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目的表达沙眼衣原体外膜蛋白2,纯化表达产物,并对其免疫性进行鉴定。方法应用聚合酶链反应(PCR)技术获得沙眼衣原体D型外膜蛋白2第167～434位氨基酸的编码基因片段,将此片段克隆于表达载体pET28b(+),转化大肠埃希菌BL21(DE3),IPTG诱导表达,SDSPAGE、蛋白印迹试验分析表达产物,亲和层析法纯化重组蛋白;重组蛋白免疫家兔以检测其免疫原性。结果重组质粒酶切分析及DNA测序证实成功构建pET28b(+)/Omp2aa167～aa434表达质粒;通过优化表达和纯化条件,获得了相对分子质量约为35.0×103纯化蛋白产物,蛋白印迹试验证实该重组蛋白能与Ct感染者阳性血清反应;ELISA法测定免疫血清特异性抗体效价在1∶1280以上。结论表达的重组外膜蛋白2aa167～aa434具有良好的免疫性。
Objective To express Chlamydia trachomatis outer membrane protein 2, and to purify the expression product, and to identify its immunity. Methods The gene fragment of amino acids 167 to 434 of D-type outer membrane protein 2 of Chlamydia trachomatis was obtained by polymerase chain reaction (PCR). The fragment was cloned into the expression vector pET28b (+) and transformed into Escherichia coli BL21 ), Induced by IPTG, expressed by SDSPAGE and Western blotting, and purified recombinant protein by affinity chromatography. The recombinant protein was immunized to detect the immunogenicity of rabbits. Results Recombinant plasmids and DNA sequencing confirmed that the recombinant plasmid pET28b (+) / Omp2aa167 ~ aa434 was successfully constructed. The recombinant protein with the molecular weight of about 35.0 × 103 was obtained by optimizing the expression and purification conditions. Western blotting confirmed that the recombination The protein reacted with positive sera from patients with Ct infection. The titer of immune serum-specific antibody was above 1:1280 by ELISA. Conclusion The expressed recombinant outer membrane protein 2aa167 ~ aa434 has good immunity.