穿透支原体P35蛋白在大肠杆菌中的表达与鉴定

Expression and Identification of Mycoplasma Penetrans 35kDa Lipoprotein

目的 为研究穿透支原体 (Mpe) 35kD脂质相关膜蛋白 (P35 )的生物学活性 ,构建其原核细胞表达载体 ,并在大肠杆菌 (E .coli)中表达。方法 PCR扩增p35基因片段 ,克隆至pQE31载体 ,用PCR介导的突变将p35基因中两个“TGA”终止密码子定点突变为“TGG” ,使其在E .coli中编码表达色氨酸。IPTG诱导pQE31 P35在E .coli中表达目的蛋白rP35 ,经Ni-NTASpin亲和纯化 ,Westernblot分析鉴定表达产物。结果 PCR扩增出约 1kb的DNA片段 ,克隆至pQE31载体 ,筛选到阳性克隆pQE31 P35 ,测序分析p35基因中两个“TGA”终止密码子成功突变成“TGG”。IPTG诱导pQE31 p35在E .coli中表达分子量约 37.4kD的蛋白质 ,Westernblot分析鉴定其为rP35。结论 构建的pQE31 p35原核表达载体 ,在E .coli中成功地表达rP35。

Objective To research the biological activities of Mycoplasma penetrans(Mpe) 35kDa lipid associated membrane protein (P35) in vitro, prokaryotic expression vector was constructed and expressed in E.coli. Methods The p35 gene amplified by polymerase chain reaction(PCR) was cloned to pQE31 and positive clone was screened. PCR-mediated mutagenesis was used to change the two “TGA” triplets to “TGG” triplets within the p35 gene. Production of recombinant protein was induced by the addition of IPTG to the E.coli culture, and the rP35 was purified with a Ni-NTA Spin Kit. Purification of rP35 was analyzed by Western blot. Results About 1kb PCR amplification was cloned into pQE31, the two “TGA” triplets within the p35 gene were successfully changed to “TGG” triplets by sequence analysis. The pQE31/ p35 encoded a protein with a calculated molecular mass of 37.4 kDa in E.coli. Western blot showed the 37.4 kDa purification was rP35. Conclusion pQE31/ p35, a prokaryotic expression recombinant, was successfully constructed and rP35 was ideally expressed in E.coli.