HCV (hepatitis c virus) recombinant fusion antigen, and expressed gene and preparation method thereof

Abstract

The invention discloses an HCV recombinant fusion antigen and an expressed gene and a preparation method thereof. Amino acids of the HCV recombinant fusion antigen are successively composed of an amino acid shown in SEQ ID No. 1, a first connexon, an amino acid shown in SEQ ID No. 2, a second connexon, an amino acid shown in SEQ ID No. 3, a third connexon and an amino acid shown in SEQ ID No. 4, wherein each connexon is composed of six flexible, polar and hydrophilic amino acids with small molecular weight. The preparation method comprises the following steps: constructing a recombinant plasmid pET41a-Core-NS3-NS41-NS42; and expressing and purifying the HCV recombinant fusion antigen. The HCV recombinant fusion antigen has high sensitivity and specificity and is applicable to the field of biological medicine, especially to colloidal gold test strips for antibodies to hepatitis c.

Description

technical field [0001] The invention belongs to the field of medicine and biology, and in particular relates to a HCV recombinant fusion antigen and its expression gene and preparation method Background technique [0002] Hepatitis C is caused by hepatitis C virus (Hepatitis c virus, HCV) infection, non-A, non-B (NANB) viral hepatitis. In 1974, Laneld first reported non-A, non-B (NANB) hepatitis after blood transfusion. In 1989, Choo et al. successfully isolated (NANB) virus from infected chimpanzee blood samples, and started research on this strange virus since then. According to the statistics of the World Health Organization, the global average infection rate of HCV is about 3%, about 11.7 to 21 billion people are infected with HCV, about 3.15 million new cases occur every year, and about 350,000 people die each year due to hepatitis C-related diseases. [0003] Hepatitis C virus is mainly transmitted through blood, accounting for 80-90% of post-transfusion hepatitis. In...

AI Technical Summary

Problems solved by technology

The antigen C100-3 used in the first generation of anti-HCV ELISA diagnostic reagents is a fusion protein expressed after the chimerism of HCV non-structural genes (NS3 / NS4) and human superoxide dismutase (SOD) genes, because C100 only contains 363 Amino acids, and the protein encoded by the HCV genome is as long as 3011 amino acids, so the C100-3 antigen-antibody system only represents a small part of the entire HCV antigen-antibody system, so the sensitivity is not high, and it is difficult to meet the needs of reagent detection; , anti-C100-3 appeared relatively late, and did not meet the basic conditions for early diagnosis

The second-generation anti-HCV ELISA diagnostic reagents added core region recombinant protein C22 and NS3 region recombinant protein C33c on the basis of the first generation, which increased the detection rate by 25% to 30% and shortened the window period for detecting antibodies , but because there are still SOD polypeptides in the recombinant gene polypeptide antigen, there are still false positives and a few missed detections caused by anti-SOD

However, the results of this study show that the positive rate of detecting HCV is as high as 90%, and the false positive rate is as high as 20%, so it is obviously unreasonable to only consider the linear epitope of the antigen

In 2010, Nie Dongsong and others also published a report on HCV fusion multi-epitope antigen, but the fusion protein is only in the stage of laboratory research, and only 10 chronic liver disease patients and 5 HCV-negative patients were detected in the sensitivity and specificity evaluation. Serum was tested, no follow-up studies involved

In 2005, Yu Longlin's research group expressed prokaryotic expression of the full-length NS5A and NS5A protein (amino acid 2212-2313) genes in the high immunogenic region required by the third-generation anti-HCVELISA diagnostic reagent, and the sensitivity test results found that the full-length NS5A gene Both the recombinant protein and the recombinant protein of the NS5A high immune region gene have good immunological detection results, but the detection sensitivity of the NS5A full-length recombinant protein is significantly higher than that of the NS5A high immune region recombinant protein

In addition, the protein expressed by the NS5A full-length gene added to the third-generation diagnostic reagent is too large, and after being expressed in tandem with the dominant epitopes of Core, NS3, and NS4, part of the epitopes cannot be exposed on the molecular surface due to the occlusion of the spatial structure In addition, according to reports at home and abroad, there is no antibody in serum that only reacts specifically with NS5

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