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DNA (Deoxyribose Nucleic Acid) vaccine of HCV (Hepatitis C Virus) and preparation method thereof

A nucleic acid vaccine and mutant technology, applied in the fields of molecular biology and infection immunity, can solve the problems of low eukaryotic expression yield, low E1E2 immune protection, difficult purification, etc., and achieve the effect of strong immunogenicity

Active Publication Date: 2012-10-24
WUHAN UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0006] Although the function and role of E1E2 have been clarified, the immune protection of E1E2 is still low
In addition, the molecular weights of the E1 and E2 proteins encoded by the HCV gene are: 31KD and 70KD, respectively. The prokaryotic expression protein cannot form protein glycosylation, and the expression is difficult, while the yield of eukaryotic expression is low and purification is difficult.
Therefore, the application of E1E2 in actual immune protection is limited, which is not conducive to popularization

Method used

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  • DNA (Deoxyribose Nucleic Acid) vaccine of HCV (Hepatitis C Virus) and preparation method thereof
  • DNA (Deoxyribose Nucleic Acid) vaccine of HCV (Hepatitis C Virus) and preparation method thereof
  • DNA (Deoxyribose Nucleic Acid) vaccine of HCV (Hepatitis C Virus) and preparation method thereof

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Experimental program
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Embodiment 1

[0022] 1. Construction of eukaryotic recombinant plasmids containing E1E2 wild type and mutants

[0023] The vector is pVAX-1, pcDNA3.1-E1E2 plasmid vector (Chen F, et al., Antimicrobial agents and Chemotherapy, 2010, p3355-3364) containing the full-length gene sequence of E1E2 of complete type 1a HCV (gene sequence number is AY958062 ), digested with BamHI+EcoRI and inserted into pVAX-1 (Invitrogen) vector. . The asparagine at the corresponding glycosylation site on E1E2 was mutated into aspartic acid by site-directed mutagenesis PCR. BamHI+EcoRI double digested pVAX-1, and E1E2 and glycosylation mutant fragments, ligated, and obtained recombinant plasmids named flat pVAX-CpG-N2N8, CpG-N8, CpG-N13, CpG-N2N4, CpG-N2N8 , CpG-N2N14. The primers required for construction are as follows:

[0024] E1E2-P1:

[0025] 5'-CGGGATCCGCCACCATGGGTTCCTCTTTTTTCTAT

[0026] E1E2-P2:

[0027] 5'-CGGAATTCTACGCCTCCGCTTGGGATA

[0028] E1E2-P3:

[0029] 5’-CAATACTCGAGTCAGGGCAATCAT

[0030...

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Abstract

The invention discloses a DNA (Deoxyribose Nucleic Acid) vaccine of an HCV (Hepatitis C Virus) and a preparation method thereof. The vaccine has CTL (Cytotoxic Lymphocyte) cell immunity and the capability of neutralizing virus infection. The preparation method comprises the following steps of: constructing a recombinant plasmid containing an E1E2 wild-type gene; and mutating a corresponding glycosylation mutant (E1-N209SS is mutated into E1-D209SS, and E2-N430AS is mutated into E2-D430AS) on site-directed mutated PCR (Polymerase Chain Reaction) mutated E1E2; and coupling a CpG sequence by adopting a PCR method, wherein a mutant is named CpG-N2N8. After a mouse is immunized with the DNA vaccine, an antiserum for neutralizing HCVcc (cell cultured Hepatitis C Virus) can be obtained. The method is easy and convenient, and is low in cost; and the DNA vaccine can be used for expressing virus genes, can be used for inducing body fluid immune response of a CTL cell immunizing and neutralizing antibody and which is stronger than wild E1E2, and has a remarkable economic benefit.

Description

technical field [0001] The invention belongs to the fields of molecular biology and infection immunity, and specifically relates to a DNA vaccine of hepatitis C virus (HCV), and also relates to a preparation method of the vaccine. Background technique [0002] my country is one of the countries with high HCV infection rate. HCV infection is one of the main factors leading to chronic hepatitis, liver cancer and cirrhosis, which seriously affects human health. There is currently no effective therapeutic and preventive HCV vaccine. [0003] Ultimate control of HCV will depend on vaccine prophylaxis. Specific neutralizing antibodies and cellular immunity play a very important role in the process of resisting virus invasion and immune resistance to HCV attacking human cells. HCV envelope (envelope) glycoproteins (E1 and E2) can induce the body to produce neutralizing antibodies to block the invasion of the virus. The envelope E2 protein is the main target antigen for inducing ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C12N15/52C12N15/63C12N15/66A61K48/00A61K39/39A61P1/16A61P31/14
Inventor 章晓联任玉珊林苗
Owner WUHAN UNIV
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