摘要
目的检测鹦鹉热嗜衣原体（Chlamydophila psittaci，c加）蛋白酶样活性因子（cMamydial protease-likeactivity factor，CPAF）免疫优势区基因在E．coli B121中高效表达的产物在印5感染诊断中的应用，为建立印s感染的快速诊断奠定实验基础。方法利用生物信息学软件筛选出CpsCPAF免疫优势区基因序列（CPAFm，A196～A450），设计特异性引物，PCR扩增目的基因，将其克隆入pGEX6p-2载体后转化E．coliB121，利用IPTG诱导表达重组蛋白，Westernblot鉴定重组蛋白。以纯化的重组蛋白作为包被抗原，建立血清学诊断的间接ELISA法；同时用商品ELISA试剂盒与建立的间接ELISA法检测180份可疑Cps感染的病鸭血清标本，将检测结果进一步用Westernblot方法验证。结果构建原核重组质粒pGEX6p-2／Cps CPAFm，表达相对分子质量约为54×10^3的目的蛋白产物。以纯化的重组蛋白为包被抗原建立间接ELISA法检测Cps参考血清，其阴、阳性符合率均为100％；与肺炎嗜衣原体（C．pneu-moniae，印n）、沙眼衣原体（C．trachomatis，Ct）无交叉反应。对180份可疑病鸭血清标本进行检测，与商品Birds Chlamydia Psittaci IgG ELISA试剂盒比较，以Westernblot方法作对照，自建的ELISA法符合率均为100％，Birds Chlamydia Psittaci IgG ELISA Kit符合率为77．5％～95．0％。结论以Cps CPAF免疫优势区（A196～A450）作为诊断抗原，建立血清学诊断的间接ELISA法具有较好的临床诊断价值。
Objective To clone and express the immunodominant domain of the chlamydial protease- like activity factor（CPAF） from Chlamydophila psittaci（ Cps ） and evaluated the diagnosing value of the recombi- nant protein in Cps infection. Methods The immunodominant region epitope of CPAF （CPAFm, A196-A450） from Cps was chosen according to bioinformatics analysis and references. The specific primer was designed and the gene was amplified by PCR and then ligated into a pGE×6p-2 vector. Recombinant protein was induced to＇ express by IPTG and analyzed by SDS-PAGE and Western blot. Indirect ELISA method of serological diagnosis was established with the reorganization protein as coating antigen. One hundred and eighty sera samples from ducks with respiratory tract infection symptoms were detected with the established indirect ELISA and a commer2 cial ELISA-kit to assess the value of the recombinant protein in serodiagnosis. The results were further identified with Western blot. Results Prokaryotic expression vector pGEX6p-2/CPAFm was constructed and a 54×10^3 fusion protein was attained. The indirect ELISA method was established with reorganization protein for envelope antigen. Using the indirect ELISA to detect Cps IgG positive and negative reference sera, the sensitivity and specificity were both 100% （20/20）. And the recombinant protein has no cross reaction with either Chlarnydophila pneumoniae or Chlamydophila trachomatis. The concordance rate between the indirect ELISA and Western blot to 180 ducks sera samples was 100% , while the concordance rate of the commercial ELISA kit was 77.5%- 95.0%. Conclusion The prepared recombinant protein of the CPAF immunodominant region epitope gene from Cps can highly benefit on developing new indirect ELISA as methods to detect specific anti-Cps antibodies.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2011年第9期781-786,共6页
Chinese Journal of Microbiology and Immunology
基金
传染病防治重大专项课题项目（2008ZXl0004-001）
国家自然科学基金项目（81001318）
关键词
鹦鹉热嗜衣原体
衣原体蛋白酶样活性因子
免疫活性
血清学诊断
Chlamydophila psittaci
Chlamydial protease-like activity factor
Immunocompetence
Serological diagnosis
