EB病毒融合基因重组BCG穿梭质粒的构建与鉴定

Construction and identification of recombinant shuttle-plasmid pMVZ2A

目的构建分泌性表达EB病毒融合基因Z2A的卡介苗(BCG)重组质粒。方法分别以BCG和EB病毒融合基因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信号肽序列与大肠埃希菌-BCG穿梭表达载体pMV261重组,得到重组质粒pMVS。再将EB病毒融合基因序列Z2A亚克隆至pMVS中,得到重组质粒pMVZ2A。结果构建的重组质粒pMVZ2A经双酶切、PCR扩增及测序鉴定证实,克隆基因BCG-Ag85B信号肽和Z2A正确插入载体pMV261。结论重组质粒pMVZ2A可望在BCG中分泌性表达,该质粒的构建成功为改造BCG、发展新型抗EB病毒疫苗奠定了基础。

Objective To construct a recombinant secretory plasmid of Bacillus Calmette-Guerin（BCG） Ag85B-fused LMP2A and BZLF1.Methods The BCG Ag85B signal sequence and Z2A gene were amplified from the genome of BCG and Z2A by PCR,respectively.The 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.Results The cloned genes BCG Ag85B and Z2A were correctly inserted into the vector pMV261,and this was confirmed by restriction endonuclease digestion and PCR amplification of Z2A and gene sequencing,respectively.Conclusion pMVZ2A should secretively express Z2A in BCG.This study provides the basis for further studies on the development of new EB virus vaccines and reconstruction of BCG.