Immune Response Induced By Micrococcus Luteus Rpf Domain And Its Mutants In Mice

BACKGROUND:Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis (MTB) is achronic respiratory infectious disease with a long history. TB still remains the secondleading cause of death which brings heavy burden for every country especiallydeveloping countries. According to WHO data and statistics, in2007, there were9.37million new cases of active TB worldwide. And the number of death reaches1.32million(unincluding HIV patients). There were1.30million new cases of active TB in China.The current situation of TB prevention and control is very serious. At present, BCG is thesole vaccine that commonly used to prevent TB. BCG can bring children satisfactoryprotection. But for adults, the pretection is very limited. Furthermore, BCG can not avoidrecurrence of TB. Therefor, a new vaccine that can protect both infant and latent infectedpopulation is urgently needed. AIM:To detect the expression level of specific antibody, IFN-γ and IL-2stimulated by Rpfdand its mutants (Rpfd1and Rpfd2) in mice, and evaluate their immunopotency.METHODS AND RESULTS:1. The purification, identification and concentration measurement of Rpfd, Rpfd1and Rpfd2proteinThe recombinant plasmids containing GST (PGEX-4T1-Rpfd, PGEX-4T1-Rpfd1andPGEX-4T1-Rpfd2) gene was constructed in previous work, and was transformed into E.coli competent cells DH5α. Expression protein was induced and purified. Westernblotting was used to identify the expression protein. The concentration of Rpfd, Rpfd1and Rpfd2protein is1.400,2.438and3.882mg/ml respectively.2. Mice were immuned by the recombinant protein. And humoral immunityresponse was observed.The Rpfd, Rpfd1and Rpfd2protein were injected to mice via tail vein. The level ofspecific antibody was measured in serum. While the level of specific antibody was alsomeasured after injection of BCG. Results revealed that the Rpfd, Rpfd1and Rpfd2protein showed good immunogenicity compared with BCG. The level of antibodyimmuned by recombinant protein was higher than that of BCG and normal saline. Thehighest antibody level was obtained by Rpfd2protein immunity.3. Mice were immuned by the recombinant protein. And cellullar immunologicresponse was observed.The Rpfd, Rpfd1and Rpfd2protein were injected to mice via tail vein. The level ofIFN-γ and IL-2was measured in serum. While the level of IFN-γ and IL-2was alsomeasured after injection of BCG. Results revealed that the level of IFN-γ and IL-2wereincreased in the mice which injected with the Rpfd, Rpfd1and Rpfd2protein.CONCLUSION:The immunity of Rpfd, Rpfd1and Rpfd2protein can promote humoral immunity andenhance cellullar immunologic response in mice, which could be a new immune strategyfor TB preventation and therapy. The immunity of recombinant protein can promote humoral immunity and enhance cellullar immunologic response. The Rpfd, Rpfd1andRpfd2protein were expected to be a good choose for new TB vaccine.