A Prelimary Study Of Immunogenicity Analysis Of The PPE Proteinfamily From Mycobacterium Tuberculosis

TB (Tuberculosis) is chronic disease caused by Mycobacterium tuberculosiswhich also makes animal disease, which is still the main threat to human healthdiseases today. The traditional method of diagnosis Tuberculosis is susceptible tomany factors interfere and produce a higher false positive diagnosis results or falsenegative diagnosis results, is not conducive to the early diagnosis and treatment oftuberculosis. There is a need to develop newer and more effcient diagnostic tests thatcan identify most of the TB cases with better specifcity and sensitivity. Althoughmany of M. tuberculosis antigens have been identified, but most of them are notsuitable to distinguish between real tuberculosis patients and healthy people whovaccinated Bacillus Calmette Guerin (BCG) Mycobacterium tuberculosis genome,encoding about10%for PE and PPE protein family, and the family of proteins areunique to Mycobacterium tuberculosis. Although some members on the PPE proteinshave been successfully identified and applied, but a more systematic analysis ofprotein immunogenicity of PPE reports is still rare.In this study, Ten of PPE protein family members were selected as the object ofstudy with phylogenetic analysis. PPE protein coding genes were amplificated withPCR,then the PCR product was cloned in pET32a or pET41a vectors to construct therecombinant plasmid in the expression vector, then transformed into E. coli BL21(DE3) expression strain and screening of genetic engineering expression strain. PPErecombinant fusion proteins were chelated nickel ion metal affinity chromatography,purified recombinant fusion protein as a target antigen PPE were coated96-wellmicrotiter plates, and a standard ELISA-based serological tests method wasestablished for early diagnosis of tuberculosis.60patients diagnosed with TB and30healthy people were used for ELISA detection of serum.We used SPSS17.0softwareto make ROC curve calculated sensitivity and specificity of ELISA, and further assessthe PPE antigen as the value of early serological diagnosis.The results show that we successfully cloned and expressed all10selected PPEantigens in E. coli.but PPE68did not purified successfully. Then, The purifiedproteins were used as coating antigen PPE and ELISA serological diagnostic methodswere established, The results was that the sensitivity of all of the PPE proteins as theELISA detection antigen were higher than Ag85A. The area under the ROC curveof Ag85A was only0.573, and all PPE proteins were greater than the area under theROC curve of0.679, indicating the accuracy of the application of PPE protein asantigen serological diagnostic was higher. This is because Ag85A not only exist inMycobacterium tuberculosis, also is present in BCG and other mycobacteria, can leadto cross reactions, and thus specificity is not high. In the PPE antigen in the selectionof the five categories, the first class of PPE protein (PPE68) failed purification;second PPE protein (PPE37) detected the highest specificity (99.3%), second only toMycobacterium tuberculosis-specific antigens ESAT-6, but its detection sensitivity (65%) and accuracy (ROC area under the curve0.738)was higher than ESAT-6, andthere are no reports at home and abroad, suggesting that as a new candidate antigensused in TB Serological diagnosis of early studies; third category PPE protein (PPE36,PPE41, PPE57, PPE58, PPE59, PPE69) detection sensitivity (65%~80%) andaccuracy (ROC area under the curve0.761~0.805) are higher then PPE37, butslightly lower specificity (80%~93.3%), and the results reported in the literaturegenerally consistent; fourth category PPE protein (PPE17) the detection sensitivity(63.3%), specificity (83.3%) and accuracy (ROC area under the curve0.679) are nothigh; fifth PPE protein (PPE64) the detection sensitivity (63.3%), specificity (90%)and accuracy (ROC area under the curve0.757) and PPE37considerable, suggestingthat it also can serve as a new serological diagnostic antigen candidate.In conclusion, this study successfully cloned, expressed and purified nine PPEantigens, and it was preliminarily verified a good PPE antigen immunogenicity byELISA method, which can be used as serological diagnosis of tuberculosis candidateantigens. In addition, the sensitivity, specificity, and accuracy of the second PPEantigen PPE37and fifth PPE antigen PPE64should be better than ESAT-6, and relatedresearch have not been reported at home and abroad, suggesting that these two PPEprotein can be used as a candidate antigen in serological diagnosis. Next, we willfurther apply these purified PPE antigen to stimulate peripheral blood mononuclearcells of TB and analyze the cause of the cellular immune response to PPEantigens,integrated evaluate of its immunogenicity and significance of vaccineresearch or diagnosis of TB.