Zhao Ming-cai, Bao Lang, Wu Yue-han, Zhang Hui-dong
Infection and Immunity Unit, West China Center of Medical Sciences, Sichuan University, Chengdu 610041, China.
Wei Sheng Yan Jiu. 2006 Mar;35(2):152-4.
To construct a prokaryotic expression vector bearing Rv2994 gene from Mycobacterium tuberculosis and provide materials for investigating the function of the gene.
The Rv2994 gene was amplified by Polymerase Chain Reaction from Mycobacterium tuberculosis H37Rv strain and cloned into prokaryotic expression vector pGEX-1lamdaXT. The recombinant plasmid pGEX-2994 was sequenced and transformed into E. coli JM109 to be induced with IPTG and expressed the 73kDa fusion protein GST-Rv2994. It's antigenicity was confirmed by Western blotting. The expression product was purified and immunized the new Zealand rabbits.
The Rv2994 gene was amplified accurately from the genome DNA of H37Rv. A recombinant fused expression vector pGEX-Rv2994 was constructed and GST-Rv2994 protein was purifiel to immunize New Zealand rabbit.
The prokaryotic expression vector pGEX-2994 was constructed, and the 73kDa fusion protein GST-Rv2994 was expressed and purified successfully. It provided the basis for the further study of the Rv2994 gene.
构建携带结核分枝杆菌Rv2994基因的原核表达载体，为研究该基因的功能提供材料。
采用聚合酶链反应从结核分枝杆菌H37Rv菌株中扩增Rv2994基因，并将其克隆到原核表达载体pGEX-1lamdaXT中。对重组质粒pGEX-2994进行测序，然后转化到大肠杆菌JM109中，用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达73kDa融合蛋白GST-Rv2994。通过蛋白质免疫印迹法确认其抗原性。对表达产物进行纯化，并免疫新西兰兔。
从H37Rv基因组DNA中准确扩增出Rv2994基因。构建了重组融合表达载体pGEX-Rv2994，并纯化了GST-Rv2994蛋白以免疫新西兰兔。
构建了原核表达载体pGEX-2994，成功表达并纯化了73kDa融合蛋白GST-Rv2994。为进一步研究Rv2994基因奠定了基础。
