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

Inactive Publication Date: 2009-10-07
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) that can express in eukaryotic cells independent of the regulatory protein Rev was obtained.

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

[0047] Collect 20 venous blood samples of HIV-1 antibody positive persons in HIV-1 endemic areas in Henan, each 5ml, with heparin anticoagulation, and use QiaAmp Blood reagent of Qiagen Company to extract cellular DNA according to the instructions, and store the nucleic acid samples at -20°C. The gag gene was amplified by nested-PCR method. Using 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 primer, the first PCR reaction was performed, The conditions are: 94°C for 5min pre-denaturation, 94°C for 45sec, 55°C for 45sec, 72°C for 210sec, 30 cycles; 72°C for 10min. Take one-tenth of the PCR product, use G-3 / G-4 (G-3: ...

Embodiment 2

[0062] Example 2 Construction and identification of DNA vaccine pVR-mod.gag containing mod.gag gene

[0063] 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 the human body, and the vector uses kanamycin resistance instead of general. The ampicillin resistance selection marker commonly used in the vector is used to screen positive clones in Escherichia coli, and it does not contain any eukaryotic selection marker, which ensures its safety in the human body.

[0064] 2.1 Construction and identification of recombinant plasmids containing mod.gag

[0065] Using primer pairs G1 (5'GAT CTG GAT CCT TAT TGT GAC 3') and G2 (5'CCG GGG ATA TCG CCACCA TGG G 3'), the pUC57-mod.gag obtained from Example 1 was used as a template to amplify The target gene is gag, and a BamH I restriction site is introduced at its 5' ...

Embodiment 3

[0080] Example 3 Immunoprotective power test of DNA vaccine

[0081] 3.1 Immunization of DNA vaccine pVR-mod.gag

[0082] 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 / mice (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 way as a control. Specific cellular immune responses and antibody response levels were detected at 4, 8, and 12 weeks after primary immunization, respectively.

[0083] 3.2 Detection of HIV-1gag-specific cellular immune responses in immunized mice by intracellular cytokine staining (ICS)

[0084] Mice were sacrificed by cervical dislocation at 4 weeks, 8 weeks and 12 weeks after the primary immunization, respectively. The spleen lymphocytes of the mice were separated with lympho...

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Abstract

The invention provides a modified HIV-1 gag gene and a DNA vaccine constructed on the basis thereof. The modified HIV-1 gag gene can be highly expressed in eukaryotic cells independently of the regulatory protein Rev, and the expression level is increased by about 20 times compared with before modification. The DNA vaccine can effectively stimulate cellular immunity and humoral immunity at the same time, and because the target gene contained therein comes from the area where the vaccine is planned to be applied, it is highly representative and pertinent.

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 Chinese epidemic strain 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 Lentiviridae, the group of primate lentiviruses. Acquired immunodeficiency syndrome (AIDS) caused by its infection in humans is the world's largest infectious disease that directly threatens human health. 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 (35.9 to 44.3 million) living HIV-infected persons worldwide, of which 4.9 million were newly infected in 2004 (http: / / www.unaids.org / wad2004 / report.html). In China, t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/49C12N15/63C07K14/155A61K48/00A61P31/18
Inventor 曾毅冯霞余双庆
Owner 曾毅
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