Expression And Purification Of Protein P116-C,P40 And Research For Serological Application Of Various Recombinant Proteins Of Mycoplasma Pneumonia

Mycoplasma pneumonia(M.pneumoniae)is a common cause of respiratory tract infection pathogens spread by droplets.In addition to causing respiratory infections in children and adults,many studies have shown that M.pneumoniae is also associated with many other extrapulmonary diseases such as coronary artery disease and respiratory diseases in HIV patients.M.pneumoniae belongs to the soft membrane body of the prokaryotes.Commonly used antibiotics such as penicillins,aminoglycosides for bacterial pneumonia treatment targeting on cell wall are invalid for M.pneumoniae due to its lack of cell wall.Similar to respiratory infections caused by other pathogens,the symtom of M.pneumoniae infection is lack of specificity.In addition,unsuccessful treatment for M.pneumoniae infections is very likely to cause recurrent respiratory tract infections,thus,early diagnosis is particularly important to avoid the clinical medication improper and delay of the disease or producing resistant bacteria.At present,the methods of detecting M.pneumoniae infections are as follows: cell culture,PCR gene fragment amplification,serological antibody detection,and etc.The serological antibody detection is the most commonly used method.Most of the existing serological detection diagnostic kit mainly considers M.pneumoniae whole cell or cell membrane as antigen.Because of the presence of non-specific cross-reaction between the mycoplasma pneumoniae cell membrane glycolipid antigen and other micro-organisms or human tissue,the specificity of existing serological detection methods is low.Therefore,clonal expression and purification of M.pneumoniae surface adhesion protein,screening a variety of recombinant proteins,and finding candidate proteins for the development of M.pneumonia diagnostic kit of high sensitivity and specificity is the focus of this experiment.Objective: This aim of this study was to obtain therecombinant M.pneumoniae adhesion P116-C protein and P40 proteins,establish ELISA detection method which used the above proteins and the other proteins of Mp as the antigen,and to establish ROC curves and demonstrate the results of Se,Sp,PPV,NPV,and AC values according to the optimal CUTOFF values on the ROC curves.Methods: P116-C gene and P40 gene were amplified by PCR.The fragments were cloned into p ET-28a(+)expression vector to construct p ET-28a(+)/ P116-C and p ET-28a(+)/ P40 recombinant plasmids;after properly transfered into BL21(DE3)engineered bacteria,P116-C and P40 proteins were obtained under IPTG induction.The production was analyzed by SDS-PAGE electrophoresis and mass spectrometry,then purified by passage of the cell lysate through a Ni-NTA column;established ELISA method which used the recombinant P1,P116-C,P40,CARDS proteins as the antigens,and detected 166 serum samples of M.pneumoniae,then compared the results with those frommarket commercialization of ELISA test kits.ROC curves were established and the results of SE,SP,PPV,NPV,and AC values according to the optimal CUTOFF values on the ROC curves were demonstrated.Results:? The fragments of P116-C protein and P40 protein genes were successfully amplified by PCR and then expressed in E.coli BL21 using p ET-28 vectors.The concentrations of P116-C and P40 were 0.3mg/ml and 1.1mg/ml respectively after purification with Ni-NTA.? The results of 166 serum samples of recombinant P116-C,P40,P1,and CARDS proteins using ELISA method are as follow: AUC values were 0.866,0.888,0.889,0.559,AC values were 0.825,0.813,0.861,0.825,Se values were 0.838,0.932,0.814,0.554,Sp values were 0.793,0.750,0.928,0.685,PPV values were 0.765,0.742,0.940,0.586,NPV values were 0.859,0.932,0.780,0.656.Conclusions: This study exploredthe possibility of M.pneumoniae P40 and P116-C proteins,along with P1 and CARDS proteins to be thecandidate antigens for serodiagnosis.The accuracy and the specificity of serodiagnosis using P1 protein as antigen was high.However,the sensitivity of serodiagnosis using P1 protein was insufficiently low.The accuracy and the sensitivity of serodiagnosis using P40 protein as antigen was high,while the specificity was insufficiently low.The accuracy of serodiagnosis using P116-C protein as antigen was high.Its specificity was lower than that of serodiagnosis using P1 protein,and its sensitivity was lower than that of using P40 protein.In general,P116-C protein performed more balanced in sensitivity,specificity,and accuracy.Furthermore,the AUC value on ROC curve for the serodiagnosis using P116-C protein as antigen was higher than other candidates.Thus,P116-C protein can be used alone as candidate antigen of M.pneumoniae serodiagnosis.