The Screening Of The Important Surface Proteins Of Mycobacteirum Tuberculosis H37rv In The Infection Stage And The Research Of Relative Immunological Response In Host

To this day, TB remains one of the leading causes of mortality. Additionally, about one thirdof the world population has latent TB and10%of them develop the disease during their lifetime.Owing to the complex pathogenic mechanism of MTB, vaccine development and diagnosticstudies have become a hot topic. Surface proteins consisting of secreted and transmembraneproteins play a central role in MTB pathogenesis, as they are used by the bacteria for nutrientuptake, adherence to host proteins and modulation of the host immune response. Surface proteinsalso allow MTB to target and proliferate inside immune cells. Understanding the interactionbetween surface proteins of MTB and the host cell is therefore likely to help us better understandthe pathogenesis of MTB.In this study, we constructed a MTB surface proteins phage display library with840positiveclones, which can be used for direct selection, identification, expression, purification andfunctional research on surface proteins of MTB. After sequence analysis of a randomly selected200clones, there were140clones with a signal sequence, and47distinct ORFs predicted toencode secretome proteins. When extrapolated to the840clones, there are about188surfaceproteins, which is similar to the number of secreted proteins with signal peptide predicted bysoftware(179),which showed the efficiency of our display system.We also made an innovative iterative screening with the phage display MTB surface proteinlibrary to identify antigens with specific reactivity against antibodies in the sera of TB patients. Asa result, we identified six antigens(MPT64, Ag85B, ESAT-6, PapA2, LpqA and CpsA),and theAg85B, PapA2, LpqA and CspA proteins sharing>99%identity with BCG are still to be identified.These antigens may be expressed differently in virulent stains of MTB and the BCG strains, whichmay contribute to higher specific reactivity with sera from active TB compared to the reactivitywith the serum from BCG vaccinations.The sensitivity of cpsA and PapA2phage display proteins in active TB patients and sputum-negative TB patients after anti-TB treatment ranged from80.1%to13.3%and79.3%to14.0%respectively. Our research revealed the dynamic changes of the antigen-specific serologicalreaction after anti-TB treatment, and provided an effective method for the screening of importantantigens on the earlier infection and incubation stage.The B-cell epitope of these six MTB surface proteins were predicted with the biologicalinformation software, then synthesised17B-cell epitope peptides. The serum reaction of these17epitope peptides between the TB patient serum and BCG immune serum were detected usingELISA method. As a result,6peptides showed a specific reaction to the sera of TB patients. Onthe other hand, the reaction sensitivity of epitope peptide A1, C3and P3to the sputum culturepositive serum were significantly higher than that of sputum culture-negative serum after anti-TBtreatment.The differences of antigen-specific IFN-γ responses of the cpsA, LpqA, PaPA2phage fusionproteins and the epitope peptides A1, C3and P3between active TB patients and sputumsmear-negative TB after anti-TB treatment were determined using flow cytometry and ELISPOT.As a result, after the treatment, the antigen specific IFN-γ response of cpsA and PapA2and theepitope peptide C3and A1decreased greatly. Our research have verified a antigen specificserological and IFN-γ response dynamic changes between the active stage and the infectionconvalescent stage, the cpsA and PaPA2should be the virulent factors of the MTB exhibits greatersecretory activity in the active stage, which would have great impact on diagnosis, monitoring oftreatment and vaccination strategies.New subunit vaccines of MTB were obtained by combining Ag85B＋ESAT-6with cpsA andPapA2phage display protein, the antigen-specific multifunctional T cell proliferation in thedifferent vaccination groups were analysised using multicolor flow cytometry, then made ajudgment on the valuable CD4+T cell marker for vaccine-take in TB vaccine trials. Aftervaccination, the combination vaccination groups induced qualitatively and quantitatively differenthumoral and cellular immune responses compared to and single component. The cpsA and PapA2phage display proteins can stimulate highly expression of IFN-γ, interleukin-12(IL-2), and tumornecrosis factor-α (TNF-α)three cytokines, IL-2and TNF-α two cytokines, and TNF-α CD4+Tcell populations, which proved that these two phage display proteins can stimulate a memoryimmune response.In this study, we successfully constructed a MTB surface proteins phage display library, which can be used for direct selection, identification, expression, purification and functionalresearch on surface proteins of MTB, and made a new iterative screening with this library toidentify antigens with specific reactivity against antibodies in the sera of TB patients. As a resultwe identified six antigens, the B-cell epitope of these six MTB surface proteins were predictedwith the biological information software, and synthesised17B-cell epitope peptides. Thedifferences of antigen-specific serological reaction and IFN-γ responses of these six phage fuseproteins and the B cell epitope peptides between active TB patients and sputum smear-negativeTB after anti-TB treatment were determined using flow cytometry and ELISPOT, hoping to revealthe change trend of the antigen-specific serological and IFN-γ reaction after anti-TB treatment,which provided a platform for the screening of the virulent factor in the TB active stage, andprovide theoretical basis for the selecting of TB vaccine relative antigens, At last, we constructedsubunit vaccine by combining Ag85B＋ESAT-6with cpsA and PapA2phage display proteins.Then a comprehensive analysis of the immune response from each combination group were madefrom the humoral immune response, cytokine expression levels, the number of multifunctionalcytokine expression and the proportion of multiple T cell subsets, which helped to make asystematic interpretation of the useful anti-TB host T cell subsets and also helped to provide aneffective platform and evaluation standards for the TB vaccine development.