肺炎嗜衣原体蛋白酶样活性因子重组蛋白的表达与临床应用

Preliminary Application of Recombinant Protein of Chlamydial Protease-like Activity Factor from Chlamydia pneumoniae in Laboratory Diagnosis

目的探讨肺炎嗜衣原体(CPN)蛋白酶样活性因子(CPAF)重组蛋白在CPN感染实验室诊断中的应用价值。方法构建CPAF免疫优势区基因重组质粒,诱导表达并纯化重组蛋白,分析其抗原特异性;间接ELISA法检测CPN参考血清、临床血清标本中的特异性IgM和IgG抗体,以及沙眼衣原体(Ct)阳性血清。结果高效表达和有效纯化出一相对分子质量(Mr)约为5.13×103的特异性重组蛋白;间接ELISA法测定用其免疫兔血清中的特异性IgG和IgM抗体效价,分别高达1∶16 000和1∶8 000;检测Cpn IgM和IgG参考血清,阴性和阳性结果符合率均为100.0%;与"金标准"方法MIF比较,检测300份呼吸道感染患者抗血清,IgMI、gG符合率分别为98.3%、99.0%;检测Ct阳性参考血清和阳性临床血清标本,无阳性结果。结论表达的CPN CPAF免疫优势区重组蛋白具有良好的抗原性,在CPN感染的实验室诊断中具有较高的应用价值。

OBJECTIVE To evaluate the application value of the recombinant protein of chlamydial protease-like activity factor（CPAF） from Chlamydia pneumoniae in laboratory diagnosis. METHODS Recombinant plasmid containing CPAF immunodominant region gene was constructed. A recombinant protein was expressed after induction by IPTG, and identified by SDS-PAGE and Western blot, and then purified with glutathion S-transferase （GST） agarose gel FF. An indirect ELISA ofter optimization was developed with the purified recombinant protein as coating antigen, and used to detect anti-Chlamydia trachomatis positive sera and C. pneumoniae-specific IgG and IgM antibodies. RESULTS A specific recombinant protein with relative molecular mass about 5.13 × 10^3 was efficiently expressed and purified. After the New Zealand rabbits were immuned with the purified recombinant protein, the titers of the specific IgM and IgG antibodies were above 1 ： 8 000 and 1 ： 16 000, respectively. C. pneumoniae IgG and IgM reference sera were detected with the indirect ELISA, both coincidence rates were 100%. Anti-C. trachomatis positive control sera and positive clinical sera were detected with the indirect ELISA based on the recombinant protein, then no positive result was found. In comparison with MIF as reference method, the serum antibodies from 300 patients with respiratory infection were detected and the IgG and IgM coincidence rate was respectively 99. 0% and 98. 0%. CONCLUSIONS The recombinant protein of the CPAF immunodominant region from C. pneumoniae showsd excellent antigenicity. It could be efficiently used as a potential antigen to diagnose C. pneumoniae infection.