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| Soluble Expression and Inhibitor Screening of the Central Core Domain of HIV-1 Integrase |
| HE Hong-qiu, JIA Yu-yue |
| Chongqing Center for Biomedicines and Medical Equipment, Chongqing Academy of Science and Technology, Chongqing 401123, China |
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Abstract In order to express the central core domain of HIV-1 integrase (IN-CCD) in a soluble form and establish a method for inhibitor screening targeting IN-CCD in vitro, the gene encoding the IN-CCD protein with F185K mutation was amplified from a plasmid containing the full length of IN gene. The gene product encoding IN-CCD protein with F185K mutation was introduced into the pET28b vector to construct a pIN-CCD recombinant plasmid, which was subsequently transformed into E. coli BL21 (DE3) and induced with IPTG. After cell lysis, the soluble IN-CCD in the supernatant was purified by Ni-affinity column chromatography. DNA substrate was modified, and streptavidin-coated magnetic beads were employed as a solid support to capture the DNA reaction product. Finally, an enzyme-linked immunosorbent assay (ELISA) was developed for the activity characterization of IN-CCD and subsequent inhibitor identification. The results showed that IN-CCD was highly expressed in soluble form; the purity of IN-CCD protein was about 95% after purification and dialysis. The ELISA established in this work was sensitive and specific for the detection of IN-CCD disintegration reaction, and could be used for the high-throughput screening of IN-CCD inhibitors. Five samples to be active IN-CCD disintegration inhibitors were screened out from 100 samples.
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Received: 08 November 2011
Published: 25 March 2012
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