Epitope Prediction,recombinant Protein Preparation And Immunological Characteristics Of Mycobacterium Tuberculosis Rv2657c

Tuberculosis is a infectious disease caused by Mycobacterium tuberculosis(MTB),disseminated mainly by breathing.It was reported that there were over 10 million cases of tuberculosis,which threatened seriously human health.When the persons were infected with MTB,there were three kinds of results as following: The first is that MTB was removed directly when the person's immunity was strong.The second is that MTB was inhibited in the body,but could not be completely eliminated,the person developed latent tuberculosis infection(LTBI).The third is that MTB rapidly proliferated in the body,and the person developed to active tuberculosis.World Health Organization(WHO)estimated that there were LTBI in 1/3 of the world's population.85%-90% of the newly discovered TB patients originated the population with LTBI.Therefore,If the person with LTBI could be early diagnosed,prevented and treated,the incidence of tuberculosis will greatly reduce.However,there is lack of effective and reliable markers for monitoring and diagnosing LTBI.The screening of LTBI will play an important role in the prevention and control of tuberculosis.The purpose of this study was to try to find a diagnostic marker for LTBI.In the LTBI stage,the expression of MTB latent infection protein in the body increased,this protein may be a potential diagnostic marker of LTBI.MTB Rv2657 c is a protein up-regulated during the lack of nutrition,its encoding gene is located in the region of difference between MTB and M.bovis Bacille Calmette-Guerin(BCG)genomes.Rv2657 c protein consists of 86 amino acids,with encoding gene length of 261 bp,the relative molecular mass of 9506.8Da,isoelectric point of 9.2211.Its function was unknown,may be a phirv2 phage protein.In this study,we used DNAStar software package to predict T cell and B cell epitopes in MTB Rv2657 c protein,used Blast method to analyze the sequence homology with the human sequence,then predicted the CTL epitopes by the combination analyses of BIMAS quantitative motif method,NetCTL prediction method and SYFPEITHI supermotif method;predicted the Th epitopes by RANKPEP and SYFPEITHI supermotif prediction method.The epitopes with higher concentration and higher scores were chosed as the candidate polypeptides.TMHMM software was used to predict the transmembrane protein,PSORT software version was used to predict the intracellular localization of the protein,and SignalP software 2 and SignalP 4 Server were used to analyze the signal peptide of the protein.The engineered Escherichia coli strain expressing MTB Rv2657 c recombinant protein was constructed,and then induced the expression of Rv2657 c protein.Rv2657 c recombinant protein was purified.The immunological characteristics of recombinant protein was studied by Western blot and enzyme-linked immunospot assay(ELISPOT).In ELISPOT assay,a proliferative phaseas protein(the culture filtration protein 10 and early secretory antigen target 6 recombinant fusion protein,CE)was used as contrast,recombinant Rv2657 c protein was used to stimulate peripheral blood mononuclear cells from the healthy TB workers group and active tuberculosis patients,and then detecting the spot number of T lymphocytes secreting interferon-gamma(SFCs)by ELISPOT,and comparing the difference between these two populations.The results of Rv2657 c protein prediction by Protean software showed that there were better hydrophilicity,flexibility and antigenicity,and weaker the surface accessibility;the alpha helices and beta folds were more in number and distributed widely,the angle and coil structures were less.There were 5 B-cell epitopes,in which the total epitope length accounted for 50% of the whole protein,B-cell epitopes were mainly concentrated after the fourteenth amino acids.There were 6 T-cell epitopes,in which the total epitope length accounted for 38.4% of the whole protein,T-cell epitopes were mainly concentrated after the tenth amino acids.This protein predicted by SYFPEITHI supermotif method,BIMAS quantitative motif method and NetCTL had 6 CTL epitopes,in which there were more mumber and high score of HLA-A2-restricted epitopes.The protein predicted by RANKPEP and SYFPEITHI supermotif had 38 Th-cell epitopes,in which there were more epitopes of HLA-DRB1*0401 and HLA-DRB1*0701 phenotypes.Rv2657 c protein predicted by TMHMM software,PSORT version software,SignalP software 2.0 and SignalP 4.0 Server was a cytoplasmic protein without signal peptide.We successfully constructed a engineered Escherichia coli strain expressing MTB Rv2657 c recombinant protein and obtained purified protein.The results of Western blot showed that the Rv2657 c protein could react with the sera from Guinea pigs with latent tuberculosis infection,did not react with the sera from healthy Guinea pig.The results of rRv2657c-ELISPOT showed that the SFCs of healthy TB workers group(11)was significantly higher than that of active tuberculosis group(1.5)(p<0.05).In summary,Rv2657 c protein is an antigen with both B cell epitope and T cell epitope,which is dominated by B cell epitopes.The molecular weight of recombinant Rv2657 c protein was 12.8 kDa.The protein was expressed in two forms: inclusion body and soluble protein,but it was mainly expressed in soluble protein.The protein was purified by metal ion affinity chromatography,and then concentrated by ultrafiltration.The rRv2657 c protein was proved to have good antigenicity in Western Blot test,and its immunogenicity was proved by Elispot test,and may be a potential marker for the diagnosis of latent tuberculosis infection.