After hepatitis C virus (HCV) is the blood transfusion, hepatitis and sending out non-A and B type hepatitiss main cause of diseaseIII. in September, 1989 convened in Tokyo in the international non-A and B type hepatitis conference named this kind of virus officially as hepatitis C virus (HCV), caused the hepatitis by HCV to call it hepatitis C (HC).

1 HCV gene characteristic

HCV is one kind of new viral type, belongs to the single laid normal chains RNA virus, its genome team codes the multi-peptides the overall homology to be small with other viral sequence, however several group of experimental results indicated that HCV is the human yellow virus. The first HCV 5 terminal regions and the acute communicable disease viruss corresponding region has many homology (45 ~49), but the HCV multi-peptides water affinity tallies very much with the yellow viral multi-proteins,And three gene structures and the function are also very similar. This and recently classifies them for the yellow virus in 3 different animal representations is consistent. HCV and the acute communicable disease virus, the yellow viruss blood relationship evolution relations, will possibly promote in the future the related HCV infection biology research, also possibly will be helpful to some important question solution,For example: In the HCV non-blood transfusion dissemination way possibly involves medium, some kind of cell as viral Chu Cunkus possibility, whether to have the viral gene, in the host genes conformity causes chronic, whether usually to exist is addicted to outside livers different cell to be addicted to the nature to cause the clinical manifestation diversification newly,As well as whether to have the immune body dependence to strengthen the phenomenon and so on. In addition, the yellow virus reduces the poisonous live vaccine the success, was the HCV recombinant vaccine development has provided the optimistic prospect.

2 HCV sequential analysis

Since 1989 Choo reported that the HCV subsequences (731Obp), many researchers have completed the many HCV entire genes or the gene fragment analysis. Looking from the existing data, these clone differently nucleotide (nt) has the very big difference, the homology from 57 ~92 different. The same-type clone the nucleic acid homology is not smaller than 80, the same-type each even identical HCV carrier separates between base ink is prosperous also has some differences.Ogata separates from the identical patient blood serum to 1979 with 1990 carries on quite discovered that has 123 difference in 4 923 nt. Some people thought that the HCV nucleic acid sequence changes along with the time lapse evades the host immune system.in 1991 hillock this announcement Et this HCV hypotype classification, the HCV each between involucrum protein and the sequence had differently 26 ~44. Therefore divides into HCJ, HCJ, HCJe and HCJ,4 plants. Et this HCJ are most, American HCJ are most.

3 HCV infection experiment diagnosis

Before El, uses in the HCV infection the experiment diagnosing the method mainly to have two kinds, examines anti-HCV and HCV RNA.

in May, 1989, American Chirob Corporation separated with the molecular cloning law to one can express NANB hepatitis virus specificity protein cDNA to clone 5-1-1. Also with 5-1-1 had 3 ORF to overlap the clone to connect together establishes another to clone C100. C 100 reorganize the expression with the hyperoxide mutase (SOD) gene fusion in the yeast the multi-peptides is C oo -. . Complete C · oo -.The multi-peptides N end has 154 SO the D amino acid, 5 attach the expression amino acid by the EcoR I spot man-power and 363 HCV C100. cDNA code amino acid; Also has 5 MS2 amino acids in its C end. HCV C oo-. The immune body examination system, is most early the application examination method. The world about HCV in each kind of crowds infection percentage, basically is comes from this reagent box the examination.The test result proved that after HCV is in the world nearly all bloods transfusion, non-A and B type hepatitis and part sending out non-A and B type hepatitiss cause of disease virus. Because Cloo only contains 363 amino acids, but the HCV genome team codes protein has is 3 010 amino acids. Therefore C1oo -. An antigen immune body system only represents the entire HCV antigen immune body system the - 4 parts, therefore the sensitivity is not high, anti-C100. -.Appears is also too late, is not suitable for the early diagnosis, and some patients simply do not appear. This method use antigen including SOD fusion protein, when therefore the human body has anti-SOD presents the false positive frequently. But uses the RIBA method to be able to remove because of C 100. -. In the antigen the false positive which causes including SOD responded that often serves as the confirmation experiment. The first generation of RIBA-1 reagent, includes C1oo -. , 5-1-1 two kind of antigen (including the SOD sequence),Compares the specificity distinct enhancement with ELISA, has avoided the false positive, but the sensitivity reduced. The second generation of RIBA-2 reagent includes four kind of antigen. In C 100-3. , in 5-1-1 foundations increased two antigen. C (NSa area) and C2z-. (After the C area) uses many antigen, not only enhanced the examination specificity, moreover enhanced the sensitivity greatly, picks out suspicious or the weak masculine sample regarding the ELISA method, especially is suitable the RIBA method to do the confirmation experiment.Along with HCV gene order exposition, the people discovered that the C area gene code protein compares the E area and certain non-structure area protein keeps. Therefore, examines the HCV immune body with the C area gene product appears more reasonable, therefore has established some new examination method one after another, attempts to overcome C100. Antigen certain flaws, call it second generation of anti-HCV reagent. The second generation of recombination gene multi-peptides examined anti-HCV the ELISA technology have joined core antigen C2z in the original envelope antigen one. , C o, C o.(C100-3) compares with the first generation of reagent, the second generation of reagent has the detection rate high (to be possible to enhance 25 ~30%), moreover the immune body appears shifts to an earlier time to 16~60 days. Because in the recombination gene multi-peptide antigen still had the SOD multi-peptides, therefore still had the false positive which anti-SO D creates, therefore the people start to use the synthesized peptide section establishment to examine anti-HCV the new reagent. With Chinese northern area HCV entire gene order,According to the literature developments forecast protein physics and chemistry nature and the secondary structure and the antigen decision bunch of possible position spot sequence software, to complete the HCV entire gene order water affinity, to be intimate with the natural, the mobilized analysis, has forecast the HCV antigen decision bunch of position spot. To the development C area 3 oligopeptides (CPA, C e, CPzo) has carried on the antigen activeness comparison first and applies initially in clinical, simultaneously expresses multi-peptide C with Japan genetic engineering C area to carry on the comparison.CP8+CP. the e masculine gender rate and the C masculine gender rate not obvious difference, the coincidence rate is 87.6. With double PCR experiment which establishes, compared with 82 example anti-CP and the HCVRNA test result, the coincidence rate is 87.5. Many countrys experiences proved that carries on a anti-HCV determination to giving blood, may reduce the blood transfusion future trouble hepatitis C greatly risk [20], cuts hepatitis Cs disease incidence rate]. Therefore the HCV examination may become the early infection the examination target [7].

3.2 HCVRNA examinations

The polymerase chain reaction is in the present molecular biology the most sensitive method, in the sample, so long as has a copy HCV sequence to have the possibility to pick out. Because HCV in is been very low by the infected person in vivo density, counter copies RNA PCR(RTPCR) to be able to examine in blood serums HCV RNA. The PCR technology has many merits: (1) specificity. A anti-HCV masculine gender cannot show directly patient in vivo at that time HCV whether to exist,But PCR examines whether there is can HCVRNA take the infectious direct indicator. (2) sensitivity is high. Detectable anti-HCV negative HCV infection. (3) masculine gender appears the morning. May also which advances in ALT pick out. After the chimpanzee infects HCV, the 3rd day then picks out HCV RNA in the blood serum. (4) application is widespread. May examine in the organization and the body fluid RNA. But the operation sequence is complex, the specification is strict, the experimental cost is high, therefore is only restricted in has the condition laboratory use.in 1991 established one item by Beijing medical department leaving class liver disease research institute to examine HCV PCR directly the dual PCR law, might also call “the one-stage process”. The characteristic is micro (may from 100~1 blood serums mention that RNA), simple (does not need equipment and so on low temperature supercentrifuge), fast (from to withdraw the RNA counter duplication to be possible to complete in 4 hours), reduces the Rnase pollution, in 1~2 days may transmit consider. As a result of PCR many merits, so long as unceasing corrective method,Causes it to hasten is simple, is fast, will certainly to become the HCV infection diagnosis the important means. Recently some findings indicated that carries on quota PCR again in the original RT-PCT foundation, its method is joins in the sample PCR response intermixture. HdTTP mixes, the fluid dodges the law determination. H mixes in the CPM value purifies the known density cDNAsfuw ( 10pg/to manage 30UI every time),

At present a various factories sells anti-HCV reagent box, during reagents sensitivity, specificity existence certain difference, are composed separately by the recombination gene multi-peptide synthesized multi-peptides, the genetic engineering multi-peptides merit is: (1) may contain many anti-home position spots. (2) once clones expresses the successful output to be high, the price is low. (3) expresses the multi-peptides the stability to be good.(4) may several fragment superposition use, have the high specificity and the sensitivity. (5) purification is simple, does not have other protein disturbance, may perform the inhibition test. The shortcoming is: (1) clone expression specification is high, is not easy to achieve the satisfactory result. (2) expresses the product purification difficulty, includes the mycelium and so on other protein disturbance. during (3) each batch of output the stability is smaller than the difference. Synthesis EP27 and the test result, has prompted it and a second generation of anti-HCV reagent diversity.

Reference

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