| 研究报告 |
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| Prokaryotic Expression of Integrase Enzyme of HIV-1 and The Function Study of Intergrase Protein |
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Abstract The pol gene of HIV1 encodes mainly three enzymes: reverse transcriptase (RT), protease (PR) and integrase (IN). Currently, FDA approved drugs targeting RT and PR are available and administered in various combinations, while no antiIN drug was approved. HIV1 integrase is an essential enzyme for the viral replication and an interesting target for the design of new pharmaceuticals for multidrug therapy of AIDS. The 288 amino acids of IN (32kDa) recognizes specific sequences in the long terminal repeats (LTRs) of the retroviral DNA. The IN protein catalyzes the 3′processing step and the 5′strand transfer step reaction in vivo, which was called integration and this reaction could be analysed by ELISA Assay in vitro. It has been reported that F185K and C280S mutations of HIV1 integrase would improve the enzyme solubility, and the catalytical activity of the enzyme was the same as that of the wildtype enzyme in vitro. In order to build the platform of screening inhibitor against integrase of HIV1 virus, the IN enzyme was expressed and the function of integrase protein was assayed. The cDNA of clade B HIV1 genome was used as a template, overlapped PCR was used to construct site mutagenesis of F185K/C280S and NdeI/Xho I restriction sites were brought in. The PCR product was cloned into the prokaryotic vector pET28a(+) to form a recombined plasmid, transferred into the host cell E.coli(BL21 DE3). The recombined clones were identified by PCR and Nde I/Xho I digestion .The positive plasmid was sequenced, and the successfully recombined plasmid in the host cell was induced by IPTG. The expressed IN protein was purified by the Co+ affinity chromatography column and SDSPAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombined IN protein. The recombinant protein was soluble, and expressed highly and stably in E.coli. The molecular weight of the expression product was identical to the expectation. The IN protein was proved to be functional in 3′ processing and 5′strand transfer by ELISA. It will be helpful to build the platform of screening inhibitors against HIV1 integrase.
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Received: 25 January 2006
Published: 25 January 2006
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