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AIDS vaccine of N1L and B8R gene deletion-based vaccinia virus vector

A technology of vaccinia virus and AIDS, applied in virus/bacteriophage, antiviral agent, genetic engineering, etc., can solve serious problems

Inactive Publication Date: 2010-03-03
NAT CENT FOR AIDSSTD CONTROL & PREVENTION CHINESE CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Recombinant vaccinia virus vaccine has the advantages of good effect, good thermal stability, convenient vaccination, and no adjuvant required. cause serious complications
Recombinant vaccinia virus vaccines expressing certain immunogens failed to achieve the desired immune effect. Scholars have made a lot of in-depth research on how to enhance the immunogenicity of recombinant vaccinia virus vaccines and further improve the safety of vaccinia virus vectors

Method used

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  • AIDS vaccine of N1L and B8R gene deletion-based vaccinia virus vector
  • AIDS vaccine of N1L and B8R gene deletion-based vaccinia virus vector
  • AIDS vaccine of N1L and B8R gene deletion-based vaccinia virus vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Construction of transfer plasmid

[0053] 1. Construction of transfer plasmid pSKN1L

[0054] The DNA of the vaccinia virus Tiantan strain (purchased from Beijing Institute of Biological Products) was used as a template to amplify the left homologous sequence N1LL fragment of the vaccinia virus Tiantan strain N1L gene (primers: SEQID NO.: 1 and 2) and the same The source sequence N1LR fragment (primers: SEQ ID NO.: 3 and 4), the N1LL fragment and the N1LR fragment were respectively digested with Bgl II and ligated, and the ligated product was used as a template for PCR amplification (primers: SEQ ID NO. : 1 and 4) The left and right homologous sequences of the N1L gene are connected into a large fragment, which is called N1LLR. The large fragment N1LLR, vector plasmid pBR-SK (restriction map see Figure 17 ) were co-digested with KpnI / SalI respectively, and after digestion, the two were ligated with T4 DNA ligase to construct a transfer plasmid pSKN1L wit...

Embodiment 2

[0101] Embodiment 2: The construction of the recombinant vaccinia virus inserted HIV-1 gag, pol, env genes of the present invention, and deleted B8R and N1L genes

[0102] 1. Preparation of recombinant vaccinia virus of the present invention by transfection

[0103] Infect 80- 90% chicken embryo fibroblasts (CEF, purchased from Beijing Experimental Animal Center) (MOI=0.01~0.1) in sheets, after 1 hour of adsorption at 37°C, the culture supernatant was discarded and replaced with serum-free Eagle's medium ( The Chinese Center for Disease Control and Prevention (Institute of Viral Disease Prevention and Control) rinsed the cells twice. Using the transfection kit (Lipofectin Reagent Cat. 18292-011) of Invitrogen Company, the above-mentioned CEF cells infected with the recombinant vaccinia virus VTKgpeΔB8R were transfected with the transfer plasmid pSKN1LLacZ according to the instructions of the kit instructions. After the transfection, the transfection mixture was frozen and th...

Embodiment 3

[0139] Example 3: Research on the Impact of Deleting N1L and B8R Genes on the Safety of HIV-1 Tiantan Strain Recombinant Vaccine

[0140] 1. Rabbit skin toxicity test

[0141] Six New Zealand rabbits (purchased from Beijing Kaiyuan Rabbit Industry Farm) (2kg) were shaved on the back, and the back of each rabbit was injected intradermally and simultaneously inoculated with 1×10 6 VTT, VTKgpe, VTKgpeΔB8R, VTKgpeΔB8RΔN1LLacZ of PFU. Observe and record the redness and swelling of the skin injection site for 14 consecutive days, and measure the infiltration diameter and necrosis diameter with a caliper.

[0142] The results showed that the skin acne infiltration toxicity of VTKgpeΔB8RΔN1LLacZ was 100 times lower than that of VTKgpe, and the scar on the skin surface formed by it was significantly smaller than that of VTKgpe and VTKgpeΔB8R (results as follows Figure 8 and Table 1), indicating that deletion of the N1L gene on the basis of VTKgpeΔB8R can significantly reduce the ski...

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Abstract

The invention relates to a replicating type AIDS live vector vaccine expressing a human immunodeficiency virus (HIV) antigen and application thereof. The vaccine is constructed based on a replicatingtype vaccinia virus such as a vaccinia virus Tiantan strain. The replicating type AIDS live vector vaccine can induce high-level anti-HIV humor immune response and cellular immune response, and provides a vector for constructing the AIDS vaccine. The invention also relates to an immunization scheme using the AIDS vaccine.

Description

technical field [0001] The invention relates to the field of antiviral immunology. More specifically, the present invention relates to a replicating vaccinia virus vector, a recombinant AIDS vaccine based on the vector, and a preparation method and application of the vaccine. Background technique [0002] Acquired Immunodeficiency Syndrome (AIDS), referred to as AIDS, is an infectious disease caused by Human Immunodeficiency Virus (Human Immunodeficiency Virus, HIV) infection. Since the discovery of the first case in 1981, AIDS has been spreading around the world at an alarming rate and has become one of the most serious viral diseases that endanger human life and health in the world. [0003] The global prevalence of HIV has led to extremely high morbidity and mortality. In order to prevent and treat HIV infection, traditional chemical drug therapy, immunotherapy and new methods have been continuously researched and developed. Highly active antiretroviral therapy (HAART) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/21A61P31/18C12N7/00C12N15/11C12N15/863
Inventor 邵一鸣黄薇刘颖刘明杰
Owner NAT CENT FOR AIDSSTD CONTROL & PREVENTION CHINESE CENT FOR DISEASE CONTROL & PREVENTION
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