| Mycobacterium tuberculosis(MTB) is the pathogen of infectious disease at the second leading cause of death, with respiratory tract infections as the main way, once invading the host body that is difficult to be removed. And due to the lack of accurate, rapid and effective diagnostic tools and a safe and effective preventive vaccine, the prevention and treatment of the disease is still one of the major problems that need to be solved in China. It is estimated that one-third of the world's population with MTB latent infection, 5% to 10% of the infected people in the future life will develop into active tuberculosis. Therefore, the effective prevention and control of tuberculosis infection latent(LTBI) is very important. Bacillus Calmette-Guerin(BCG) is currently the main vaccine to prevent TB, but its immune effect is uncertain, and can not effectively prevent LTBI development into TB. Therefore, the development of the vaccine for LTBI has important significance for the control of TB. The expressions of latency-associated proteins were upregulated during latent MTB infection, it is likely to become markers of LTBI and develop new vaccine for LTBI.In this study, the epitopes of M.tb Rv3287 c protein were predicted using bioinformatical softwares, and then its antigenicity was understood. Moreover, recombinant Rv3287 c protein was prepared by molecular cloning technique, and studed its immunogenicity.1. Epitope prediction, characteristics analyses and codon optimization of M.tb Rv3287 c proteinThe amino acid sequence of Rv3287 c protein was input and predicted B-cell and T-cell epitopes using multi-parameters including the secondary structure, hydrophilicity, antigenicity, surface probability, flexibility and charge distribution by Protean software of DNAStar software package. The CTL epitopes of Rv3287 c protein were predicted on line by means of combination analyses of SYFPEITHI super motif method, quantitative BIMAS motif method and Net CTL method. MTB Rv3287 c protein had abundant secondary structures,contained a few proportion of potential B cell epitopes at 1-20, 78-88, 112-130, 137-145 amino acid residues or nearby, which basically contained the beta angle structure, the indexes of antigenicity, hydrophilic, surface potential and flexibility were higher. There were more potential T cell epitopes, located at 16-19, 24-25, 28-44, 47-52, 54-68, 71-79, 84-88, 91-101, 107-109, 111-124, 126-129, 140-143 amino acid residues or nearby. The the CTL epitopes were predicted on line using three methods mentioned above, the results of SYFPEITHI prediction showed that Rv3287 c protein in HLA-A*0201, HLA-A*03 and HLA-B7 score of more than 18 had 22, 19 and 4 epitopes, respectively; the results of BIMAS prediction showed that the HLA-A*0201, HLA-A*03 and HLA-B7 in the score of more than 30 had 4, 0 and 3 epitopes respectively; the results of Net CTL prediction showed that the HLA-A*0201, HLA-A*03 and HLA-B7 in the score of more than 0.75 had 6, 1 and 7 epitopes respectively. According to the preferred codon of Escherichia coli, the rv3287 c nucleotide sequence was optimized. The transmembrane characteristics of Rv3287 c protein was predicted using TMHMM software, the signal peptide was predicted by the Signal P software version 2, and found that the Rv3287 c protein was a non-secretory cytoplasmic protein without signal peptide.2. MTB Rv3287 c cloning, expression and purificationThe gene engineering Escherichia coli containing recombinant plasmid puc57-rv3287 c synthetized was prepared, and then recombinant plasmid puc57-rv3287 c was extracted with a high purity plasmids small extraction kit. The recombinant plasmid and vector p ET30 a were digested by restriction enzyme Nhe I and Eco R I, a large number of rv3287 c gene fragment recovered was connected with vector p ET30 a cut by enzyme by T4 DNA ligase, and then the products were transformed into Escherichia coli BL21 competent cells, which were smeared and cultured on the medium plates, single clone was selected and identified by enzyme digestion and DNA sequencing. The prokaryotic expression plasmid containing correct rv3287 c gene sequence was extracted and named as p ET30a-rv3287 c. Protein expression in Escherichia coli BL21 containing plasmid p ET30a-rv3287 c was induced by IPTG, the recombinant protein was purified by nickel absorption chromatography. The results showed that the sequence of recombinant plasmid p ET30a-rv3287 c was correct, which was constructed successfully. The recombinant protein Rv3287 c in E. coli BL21(DE3) was expressed in the form of inclusion body, its molecular weight was about 14.89 k Da. The purified recombinant protein Rv3287 c was obtained.3. Study on the immunological characteristics of recombinant MTB Rv3287 c proteinUsing proliferative protein(CFP10-ESAT6) as control, the ELISPOT method with latent infection-related protein Rv3287 c as the stimulant was established. The spot-forming cells(SFCs) of IFN-? production in effector T cells were detected in PBMCs from 324 cases(including 128 uninfected healthy controls, 63 LTBI persons, 55 tuberculosis patients and 48 non-tuberculous respiratory disease patients), compared the difference among different populations, to evaluate the effect of Rv3287 c protein in the cellular immune response duing anti-tuberculosis latent infection. Serum anti-Rv3287 c antibody Ig G levels in uninfected healthy control group(30 cases), LTBI group(35 cases), TB patient group(30 cases) and non-tuberculosis respiratory disease group(30 cases) were detected by ELISA, and compared the differences among 4 groups to understand their humoral immune responses. The SFC values of effector T cells in LTBI group were taken as the positive group, and those in the TB patients group were used as negative group, a ROC curve was drawed to identify LTBI with active TB, and the diagnostic efficiency of the Rv3287 c antigen was evaluated when SFC values of 80% and 90% specificity were taken as the cut-off value. The results of ELISPOT showed as follow: the SFCs stimulated by Rv3287 c protein in LTBI group was higher than that in uninfected healthy control group, but there was no significant difference between these two groups(p>0.05); the SFCs in LTBI group was significantly higher than that in the active tuberculosis group(p<0.05); the SFCs in non-tuberculosis respiratory disease group was significantly lower than that in LTBI group and in healthy control group(p<0.05). The results of ELISA showed that the serum Ig G levels against r Rv3287 c had not significant difference among four groups(p>0.05). The results of the ROC curve were showed as follow:the area under the curve of r Rv3287 c was 0.727, when the specificity was 83% and the cut-off value was 6 SFCs, the positive rate in the uninfected healthy group was 39%, in LTBI group was 50.8%, in active tuberculosis group was 14.5%, in non-tuberculosis respiratory disease group was 29.2%; when the specificity was 91% and the cut-off value was 11 SFCs, the positive rate in the uninfected healthy group was 20%, in LTBI group was 38.1%, in active tuberculosis group was 7%, in non-tuberculosis respiratory disease group was 12.5%.In summary, Rv3287 c protein is a T-cell-epitope dominant antigen, B-cell epitopes also exists but was less than T-cell epitopes, and is a non-secretory cytosolic protein without signal peptide through bioinformatics prediction,. Recombinant Rv3287 c protein was prepared successfully, and the efficiency of Rv3287 c protein expression was higher after codon optimization. The r Rv3287 c could be recognized by LTBI persons, and induced to produce stronger immune responses of effector T cells than those in active tuberculosis group and in the uninfected control group, but could not distinguish between uninfected control group and latent infection group. The r Rv3287 c protein could not induce the humoral immune response. |
PDF Full Text Request