| Tuberculosis(TB)is one of the most serious infectious diseases worldwide.According to the annual statistic report of the World Health Organization(WHO),in 2016,there were at least 1040 new TB cases and 1.67 million people die caused by TB worldwide,by which human health and socio-economic development had been affected seriously.With the increase of the floating population,the increase of the co-infection of human immunodeficiency virus and Mycobacterium tuberculosis(M.tuberculosis),the occurrence and epidemic of multidrug-resistant tuberculosis,the control of the epidemic of tuberculosis is still facing a severe test at present.Bacillus Calmette-Guérin(BCG)is currently the only vaccine to be used to prevent tuberculosis in the world.All the time the immune protection effectiveness of BCG has always been controversial.Therefore,in order to achieve the goal of tuberculosis prevention and control,It is imminent to develop a new and highly effective anti-tuberculosis vaccine.Relevant literatures reported that the reconstituted BCG had been considered to be great potential research and development because of the advantages of safety,high efficiency,wide application,long-lasting immunity,low cost,high thermal stability,easy operation,for which the key technology is to find specific and efficient antigen in BCG lacking or low expression.In this study,we studied the polymorphisms of the gene sequences including their human T/B cell epitope of the 12 proteins(Rv0222,Rv0309,Rv1255 c,Rv1256c,Rv1977,Rv1979 c,Rv1984c,Rv1985 c,Rv1987,Rv3871,Rv3878,and Rv3879c)in 6 RD regions of M.tuberculosis to provide the foundation for the subsequent developing the novel anti-tuberculosis vaccines(eg recombinant BCG).We selected 180 clinical M.tuberculosis strains including different Spoligotyping genotypes from different provinces,autonomous regions or municipalities in China,11 BCG strains from different countries and regions.The gene sequences of the 12 proteins in 6 RD regions of of the strains were respectively tested by means of PCR and DNA sequence methods.The sequence polymorphisms of the coding genes of the 12 proteins were conducted comparative analysis with the whole genome data of M.tuberculosis complex(MTBC)strains downloaded from the NCBI website as the reference sequences.The variability of cell and non-cell epitope regions of the 12 proteins were analyzed with the human T/B cell epitope sequences from the 12 protein antigen genes obtained by searching the IEDB(Immune Epitope Database,IEDB)database.And,the d N value,d S value and d N/d S of the entire gene coding region and T/B cell and non-T/B cell epitope sequence regions which were calculated with Mega software were used to evaluate whether the 12 protein antigen genes were conserved in M.tuberculosis genes and the different sequence regions were affected by the selective pressure of the host immune system.The results showed that the encoding genes of the investigative proteins were highly conserved in the clinical M.tuberculosis strains,but there were also genetic mutations in some of the strains.Of the 180 strains of clinical M.tuberculosis strains,4(2.22%)had a specific mutation(G168T,Gln159His)in Rv0309 gene;2(1.11%)had a G203 deletions in Rv1977 gene;4(2.22%)had a synonymous SNP(C1443T,Arg481Arg)site in Rv1979 c gene;2(1.11%)had a mutation(C592T,CAG198TAG)in Rv1984 c,because TAG is the stop codon,this mutation is a stop mutation;92(51.11%)had a synonymous mutation(G102C,Pro34Pro)in Rv1985c;105(58.33%)had a non-synonymous mutation(G130A,Asp44Asn)in Rv3879 c.In addition,some strains had separate mutation of non-synonymousand insertions or deletions of single bases in the different genes,which also affected the amino acid encoded by their corresponding codons.At the same time,using Mega software to calculate,we discovered that the d N/d S values of entire coding region,the T/B cell and non-T/B cell epitope regions of Rv0309,Rv1256 c,Rv1977,Rv1984 c,and Rv1987 genes were all >1.The d N/d S value of the T cell epitope region was lower than that of the non-T cell epitope region of Rv1987 gene,while the d N/d S value of the T cell epitope region was higher than that of the non-T cell epitope region of Rv0309,Rv1256 c,Rv1977,and Rv1984 c.The d N/d S value of entire coding region,the T cell epitope region of Rv1979 c,Rv1985c,Rv3871,Rv3878,and Rv3879 c genes were all <1,and the d N/d S values of the non-T/B cell epitope regions were >1.Of 12 protein ecoding genes of 6 RD regions in M.tuberculosis genome involved in this study,no sequence polymorphisms was found in Rv0222 and Rv1255 c genes,and all the others had varying degrees of mutations.According to the calculated d N,d S and d N/d S,we could judge Rv1979 c,Rv1984c,Rv1985 c,Rv3871,Rv3878 and Rv3879 c genes are well conserved,which may be candidates for the development of a new type of anti tuberculosis vaccine through further study;while there were significant mutations in Rv0309,Rv1256 c,Rv1977,and Rv1987 genes,suggesting that they had a positive selection effect and a certain degree of immune escape in the process of co-evolution with the host immune system. |
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