乙型肝炎病毒全X基因的克隆及原核表达

Cloning and prokaryotic expression of HBV Whole-X gene

目的 :构建乙型肝炎病毒全 X基因原核表达载体并在细菌中表达、纯化 .方法 :利用基因重组技术 ,将全 X基因定向克隆于原核表达载体 pET 32a(+)多克隆位点中 ,限制性酶切和测序鉴定 .细菌表达产物及纯化后的产物经SDS PAGE和Western免疫印迹分析 .结果 :经限制性酶切分析及测序鉴定 ,证实成功构建全 X原核表达质粒 .SDS PAGE和Western免疫印迹分析表明重组质粒在大肠杆菌中表达 .结论 :本研究将全 X基因定向克隆于原核表达载体 ,并在大肠杆菌中成功表达、纯化 .为进一步研究全 X的功能及制备相应抗体奠定了基础 .

AIM: To construct the prokaryotic expression plasmid pET-32a(+)-Whole-X. METHODS: The Whole-X gene was cloned into EcoR I/Sal I site of pET-32a(+) and was sequenced. The fusion protein was expressed in E. coli with IPTG induction. After the induction and purification, the protein was analyzed in SDS-PAGE and identified by Western blotting. RESULTS: Endonucleases digesting and sequencing confirmed that the Whole-X gene was correctly inserted into the vector. The Whole-X gene was successfully expressed in E.coli. The fusion protein was confirmed by SDS-PAGE and Western blotting. CONCLUSION: The prokaryotic expression plasmid pET-32a(+)-Whole-X has been successfully reconstructed, which provides the experimental base for study on its biological functions and antibody preparation.