Preparation Of Mycobacterium Tuberculosis Rv3407 Recombinant Protein, DNA Vaccine And The Research Of Its Immunogenicity

About one third of the global population were infected by Mycobacterium tuberculosis(M.tb), in which 5%~10% of the persons with latent tuberculosis infection(LTBI) will develop to active tuberculosis(TB).Therefore, the effective prevention and control of LTBI is crucial. At present, BCG vaccine is one and only vaccine for the prevention of tuberculosis, but it can not prevent the development of LTBI to TB effectively. The immune efficacy of BCG on LTBI is not very sure. Therefore, the research of vaccines against LTBI is vital to the control of TB. M.tb Rv3407 protein is related to the resuscitation and reactivation of M.tb, and a marker of the activity of M.tb. Preparation of rv3407 DNA vaccine may act on the immune elimination and the inhibition of resuscitation of latent M.tb which can provide the immunoprotection on LTBI. Therefore, rv3407 DNA vaccine might have the potential to be a new candidate vaccine against LTBI, and lay the experimental foundation on the prevention and control of TB.In this study, we predicted the epitopes of M.tb Rv3407 protein with bioinformatical softwares, and get to know its antigenicity. Moreover, we used molecular cloning techniques to prepare Rv3407 recombinant protein and construct rv3407 DNA vaccine. Finally, we evaluated the immunogenicities of the vaccine in mice.1. Prediction of epitopes of M.tb Rv3407 proteinThe amino acid sequence of Rv3407 protein was input and predicted B-cell and T-cell epitopes using multi-parameters including the secondary structure, hydrophilicity, antigenicity, surface probability, flexibility by Protean software of DNAStar software package. The results showed that the Rv3407 protein had rich secondary structure and multiple sections with higher antigenicity. There were rich potential B-cell epitopes at 11~26, 28~43, 50~61, 66~74, 76~99 amino acid residues or nearby, theses epitopes had better antigenicity, contained beta angle structure mostly, presented in the surface probability and had larger flexibility. There were more potential T cell epitopes at 2~12, 22~26, 34~37, 40~44, 56~60, 75 ~79, 86 ~ 93 amino acid residues or nearby. HLA-DRB1*0101 、HLA-DRB1*0301 、 HLA-DRB1*0401 、 HLA-DRB1*0701 、HLA-DRB1*0801、HLA-DRB1*1101、HLA-DRB1*1501 restricted Th epitopes were predicted by SYFPEITHI and RANKPEP. The results showed that there were about 22 Th cell epitopes predicted. There was low homology between Rv3407 protein and human proteins by BLAST. HLA-A* 0201, HLA-A*03 and HLA-B7 restricted CTL epitopes were predicted by SYFPEITHI, BIMAS and Net CTL. The results showed that there were about 20 CTL epitopes predicted.2. Cloning, expression and purification of M.tb Rv3407 protein using affinity chromatograghyThe gene encoding Rv3407 protein was amplified by PCR. The PCR fragment was linked with expression vector p ET30 a to form recombinant plasmid, and then transferred into E. coli BL21(DE3). After E. coli BL21(DE3) containing recombinant plasmid p ET30a- rv3407 was inducted with IPTG, the expression quantity and expression form of target protein were identified by SDS-PAGE. The recombinant protein was purified using Protein Iso Ni-NTA Resin. The results showed that the PCR product was successfully amplified. The sequence ofp ET30a- rv3407 was consistent with that reported in the Gen Bank. The Rv3407 recombinant protein was expressed in the soluble form in E. coli BL21(DE3), its molecular weight was about 11 k Da. The purified Rv3407 recombinant protein was obtained.3. The preparation of rv3407 DNA vaccineThe rv3407 gene fragment was amplified from genome of M.tb H37 Rv by PCR. The amplified fragment was purified and inserted into vector p GEM-T. The recombinant plasmidp GEM-T-rv3407 was transferred into E. coli DH5α. The recombinant plasmid was extracted and identified by PCR amplification, DNA sequencing and digesting with restriction enzyme Nhe Ⅰ and Eco R Ⅰ. The correct plasmid p GEM-T-rv3407 was digested by enzyme NheⅠand Eco RⅠ. The producingrv3407 fragment was purified, and then connected with vector p VAX1 digested by same enzymes. The recombinant plasmid p VAX1-rv3407 was transformed into E. coli DH5α. The E. coli DH5α colony containing plasmid p VAX1-rv3407 was identified by colony PCR and sequencing. The results confirmed that rv3407 DNA vaccine was successfully constructed. The recombinant plasmid p VAX1-rv3407 was extracted and purified by QIAGEN Plasmid Maxi Kit. The expression of p VAX1-rv3407 DNA vaccine was detected in HEK293 cells by means of cell culture and RT-PCR. The results showed that the aim gene fragment can be amplified successfully. It confirmed that the vaccine can expressed in eukaryotic cell at transcriptional level.4. Immunogenic evaluation of rv3407 DNA vaccine in miceFifty BALB/c mice were randomly divided into five groups: the sterile water group, the p VAX1 plasmid group, the M. vaccae group, the Ag85 A DNA vaccine group and the rv3407 DNA vaccine group, 10 mice per group. The mice were immunized with muscular injection at the bilateral pretibial muscle for three times, at two weeks interval, and the mice were sacrificed at the three weeks after the third immunization. The percentages of Th1, Th2, Tc1 and Tc2 cells, and the ratio of Th1/Th2, Tc1/Tc2 in the whole blood were determined by flow cytometry. The antibody Ig G against Rv3407 in the sera and IFN- γ in the supernates of cultured splenolymphacytes were detected by ELISA method. The result showed that the percentage of Th1 cells and the ratio of Th1/Th2, inrv3407 DNA vaccine group were significantly higher than those in the sterile water group, the p VAX1 plasmid group and the M. vaccae group(p<0.01), but had no significant difference with that in Ag85 A DNA vaccine group. The percentages of Tc1 cells, Tc2 cells and Th2 cells, and the ratio of Tc1/Tc2 inrv3407 DNA vaccine group had no significant difference with other groups. The specific Ig G levelsinrv3407 DNA vaccine group and Ag85 A DNA vaccine group were significantly higher than those in the sterile water group, the p VAX1 plasmid group and the M. vaccae group(p<0.01). The IFN-γlevel in 3/10 mice from the rv3407 DNA vaccine group and in 1/10 mice from the p VAX1 plasmid group were higher, whilenone of the mice from sterile water group, the p VAX1 plasmid group and the M. vaccae group produced IFN-γ.In summary, Mycobacterium tuberculosis Rv3407 protein was an antigen with rich B-cell-epitope and T-cell-epitope. We prepared Rv3407 protein and rv3407 DNA vaccinesuccessfully. Rv3407 DNA vaccine had good immunogenicity that could induce both humoral immune response and cellular immune response. In the future, we will further study its anti-tuberculosis effect.