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HIV-1 membrane fusion inhibitor and application thereof

A technology of HIV-1 and membrane fusion, applied to antiviral agents, medical preparations containing active ingredients, and the use of vectors to introduce foreign genetic materials, etc.

Inactive Publication Date: 2010-12-15
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Research on the protein binding properties and functions of gp41 has not been interrupted at home and abroad

Method used

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  • HIV-1 membrane fusion inhibitor and application thereof
  • HIV-1 membrane fusion inhibitor and application thereof
  • HIV-1 membrane fusion inhibitor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The preparation of embodiment 1, P20 and P20-A

[0055] 1.1 Materials

[0056] Enzymes and dNTPs amplified by PCR: purchased from Takarra Company;

[0057] PCR amplification primers: synthesized by Shanghai Shenggong Company, purified by HAP method;

[0058] Takara restriction endonuclease (BamH I, Xho I): purchased from Dalian Bao Biological Company;

[0059] TakaraT4 ligase: purchased from Dalian Bao Biological Company;

[0060] 18% Tris-glycine gel: purchased from Invitrogen (Carlsbad, CA);

[0061] Glutathione Sepharose 4B affinity column: purchased from Amersham Company (Sweden);

[0062] Prokaryotic expression vector pGEX-6p-2, PreScission protease (PPase): purchased from Amersham Biosciences (Sweden).

[0063] Escherichia coli DH5 and BL21 were purchased from Invitrogen. The rest of the conventional chemical reagents were of domestic analytical grade, unless otherwise specified.

[0064] 1.2 Experimental steps

[0065] 1.2.1 Plasmid construction

[0066]...

Embodiment 2

[0091] Example 2: P20 inhibits cell fusion

[0092] 2.1 Materials

[0093] Cell line: 3T3.T4.CXCR4 cell line can stably express CD4 and CXCR (receptor and co-receptor for HIV envelope protein). CHO-WT cell line was stably transfected with HIV-1HXB2 Env expression plasmid pEE14, which can express HIV-1HXB2 Env gp160. The cell lines were purchased from NIH AIDS Research and Reference Reagent Program.

[0094] Preparation of GMEM-S selective medium.

[0095] 1 liter of GMEM medium contains: 914ml deionized water, 40g MEM w / o L-glutamine (Gibco), 36ml 7.5% sodium bicarbonate (Gibco), 20ml 50-fold concentrated nucleoside stock solution, 10ml 100-fold concentrated Glutamic acid + asparagine stock solution, 10ml 100mM sodium pyruvate (Gibco), 10ml non-essential amino acids (Gibco).

[0096] The preparation method of the 50-fold concentrated nucleoside stock solution is as follows: Weigh 35mg adenosine, 35mg guanosine, 35mg cytidine, 35mg uridine, 12mg thymidine, add 100ml deioniz...

Embodiment 3

[0108] Example 3: P20 and P20-A inhibit HIV-1 strains

[0109] 3.1 Materials

[0110] HIV-1 laboratory-adapted strains (NL4-3, IIIB, Bal) and detection cells (TZM-bl, MT-2) were purchased from NIH AIDS Research and Reference Reagent Program. Luciferase detection kit was purchased from Promega. HIV-1 primary virus strain (94UG103 (A, X4R5), 92RW008 (A, R5), 92US657 (B, R5), 92BR014 (B, X4R5), 93MW959 (C, R5), 92IN101 (C, R5), 92BR025(C, R5), 94UG118(D, R5), 92UG001(D, X4R5), 92TH009(A / E, R5), 92TH023(E, R5), 93BR020(F, X4R5), RU570(G, R5) , BCF02(O, R5)) According to Li, L. et al., "3-hydroxyphthalic anhydride-modified chicken ovalbumin exhibits potent and broad anti-HIV-1 activity: a potential microbicide for preventing sexual transmission of HIV-1", Antimicrob Agents Chemother 54: 1700-11 (2010) described method isolated and tested.

[0111] 3.2 Detection methods of laboratory-adapted strains

[0112] Take 100 μl of TZM-bl cells in good condition, the concentration is ab...

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PUM

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Abstract

The invention relates to a separated peptide for inhibiting HIV-1 mediated membrane fusion, which is characterized in that the peptide comprises an amino acid sequence of WGRLEGRRT (SEQ ID NO: 3). The peptide can react mutually with gp41, effectively prevent cell fusion mediated by viral envelope protein, and neutralize HIV-1.

Description

field of invention [0001] The invention relates to the field of prevention and treatment of human immunodeficiency virus (Human Immunodeficiency Virus, HIV), in particular to an HIV virus membrane fusion inhibitor. Background of the invention [0002] Acquired Immunodeficiency Syndrome (AIDS), also known as AIDS, is an acquired acquired human immunodeficiency disease, which has brought serious social and economic problems to China and the world. The most effective way to deal with AIDS in the past was "highly active antiretroviral therapy" (HAART), which is called "cocktail therapy", but now the process of HIV virus entering target cells has become a popular target for fighting HIV virus and defeating AIDS (Jiang , S., Lin, K., Strick, N. & Neurath, A.R. HIV-1 inhibition by a peptide. Nature 365, 113 (1993); Liu, S., Wu, S. & Jiang, S. HIV entry inhibitors targeting gp41: from polypeptides to small-molecule compounds. Curr Pharm Des 13, 143-162 (2007)). The membrane protei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12P21/02A61K38/16A61K39/00A61K48/00A61P31/18
Inventor 陈应华朱赟陆路杨恒文徐利玲
Owner TSINGHUA UNIV
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