Study On Epitopes Polymorphisms In Eight Important Antigenic Protein Genes Of Mycobacterium Tuberculosis

Tuberculosis (TB) remains a major public health problem threating to the health of the people worldwide. World Health Organization (WHO) estimated that almost8.6million people developed active TB and1.3million people died of TB in2012, India and China alone accounted for26%and12%of total cases, respectively. The emerging of co-infection with both Mycobacterium tuberculosis (M. tuberculosis) and the human immunodeficiency virus (HIV), and the drug resistance TB bave made more difficult for TB control and treatement. Current efforts to reduce this global problem are focus on improving diagnosis and developing effective vaccines. Biochemical, immunological, and molecular biological characterization of M. tuberculosis has led to the identification of several antigens that may be useful in the development of improved diagnostic methods or vaccines.Recombination BCG, protein subunit vaccine, DNA vaccine, and attenuated live Mycobacterium vaccine gained extensive attention in many recently posted reseach reports, and as the recombination BCG is more safety, cost effective, easy to inoculate and could stay alive in the vaccinated host which may induce long term immune protection, make it more suitable for developing a new TB vaccine. But, some researchers have reported that M. tuberculosis was evolutionarily conserved as expected, and in most of the experimentally confirmed human T cell epitopes there even showed little sequence variation and had a lower ratio of nonsynonymous to synonymous changes than seen in essential and nonessential genes, purifying selection may acting on these epitopes, implying that MTBC isolates might benefit from recognition by human T cells. This gives some challenges to the past theory for developing new TB vaccines. The aims of this study were to investigate the mutation profiles of the selected eight important M. tuberculosis antigenic protein’s coding genes, and the status of the human T and B cell epitopes in them.180clinical MTBC isolates from China and11different originality BCG stains were selected, from which the8important antigenic proteins’(MPT64, pstSl, Ag85A/B/C, ESAT6, CFP10and14kDa) genes were amplified and sequenced respectively, and then compared the sequences together with the gene sequences downloaded from NCBI website, to acquire the mutation status of each gene. The epitope sequences in M. tuberculosis were obtained from the Immune Epitope Database (IEDB), and the distributions of them in the reference strain M. tuberculosis H37Rv genome were determined by using the BLAST method. Finally, the mutations in the target genes and the human T/B cell epitope regions were obtained by using personalized Perl scripts and ClustalW software, the dN and dS calculated by using Mega5software were used for evaluating the environment pressure.Took all of the coding sequences acquired in this study into analysis, and found that except the CFP10gene, mutations were found in all other genes, some mutations were found commonly in some strains of the target genes. The main results were:(1)8of the180clinical isolates harbored a63bp deletion in MPT64gene;(2) All of the Mycobacterium bovis (M. bovis) and BCG strains contained a nonsynonymous SNP (T1055C, Va1352Ala) in pstSl;(3) In Ag85C gene,93of the180clinical isolates showed a synonymous SNP (C714A, Pro238Pro), and6strains had a SNP (G472A, Gly158Ser). Besides, some unique mutations were also found in some strains of the studied samples, which contained synonymous and nonsynonymous SNPs, mono base insertion and deletion. The dN/dS of MPT64and pstSl genes, and that of the human T/B cell epitopes areas were greater than1, but that of the non-epitope regions of the two genes were little than1. In the Ag85C gene, the dN/dS of the whole gene coding sequences (CDS) and non-epitope regions were general than1while the human T cell epitope regions were little than1. The dN/dS for the rest genes were all little than1, and no difference was found between the human T/B cell epitopes areas and the non-epitope regions.Some antigenic proteins, like MPT64and pstSl, in M. tuberculosis genome showed highly variability while other may highly conserved. The polymorphisms of the genes and the human T/B cell epitope regions may indicate that immune evasion of M. tuberculosis was also actually exists, while the conserved proteins, like14kDa and Ag85A/B, may serve as the candidate proteins for developing recombinant BCG.