Preparation Of Recombination Protein PIP-Linker-MOMP And Study On The Serodiagnosis Of Legionella Pneumophila

Objective To construct the recombinant plasmid pET-plm with the pip gene and mompgene of Legionella pneumophila，to induce expression of the recombinant plasmid pET-plmand build indirect-ELISA method with the recombination PLM as a diagnostic antigen todetect antibody (IgG, IgM, IgA)during legionella pneumophila infection and evaluate thefeasibility and value in the serological diagnosis.Method The pip gene and momp gene was amplified by PCR from the recombinantplasmid pET-pip and pET-momp,then inserted into the prokaryotic expression vectorpET-32a(+).The recombinant plasmid pET-plm was constructed.The recombinant plasmidwas identified by restriction-endonuclease digestion，PCR and DNA sequencing analysis andtransferred into BL21.The expression of pET-plm was induced with IPTG. The fusion proteinwas analyzed with SDS-PAGE and purified by affinity chromatography. The PLM was usedas coating protein and the optimal concentration of coating antigen was selected by criss-crossserial-dilution analysis.32healthy human and58cases of patients with Legionnaires’ diseasesuspected serum (IgA, IgG, IgM) were detected by the PLM ELISA method. The ROC curveof the paired data was used to compare the test results of four method（DRG ELISA Kit, R&DIgA ELISA Kit, R&D IgG ELISA Kit, R&D IgM ELISA Kit）and to evaluate the feasibilityof the PLM ELISA method for Legionnaires disease immunodiagnostics.Results The pip gene of723bp and momp gene of830bp in length ware amplified andthe recombinant plasmid pET-plm was constructed．The gene sequence of pET-plm has98% homology with the published sequence in the GenBank. Eleven nucleotide sites werechanged，which made an amino acid change.The changed amino acid didn’t affect the proteinspatial structure and function，so these almost didn’t affect the antigen epitopes. The PLMprotein of approximately67KD in size was expressed in E. coli and purified, the proteinconcentration was728mg/L. The purified PLM protein was the coating antigen to develop anindirect ELISA.The Lp antibody IgG,IgM and IgA in blood serum were detected,respectively.Compared with DRG(Germany, IgG/IgM/IgA) Lp kit, the specificity was92.1%and thesensitivity was92.6%, the Kappa value was0.821(P＜0.05), the area of under the ROC curvewas0.923; Then compared with R&D Lp kit, the sensitivity of IgG was91.7%and thespecificity was90.9%, the Kappa value was0.784(P<0.05), the area under the ROC curvewas0.913; the sensitivity of IgM was90.6%and the specificity was93.1%, the Kappa valuewas0.831(P<0.05), the area under the ROC curve was0.919; the sensitivity of IgA was88.9%and the specificity was92.1%, the Kappa value was0.793(P<0.05), the area underthe ROC curve was0.905.Conclusion The pip gene of723bp and momp gene of830bp in length ware successfullycloned and the recombinant plasmid pET-plm was constructed successfully and expressedefficiently．We used the PLM as a diagnostic antigen after purification and compared withDRG’s ELISA kit and R&D’s ELISA kit to evaluate its value in serodiagnosis of Legionnairesdisease, the result shows high specificity, sensitivity and consistency, which laid a goodfoundation for the research of Legionella diagnostic kit.