Expression And Purification Of Mycobacterium Tuberculosis Antigenic Proteins Rv1738, TB31.7, RPFE And Their Immunologic Characteristics Analysis

Tuberculosis (TB) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). It has been one of the most important contributory and mortiferous factors in the world. It is estimated by WHO that one third population was infected by MTB.The number of TB patients in China is the second in the world, about400million people infected, and10%of them will develop to active TB. The majority of infected individuals exist in condition of dormant infection. M.bovis Bacille Calmette and Guerin (BCG), the only one vaccine available for TB has several disadvantages, such as ineffective to subclinical infection, short period of protect and low immune response. In order to reduce the immense burden of tuberculosis, new vaccines or vaccination strategies, or both, are urgently needed.Foreign researchers have predicted the candidate proteins with high antigenicity by using the bioinformatics methods. In this study, Rv1738、TB31.7and RPFE of antigenic proteins from MTB were expressed through the prokaryotic expression system, and their immunologic characteristics were analyzed by mice vaccination.1Expression, purification and identification of Rv1738、TB31.7and RPFE antigenic proteins from Mycobacterium tuberculosisThe coding gene of Rv1738、TB31.7and RPFE were amplified from H37Rv genome DNA by PCR, and then cloned into the prokaryotic expression vector pET30a or pET32a. The recombination expression plasmid were transformed into E. coli BL21(DE3), respectively. The positive clones were screened and induced by IPTG, the expressed fusion proteins were purified by affinity chromatography assays as well as identified by Western blotting assay. SDS-PAGE analysis demonstrated that all fusion proteins were successfully expressed after IPTG induced, Rvl738and RPFE were expressed mainly in soluble form, but TB31.7in inclusion bodies, It was adopted that the method of slow refolding inclusion body proteins into a soluble form. Their results of western blotting assay indicated that the purified proteins could well react with anti-H37Rv polyclonal antibodies and proved their immunoreactivity.2Immunological properties of Rv1738、TB31.7and RPFE fusion proteinsThe6-week-old C57BL/6mice were immunized subcutaneously with the fusion protein Rv1738, TB31.7or RPFE, emulsified in equal volume of DDA adjuvant, and sacrificed two weeks post third immunization. The serum samples and lymphocytes were collected for the assessment of humoral and cellular immune responses in sandwich ELISA assays. The significant higher levels of specific antibodies elicited by the fusion protein were detected. Meanwhile, it was shown that the higher levels of IFN-y and IL-2were elicited by three fusion proteins, respectively. The level of IFN-y was significantly higher than that of IL-2. Three fusion proteins could not stimulate high level of IL-4. All the results suggested that these three fusion proteins had good immunogenicity, could induce Thl-bias immune responses, and had potential use as target antigens for vaccine development of TB.