肺炎嗜衣原体蛋白酶样活性因子免疫优势区基因重组蛋白在早期诊断中的应用

Early diagnosis using recombinant protein of immunodominant region gene of chlamydial protease-like activity factor from Chlamydophila pneumoniae

【目的】克隆和表达肺炎嗜衣原体(Chlamydophila pneumoniae,Cpn)蛋白酶样活性因子(CPAF)免疫优势区基因,评价重组蛋白在早期感染诊断中的应用价值。【方法】挑选并克隆出Cpn CPAF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,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抗体,以及呼吸道感染患者痰咽拭子中的Cpn抗原;检测沙眼衣原体(Chlamydia trachomatis,Ct)临床阳性血清和泌尿生殖道分泌物。【结果】高效表达和纯化出一相对分子量约51.3kDa的重组蛋白;Westernblot证明其只与人抗Cpn抗血清发生特异性反应;间接ELISA法检测40份CpnIgM参考血清,阴性和阳性结果的一致率均为100%(40/40);与"金标准"方法MIF对照,检测300例临床血清标本中的IgM抗体,符合率为98.3%;与PCR试剂对照,检测120份呼吸道感染患者痰咽拭子中的Cpn抗原,符合率为88.3%;检测Ct阳性血清和泌尿生殖道分泌物,与Ct没有交叉反应。【结论】制备的CPAF免疫优势区基因重组蛋白具有良好的抗原性,在Cpn感染早期诊断中具有较高的利用价值。

[Objective] To clone and express the immunodominant domain gene of the chlamydial protease-like activity factor（CPAF） from Chlamydophila pneumoniae.The value of the recombinant protein was evaluated for diagnosing early infection. [Methods] According to bioinformatics analyses and references, we chose the immunodominant region epitope of chlamydial protease-like activity factor（CPAF） from Chlamydophila pneumoniae, then amplified the genes of the epitope by PCR, and then ligated the genes into a pGEX6p-2 vector. We purified the expressed recombinant protein with glutathione S-transferase （GST） agarose gel FF, then identified it by SDS-PAGE. By immuning New Zealand rabbits evaluated the immunogenicity of the recombinant protein, and analyzed its antigenicity with Western blot. The specific IgM antibodies in 300 clinical sera samples and C. pneumoniae reference sera, the antigen of C. pneumoniae in 120 sputum and throat swabs were detected with the developed indirect ELISA. At last, we investigated the cross-reactivity against Chlamydia trachomatis with the developed ELISA method to detect anti-C, trachomatis positive antisera and secretions in genitourinary tract. [Results] We attained successfully a 51.3kDa recombinant protein. Western blot assay proved the recombinant protein could only specifically react with human anti-C, pneumoniae antisera. The titer of the specific IgM antibodies in the immuned New Zealand rabbits was above 1：8000. The developed ELISA detected anti-C. pneumoniae IgM positive and negative reference sera, the sensitivity and specificity were both 100% （40/40）.The concordance rate between the indirect ELISA and the MIF to 300 clinical sera samples was 98.3%. The concordance rate of antigen detection between the new ELISA and the PCR reagent to 120 sputum and throat swabs was 88.3%. When detecting anti-C.trachomatis positive antisera and secretions in genitourinary tract with the developed ELISA, we didn＇t found any cross reaction against C.trachomatis. [Conclusion] The prepared recombinant protein of the CPAF immunodominant region epitope gene from C. pneumoniae shows excellent antigenicity and can highly benefit on developing new indirect ELISA as methods to detect IgM antibodies and antigen of C. pneumoniae for diagnosing early infection.