Screening And Analysis Of In Vivo Induced Genes Of Mycobacterium Tuberculosis

Mycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, infects one-third of the world's population and kills 3 million people each year. Tuberculosis is the largest cause of death caused by a single infectious organism. In recent years, the rapid development of multidrug resistant strains and the dubious protective efficacy of BCG made the two important strategies to control tuberculosis become ineffective. So it is very imminent to find new drugs and vaccines to deal with the crisis.After M.tb invades the human body, a series of bacterial genes will start up and express to adapt the internal environment. These genes may be the key ones to escape the immune attack, enter and multiply in the phagocyte, and cause the disease. In vivo induced antigen technology (IVIAT) provides a simple method for identifing genes specifically expressed in vivo, not depending on the animal model of infection. These genes are possible molecular targets of the immunological and therapeutic research.Genomic DNA from M.tb strain H37Rv was extracted. The DNA was partially digested with Sau3A I and the purified fragments were inserted into the pET30a(+), pET30b(+) and pET30c(+) expression vectors to construct the genomic library. The library was induced with IPTG and then was screened with pooled tuberculosis patient sera preabsorbed with in vitro grown M.tb strain H37Rv and Escherichia coli BL21(DE3). The inserts of positive clones were sequenced with primer T7 promoter. The sequences were aligned in the genomic database of M.tb strain H37Rv (http://genolist.pasteur.fr/Tuberculist) to identify the open reading frames. PPE15 gene knockout vector was constructed and electrotransformed into strain H37Rv. Transformants were selected with kanamycin and sucrose. The PPE15 mutagenesis strains were preliminarily identified using PCR.We constructed the genomic expression library of strain H37Rv. The library included 4.3×104 clones, and more than eighty percent were recombinated plasmids. The library had reached the theoretic requirement.Fifty-one open reading frames were obtained in this study including one virulence gene, thirteen cell wall and cell processes genes, eleven intermediary metabolism and respiration genes, seven lipid metabolism genes, two information pathways genes, three PE/PPE genes, twelve conserved hypotheticals and two conserved hypotheticals with an orthologue in M. bovis.We successfully constructed PPE15 gene knockout vector(pJQ200-XylE∷ΔPPE15∷Km)and electrotransformed it into strain H37Rv. Yet we haven't obtained PPE15 mutagenesis strains, and this work is underway.In conclusion, this study identified some in vivo induced genes of M.tb by IVIAT approach and did some initial work about the gene function. We marched one step on the road of exploring the key genes which help M.tb survive in the body and result in tuberculosis. And the study also provided some new molecular targets data of prophylaxis and therapy of tuberculosis.