目的  构建能分泌性表达EB病毒（Epstein-Barr virus，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cDNA为模板，通过PCR扩增得到139 bp的BCG-Ag85B信号肽序列和2291 bp的Z2A基因序列。将BCG-Ag85B信号肽序列与大肠杆菌-BCG穿梭表达载体pMV261重组，得到重组质粒pMVS。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-聚丙烯酰胺凝胶电泳（SDS-PAGE）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 LMP2A and BZLF1, 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 in E.coli-BCG shuttle-vector  pMV261 to obtain pMVS. Then a new recombinant plasmid pMVZ2A was constructed by inserting 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 vector 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.