Study On The Biological Activity Assays For HIV-1 Integrase Inhibitor

Acquired immunodeficiency syndrome(AIDS), caused by the human immunodeficiency virus(HIV), is one of the most serious infectious diseases harmful to people's healths. The integrase(IN) is one of the enzymes necessary for viral replication. The selective inhibition of IN activities can effectively block the viral replication cycle of HIV in host cells. Since there is no counterpart of IN in human cells, inhibitors specifically targeting IN have little side effect on human body. Therefore, IN is considered to be an ideal target for the research of anti-HIV drugs.IN can catalyze the integration of the viral DNA into the host genome by two successive reactions: termed 3?-processing and strand transfer in vivo. Both the above reactions can be modeled in vitro using the liquid solution, purified recombinant IN protein, divalent cationic cofactor and oligonucleotide DNA substrates.In this study, Sso7 d and IN gene were conneted by overlaping PCR, then the product were ligated to T vector. After double digesting, Sso7d-IN gene was ligated to a pET-15 b expression plasmid and IN was over-expressed in Escherichia coli strain BL21 as a soluble protein. After purification using Histrap column, we obtained the highly purified recombinant Sso7d-IN protein was obtained which had activities in 3?-processing and strand transfer reactions. Compared with the wild-type IN, the activity of Sso7d-IN was significantly increased. The protein have also been applied in the IN inhibitors screening.The assay which based on the principle of molecular beacons was applicable in the high-throughput screening of inhibitors in 3?-processing reaction. 57 componds had been screened and the best IC50 is 3.9 ?M. The high-throughput enzyme-linked immunosorbent assay was applied to screening the inhibitors targeting IN in the strand transfer reaction. 12 componds had been screened and the best IC50 is 7.4 ?M. It provides valid foundation for further study.