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DNA (deoxyribonucleic acid) vaccine of HCV (hepatitis C virus) and preparation method thereof

A DNA vaccine and envelope glycoprotein technology, which is applied in the fields of molecular biology and infection immunity, can solve the problems of low immune protection of HCVE2 protein, difficulty in purification, difficulty, etc., achieves neutralization of further infection by virus, overcomes difficulties, and has a simple method. Effect

Active Publication Date: 2013-12-11
WUHAN UNIV
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AI Technical Summary

Problems solved by technology

However, it is still unclear whether the removal of the glycosylation site N1 / N2 / N4 / N6 / N11 associated with the neutralizing antibody epitope on E2 can induce the production of anti-HCV neutralizing antibody (neu-IgG) in vivo , and whether the deletion of N-glycans in these sites can mediate a strong cellular immune response in vivo has not been reported at home and abroad
[0005] E2 is crucial to the entire life cycle of HCV. However, there are still no small molecule drugs or effective broadly neutralizing antibodies against HCV E2, and the treatment options for HCV are very limited.
The method of using classical protein immunization to obtain antibodies is difficult in practical application. On the one hand, the yield of eukaryotically expressed E2 protein (~70KD) with complete glycosylation modification is extremely low and purification is difficult, while the use of prokaryotic expression system to obtain The E2 protein of HCV is only ~35KD, lacks glycosylation modification, and cannot maintain the original configuration. It is used as an antigen and lacks some key epitopes. On the other hand, the immune protection of HCV E2 protein is low, and further research is needed transform

Method used

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  • DNA (deoxyribonucleic acid) vaccine of HCV (hepatitis C virus) and preparation method thereof
  • DNA (deoxyribonucleic acid) vaccine of HCV (hepatitis C virus) and preparation method thereof
  • DNA (deoxyribonucleic acid) vaccine of HCV (hepatitis C virus) and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Construction of eukaryotic recombinant plasmids for E2 mutants with secretory glycosylation single-point deletion mutations

[0029]The E2 gene sequence that can express the secreted E2 protein (Secreted E2, sE2) was amplified from the HCV type 1a genome sequence (GenBank Acc.No.AY958062) by PCR, and at the same time, it started at the N-terminal of the sequence The upstream of the codon (ATG) introduced a bacterial-derived non-methylated CpG sequence (GACGTT), and further constructed the fragment into the eukaryotic expression vector pcDNA3.1(-) (Invitrogen). Then, the asparagine at the second N-glycosylation site (N423RT) on sE2 was mutated to aspartic acid (D423RT) by the PCR site-directed mutagenesis method, and the recombinant plasmids obtained were named PingN2.

[0030] The primers used in this example are shown in the table below:

[0031]

[0032] (The italics in the table indicate the restriction site, the underlined site is the non-methylated CpG sequence...

Embodiment 2

[0041] Material preparation and mouse immunization process

[0042] The endotoxin-free plasmid DNA extraction kit (Endo Free Plasmid MaxiKit) from OMEGA Biotek was used to obtain the recombinant sE2 wild-type plasmid pcDNA3.1-sE2 (WT) and the N-glycosylation site mutant plasmid N2, Perform concentration determination and adjust the plasmid concentration of each group to 2 μg / μL. Further use Ao reagent (Zhanjiang Andus Biological Co., Ltd.) to test the residual endotoxin in the obtained recombinant plasmids. The test results show that the endotoxin content in the obtained plasmid DNA is less than 0.06Edotoxin Unit / μg DNA, which meets the requirements of plasmid DNA products for gene therapy. Bacterial endotoxin residue standard (less than 0.1EU / μg).

[0043] Preparation of prokaryotic expression sE2 protein: The 384-661 bases on the HCV genome were constructed into the prokaryotic expression vector pET28a vector (Invitrogen) using molecular cloning technology, and the expresse...

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Abstract

The invention discloses a DNA (deoxyribonucleic acid) vaccine of a HCV (hepatitis C virus) and a preparation method thereof, belonging to the field of molecular biology and infection immunity. The DNA vaccine is characterized in that that the second N-glycosylation site of HCV-E2 is mutated, that is, N423RT is mutated into D423RT. A nucleotide sequence of the mutant is shown in SEQIDNO.1. The DNA vaccine capable of generating the E2 mutant is obtained by introducing CpG sequences to the two terminals of an E2 gene and constructing mutated fragments onto a eukaryotic expression vector. The method is simple and convenient and is low in cost. The prepared DNA vaccine can induce stronger specific cellular immunity and neutralizing antibody than wild type HCVE2, enhance CD8+T specific killing activity, induce stronger IFN (interferon)-gamma and IL4 release and effectively neutralize further infection of the virus and overcomes the expression difficulty of glycosylated proteins.

Description

technical field [0001] The invention belongs to the fields of molecular biology and infection immunity, and in particular relates to a DNA vaccine of hepatitis C virus and a preparation method thereof. Background technique [0002] Hepatitis C virus (HCV) infection is one of the main factors leading to chronic hepatitis, liver cancer, and cirrhosis, which seriously affects human health. my country is one of the countries with a high infection rate of HCV. The current treatment methods are limited and there is no Effective therapeutic and prophylactic HCV vaccines. [0003] HCV is a single-stranded positive-sense RNA virus, and its gene expression product is a polyprotein containing 3011 amino acids, which is cleaved by protease to form various proteins (nucleocapsid protein Core, envelope protein E1 and E2; ion channel protein P7; Nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A, NS5B) (Pwalotsky JM, Gastroenterology, 2002, 122(6):1554-1568). The amino acid sequence of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C12N15/51A61K48/00A61P31/20C12N15/85C12N15/66
Inventor 章晓联闵远琴
Owner WUHAN UNIV
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