肺炎嗜衣原体CPAF原核表达载体的构建及其在大肠杆菌中的高效表达

Construction of Prokaryotic Vector Encoding Chlamydophila Pneumoniae CPAF and Its Effective Expression in E.coli

目的对肺炎嗜衣原体蛋白酶样活性因子(CPAF)基因进行原核表达,为肺炎嗜衣原体的诊断及疫苗研究奠定基础。方法利用PCR技术从肺炎嗜衣原体标准株AR-39中扩增CPAF基因,将其克隆于原核表达载体pGEX-6p-2中,构建重组表达载体。并用双酶切、PCR及序列测定等方法进行鉴定后,在大肠杆菌中诱导表达GST融合蛋白。结果重组质粒中CPAF基因序列与肺炎嗜衣原体AR-39的同源性达到100%,CPAF在大肠杆菌中表达高效的GST融合蛋白。结论成功构建肺炎嗜衣原体CPAF基因的原核表达系统,并成功表达重组蛋白,为肺炎嗜衣原体疫苗的研究和临床检测试剂盒的研制打下基础。

Objective To express the recombinant protein of chlamydial protease-like activity factor CPAF from Chlamydophila pneumoniae for using in diagnosis and vaccine development. Methods The CPAF gene was amplified from the genome of C. pneumoniae strain AR-39,cloned into prokaryotic expression vector pGEX-6p-2 for fusion expression with GST. With identification by sequencing,the recombinant plasmid was transformed into Escherichia coli XL1-Blue and the recombinant proteins fused with GST was analyzed by SDS-PAGE. Results Sequence of the cloned CPAF gene was correct. The CPAF gene could be expressed in E. coli XL1-Blue which was transformed with pGEX-6p-2/CPAF but could not be expressed in E. coli XL1- Blue with pGEX-6p-2 or untransformed cells. Conclusion The Prokaryotic expression plasmid pGEX-6p-2/CPAF was successfully constructed and expressed in E. coli XL1-Blue,which is useful in C.pneumoniae diagnosis and vaccine development.