梅毒螺旋体重组蛋白Tp0136的表达、纯化及免疫活性分析

Expression, purification and immunocompetence analysis of a recombinant membrane protein, Tp0136, of Treponema pallidum

目的 克隆并表达梅毒螺旋体Tp0136基因，对其表达产物进行纯化以及免疫活性分析。方法 全基因合成Tp0136基因，构建E.coli表达载体；诱导表达并纯化重组蛋白，用SDS-PAGE和Western印迹鉴定。用纯化的重组蛋白Tp0136免疫新西兰兔，并以重组Tp0136作为包被抗原建立间接酶联免疫吸附试验（ELISA）以鉴定其免疫原性，Western印迹检测梅毒阳性血清鉴定其免疫反应性。结果 成功构建E.coli表达载体pET28a-Tp0136，并表达和纯化出膜蛋白Tp0136，相对分子质量为50 000，纯度 〉 95%。用重组蛋白Tp0136免疫新西兰兔，能刺激其产生高滴度抗体，具有较强的免疫原性。Western印迹显示其能与梅毒阳性血清发生特异性反应，具有较好的免疫反应性。结论 成功表达具有Tp0136全基因和较强免疫活性的膜蛋白，Ｔp０１３６可能在梅毒螺旋体的免疫致病机制中起到重要作用。

Objective To clone and express the Tp0136 gene of Treponema pallidum, to purify the recombinant protein and to evaluate its immunocompetence. Methods The full-length Tp0136 gene was synthesized, then subcloned into the expression vector pET28a to construct a recombinant plasmid, pET28a-Tp0136, which was subsequently transfected into E.coli Rosetta for protein expression. The recombinant protein was purified with nickel-nitrilotriacetic acid （Ni-NTA） affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis（SDS-PAGE） and Western blot. New Zealand rabbits were immunized with the recombinant Tp0136 （rTp0136）protein, and anti-Tp0136 polyclonal antibodies in sera of the rabbits were examined by indirect enzyme linked immunosorbent assay （ELISA） with rTp0136 protein as the coating antigen. Also, positive sera were obtained from patients with syphilis and examined by Western blot to identify the immunoreactivity of the rTp0136 protein. Results The E.coli expression vector pET28a-Tp0136 was constructed successfully, and rTp0136 protein was also successfully expressed with a molecular weight of about 50 kD and a purity above 95%. High titres of anti-rTp0136 antibodies were detected in sera of rabbits immunized with the rTp0136 protein, and Western blot showed that the rTp0136 could specifically react with the sera from syphilitic patients, which proved the high immunogenicity and immunoreactivity of the recombinant protein. Conclusions The full-length Tp0136 membrane protein is successfully expressed with a high immunocompetence, and Tp0136 membrane protein may play an important role in the pathogenic mechanisms of Treponema pallidum.