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Multi-type HCV-E1 epitope complex immunogen, encoding gene and application thereof

A compound antigen and epitope technology, applied in the field of compound immunogens, to overcome the effect of variation

Inactive Publication Date: 2008-12-10
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is also one of the reasons why the existing E1-based vaccine research is often only effective for some patients

Method used

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  • Multi-type HCV-E1 epitope complex immunogen, encoding gene and application thereof
  • Multi-type HCV-E1 epitope complex immunogen, encoding gene and application thereof
  • Multi-type HCV-E1 epitope complex immunogen, encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Screening of HCV E1 epitope representative sequences

[0061] 1. Screening of HCV E1 neutralizing epitopes In the HCV Immunology Database, there is a summary of HCV E1 antibody epitopes, among which the only human antibodies are H111 and U1 / 30-33 series antibodies. U1 / 30-33 series antibodies are early years (Siemoneit K, Cardoso Mda S, et al. Human monoclonal antibodies for the immunological characterization of a highly conserved protein domain of the hepatitis Cvirus glycoprotein E1. Clin Exp Immunol. 1995; 101(2): 278-83) The human monoclonal antibody studied was the result of screening with synthetic peptides (314-330), but the binding effect of this antibody on recombinant E1 antigen was not clear, and whether it had neutralizing activity was not studied. Most other antibodies are obtained in animal experiments. Therefore, the present invention selects the action epitope of the neutralizing antibody H111. In addition, there are A4(m) and 159 antibody epi...

Embodiment 2

[0065] The construction of embodiment 2 recombinant polytype HCV E1 expression plasmid

[0066] The representative sequence of HCV E1 has been selected above, and the universal TH epitope sequence PADRE has also been determined, but they are all amino acid sequences. If they are to be expressed in the form of recombinant proteins, they must first be converted into gene sequences and cloned into expression vectors. The following studies were carried out for this purpose:

[0067] 2.1 The design of synthetic gene primers: According to the sequence 10, 11, 12, the gene sequence was deduced according to the biased codon of Escherichia coli, and three sets of primers for gene synthesis were respectively designed, and connecting arms were added to the 5' and 3' ends respectively, In order to add enzyme cutting sites after amplification with universal primers, it can be connected with the carrier. The specific sequences of the three sets of designed primers are shown in Sequences 13...

Embodiment 3

[0160] Example 3 Recombinant Multitype HCV / E1 Epitope Compound Antigen Immunogenicity Analysis

[0161] Through the above-mentioned Examples 1 and 2, the recombinant multi-type HCV E1 complex epitope antigen has been obtained, but whether the antigen is immunogenic, that is, whether it can effectively stimulate the body to produce antibodies, further research is needed. protocol, mice were immunized three times. Blood was collected half a month after each immunization to determine the antibody level of the immunized mice. In order to avoid the interference of part of the IL-1 molecule fused in the antigen when measuring the antibody titer, the pBV / E1s-PADRE expression plasmid was also constructed, which can express multitype HCV / E1 epitopes without IL-1 The composite antigen (refer to the above examples for the preparation steps, except that IL-1 was not introduced during the construction of the plasmid) was used as the antigen for antibody determination. The specific operat...

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Abstract

The invention discloses an epitope antigen gene containing E1 in different HCV types and a prokaryotic expression plasmid pBVIL1 / E1s-PADRE of general T auxilliary cell epitope gene. Engineering bacteria obtained by transforming the plasmid into escherichia coli can efficiently express recombinant multi-type HCV / E1 epitope complex antigens through induction culture, as well as the application of the multi-type HCV / E1 epitope complex recombinant protein of the expression in the preparation of HCV vaccines and diagnostic agent. Through the strategy of selecting a multi-type epitope antigen, complex immunogen containing HCV 1a, 1b, 2, 3, 4 and 6 type HCV / E1 main neutralizing epitopes is successfully constructed, which can be used as a component part of HCV vaccine immunogen so as to overcome the variation problem of HCV; meanwhile, the antigen can also be used for the antigen of HCV diagnostic agent.

Description

Technical field: [0001] The invention relates to a composite immunogen containing multiple types of HCV / E1 epitopes, a preparation method of the expression plasmid pBVIL1 / E1s-PADRE and its application in the preparation of vaccines and diagnostic reagent research. Background technique: [0002] Hepatitis C is a chronic infectious disease caused by hepatitis C virus (HCV). Because its early symptoms are not obvious, people often don't pay attention to it. In fact, the HCV infection rate is relatively high in my country, accounting for about 3.2% of the population, and the number of infected people in the country is estimated to reach 40 million. There are about 170-200 million HCV-infected people in the world. In most western countries, HCV infection has accounted for more than half of patients with chronic hepatitis, and is the main cause of liver cancer after hepatitis. Since there is no effective treatment and no vaccine has been developed, most of the infected people (a...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N1/21C12P21/02A61K39/29A61P1/16A61P31/12
Inventor 凌世淦贺锋宋晓国
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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