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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 BCG recombinant plasmid secreting the EBV fusion gene Z2A. Methods The BCG-Ag85B signal peptide sequence of 139 bp and the Z2A gene sequence of 2291 bp were amplified by PCR using the BCG and Epstein-Barr virus fusion gene cDNA as template respectively. The BCG-Ag85B signal peptide sequence was recombined with the Escherichia coli-BCG shuttle expression vector pMV261 to obtain the recombinant plasmid pMVS. The EBV fusion gene sequence Z2A was subcloned into pMVS to obtain the recombinant plasmid pMVZ2A. Results The constructed recombinant plasmid pMVZ2A was confirmed by double enzyme digestion, PCR amplification and sequencing. The cloned BCG-Ag85B signal peptide and Z2A were correctly inserted into vector pMV261. Conclusion The recombinant plasmid pMVZ2A is expected to be secreted in BCG. The successful construction of this plasmid lays the foundation for the transformation of BCG and the development of new anti-EBV vaccine.