MgPa重组蛋白在生殖支原体血清学诊断中的应用

摘要

[目的]表达、纯化生殖支原体 (Mycoplasma genitalium,Mg) 黏附蛋白(MgPa)优势表位基因(MgPa',1075~1364aa),探讨MgPa'重组蛋白在Mg血清学诊断中的应用.[方法]IPTG诱导表达重组质粒pET-30a(+)/MgPa',SDS-PAGE和免疫印迹鉴定表达结果;Ni-NTA亲和层析法纯化重组蛋白,间接酶联免疫吸附试验(Enzyme link immunosorbent assay,ELISA)检测人血清中特异性IgG抗体.[结果]SDS-PAGE检测诱导产物显示有一分子量约为37 000的特异蛋白带,免疫印迹检测其只与Mg抗血清发生特异反应;在311例有临床症状性传播疾病(STD)患者中用PCR法筛查出Mg阳性42例,阳性率13.5%(42/311).重组蛋白用作ELISA包被抗原检测Mg阴阳性血清,重组蛋白能和Mg感染者阳性血清特异性结合,重组蛋白有良好的免疫反应性.[结论]表达的MgPa'重组蛋白具有较好的免疫反应活性,可望用于生殖支原体的血清学诊断.%[Objective] To express and purify dominance epitope gene(MgPa ) of attachment protein(Mg-Pa) of Mycoplasma genitalium(Mg) and explore MgPa recombinant protein used in the serodiagnosis of Mg. [Methods] The expression of recombinant plasmid pET-30a( + )/MgPa was induced by IPTG. SDS-PAGE and Western blot were used to identify the expression result. Ni-NTA affinity chromatography was used to purify recombinant protein. The specific antibody to MgPa in human sera was detected by indirect ELISA. [Re-sults!A specific protein with molecular weight about 37KDa was detected by SDS-PAGE after induced expression. Western blot proved that the recombinant protein could specifically react with Mg-positive sera. Forty two Mg-positive specimens of 311 patients with sexually transmitted disease(STD) were detected by PCR, and the Mg-positive rate was 13. 5% (42/311). Recombinant protein as the coating antigen of ELISA was used for measuring the Mg-positive or Mg-negative sera. Recombinant protein could specially react with the sera from Mg-positive infected patients, and it showed excellent immunoreactivity. [Conclusion] The recombinant protein expressed MgPa shows excellent immunoreactive activity. It is hopeful to be used in the serodiagnosis of Mg.