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HCV (hepatitis C virus) recombinant fusion antigen and application thereof

A fusion antigen and C-terminal technology, applied in the direction of fusion peptide, recombinant DNA technology, hybrid peptide, etc., can solve the problems of difficult adsorption, low specificity, and small molecular weight of synthetic peptides, and achieve the effect of easy renaturation

Active Publication Date: 2017-09-05
SICHUAN ANKERUI NEW MATERIAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the first-generation antibodies have several shortcomings: 1. Low sensitivity and false negatives; 2. Low specificity and prone to false positives; 3. Cannot be used as an indicator of infectivity
The artificially synthesized polypeptide antigen has the advantages of good specificity and no infectivity, but there are also some disadvantages. The synthetic polypeptide has a small molecular weight and is not easily adsorbed to the solid phase; the structure is short, has low affinity with antibodies, and contains fewer epitopes. It is easy to miss detection; the cost of synthetic peptide is high, which is not conducive to promotion, etc.
In addition to the risk of missed detection, since the main raw material recombinant antigen used in the hepatitis C virus diagnostic reagent is a biologically active substance, its activity is easily inactivated by temperature, and the thermal stability of the antigen is poor. The detection kits are required to be stored at 2-8°C afterward, and are greatly affected by the ambient temperature

Method used

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  • HCV (hepatitis C virus) recombinant fusion antigen and application thereof
  • HCV (hepatitis C virus) recombinant fusion antigen and application thereof
  • HCV (hepatitis C virus) recombinant fusion antigen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design of HCV recombinant fusion antigen

[0042] Because the antigens of viral infectious diseases are often large, it is difficult to express the complete antigenic protein in vitro, or even if it is expressed in vitro, the antigenic protein with a relatively large molecular weight often cannot be folded correctly, resulting in difficulties in later renaturation, or renaturation. The antigenic activity of the poor, restricting the development of detection reagents. Therefore, using genetic engineering technology, the fusion expression of multiple dominant antigen epitope genes has become the direction of antigen research. Through the screening of a large number of experiments, the present invention finally determines to select two dominant epitopes of core (2-102aa) and NS5 (2322-2422aa), optimize the codons and connect them in series through the flexible link GGGSGG with strong hydrophilicity . The epitope of the selected Core is 2-102aa, its amino acid s...

Embodiment 2

[0048] Example 2 Construction of HCV Recombinant Fusion Antigen Expression Vector

[0049] The target gene fragment 1025 (SEQ ID NO.11) obtained in Example 1 and the expression vector pET28a were subjected to double digestion with restriction endonucleases EcoRI and XhoI, respectively.

[0050] The enzyme digestion reaction system of target gene fragment 1025 is:

[0051]

[0052] The enzyme digestion reaction system of the expression vector pET28a is:

[0053]

[0054] Enzymatic digestion in a water bath at 37°C overnight. The digested products were detected by 1% TAE agarose gel electrophoresis. Recover with Axygen Mini Gel Recovery Kit. Ligated at 16°C for 4 hours, and transferred the ligated product into competent E.coli DH5α cells by heat shock method, and spread it on a medium containing 50 μg / ml Kan + On the LB agar medium, under the condition of 37 ℃, culture upside down overnight or 12-14 hours until a single colony grows.

[0055] Initial screening was car...

Embodiment 3

[0066] Example 3 Induced Expression of HCV Recombinant Fusion Antigen

[0067] Transform Escherichia coli E.coliBL21 (Rossetta) competent cells with the positive recombinant plasmid pET28a-1025 that has been sequenced and identified by restriction enzyme digestion by heat shock method, and spread it on a medium containing 50 μg / ml Kan + and 25 μg / ml Chl + On the LB agar medium, under the condition of 37 ℃, culture upside down overnight or 12-14 hours until a single colony grows.

[0068] Pick 2 single colonies and inoculate to contain 50μg / ml Kan + and 25 μg / ml Chl + In 5ml LB liquid medium, culture overnight at 37°C, 200r / min. On the next day, transfer 200 μl of bacterial solution to 5 ml of fresh 50 μg / ml Kan + and 25 μg / ml Chl + LB liquid medium, 37°C, 200r / min culture to OD600≈0.8. Add IPTG to a final concentration of 1 mM, and continue shaking culture at 37° C. for 4 hours. SDS-PAGE electrophoresis was performed after induction (see figure 2 ). The cloned bacter...

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Abstract

The invention relates to an in-vitro detection diagnosis reagent and specifically discloses a recombinant fusion antigen for detecting HCV (hepatitis C virus), wherein the recombinant fusion antigen is composed of a Core antigen (2-102aa), a flexible connexon and an NS5 antigen (2322-2422aa). According to the recombinant fusion antigen disclosed by the invention, by optimizing an antigen area of nucleocapsid protein, redundant amino acids at a terminal C are removed; a 2-102aa antigen area is selected to reduce the false positive rate without impairing sensitivity. Moreover, the nucleocapsid protein is added to a 2322-2422aa amino acid area of non-structural protein NS5 so that the provided fusion antigen has remarkably high sensitivity, fairly strong specificity and high thermal stability.

Description

technical field [0001] The invention relates to an in vitro detection and diagnosis reagent, in particular to a recombinant fusion antigen for detection of HCV. Background technique [0002] Hepatitis C virus (Hepatitis c virus, HCV) infection is a major health problem, and the global average infection rate is about 3%. More than 75% of acutely infected individuals eventually develop a chronic carrier state that eventually leads to cirrhosis, liver failure, and liver cancer. HCV is mainly transmitted through blood and accounts for 80-90% of post-transfusion hepatitis. The current treatment for HCV infection is a drug combination of interferon and ribavirin, but this treatment is not effective in all cases. Since there is currently no effective drug for the treatment of hepatitis C, early detection of HCV infection, accurate judgment of virus subtype and patient infection status are important means for the prevention and control of hepatitis C. Therefore, the use of diagno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62G01N33/68G01N33/569
CPCC07K14/005C07K2319/00C12N2770/24222G01N33/56983G01N33/6854G01N2333/186
Inventor 干盈盈王磊李岚敏张兵何涛龙腾镶
Owner SICHUAN ANKERUI NEW MATERIAL TECH CO LTD
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