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HIV-1 virus-like particle and its prepn and use

An HIV-1, virus-like technology, applied in biochemical equipment and methods, viruses/phages, viral antigen components, etc., can solve the problems of low immune response, difficulty in continuous production of VLP, rapid decline in expression, etc., to achieve strong immunity. Originality, good safety, the effect of improving the possibility

Inactive Publication Date: 2006-05-17
JILIN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] There have been many reports on HIV-1 virus-like particle vaccines in previous studies. People have used different expression systems to form VLPs. Malcolm Haddick used the pBCCX-CSF plasmid to produce VLPs except for the HIV-1 Nef gene, expressed them with COS cells, and produced Particles with a structure similar to mature viruses, but the expression level decreased rapidly after 3 days; C. JANE PALE constructed a chimeric VLP of HPV and SHIV (SIV gag27, HIV Tat Rev), and immunized animals through the whole body and mucosa, although antibodies and T cell immune response, but the immune response is low; Shiwen Peng et al. constructed HPV and HIV gp160 or gp120 chimeric virus to express VLP, and after immunizing animals, antibodies against HPV can be produced, while antibodies against HIV are relatively few; other virus expression systems also have There are attenuated vaccinia virus, canarypox, COXACKIE, FOWLPOX, VSV, etc., but their safety and effectiveness are still worthy of consideration. It is also difficult for these systems to continuously produce VLP. In addition, DNA vaccine must be used for primary immunization to obtain high-titer antibodies; Therefore, how to overcome the above-mentioned difficulties and establish a safe, effective and durable system for producing VLPs is a key issue that HIV-1 VLP vaccine researchers are facing and waiting to be resolved.

Method used

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  • HIV-1 virus-like particle and its prepn and use
  • HIV-1 virus-like particle and its prepn and use
  • HIV-1 virus-like particle and its prepn and use

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1. Selection of plasmids and resistance selection markers

[0040] D-GPEi plasmid: We chose the eukaryotic cell expression plasmid D-GPEi, which was constructed by our laboratory, and cloned the main structural protein genes Gag, Pol and Env of the epidemic strain (regional) HIV-1 in China into a self-developed construction The new-generation expression vector pVR vector (see Patent Publication No. CN1631441A for specific construction methods). The vector is composed of CMV promoter, kanamycin resistance gene, prokaryotic cell high copy factor, intron A and other conventional components. It meets the safety standards of the US FDA for human clinical trials, and has the ability to stably and efficiently express foreign genes Features. The antigen genes contained in the constructed plasmid are GagPol and Env of the modified HIV-1 epidemic strain in China. The gene expression products are the same as the original gene, but a large number of expression inhibitors i...

Embodiment 2

[0042] Embodiment 2. Construction of 293 stable cell lines

[0043] 1. Materials, reagents and instruments

[0044] 25cm 2 Cell culture flask, six-well plate, 96-well plate, CO2 incubator (Thermoforma), 293 cells, plasmid D-GPEi (constructed in our laboratory), vector pcDNA3.1 (purchased from Invitrogen), lipofectmine2000 (purchased from Invitrogen) , polyacrylamide gel electrophoresis (Bio-Rad), G418 (purchased from Dingguo, repackaged by gemview); HIV positive serum (collected from positive patients in Guangxi); HRP-IgG (Jackson ImmunoResearchlaboratories, INC).

[0045] Cell lysate: DTT 0.617g, SDS 0.8g, 1M Tris-HCl pH6.83.2ml, glycerin 4ml, bromofin 10.08g, water 12.8ml;

[0046] 5xTris-glycine electrophoresis buffer: 15.1g Tris base, 44g glycine, 5g SDS, add water to 1000ml;

[0047] Transfer buffer: dissolve 2.9g of glycine, 5.8g of Tris base, 0.37g of SDS, and 200ml of methanol in 800ml of water, and set the volume to 1000ml.

[0048] 2. Method steps

[0049] 2.1. ...

Embodiment 3

[0060] Construction of embodiment 3.Vero stable cell line

[0061] 1. Materials, reagents and instruments

[0062] 25cm 2 Cell culture flask, six-well plate, 96-well plate, CO2 incubator (Thermoforma), Vero cells, plasmid D-GPEi (constructed in our laboratory), vector pcDNA3.1 (purchased from Invitrogen), lipofectmine2000 (purchased from Invitrogen) , polyacrylamide gel electrophoresis instrument (Bio-Rad), G418 (purchased from Dingguo, gemview repackaged) HIV positive serum (collected from Guangxi positive patients); anti-human IgG (Jackson ImmunoResearchlaboratories, INC).

[0063] Cell lysate: DTT0.617g, SDS0.8g, 1M tris-HCl pH6.83.2ml, glycerin 4ml, bromphenol0.08g, H 2 O12.8ml;

[0064] 5xTris-glycine electrophoresis buffer: 15.1g Tris base, 44g glycine, 5g SDS, add water to 1000ml;

[0065] Transfer buffer: dissolve 2.9g of glycine, 5.8g of Tris base, 0.37g of SDS, and 200ml of methanol in 800ml of water, and set the volume to 1000ml.

[0066] 2. Method steps

[00...

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Abstract

The present invention relates to one kind of and its preparation and use, and belongs to the field of biotechnology. The HIV-1 virus-like particle contains complete core protein gag, encoding enzyme protein pol and outer membrane protein env of HIV-1, and is nonreplication type. The present invention is superior in that stably expression cell line is established through cotransfection and monoclonal cell screening. The cell line can secrete HIV-1 virus-like particle stably and continuously, and the HIV-1 virus-like particle contains no virus nucleic acid and has high safety. The constituted VLP has reasonable assembling form and high similarity with natural virus in structure, and may be used as HIV-1 treating vaccine.

Description

technical field [0001] The invention relates to an HIV-1 virus-like particle and its preparation method and application. The invention also relates to a cell line stably expressing human immunodeficiency virus-1 (HIV-1) structural protein, its construction method and application. The cell line can continuously express and secrete virus-like particles with artificially modified gag, pol and env nucleotide sequences, and can be used as stromal cells for HIV-1 virus-like particle vaccines. Background technique [0002] AIDS is considered to be the first major infectious disease that directly threatens human health in the world. The "2004 Global AIDS Epidemic Report" released by the World Health Organization (WHO) and the United Nations Program on AIDS (UNAIDS) on November 23, 2004 pointed out that, At present, the number of AIDS-infected people in the world has exceeded 39 million. This year, 4.9 million newly-infected people will be newly infected, and more than 3 million pati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/12C12N7/01C12N15/65C12N15/79
Inventor 孔维张喜珍于湘晖赵东海田春娟于晓方
Owner JILIN UNIV
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