[Early diagnosis using recombinant protein of immunodominant region gene of Chlamydial protease-like activity factor from Chlamydophila pneumoniae].
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.


CONCLUSIONS
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.
Zheng
Jianghua
J
Institute of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China. jhzheng2005@21cn.com
Wu
Yimou
Y
Liu
Jiaqiang
J
Liu
Guangchao
G
Chen
Chaoqun
C
D015153:Blotting, Western; D023521:Chlamydophila Infections / Q000175:diagnosis* / Q000276:immunology; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000276:immunology*; D003429:Cross Reactions; D042241:Early Diagnosis; D010450:Endopeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007106:Immune Sera / Q000032:analysis / Q000276:immunology; D016056:Immunodominant Epitopes / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology
D007106:Immune Sera; D016056:Immunodominant Epitopes; D011994:Recombinant Proteins; D010450:Endopeptidases; C425923:CPA factor