摘要
目的构建EB病毒基因LMP2A、BZLF1重组分泌性表达质粒Ag85B,然后将它成功转化成卡介苗。方法分别用PCR扩增Ag85B信号序列和LMP2A和BZLF1融合基因,将Ag85B信号序列与大肠杆菌-卡介苗穿梭质粒pMV261重组构建获得质粒pMVS。然后将LMP2A和BZLF1融合基因Z2A与质粒pMVS重组获得质粒pMVZ2A,将其电转化卡介苗,用SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）对其结果进行分析。结果信号序列与LMP2A和BZLF1融合基因被正确插入分泌表达载体pMV261。重组质粒经限制性内切酶双酶切、PCR扩增、基因测序等鉴定。蛋白纯化后可得到一条带。结论 pMVZ2A在BCG中能够分泌表达,本研究为获得抗结核杆菌和EB病毒的双价疫苗奠定了基础。
Objective To construct a recombinant secretory plasmid of Bacillus Calmette-Guerin（BCG） Ag85B-fused EB virus LMP2A and BZLF1 genes,and then transform it into BCG.Methods BCG-Ag85B signal sequence and Z2A gene were amplified from the genome of BCG and Z2A by PCR,respectively.BCG-Ag85B signal sequence was cloned into E.coli-BCG shuttle-vector pMV261 to obtain pMVS.The new recombinant plasmid of pMVZ2A was constructed by inserting the Z2A gene into pMVS and transformed into BCG by electrotransformation.The recombinant proteins were analyzed by SDS-PAGE.Results The cloned genes of BCG-Ag85B and Z2A were correctly inserted into the vector pMV261.The recombinant plasmid of pMVZ2A was identified by double restriction enzyme digestion,PCR amplification and gene sequencing.Conclusions pMVZ2A effectively expressed Z2A protein in BCG.The study provided a basis for BCG reconstruction and the development of new bivalent vaccine agaisnt EB virus and mycobacterium tuberculosis.
出处
《中国病毒病杂志》
CAS
2011年第4期282-286,共5页
Chinese Journal of Viral Diseases
基金
山东省卫生厅基金（2007HW035）
济宁医学院青年基金项目资助
