Evaluation Of Mycobacterial Virulence Using Rabbit Skin Liquefaction Model And Study On Mycobacterium Tuberculosis Fusion Protein Subunit Vaccine

1. Evaluation of mycobacterial virulence using rabbit skin liquefaction modelObjective:Using M. bovis BCG, M. tuberculosis avirulent strain H37Ra, M. smegmatis, and the H37Ra strains complemented with 11 selected genes from virulent M. tuberculosis strain H37Rv to infect rabbit skin and observing the skin pathological changes. Then evaluating mycobacterial virulence and difference in production of liquefaction.Methods:In the first experiment, the rabbits were divided into 6 groups, and each group injected intradermally with about 5x106 CFU of both live and heat-killed mycobacteria (BCG, H37Ra, M. smegmatis) respectively. After 42 days, a second same dose of same bacteria was injected intradermally. And we observed the skin pathological changes. In the second experiment, there were 13 groups, the H37Ra and pOLYG vector were contol, and the experiment groups contained H37Ra complement strains:P10, P11, P12...P20. Each group was injected intradermally with 5×107 CFU,5×106 CFU and 5×105 CFU in multiple sites respectively. Six weeks later, a second same dose of same bacteria was injected intradermally. The animals were observed.Results:In the first experiment, we found that the boosting induced stronger liquefaction and more severe lesions in shorter time compared with the prime injection. The skin lesions caused by high dose live BCG (5×105 CFU) were the most severe followed by live M. tuberculosis H37Ra with M. smegmatis being the least pathogenic. But all the heatkilled mycobacteria induced no liquefaction. In the second experiment, some of the complemented H37Ra strains produced more severe skin lesions than H37Ra. P10, which is H37Ra complemented with pstAl from H37Rv, caused the largest tubercles, followed by P19 (1pdA),P17 (pabA) and P15 (mazG), P18 (nrdH), P14 (pknH), which all induced obvious bigger tubercle than H37Ra did (*p= 0.05). However, P11 (phoP), P12 (marR family), P13 (luxR/uhpA family), P16 (nadD) and P20 (ilvD) produced similar lesions as the H37Ra transformed with vector pOLYG control and H37Ra. P14 (pknH) and P18 (nrdH) induced the most obvious liquefaction among all groups. The lesions of P10 (pstA1), P11 (phoP), P13 (luxR/uhpA family), P20 (ilvD), and P16 (nadD)were similar in liquefaction to H37Ra vector pOLYG control.Conclusion:High dose (5×106) of live BCG, H37Ra and M. smegmatis could induce liquefaction in rabbit skin, and the pathological reaction induced by BCG was stronger than that caused by H37Ra and M. smegmatis. The heat-killed bacteria lost the ability to induce liquefaction. It was demonstrated that the rabbit skin model could tell differences in pathology of complemented H37Ra strains. The rabbit skin model may be a useful model for studying the mechanism of tuberculosis liquefaction, rapidly assessing mycobacterial virulence, and testing new vaccines and inhibitors.2. Cloning, constructing and immunologic reactivity of Mycobacterium tuberculosis fusion proteins ESAT6-Ag85B/TB10.4-Ag85B/ESAT6-TB8.4/TB10.4-TB8.4 with no tagObjective:Cloning and constructing the fusion proteins of Mycobacterium tuberculosis ESAT6-Ag85B/TB10.4-Ag85B/ESAT6-TB8.4/TB10.4-TB8.4 in this study. Using the animal experiment to evaluate the immunologic reactivity of these proteins preliminarily.Methods:Based on molecular cloning techniques, we designed primers with different restriction sites of ESAT6, TB10.4, TB8.4 and Ag85B, and H37Rv-DNA was used for the PCR template. The size of the PCR amplified results were in line with the design value before, according to the sequences in GenBank. Then we obtained the recombinant plasmids pET30a-ESAT6-Ag85B/pET30a-TB10.4-Ag85B/pET30a-ESAT6-TB8.4/pET30a-TB10.4-TB8.4 by the genetic engineering techniques. The recombinant genes were expressed by IPTG (Isopropyl-β-D Thiogalactoside) induction, and then we used the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the expression results. These fusion proteins were purified by IEX and HIC effectively. At last, we mixed the proteins and adjuvant DDA together in animal expriement. The C57BL/6 mouse were divided into 9 groups, and control were PBS, BCG and single antigens groups. The experiment groups were injected with fusion proteins.14 weeks after immunization, we used ELISA to measure INF-γ.Results:It is found that all the fusion proteins have been expressed in E. coli with the expected size. For the fusion proteins ESAT6-Ag85B with about 38KD and TB10.4-Ag85B with about 42KD expressed in the form of inclusion bodies, and finally purify them by ion exchange and hydrophobic chromatography. Meanwhile, we have purified the last two soluble proteins ESAT6-TB8.4 with about 18KD and TB10.4-TB8.4 with about 19KD by the same two chromatography methods with different medium effectively and successfully. The result of ELISA shew:When the spleen lymphocytes were stimulated by single protein ESAT6 or TB8.4, the ESAT6-TB8.4 group can induce significant production of IFN-y compared to BCG and ESAT6(**ρ<0.05 vs. BCG and ESAT6) with lower IFN-γprduction than TB8.4 group. When the spleen lymphocytes were stimulated by single protein TB8.4 or peptide of TB10.4, the group of TB10.4-TB8.4 can induce significant production of IFN-y compared to BCG and TB8.4(**ρ<0.05 vs. BCG and TB8.4). When the spleen lymphocytes were stimulated by single protein Ag85B or ESAT6, the ESAT6-Ag85B group can induce significant production of IFN-y compared to BCG(*ρ<0.05 vs. BCG). Lastly, after stimulated by single protein Ag85B or peptide of TB10.4, the TB10.4-Ag85B group can induce significant production of IFN-y compared to BCG(*ρ<0.05 vs. BCG).Conclusion:The four fusion proteins of Mycobacterium tuberculosis ESAT6-Ag85B /TB10.4-Ag85B/ESAT6-TB8.4/TB10.4-TB8.4 were constructed and purified by different chromatographic columns successfully. The animal experiment shew that all the fusion protein could induce stronger IFN-y reaction than single antigen. All these recombinant fusion proteins without tag are expected to become candidate vaccines to prevent tuberculosis, and we should evaluate the immunoprotection of these vaccines in the future.