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Chimeric simian/human immunodeficency virus strain and application thereof

A technology of immunodeficiency virus and AIDS, which is applied in the field of viruses to achieve the effect of improving efficiency and strong infection ability

Inactive Publication Date: 2012-05-23
NANKAI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, constructing the SHIV virus of the B' / C recombinant subtype will face more severe challenges

Method used

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  • Chimeric simian/human immunodeficency virus strain and application thereof
  • Chimeric simian/human immunodeficency virus strain and application thereof
  • Chimeric simian/human immunodeficency virus strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Acquisition of HIV-1 B' / C recombinant subtype envelope protein gene in China.

[0077] The purpose is to amplify the envelope protein gene for SHIV-XJN0363B8 clone construction from HIV-1 positive blood samples by PCR method.

[0078] The operation protocol includes extraction of total genomic DNA from HIV-1 positive blood samples and PCR amplification of envelope protein gene. details as follows:

[0079] First, the extraction protocol of total genomic DNA in HIV-1 positive blood samples:

[0080] After the HIV-1 positive blood samples were thawed at room temperature, total genomic DNA was extracted using the QIAGEN Whole Blood Nucleic Acid Extraction Kit. Extracted products were stored at -80°C, and repeated freezing and thawing of nucleic acid samples should be avoided as much as possible.

[0081] Second, the PCR amplification scheme of the envelope protein gene:

[0082] Using the total genomic DNA in the extracted HIV-1 positive blood sample as a template, ne...

Embodiment 2

[0093] Construction of infectious full-length clones of SHIV-XJN0363.

[0094] The purpose is to construct a full-length SHIV-XJN0363 clone carrying the Chinese HIV-1 B' / C recombinant subtype envelope protein gene by molecular cloning. SHIV-KB9 was selected as the backbone, and the envelope protein gene region to be replaced was located in the 3'SHIV-KB9, and the 2.1kb fragment of the envelope protein gene was replaced by the existing restriction site. Afterwards, the constructed 3'SHIV half-length clone was ligated with the 5'SHIV half-length plasmid through restriction sites to obtain several full-length SHIV-XJN0363 clones.

[0095] Specific operations include: purification of PCR products, cloning of PCR products, screening and identification of half-length SHIV clones, construction and identification of full-length SHIV clones, and mass preparation of full-length SHIV plasmids.

[0096] First, the purification scheme of PCR products:

[0097] After the PCR product was e...

Embodiment 3

[0107] Screening and phenotype analysis of human simian immunodeficiency virus SHIV-XJN0363B8 capable of infecting TZM-bl cells.

[0108] The purpose is to test the virus activity of all constructed full-length SHIV-XJN0363 clones in a biosafety level 3 laboratory.

[0109] Specific operations include: transfection of SHIV plasmid into 293T cell line, determination of SHIV virus SIV p27 concentration, replication of SHIV virus in TZM-bl cells, SHIV virus TCID 50 Titration and cell tropism assay.

[0110] First, SHIV-XJN0363 plasmid transfected 293T cell line:

[0111] In the transfection experiment, Lipofectamine 2000 was used to transfect the full-length plasmid into 293T cell line, and the culture supernatant was collected after 40 hours for detection of SIV p27 antigen, or for TZM-bl infection experiment.

[0112] Second, determination of SIV p27 antigen concentration in SHIV-XJN0363 culture supernatant:

[0113] SIV p27 core antigen detection was performed using the Cou...

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Abstract

The invention discloses a chimeric simian / human immunodeficency virus strain and application thereof, relating to a virus modified with an introduced exogenetic gene material. The virus is a simian / human immunodeficency virus SHIV-XJN0363B8 carrying a China HIV-1 (Human Immunodeficiency Virus) B' / C recombinant subtype envelope protein gene segment, has the classification name as immunodeficency virus and the Latin name as Lentivirus, and is preserved with the collection number of CGMCC No. 4243 on Oct. 15th, 2010 in China General Microbiological Culture Collection Center with the address of No.3, yard 1, Beichen west road, Chaoyang District, Beijing. An SHIV-XJN0363B8 / China rhesus monkey AIDS (Acquired Immune Deficiency Syndrome) model built by using the virus strain SHIV-XJN0363B8 can beused for effectively simulating the virus replication characteristic at the early stage of HIV-1 infection, and play roles in researching the in-vivo pathogenic mechanism of HIV-1 and evaluating the effectiveness of biological agents, such as AIDS vaccines, AIDS antiviral medicaments and the like, in China.

Description

technical field [0001] The technical scheme of the present invention relates to a virus modified by introducing foreign genetic material, specifically a human-monkey chimeric immunodeficiency virus strain and its application. Background technique [0002] AIDS, Acquired Immunodeficiency Syndrome (AIDS), is caused by Human Immunodeficiency Virus (Human Immunodeficiency Virus, HIV) and is an infectious disease characterized by severe damage to the systemic immune system. In 2007, the Ministry of Health and UNAIDS estimated that there were 700,000 HIV-infected people in China. The main epidemic strain in my country is the HIV-1 B' / C recombinant subtype, which is mainly distributed in Yunnan, Guizhou, Sichuan, Xinjiang and Northwest China where there are many drug users, and spread to East China and South China with the floating population of Xinjiang. [0003] For more than 20 years after the discovery of HIV-1 virus, researchers from various countries have conducted in-depth ...

Claims

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Application Information

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IPC IPC(8): C12N7/01A01K67/027A61K49/00C12R1/93
Inventor 李悦陈启民杨贵波乔文涛王金忠邵一鸣
Owner NANKAI UNIV
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