Establishment Of A Nested PCR Assay For Detection Of Mycoplasma Gallisepticum And Procaryotic Expression Of Mgc2 Gene

Infectious of mycoplasma gallisepticum was one of the chronic respiratory diseases which often complicate Newcastle disease andother infectious bronchitis resulting in huge difficulty on clinical diagnosis.To explore the response of MG adhesion proteins and establish a specific,highly sensitive method for the diagnosis of MG infection,in this study,the pathogen was isolated and identified and the diagnosis method of PCR was established,and the adhesion proteinsof mgc2 was cloned and expressed.Methods: The lung,airbags,tracheal secretions of chicken with respiratory disease were collected,and the pathogens was isolated and cultured.The preliminary identification including was made by L type bacteria identification,colony morphology,dyeing microscope examination and erythrocyte adsorption,and the MG pathogen was identified by PCR amplification and sequencing.A nested PCR detection method was established by screening the highest sensitivity primers which compare the senstivty with the 5 common pairs of primers including gapA-F/ gapA-R,16SrRNA-F/16 S rRNA-R,mgc2-F1/mgc2-R1,mgc2-F2/mgc2-R2,LP-F/LP-R,and the specificity and sensitivity were detected.The mgc2 was cloned and expressed in prokaryotic expression,and the Western blot was used to analyze whether the adhesion protein was reactive or not.Results:(1)Isolation and identification of MG: 36 strains MG were isolated,3strains of MG was isolated from single MG infection case which come from broiler farms during 3~20 days.33 strains of MG was isolated from MG mixed infection case which come from the broiler farms during 21~40 days.(2)Detection result of mycoplasma gallisepticum nested PCR: The sensitivity of mgc2 gene was the highest by comparing the sensitivity of 5 pairs of primers to MG.Its detection limits was 57.6 pg/?L,and the longth of the gene which could amplificate was 300 bp.The homologous sequence of correlation with GenBank was98%,and there was no cross reaction with Escherichia coli,Salmonella,Mycoplasma synoviae.The minimum amount of DNA which could detecte was 0.18 fg/?L.The positive rate was 80% by the detecting 50 dead chicken in some areas of Anhui and Shandong Province.(3)Mycoplasma gallisepticum adhesion protein mgc2 prokaryotic expression results: a condon which encoding Trp was mutagenized from TGA to TGG.The genewas inserted into expression vector pET-30a(+),constructed the recombinant plasmid pET/mgc2.When it was successful identified,the fusion protein with 39.6kDa was induced and expressed in E.coli BL-21(DE3)with IPTG.Western-blot analysis proved that the fusion protein had immunogenicity.Conclusion:(1)The method of nested PCR detection has strong specificity and high sensitivity.(2)The expression of adhesion protein mgc2 which had good antigencity was successfully expressed.