梅毒螺旋体特异抗原TP0684的克隆和表达

目的克隆并表达梅毒螺旋体外膜蛋白TP0684,为研制早期诊断试剂盒提供依据。方法PCR法扩增TP0684编码基因。T-A克隆后构建表达质粒pET-22b(+)-TP0684,经酶切鉴定后,转化E.coli BL21(DE3),IPTG诱导表达,表达产物经Western blot鉴定。结果PCR法扩增出了1212bp的目的片段,原核表达质粒pET-22b(+)-TP0684酶切鉴定正确,测序结果与GenBank上公布的该基因的CDS序列完全一致。转化E.coli BL21(DE3)后,IPTG诱导目的蛋白表达率为25%,SDS-PAGE初步测定目的蛋白的相对分子质量约43000,Western blot证实该蛋白能与梅毒患者阳性血清发生特异性反应。结论所表达的TP0684重组蛋白具有良好的免疫反应性,为进一步研究梅毒血清学诊断试剂奠定了基础。

Gene Cloning and Expression of Outer Membrane Protein TP0684 of Treponema pallidum

Objective To cloning and express the gene encoding the outer membrane protein TP0684 of Treponema pallidum(TP) and provide a basis for development of kit for early diagnosis of TP infection.Methods The gene encoding TP0684 was amplified by PCR and,after T-A cloning,inserted into expression vector pET-22b( ).The constructed recombinant plasmid pET-22b( )-TP0684 was transformed to E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was identified by Western blot.Results The target gene at a length of 1 212 bp was amplified.Restriction map proved that recombinant plasmid pET-22b( )-TP0684 was correctly constructed.The sequence of target gene inserted into the recombinant plasmid was completely identical to that reported in GenBank.The expressed product,with a relative molecular of about 43 000,contained about 25% of total somatic protein.Western blot showed specific reaction of expressed protein with TP positive serum.Conclusion The expressed TP0684 showed good immunoreactivity.It laid a foundation of further development of serologically diagnostic kit for TP infection.