Preparation and analysis of immunocompetence of recombinant fusion protein of the immunodominant region in chlamydial protease-like activity factor from Chlamydophila pneumoniae and its application in serodiagnosis  

作　　者：JIANG HUA ZHENG YI MOU WU TAO DING LI LICHEN JIA QIANG LIU SHUANG QUAN LIU 

机构地区：[1]Institute of Pathogenic Biology, Medical College [2]the first affiliated hospital, University of South China, Hengyang, P. R. China

出　　处：《Journal of Microbiology and Immunology》2007年第2期107-115,共9页中华微生物学和免疫学（英文版）

摘　　要：To clone the gene coding the immunodominant region in the chlamydial protease-like activity factor （CPAF） from Chlaroydophila pneumoniae, to analyze immunoeoropetenee of the expressed protein, and to evaluate its value in serodiagnosis, the CPAF immunodominant region gene was amplified, ligated into a pGEX6p-2 vector, and then the expressed recombinant protein was purified with glutathione Stransferase （GST） agarose gel FF after renaturation, then identified by SDS-PAGE and Western blot. A new indirect ELISA was developed with the purified protein as coating antigen. The immunogenicity of the recombinant protein was evaluated by immunization to New Zealand rabbits, and its immunoreactivity was analyzed by reacting with anti-C, pneumoniae antibody. 300 clinical sera samples were respectively detected by mieroimmunofluorescenee （MIF） as reference method and the indirect ELISA, and the difference between the two methods was analyzed. Cross-reactivity against Chlamydia trachomatis was investigated with the indirect ELISA to detect anti-C, trachomatis positive antisera. The results indicated that a 51.3 kDa recombinant protein was obtained. Western blot assay proved that the recombinant protein could merely specifically react with human anti- C. pneurnoniae antisera. The titers of the specific IgG antibodies in the immunized New Zealand rabbits were above 1 ： 16 000. Anti- C. pneumoniae IgG positive and negative reference sera were detected with the indirect ELISA, and the concordance rate of negative and positive results were both 100% （40/40）. The sensitivity and specificity of the indirect ELISA in comparison with MIF were 93.8% （45/48） and 100% （252/252） separately by detecting 300 clinical sera samples, and the concordance rate between the two methods was 99.0%. No cross reaction against C. trachomatis was found with the indirect ELISA to detect anti-C, trachomatis positive antisera. In conclusion, the prepared recombinant protein of the CPAF immunodominant region shows excellent immunocTo clone the gene coding the immunodominant region in the chlamydial protease-like activity factor(CPAF)from Chlamydophila pneumoniae,to analyze immunocompetence of the expressed protein, and to evaluate its value in serodiagnosis,the CPAF immunodominant region gene was amplified,ligated into a pGEX6p-2 vector,and then the expressed recombinant protein was purified with glutathione S- transferase(GST)agarose gel FF after renaturation,then identified by SDS-PAGE and Western blot.A new indirect ELISA was developed with the purified protein as coating antigen.The immunogenicity of the recombinant protein was evaluated by immunization to New Zealand rabbits,and its immunoreactivity was analyzed by reacting with anti-C,pneumoniae antibody.300 clinical sera samples were respectively de- tected by microimmunofluorescence(MIF)as reference method and the indirect ELISA,and the differ- ence between the two methods was analyzed.Cross-reactivity against Chlamydia trachomatis was investi- gated with the indirect ELISA to detect anti-C,trachomatis positive antisera.The results indicated that a 51.3 kDa recombinant protein was obtained.Western blot assay proved that the recombinant protein could merely specifically react with human anti-C.pneumoniae antisera.The titers of the specific IgG an- tibodies in the immunized New Zealand rabbits were above 1:16 000.Anti-C.pneumoniae IgG positive and negative reference sere were detected with the indirect ELISA,and the concordance rate of negative and positive results were both 100%(40/40).The sensitivity and specificity of the indirect ELISA in comparison with MIF were 93.8%(45/48)and 100%(252/252)separately by detecting 300 clinical sera samples,and the concordance rate between the two methods was 99.0%.No cross reaction against C.trachomatis was found with the indirect ELISA to detect anti-C,trachomatis positive antisera.In con- clusion,the prepared recombinant protein of the CPAF immunodominant region shows excellent immuno- competence and can be used to develop a new indirect ELISA

关 键 词：Chlamydophila pneumoniae Chlamydial protease-like activity factor Recombinant protein Immunocompetence ELISA 

分 类 号：Q51[生物学—生物化学]