| HIV-1 integrase enzyme is a 32 kDa protein encoded by HIV pol gene. It is responsible for integration of viral cDNA into host chromosomal DNA, which is indispensable for HIV replication. The inhibition of integrase activity is an effective measure to prevent HIV-1 replication. Moreover, integrase has no known functional analogue in human cells. Therefore, integrase becomes an ideal target for anti-HIV therapy. The HIV-1 integrase possesses two different activities: 3′-processing and strand transfer. After binding to the double-stranded blunt-ended viral DNA integrase cleaves the two 3′terminal bases GT and then joins the ends to acceptor DNA molecule,the strand transfer reaction is reversible, when presented with a substrate similar to 3′-end joining product. Crystal structure analysis defines the three main domains within the HIV-1 integrase protein. These are the N-terminal domain including a zinc finger like region, the central region containing the catalytic site, and the C-terminal region with a DNA-binding domainBecause of the high rate of DNA mutation in HIV-1 repliacation, the virus is tend to aquire the ability to resistant drug, which is called drug resistance. In 1996, doctor Da Yi He raised a new methed to unite multiple agents to cure AIDS, which has a name of HAART. The recipe usually includes two RTIs and one PI. HAART can inhibit the replication of the virus efficiently, which promise patients a longer life. But this method has its own defect, such as the side effect and the restoration of virus when holding cure. In October, 2007, the first drug raltigravir setting integrase as its target spot acquire the authorization by FDA. Another inhibitor, Elvitegravir which has lower IC50 than raltigravir is on its stage of clinical trial. But some trials show that the two drugs also confront the problem of drug resistance, so the research of mechanism of drug is a new hot spot nowadays.The objectives in this study are as follows: (1) To construct an integrase recombinant vector with devised mutation pET-28a(+)/IN(T66I), pET-28a(+)/IN (G118A), pET-28a(+)/IN(T66I/G118A). (2) To express and purify the 3 mutant IN. (3) To identify the strand transfer activity of the 3 mutant IN by the magnetic beads-based ELISA. (4) To research of the improvement of soluble expression of HIV-1 integrase induced by T66I mutation.The following methods are used in this study: (1) Information about mutual reaction between amino acids was acquire from the result of molecular simulation. The 3 mutations (T66I, G118A, T66I/G118A) were induced by overlapping PCR. The recombinant pET-28a(+) vector with devised mutations was expressed in in E.coli BL21(DE3). Highly pure and soluble integrase was acquired by affinity chromatography on a Ni2+-charged chromatography resin. An in vitro integrase assay about the strand transfer activity was carried out by an advanced ELISA based on magnetic beads. (2) Under the same condition, IN (F185K/C280S) and IN (T66I) were expressed and purified. The sediment was eliminated by centrifugation for several times. The difference between IN (F185K/C280S) and IN (T66I) in the aspect of solubility was analysed by SDS-PAGE and His-tagged protein staining kit.The results of this study are as follows: Three integrase recombinant vectors pET-28a(+)/IN(T66I), pET-28a(+)/IN(G118A), pET-28a(+)/IN(T66I/G118A) were successfully constructed based on the results of molecular simulation. (2) Result shows that the solubility of IN1–288/T66I is 2-3 fold of WT. 4.72mg protein was obtained from 600ml of cells expressing IN(T66I). The strand transfer activity of IN(T66I) and IN(F185K /C280S) were analyzed by advanced ELISA, which shows that two proteins are almost equal in catalytic activity. This research has provided another method, being different from F185K /C280S mutation, to improve the soluble expression of integrase. IN1–288/T66I can also be used to screen integrase inhibitor, which can avoid the drug-resistance brought by T66I mutation.
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