结核分枝杆菌Rv2994基因原核表达及鉴定

目的构建结核分枝杆菌Rv2994基因原核表达载体并进行表达,为进一步研究奠定基础。方法PCR扩增Rv2994基因编码序列,定向克隆入融合蛋白原核表达载体pGEX-1λT获得重组表达质粒pGEX-2994,转化大肠杆菌测序后诱导表达,纯化目的蛋白并免疫新西兰大白兔获取多价抗体。结果从结核分枝杆菌H37Rv株基因组DNA中扩增出Rv2994基因,成功构建了融合表达质粒pGEX-Rv2994,转化大肠杆菌JM109后,经IPTG诱导,表达出约73kDa重组融合蛋白,用Western blotting证实其有良好的抗原性,纯化蛋白免疫新西兰大白兔,获得了高效价的抗体。结论成功构建了原核表达载体pGEX-2994,获得并纯化了GST-Rv2994融合蛋白,制备了高效价的多价血清,为Rv2994基因的功能研究奠定了基础。

Prokaryotic expression and characterization of the Rv2994 gene from Mycobacterium tuberculosis

OBJECTIVE: To construct a prokaryotic expression vector bearing Rv2994 gene from Mycobacterium tuberculosis and provide materials for investigating the function of the gene. METHODS: 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. RESULTS: 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. CONCLUSION: 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.