Cloning And Expression Of Orf6 Gene From Mycoplasma Pneumonia And Its Preliminary Application In Serological Diagnosis

Mycoplasma pneumoniae is an important etiologic agent of tracheobronchitis and primary atypical pneumonia. It is responsible for 10%-30% of community acquired pneumonias overall. Because the organism grows slowly and is difficult to isolate from clinical specimens, culture is rarely undertaken in a routine context. In addition , Because M. pneumoniae lacks a cell wall, it does not respond to penicillins and other beta-lactams commonly used for the treatment of bacterial pneumonia.A reliable and sensitive serologic test for use in the early stages of M. pneumoniae infection is needed to confirm the clinical diagnosis and to ensure that the appropriate antibiotic therapy is used.P90 is a highly immunogenic protein in diagnosis and vaccine.In our research ,ORF6 gene was amplified by PCR from M. pneumoniae FH and a DNA fragment about 1003bp in length was obtained. The product was cloned into pMD18-T vector by TA clone. The recombinant plasmid was amplified by PCR again to get more ORF6 gene. The PCR product was inserted into prokaryotic expression vector pET32a.The recombinant plasmid that had correct ORF was introduced into competent cells of E.coli BL21(DE3) and then induced by IPTG, the transformant express the recombinant protein. The recombinant protein was analyzed by SDS-PAGE and Western-blotting. A specific protein band of relative molecule weigh to 56.8kDa was found by SDS-PAGE. The recombinant protein was verified to react with antiserum of M. pneumoniae . With a good immunogenicity, the recombinant protein can be used as an antigen in ELISA to detect M. pneumoniae antibodies. The recombinant protein was purified by electroelution. Then the purified recombinant protein as an antigen was applied in indirect ELISA . Coat the polystyrene 96 well plate with the optimal concentration of the antigen which was defined through the way of block titration. An indirect ELISA was established to detect IgM in M. pneumoniae infection. The results were compared with two imported commercialization kits to check the specificity and sensitivity of diagnosis method initially. The results suggested that the diagnosis method was specific and sensitive. There was an application prospect for the M. pneumoniae ELISA kit.