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Reconstruction of HIV-1 Chinese epidemic strain gag gene and recombination DNA vaccine thereof

A DNA vaccine and gene technology, applied in the direction of recombinant DNA technology, plant gene improvement, gene therapy, etc., can solve the problems of long expression time and unsuitable application

Inactive Publication Date: 2007-02-28
曾毅
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

②Long expression time
[0007] Most of the HIV vaccines used in preclinical and clinical research in foreign countries are based on the epidemic strains in Europe, America and Africa, which are not suitable for application in my country. Therefore, it is necessary to develop HIV vaccines based on the epidemic strains in my country

Method used

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  • Reconstruction of HIV-1 Chinese epidemic strain gag gene and recombination DNA vaccine thereof
  • Reconstruction of HIV-1 Chinese epidemic strain gag gene and recombination DNA vaccine thereof
  • Reconstruction of HIV-1 Chinese epidemic strain gag gene and recombination DNA vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Preparation of the modified gag gene (mod.gag)

[0045] In this example, through codon optimization, a modified gag gene (mod.gag) capable of expressing in eukaryotic cells independent of the regulatory protein Rev was obtained.

[0046] 1.1 Cloning of HIV-1 gag gene and acquisition of consensus sequence

[0047] Collect 20 samples of venous blood from HIV-1 antibody-positive patients in HIV-1 endemic areas in Henan, 5ml each, anticoagulate with heparin, use Qiagen’s QiaAmp Blood reagent, extract cellular DNA according to the instructions, and store nucleic acid samples at -20°C. The gag gene was amplified by nested-PCR method. Use G-1 / G-2 (G-1: 5'CGA CGC AGG ACT CGG CTT GC 3'; G-2: 5'CCT GGCTTT AAT TTT AC 3') as the outer primers for the first PCR reaction, The conditions are: pre-denaturation at 94°C for 5 min, 30 cycles at 94°C for 45 sec, 55°C for 45 sec, and 72°C for 210 sec; 72°C for 10 min. Take one tenth of the PCR product and us...

Embodiment 2

[0060] Example 2 Construction and Identification of DNA Vaccine pVR-mod.gag Containing mod.gag Gene

[0061] Considering that the final purpose of the constructed DNA vaccine is to be applied to clinical trials, the present invention uses a plasmid vector pVR (gifted by Professor Kong Wei of Jilin University) that can be applied to humans. This vector uses kanamycin resistance instead of general The ampicillin resistance screening marker commonly used in the vector can screen positive clones in Escherichia coli, and does not contain any eukaryotic screening markers, which ensures its safety in the human body.

[0062] 2.1 Construction and identification of recombinant plasmid containing mod.gag

[0063] template

Primer G1

Primer G2

dNTP (2.5mM)

10×PCR buffer

Taq enzyme

h 2 o

2μl

2μl

2μl

4μl

5μl

2μl (5U)

33μl

total capacity

50μl

[0064] The amplification conditions were...

Embodiment 3

[0069] Example 3 The immune protection test of DNA vaccine

[0070] 3.1 Immunization of DNA vaccine pVR-mod.gag

[0071] Thirty female BALB / c mice, 4-6 weeks old, weighing about 18-25 grams, were randomly divided into 2 groups (vaccine group and control group), 15 mice in each group. The mice in the vaccine group were injected with 100 μl / mouse (100 μg) of pVR-mod.gag into the unilateral tibialis anterior muscle, and each mouse in the control group was injected with an equal volume of PBS in the same manner as a control. The levels of specific cellular immune response and antibody response were detected at 4th, 8th and 12th weeks after the initial immunization, respectively.

[0072] 3.2 Detection of HIV-1 gag-specific cellular immune response in immunized mice by intracellular cytokine staining (ICS)

[0073] At 4 weeks, 8 weeks and 12 weeks after the initial immunization, the mice were killed by cervical dislocation, and the spleen lymphocytes of the m...

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Abstract

The invention provides the modified HIV-1gag gene and DNA vaccine on the basis of the gene. The expression content of the modified HIV-1gag gene is improved by 20 times. The DNA vaccine can effectively agitate cell-mediated immunity and humoral immunity.

Description

field of invention [0001] The invention belongs to the fields of bioengineering and AIDS prevention and treatment. Specifically, the present invention relates to the modification of gag gene of HIV-1 epidemic strain in China and its recombinant DNA vaccine. Background of the invention [0002] HIV, the human immunodeficiency virus (HIV), is a member of the family Retroviridae, the genus Lentivirus, and the group Primate Lentivirus. Acquired Immunodeficiency Syndrome (AIDS) caused by its infection to humans is the largest infectious disease that directly threatens human health in the world. Since the HIV epidemic, it is estimated that more than 60 million people have been infected and more than 20 million people have died from HIV / AIDS. According to WHO statistics, as of December 2004, there were 39.4 million (35.90-44.3 million) living HIV-infected people in the world, of which the number of new infections in 2004 reached 4.9 million (http: / / www.unaids.org / wad2004 / report...

Claims

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Application Information

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IPC IPC(8): C12N15/49C12N15/63C07K14/155A61K48/00A61P31/18
Inventor 曾毅冯霞余双庆
Owner 曾毅
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