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Yeast Two-hybrid Based Rapid Screening for Anti-HIV Chinese Herbal Medicine

Author: HuYanYan
Tutor: LiFen
School: Henan Normal
Course: Cell Biology
Keywords: Human Immunodeficiency Virus P-TEFb complex Tat Human cyclinT1 Yeasttwo-hybrid system
CLC: R285
Type: Master's thesis
Year: 2012
Downloads: 66
Quote: 0
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Abstract


AIM: HIV, which is a retrovirus, is the causative agent of AIDS. According to the2010UNAIDSReport on the Global AIDS Epidemic, there are33.3million people who lived with HIV at the end of2009,and approximately1.8million died of AIDS. To date, enormous scientific, social and political efforts havebeen taken to intervene the AIDS epidemic, but there is almost no region which has been spared in theworld. It is even more devastating that the rates of infection are increasing in some region of the world.Currently, highly active antiretroviral therapy(HAART) also known as combined therapy, applies thecombination of inhibitors of the reverse transcriptase and inhibitors against the HIV protease to the patients,improving the clinical condition and the quality of the patients, and also extending the life span of thepatients enormously. However, it use in AIDS patients leads to the formation of mutants that are resistantto the drug, apart from the severe side effects. In brief, there is urgent need for novel drugs against thereplication of HIV. It has been known that HIV protein Tat bind CDK9via human cyclinT1,and theinteraction between P-TEFb complex and TAR leads to the phosphorylation of the RNA polymeraseⅡCTD.The phosphorylated RNAPⅡproductively elongates the transcripts of HIV, ensuring HIV replication. Inthis thesis, we built an screening for discovery of anti-HIV Chinese herbal medicine via yeast two-hybridsystem based on P-TEFB as a target. In the system, human cyclinT1interact with HIV Tat, so we caninvestigate the impact of herb on the interaction between human cyclinT1and HIV Tat, screening theeffective herb fast.METHODS: a fragment of the coding region of human cyclinT1and HIV Tat were amplified byPCR from the plasmids pCDNA3-hcyclinT1and pEGFP-Tat. The DNA fragments were inserted intoeukaryotic expression vectors pGBKT7and pGADT7-Rec and then transformed into AH109and Y187yeast strains correspondingly by LiAc method; then mate the bait strain with prey strain and select for yeastdiploids. Use the diploids to screen the herb which have the inhibition.CONCLUSIONS: We successfully constructed the eukaryotic expression vectors and the yeasttwo-hybrid system. During the more than twenty kinds of Chinese herb which were screened, wesuccessfully find four useful Chinese herb medicine which can inhibit the interaction between the two proteins. This work will lay foundation for the compatibility with the herb and the in vitro(cellular)experiment to convince the inhibition of HIV.

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