梅毒螺旋体Tp0319重组蛋白的表达及免疫活性研究

Expession of Tp0319 recombinant protein from Treponema pallidum and analysis of its immunocompetence

目的 克隆和表达梅毒螺旋体Tp0319基因,并对其表达产物进行免疫活性分析.方法 挑选并克隆出Tp0319免疫优势区基因,构建原核表达载体 诱导表达并纯化重组蛋白,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和Western印迹法鉴定 用纯化的重组蛋白免疫新西兰兔 间接ELISA法检测梅毒螺旋体参考血清及临床标本.结果 成功构建原核表达载体pQE32/Tp03 19 高效表达和纯化出一相对分子质量约30 000的重组蛋白.用重组蛋白免疫新西兰兔,能刺激其产生高水平抗体滴度.Western印迹证明其能与梅毒患者血清发生特异性反应,阴性对照菌未见目的 表达条带.间接ELISA法检测80份梅毒螺旋体参考血清（阴性、阳性各40份）,阳性和阴性结果的符合率均为100%.检测200份临床梅毒血清标本及200份正常人血清,结果与梅毒螺旋体明胶凝集试验相比,灵敏度和特异度分别为92.6%和100%,符合率为96%.结论 制备的Tp0319重组蛋白具有良好的免疫活性,为进一步研究其在梅毒血清学诊断中的应用奠定一定的基础.

Objective To clone, express Tp0319 gene from Treponemapallidum （T. pallidum）, and to assess the immunocompetence of recombinant protein. Methods The immuno-dominant region of Tp0319gene was chosen by computer analysis, amplified from T. pallidum complete genome by PCR, subcloned into the expression vector pQE32 to construct a recombinant plasmid, pQE32/Tp0319, which was then expressed in E. coli M15. The recombinant protein was purified with 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 protein, and the titer of anti-Tp0319 antibodies in sera from immunized rabbits were measured with indirect ELISA. Also, indirect ELISA with the recombinant Tp0319 as coating antigen was performed to detect the anti-Tp0319 antibody in sera from 200 normal human controls and 200 patients with syphilis. Results The prokaryotic expression vector pQE32/Tp0319 was constructed successfully, and the recombinant protein Tp0319 with a molecular weight of about 30 000 was attained. Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the specific antibody titer was more than 1： 10 240 after immunization for 3 times. Western blot proved that the recombinant protein could specifically react with anti-T. pallidum IgG antibody-positive sera. Indirect ELISA was successfully developed with the recombinant Tp0319, and detected antibodies to T. pallidum in control sera with a sensitivity and specificity of 100% （40/40）, respectively. Compared with T. pallidum particle agglutination （TPPA） assay, the sensitivity and specificity of the indirect ELISA were 92.6% and 100%, respectively, in the detection of T. pallidum in sera from patients and controls, and the concordance between the indirect ELISA and TPPA was 96%. Conclusions The prepared recombinant protein shows a satisfactory immunocompetence, which may lay a foundation for its further application in the serodiagnosis of syphilis.