肺炎支原体P1蛋白抗原决定簇在大肠杆菌中的表达与纯化

Expression and cloning of the P1 protein epitopes of Mycoplasma pneumoniae in Escherichia coli

目的 得到肺炎支原体主要粘附蛋白 (P1蛋白 )的表达蛋白。材料与方法 用PCR产物限制性片段长度多肽性(RFLP)分析等方法鉴定实验用肺炎支原体菌株。针对P1蛋白基因设计上、下游引物 ,进行PCR扩增。对PCR产物进行回收、纯化 ,克隆入PET - 2 8C(+)表达载体 ,并转化入宿主菌BL2 1。对重组质粒进行酶切及PCR鉴定后 ,IPTG诱导目的蛋白在大肠杆菌BL2 1(DE3)中表达 ,并以Ni-Agarose亲和层析柱纯化。结果 1)本实验所用菌株为肺炎支原体 2型。 2 )经PCR扩增得到预期含有BamHI和XhoI的 5 33bp的目的片段。 3)完成目的基因片段的克隆与表达 ,得到 2 0kDa左右表达蛋白。经诱导阳性质粒的蛋白表达 ,得到较大量纯化的目的蛋白。结论 本研究以国际标准株FH为模板 ,成功获得纯化的近 2 0kDa的P1蛋白片段 ,其大小与目的片段理论值相符 ,且此片段在肺炎支原体 1型与 2型中的基因同源性为 98%。为在基因和蛋白水平进一步研究肺炎支原体P1蛋白成分在肺炎支原体粘附和引起机体免疫反应中的作用机制 ,打下了良好的基础。

To express a surface protein designated P1 of Mycoplasma pneumoniae in Escherichia coli,the stocked experimental strain of FH(ATCC15531) was used and identified by restriction fragment length polymorphism(RFLP) analysis.The gene encoding for the P1 protein was amplified with up and down stream primers,and the amplified products(533 bp) and the plasmid PET-28C(+) were digested with BamH1 and Xhol restriction endonuclease respectively.DNA was ligated with the vector and transformed into E.coli BL21.And the target protein was purified by a kit from QIAGEN.It was found the that the RLFP pattern of the strain was group II and the PCR product was a fragment of 535 bp that contained the restriction sites of BamHI and Xho I.The size of the expressed protein identified by PCR and endo-proteolysis was about 20 kDa.A fragment of P1 gene near the C-terminal had been inserted into the expression vector PET28C(+).In addition,the vector contained all the necessary signals for transcription and translation of the inserted fragment.The homology of the gene fragments between group I and II of M.pneumoniae was 98%.It concludes that the P1 gene fragment is cloned and expressed in E.coli successfully in the present study.