EB病毒基因Z2A原核表达载体的构建及表达

Construction and expression of prokaryotic expression vector with EB virus gene Z2A

目的构建能分泌性表达EB病毒（Epstein—Barrvirus，EBV）融合基因Z2A的BCG重组质粒并导入BCG。方法分别以BCG和EBV融合基因eDNA为模板，通过PCR扩增得到139bp的BCG—Ag85B信号肽序列和2291bp的Z2A基因序列。将BCG-Ag85B信号肽序列与大肠杆菌一BCG穿梭表达载体pMV261重组，得到重组质粒pMVSo再将EBV融合基因序列Z2A亚克隆至pMVS中，得到重组质粒pMVZ2A，电转化导入BCG，采用十二烷基硫酸钠一聚丙烯酰胺凝胶电泳对表达产物进行分析。结果构建的重组质粒pMVZ2A经双酶切、PCR扩增及序列测定证实，克隆基因BCG-Ag85B信号肽和Z2A正确插入载体pMV261。重组质粒pMVZ2A电转化导入BCG后能在BCG中有效表达相应蛋白。结论构建的重组质粒pMVZ2A能在BCG中表达相应蛋白。这一结果为改造BCG及发展新型抗EBV和结核杆菌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 genes, and then transform it into BCG. Methods BCG-Ag85B signal sequence and Z2A gene were amplified by PCR, respectively. BCG-Ag85B signal sequence was cloned in E. coli-BCG shuttle-vector pMV261 to obtain pMVS. Then a new recombinant plasmid pMVZ2A was constructed by inser- ting the Z2A gene into pMVS and transformed into BCG by electrotransformation. The expressed products were analyzed by SDS-PAGE. Results The cloned genes BCG-Ag85B and Z2A were correctly inserted into the vec- tor pMV261. The recombinant plasmid pMVZ2A was identified by double restriction enzyme digestion, PCR amplification and gene sequencing, respectively. The recombinant plasmid pMVZ2A effectively expressed Z2A in BCG after transformation. Conclusions pMVZ2A can effectively express Z2A in BCG. This study lays down the foundation for BCG reconstruction and development of new bivalent vaccine agaisnt EB virus and Mycobacterium tuberculosis.