穿透支原体主要外膜蛋白pET20b（＋）／p35重组载体的构建、表达及鉴定

目的构建高表达且稳定的pET20b( )/p35重载体,能在其中进行诱导高表达并得到纯化的p35蛋白.方法运用Blast、Pfam等生物信息学手段对穿透支原体(Mpe)p35基因序列结构与功能预测,将p35基因全长插入原核表达载体pET20b( )中,在BL21Star^TM(DE3)进行诱导表达、用Ni柱纯化,在GSH-GSSH体系中逐渐降低其变性剂的方法复性蛋白,Western-blot进行鉴定.结果成功的构建了pET20b( )/p35重组载体,并能在宿主菌中较高表达;此蛋白以包涵体的形式进行表达.结论得到了纯化的p35蛋白,为进一步研究其生物学功能及其空间结构奠定了基础.

Construction,Expression and Identification of Major Outer Membrane Protein of Mycoplasma Penetrans pET20b(+)/p35

Objective To clone Mpe p35 gene and construct recombinant plasmid pET20b(+)/p35, induce its high expression and to purify the protein product. Methods The full sequence of Mpe p35 gene was analyzed with different bioinformatic tools including Blast, Pfam etc. PCR amplified the Mpe p35 gene fragment from pQE31/ p35.The purified PCR products were directly ligated into pUCm-T vector.After being digested with BamH1+SaL1 and purified,the Mpe p35 gene fragment was inserted into the compatible site of prokaryotic expression vector, and the constructed recombinant plasmid was introduced into competent E.coli TG1.After restriction enzymes cleavage analysis and sequencing,the recombinant plasmid pET20b(+)/ p35 was transformed into an expression stain BL 21 Star TM (DE 3).The host bacteria harboring the expression plasmid was induced by IPTG, the product was confirmed by SDS-PAGE and puried by Ni-NTA column.The renatured protein was refolded in the GSH-GSSH buffer gradually and identified by Western-blot. Result The p35 gene was cloned. Most of the expressed recombinant proteins were presented in form of inclusion bodies. Conclusions The p35 gene protein is successfully expressed and purified, which lays a foundation of further study for its biology such as the role of protein played in pathology and crystalline structure..