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A method and application of high-efficiency expression of recombinant hepatitis C virus multi-epitope antigen

A high-efficiency expression technology of hepatitis C virus, applied in the field of bioengineering, can solve problems such as high background value, poor uniformity, and misjudgment of results, and achieve the effect of increasing production and avoiding negative effects

Inactive Publication Date: 2011-12-21
天津迈迪瑞康生物医药科技有限公司
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  • Description
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Problems solved by technology

[0005] The combined expression of Core and NS3 proteins has certain cytotoxicity to Escherichia coli, and the product is easily degraded by proteases in Escherichia coli. When the target protein is expressed by the conventional molecular biology methods disclosed above, the product yield is low and uniform. Poor performance, which brings difficulties to downstream separation and purification, and when the obtained antigen is used for Elisa detection, the background value is high, which may easily cause misjudgment of results

Method used

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  • A method and application of high-efficiency expression of recombinant hepatitis C virus multi-epitope antigen
  • A method and application of high-efficiency expression of recombinant hepatitis C virus multi-epitope antigen
  • A method and application of high-efficiency expression of recombinant hepatitis C virus multi-epitope antigen

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Experimental program
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Embodiment 1

[0030] By conventional means of molecular biology, construct the expression vector containing Core protein, and contain Core-NS3 (such as figure 1 Shown) the expression vector of protein, two target proteins all use pET21b plasmid, Bl21 (DE3) Rosctta competent cell as expression strain. Specific operation: Transform pET21b-Core and pET21b-Core-NS3 plasmids into Bl21(DE3) Rosctta competent cells (double resistance to ampicillin and chloramphenicol). When cultured in LB medium at 37 degrees and 200 rpm to OD600 of about 0.6, 1 mM IPTG was added for induction for 2 hours. figure 2 Lanes 2 and 4 are the results of Core overexpression, lanes 6 and 8 are the results of Core-NS3 overexpression. 1, 3, 5, 7 are the controls before induction of 2, 4, 6, and 8, respectively. It can be seen from the SDS-PAGE figure that after induction, the target protein is expressed in large quantities, and the expression product is uniform. The Core protein sequence is SEQ ID NO.3, and the NS3 prot...

Embodiment 2

[0033]

[0034]

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[0036]

[0037]

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Abstract

The invention belongs to the technical field of bioengineering, and relates to a method and application for highly expressing recombinant hepatitis C virus (HCV) multi-epitope antigens. Specifically, the recombinant HCV multi-epitope antigen includes the dominant epitope of the Core protein and / or NS3 protein in the HCV genome, and is characterized in that, by introducing an oligomeric (PDDDDPG) structure, the isoelectric point of the fusion protein is adjusted, Promote the formation of inclusion bodies to avoid the cytotoxicity of the target protein and the degradation of the target protein by E. coli protease. After the oligomeric structure, a factor Xa enzyme cleavage site (IEGR) is introduced, and a His tag is added to the C-terminus of the target protein. The product provided by the invention has the advantages of high expression level, strong product uniformity, simple purification steps and easy operation.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a method and application for highly expressing recombinant hepatitis C virus multi-epitope antigens. Background technique [0002] my country is a high-incidence area of ​​hepatitis C (HCV), with about 50 million infected persons, accounting for 40% of the global share. Hepatitis C is the main cause of liver cirrhosis and liver cancer, and has a very high infection rate in blood transfusion and other treatments. The public's awareness of hepatitis C is also low. Among the 83% of high-risk groups with hepatitis C, only 5% have been tested for hepatitis C. At present, there is neither a very clear drug nor an effective vaccine to prevent and prevent its further spread. Therefore, the early diagnosis of HCV is of great significance for screening the source of HCV infection, guiding clinical treatment and prognosis. Among them, the blood source screening of HCV is the most imp...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K48/00A61K39/29A61P31/14G01N33/569
Inventor 杨杰雷荣悦周强
Owner 天津迈迪瑞康生物医药科技有限公司
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