HIV-1蛋白酶的表达、纯化和其抑制剂体外筛选方法的建立.pdfVIP

21 2 21 2 126-130 2006 3 VIROLOGICA SINICA March 2006 HIV-1 * 1,3 1,3 1 2 2 1,** (1 650223; 2 650231; 3 100039) Expression and Purification of HIV-1 Protease and the Establishment of a Method for Protease Inhibitor Screening 1,3 1,3 1 2 WANG Yun-hua , WANG Rui-rui , YANG Liu-meng , Li Jian-feng XU Wei-ming2 ZHENG Yong-tang1, (1. Laboratory of Immunopharmacology, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223,China; 2. Institute of Medical Biology, Chinese Academy of Medical Sciences, Kunming, Yunnan 650231China ; 3. Graduate School of Chinese Academy of Sciences, Beijing 100039, China ) Abstract HIV-protease coding sequence was amplified from HIV-1IIIB RNA by one- step RT-PCR. The PCR product was inserted into the pet28a expression vector with an additional ATG start codon before and TAA stop codon after the coding sequence. Positive clone was selected and transformed into E. coli BL21 DE3. Cells were cultured in LB medium and induced by IPTG. The recombinant protease was expressed in the form of inclusion body and was up to 40% of total bacterial protein. The inclusion body was washed by Triton X-100 before dissolved in 8M urea and then applied on sephacyl s-200 H.R column. The purity of the protease reached 90% after purification. Protease fraction was collected and diluted in refolding buffer and left at 4 for 24 h. The diluent was concentrated by ultrafiltration. The activity of recom