Development And Identification Of Monoclonal Antibodies Against TB10.4Protein Of Mycobacterium Tuberculosis M.S Candidate:Zhen Ou

Human have being made a great effort to undstand of pathogenesis and interaction between host and pathgoen of tuberculosis (TB), trying to find new strategy to solve the problem on preventing and controlling of TB. It is thought that the early secreted culture filter proteins (SCFP) play an essential role in early infection event which stimulate innate immune and influence acquired immune responses. The intensive study of SCFP will have insight into interaction between pathogen and host in the early infection event, and it is helpful to treat, diagnose, design vaccine against TB. ESX family is a group of small proteins in SCFP which is always attracting much interest due to being the strong T cell immunogen in TB patients. Some members of ESX family have been applied in diagnosis or testing in clinic as candidate vaccine. It is clear that they play a significant role in pathogenesis and are key virulence factors, but limited knowledge about their biological function in pathogensis. It also implies that they have universal biological meaning since they have similar genetic character and largely distribute in G+bacteria. Recently, they became interesting scientific topic again because some members secreted through special passway that represent of new discovered typical Ⅶ secretion mechanism (T7S). TB10.4protein is a member of ESX family, and it is necessary for Mycobacterium tuberculosis survival and confers vital role on pathgensis. TB10.4is also a core secreted substrate of little knowledgeable T7S/ESX-3system.In this study, the prokaryotic expression systems were used to express different TB10.4fusion proteins as immunogen and detective antigen for the development of monoclonal antibodies against TB10.4protein. Following the typical procedure of murine B lymphocyte hybridoma technique, eight of stable TB10.4hybridomas cell lines have been established, and secreted by four of these hybrids can recognize the expressed TB10.4protein from recombinant adenovirus. These McAbs were expected to supply vaulable tool for studying TB10.4biological function in early infection event and the mechanism of ESX-3system secretion.1. Prokaryotic expression and identification of TB10.4protein of Mycobacterium tuberculosisThe prokaryotic recombinant expression plasmids pET-30(a)+-esxH and pGEX-6p-l-esxH with different fusion tag were constructed respectively and further identified by sequencing analysis and restriction enzyme digestion. The positive recombinant plasmids were extracted, and then transformed into host bacteria BL(DE3). After screening on selective medium plates containing antibiotics, the recombinant bacteria were obtained, and named as BL21(DE3)(pET-30(a)+-esxH) and BL21(DE3)(pGEX-6p-1-esxH), and further identified by SDS-PAGE for the expression of TB10.4fusion proteins from these two recombinant bacteria strains induced by IPTG in small scale. In order to prepare the large amount of expressed proteins, their inductive expression conditions were optimized, and the expressed HIS-TB10.4and GST-TB10.4proteins were purified through the affinity chromatography column, respectively. These two fusion proteins could be used as immunogen and dectective antigen for the development of monoclonal antibodies against TB10.4.2. Development and identification of monoclonal antibodies against TB10.4proteinIn order to develop monoclonal antibodies specific for TB10.4protein, an indirect ELISA was firstly established using murine antisera prepared after HIS-TB10.4protein immunization. Secondly, BALB/c mice were immunized subcutaneously with100μg of HIS-TB10.4emulsified in the Complete Freund’s adjuvant, and boosted through intrasplenic immunization with20μg of HIS-TB10.4. at21days post first injection, Using the B lymphocyte hybridoma technique,8of hybrid cell lines, secreting specific McAbs against TB10.4protein, were obtained, and named as B1, C5, F5, C12, G2, E11and B3, respectiviely. All of these McAbs reacted with TB10.4expressed in recombinat bacteria in Western-blot assay, and4of them could recognize the expressed TB10.4from Ad293cells infected with recombinant adenovirus rAd-Ag85B-TB10.4in indirect immunofluorescent assay. The ELISA titers of their ascites were up to1:10-5～1:10-6, and the isotype of them are IgG1. These McAbs would be very useful in the preparation of native TB10.4protein, determination of the molecule events during the early stage of infection, and definition of the construction and function of the ESX-3system.