is possible to isolate. First, bile is diluted with physiological saline and tissue pieces are removed by low-speed centrifugation. Next, it is layered on physiological saline containing 20 g of sucrose and subjected to ultracentrifugation. This allows virus particles to be collected in the pellet. RNA was extracted by treating the virus particles with guanidine thiocyanate, and this was used as a template to generate cDNA.
Synthesize DNA.

However, the amount of target non-A, non-B hepatitis virus in the sample is small, and it is not easy to synthesize and isolate cDNA from RNA derived from this virus. To overcome this problem, we developed a synthetic linker-DNA
Polymerase chain reaction (PCR) was applied.

That is, the present inventors added synthetic linkers to both ends of the non-A, non-B hepatitis virus gene cDNA based on the previously developed method for amplifying unknown nucleic acid sequences (Japanese Patent Application No. 1-189875). , PCR is performed using the base sequence of a portion of the linker as a primer. By this operation, it is possible to amplify the non-A, non-B hepatitis virus gene fragment, which existed only in trace amounts, to an amount that can be isolated. Next, the restriction enzyme Not included in the linker site
Using the l recognition site, the plasmid vector pB]ue
It is possible to insert and isolate scriptIr (KS+) [Strategene] into the Not 1 site. However, in this method, DNA fragments derived from coexisting cynomolgus monkey chromosomes are also amplified in the same way as viral gene fragments, so in the present invention, DNA fragments derived from cynomolgus monkey chromosomes can be removed by the following three steps. . (1) First, colony hybridization is performed using a repetitive sequence unique to monkey chromosomal DNA as a probe. (2) Next, insert DNA is collected from plasmid clones that did not react in the colony hybridization described above, and Southern hybridization of normal cynomolgus monkey liver DNA is performed using this as a probe. Probes that test positive are considered to be derived from monkey chromosomes and are therefore removed. (3) Determine the base sequence of the insert DNA of the remaining plasmid clone. Primers are synthesized based on the obtained base sequence, and PCR is performed using normal cynomolgus monkey liver DNA as a sample. Clones in which a positive band appeared by ethidium bromide staining and Southern hybridization using the entire length of the insert as a probe were excluded. The repetitive sequence unique to monkey chromosomal DNA used here was isolated from normal monkey liver DNA on another occasion, the base sequence was determined, and the high homology was confirmed in the database. Proc, Na
tl, Acad, Sci.

USA vol, 77 p2129-2133 (1
980), Nucl, Ac1dsRes, Vo
l, 13 p7813-7827 (1985),
Same Vo1.12 p5823-5836 (+984)]
. Regarding the plasmid clone pWB6-352 that remained after the above steps (2) and (3), the non-A, non-B hepatitis virus specificity of its insert DNA was examined, and the following was found.

That is, a primer was prepared based on the base sequence of pWB6-352, and used to infect various cDNAs with pWB6-352.
When performing a CR reaction and performing Zazan hybridization using a partial base sequence of pWB6-352 as a probe, no positive 2- band was detected from normal monkey bile-derived cDNA, but a positive band was detected from infected monkey bile-derived cDNA. Detected. Furthermore, no positive band is detected by Southern hybridization in cDNA derived from normal monkey feces, but a positive band is detected in cDNA derived from infected monkey feces.

In addition, cDNA was prepared from the feces of 10 non-A, non-B hepatitis patients (Myanmar) and 5 normal individuals (Japan).
When CR was performed, positive signals were detected by Southern hybridization in 7 out of 10 patients with non-A, non-B hepatitis, and were not detected in 5 normal patients.

Furthermore, when the cDNA of the present invention isolated in this way was searched for homology with the base sequences of genes that have been reported so far using a database, it was found that the cDNA of the present invention isolated in this way has a pomology with the base sequences of genes that have been reported so far. There was no.

The above results revealed that the insert DNA contained in plasmid pWB6-352 was a gene fragment derived from non-A, non-B hepatitis virus.

The base sequence of such a non-A, non-B hepatitis virus gene fragment can be used for genetic diagnosis of non-A, non-B hepatitis virus by creating a PCR primer probe based on this sequence.

Furthermore, for specimens containing a relatively large amount of virus, it is also possible to directly detect non-A, non-B hepatitis virus genes using a probe based on this base sequence without going through PCR. The substances to be measured used in this case are:
Examples include serum, feces, liver and bile.

The PCR method can be carried out by a commonly used method, as described by Saiki et al., 5science, V.
ol.

239, p487-491 (1988)]. When PCR primers are synthesized based on the base sequence of the non-A, non-B hepatitis virus gene fragment shown in the present invention, two sites each having 10 or more bases can be freely selected.

This embodiment shows an example of this.

Furthermore, using the peptide encoded by the base sequence of this non-A, non-B hepatitis virus gene fragment, it is also possible to screen for antibodies against the non-A, non-B hepatitis virus using antigen-antibody reactions. It is.

Regarding virus detection methods using such antigen-antibody reactions, methods using ordinary enzymes and other labeling substances,
For example, ELISA etc. can be applied.

Such peptides can be expressed and recovered in large amounts using a suitable vector and a suitable host cell according to conventional methods of genetic recombination technology. Additionally, chemically synthesized peptides can also be used.

Furthermore, the present invention discloses that various virus measurement methods are possible using blood as a measurement target as a method for detecting non-A, non-B hepatitis virus. In other words, although previous reports have shown that the non-A, non-B hepatitis virus, which is the subject of the present invention, exists in the feces and bile of infected patients, it has been reported that virus particles appear in the blood. had not been confirmed at all. This fact is reported for the first time by the present invention, and by using patient serum as the measurement target, it is possible to easily confirm the presence of non-A, non-B hepatitis virus. With the disclosure that non-A, non-B hepatitis viruses can also be detected in blood, it has been discovered that non-A, non-B hepatitis viruses can be detected in blood, which could not be completely removed by assays for hepatitis A, B, and C viruses. It has become possible to further remove hepatitis-causing viruses.

By applying means such as PCR using this knowledge and the non-A, non-B hepatitis virus-specific DNA sequence disclosed by the present invention, it has become extremely easy to identify viruses in samples such as blood and feces. Ta.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

When a virus solution prepared from acute phase feces of a patient with non-A, non-B hepatitis was inoculated into the vein of a cynomolgus monkey, infection was established. Furthermore, passage infection was also established using the feces of infected monkeys [Liver 9. p135-145 <
1989)].

When the bile of infected monkeys was observed under an electron microscope, virus particles with a diameter of 27 nm were detected (Gendai Kagaku July 1989 issue, p. 62-67). Virus particles were purified from approximately 2 ml of this bile. and used in the following experiments.

The virus particles obtained in (1) were treated with 1 to 5,5M guanidine thiocyanate to extract nucleic acids derived from the virus genome. Synthesize cDNA using reverse transcriptase and random primers and convert it into double-stranded linear DNA. Synthetic linkers at both ends - [DNA15'-ATTGCGG
CCGCTTAA-3° DNA2・5°-CCCTT
TAAGCGGCCGCAAT-3'], a portion of the linker was linked to the primer [DNA3; 5''~T
TAAGCGGCCGCAAT-3'] was subjected to polymerase chain reaction (PCR) [Patent application No. 1-189
No. 875]. The obtained PCR product was treated with restriction enzyme 1Jotl.
plasmid vector pB]uescript
A cDNA library was created using Ir [Strategene]. This plasmid library was screened for virus-specific nucleic acids using the following procedure.

First, plasmids showing positive results in colony hybridization using a repeat sequence specific to monkey chromosomal DNA as a probe were excluded. Monkey chromosome D used here
The NA-specific repeat sequence is found in normal cynomolgus monkey liver f.
The DNA was isolated from iDNA and the base sequence was determined. A mixture of three types of repeat sequences that were confirmed to have high homology with previously reported repeat sequences was used [Proc, N1t1. Acad, Sci,
USA Vol, 77 p21292133 (1
, 980), Nucl, Ac1d Res,, V
ol, 12 p58235836 (1984), N
ucl, Ac1d, Res, Vol, 13 p
78137827 (1985)]. The insert size of the remaining plasmid was examined, and insert DNA was prepared from the plasmid with a long insert. Using the obtained insert DNA as a probe, normal cynomolgus liver DNA was detected.
Southern hybridization of A was performed. As a result, probes that showed positive results were excluded because they were considered to be derived from monkey chromosomes.

Furthermore, the nucleotide sequence of the insert DNA of the remaining plasmid was determined. Primers were synthesized based on the obtained base sequence, and PCR was performed using normal monkey liver DNA as a sample. Plasmids in which a positive band appeared by ethidium bromide staining and Southern hybridization using the full length insert as a probe were excluded.

In this way, plasmid pWB6-352 into which a 360 bp gene fragment was inserted remained.

First, RNA was extracted and cDNA was synthesized from non-A, non-B hepatitis virus-infected monkey bile and normal monkey bile, respectively.

Based on the insert base sequence of plasmid pWB6-352? -352-A[5°-ACCTGTGGT
GAACTTGTT3° and 352-B [5'-
AACCAGCGCAAGGCCGTG-3'] was used to perform PCR, and 352-C[5'-GCTCG''T
, ACAGTTCACAAGTT-3′] was used as a probe. As a result, no positive band was detected from cDNA derived from normal monkey bile.

On the other hand, a positive band was detected in Southern 9-in hybridization of cDNA derived from infected monkey bile, and the band had the expected molecular size (approximately 280 b).
p).

Next, cDNA was extracted from infected monkey feces and normal monkey feces, respectively.
A was prepared and PCR was performed in the same manner as above. For cDNA derived from normal monkey feces, no band that hybridized with probe 352-C was detected by Southern hybridization, but for cDNA derived from infected monkey feces, a positive band of the expected size was detected by Southern hybridization. It was done.

Finally, cDNA was prepared from the feces of 10 non-A, non-B hepatitis patients (Myanmar) and 5 normal individuals (Japan), and PCR was performed. As a result, positive signals were detected by Southern hybridization in 7 out of 10 non-A, non-B hepatitis patients, and not in 5 normal patients.

Furthermore, the insert DNA nucleotide sequence of pWB6-352 had no pomology with any gene nucleotide sequence reported in the database.

20- From the above results, it was revealed that the gene contained in plasmid pWB6-352 was derived from non-A, non-B hepatitis virus.

An attempt was made to detect the virus in the blood of infected cynomolgus monkeys based on the base sequence of the non-A, non-B hepatitis virus gene fragment obtained by PCR. First, four cynomolgus monkeys (No. 5578.5) were tested for infection with non-A, non-B hepatitis virus.
975.6201 and 6205) from before virus inoculation to after recovery from hepatitis.

Next, 100A of each serum was added with 5M Na, 10.7% and 40%
%PE023, 4d was added, and after standing at 0°C for 1 hour, the pellet was collected by centrifugation. Viral nucleic acid was extracted with 5.5M guanidine thiocyanate, and cDNA was synthesized using reverse transcriptase and random primers. 352
After 35 cycles of PCR using A and 352-II as primers, 2x agarose gel electrophoresis and ethidium bromide staining were performed. 10 more 352-C
- Approximately 2
An 80 bp band was detected. The results are shown in Figure 2.

Figure 2 shows the GOT value (b) and GPT value (◆) in the cynomolgus monkey infection experiment, the appearance of virus particles in bile by electron microscopy (+), and the detection results of viral nucleic acid in serum by PCR (P: positive, N negative).

It was found that viral nucleic acid was detected in the serum of all four animals from around 7 days after vaccination, and was no longer detected after recovery from hepatitis.

This change corresponded to the appearance of virus particles in the bile as observed by electron microscopy.

From the above results, it is clear that the epidemic non-A, non-B hepatitis virus, which is mainly transmitted through water, also exists in the blood at the early stage of infection, and that the presence of the virus in the blood can be confirmed by the present invention. Based on the base sequence of the non-A, non-B hepatitis virus gene fragment, it was found that it could be detected by PCR method.

[Brief explanation of drawings]

Figure 1 shows pWI1 cloned in the present invention.
The nucleotide sequence of 6-352 and the amino acid sequence that can be encoded by this gene are shown in single letter notation. 21 2- Figure 2 shows the GOT value (b) and GPT value (◆) in the infection experiment using four cynomolgus monkeys in Example (4).
), the appearance of virus particles in bile by electron microscopy (10), and the detection results of viral nucleic acids in serum by PCR method (P: positive, N: negative). (nx) ldυ' Engineering 09 -648 (nx ) 1dE) 'l0E) (n) () 1dE)'10E)

Claims (7)

[Claims] (1) Gene fragment specific to non-A, non-B hepatitis virus. (2) The gene fragment according to item (1) above, wherein the gene fragment contains the following base sequence or a part thereof. [There is a gene sequence] (3) A non-A, non-B hepatitis virus peptide encoded by the gene fragment described in item (2) above. (4) A method for detecting non-A, non-B hepatitis virus, which comprises detecting a gene complementary to the gene fragment described in item (1) above. (5) Non-A contained in blood as a test object
A non-A, non-B hepatitis virus detection kit characterized by detecting a non-B hepatitis virus. (6) The detection kit according to item (5), wherein the method for detecting non-A, non-B hepatitis virus is to detect a gene complementary to the following gene sequence or a partial sequence thereof. [There is a gene sequence] (7) The method for detecting non-A, non-B hepatitis virus is to detect a peptide encoded by the following gene fragment or a partial sequence thereof by antigen-antibody reaction.
Detection kit described in section ). [There is a gene sequence]