沙眼衣原体CPAF免疫活性区域重组蛋白的表达及在临床诊断中的应用

Expression, purification and clinical use of CPAF immune dominant domain recombinant protein of Chla.nydia trachomatis

目的分析沙眼衣原体（Ct）蛋白酶样活性因子（CPAF）免疫优势区的免疫活性，探讨其在Ct感染诊断中的应用价值。方法构建原核表达重组质粒pGEX-6p-2／CPAF’，异丙基硫代半乳糖苷（IPTG）诱导表达，用该纯化的表达产物包被酶标板，建立间接ELISA检测病人血清中的Ct特异性IgM和IgG抗体，同时与晶美公司Ct ELISA试剂盒检测结果进行对比分析。结果含pGEX-6p-2／CPAF’的大肠杆菌BL21大量表达相对分子质量（Mr）约为43×10^3的目的蛋白，用该重组蛋白包被酶标板，建立间接ELISA检测Ct IgM、IgG阴性和阳性参考血清各50份，符合率均为100％；同时检测250份临床血清标本，与晶美公司ELISA试剂盒检测结果的符合率IgM为96．8％，IgG为98．4％。结论表达的Ct CPAF免疫优势区（AA200～AA388）重组蛋白具有良好的免疫反应活性，可望用于Ct感染的临床诊断。

Objective To clone and express the chlamydial protease-like activity factor（CPAF） immune dominant domain gene of Chlamydia trachomatis（Ct ）, and to assess the immunocompetence of expressed product. Methods The immune dominant domain gene of Ct CPAF was amplified by PCR and cloned into the express vector pGEX-6p-2. The recombinant plasmid pGEX-6p-2/CPAF＇ expressed in E. coli BL21 and the recombinant protein was purified with GST agarose gel FF after renaturation. The immunoreactivity was evaluated by Western blot and indirect ELISA. Indirect ELISA was developed to detect control sera and the antibodies to Ct in human sera. Results Recombinant plasmid pGEX-6p-2/CPAF＇ was constructed succesfully. SDS-PAGE test proved that the recombinant protein with relative molecular mass （Mr） about 43×10^3 was expressed effectivly. Western blot proved that the recombinant protein can specifically react with GST mouse McAb and Ct IgG positive sera. Specific humoral response was elicited after introducing recombinant protein in New Zealand rabbits and the specific antibody titer of IgM and IgG was above 1 ： 8000 and 1 ： 16 000, respectively. The sensitivity and specificity of the indirect ELISA detected by 100 control sera both were 100% （50/50）. The concordance of IgM results between the developed ELISA test and the ELISA test by Jingme icorporation was 96.8 % and IgG was 98.4 %. Conclusion The expressed recombinant protein showed excellent immunocompetence, had a better effect in clinical serodiagno-sis and could be used to develop the indirect ELISA to detect Ct antibodies in human sera.