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

A technique of fusing antigens and expressing genes, which is applied in the field of medicine and biology, can solve the problems of late emergence of anti-C100-3, low sensitivity, large protein, etc., achieve high sensitivity and specificity, improve hydrophilicity, and good solubility Effect

Active Publication Date: 2014-05-28
GUANGZHOU WONDFO BIOTECH
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  • Abstract
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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 als

Method used

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

Examples

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Effect test

Embodiment 1

[0036] Example 1: Design of HCV Fusion Antigen

[0037] Because the antigens of viral infectious diseases are often large, it is difficult to express the complete protein in vitro. Even if the complete protein expression can be achieved, due to species differences, proteins with relatively large molecular weights often cannot be folded correctly, and later renaturation is difficult, which restricts Development of detection reagents. Therefore, using genetic engineering technology, the fusion expression of multiple dominant surface antigen peptide genes has become the direction of antigen research. Through the screening of a large number of experiments, the present invention finally selects four dominant epitopes of core (2-125aa), NS3 (1192-1459aa), NS4 (1694-1735aa), and NS4 (1901-1936aa), and performs codon After optimization, they were connected in series through soft linkers GGGGGG, SGGGGS, and GGGSGG with strong hydrophilicity. The antigenic epitope of the selected Core...

Embodiment 2

[0039] Example 2: Construction of HCV Fusion Antigen Expression Vector

[0040] According to the analysis of biological software, it is found that the recombinant protein has strong hydrophobicity. In order to increase the water solubility of the protein, the present invention preferentially selects the main epitope, and removes the non-antigenic epitope region with strong hydrophobicity as much as possible; at the same time, pET41a is preferentially selected As an expression vector, the soluble expression of the protein is made by using the highly hydrophilic solubilizing tag GST on the expression vector.

[0041] The target gene fragment (SEQ ID NO.26) obtained in Example 1 and the expression vector pET41a were double-digested with restriction endonucleases BamHI and HindIII respectively, ligated overnight at 16°C, and the ligated product was transfected by heat shock method. Into the competent DH5α, coated with 50μg / ml Kan + On the LB agar medium, positive plasmids were sc...

Embodiment 3

[0042] Example 3: Induced expression and purification of HCV fusion antigen

[0043] 3.1 Induced expression of the fusion antigen: Transform the competent cells of Escherichia coli E.coli BL21(DE3) with the positive recombinant plasmid identified by the correct sequence restriction enzyme digestion, and spread it on Kan + On the LB agar medium with a concentration of 50 μg / ml, culture at 37°C for 16 hours; pick 4 single colonies and inoculate to the concentration of 50 μg / ml Kan + In 5ml of LB liquid medium, cultivate overnight at 37°C, 200r / min; the next day, transfer to 5ml fresh containing 50μg / ml Kan at a ratio of 1:100 + In LB liquid medium, 37°C, 200r / min cultured to OD600≈0.6, the expression factors were screened according to the orthogonal test, including temperature, induction dose, induction time, OD value of host bacteria and pH value of the medium, etc.; the results It was found that when IPTG was added to a final concentration of 0.5mM and cultured at 25°C for 5 ...

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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...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70G01N33/569
Inventor 任艳娜才蕾唐时幸王继华
Owner GUANGZHOU WONDFO BIOTECH
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