| Tuberculosis remains one of the most common chronic diseases to cause morbidity and mortality. T lymphocytes mediating immunity and delay type hypersensitivity have a determining effect on the pathogenesis, evolution, and outcome of tuberculosis. As the cross-link of native and adaptive immunity, γδT cells play an important role in the immune response against mycobacterium tuberculosis. On coupling with the ligands such as ULBP (UL16binding protein) and the antigens of mycobacterium tuberculosis, the receptors on the surface of γδT cells mediate the process of immunity.Our previous research indicated the probability of γδT cells recognizing the protein Rvl194c through CR3(Complementary determining region3), so we decided to express the protein for a full elucidation of this mechanism. At the same time, we established Multl (Murine ULBP like transcript1) transgenic mice, which were intended to overexpress MULT1, the analogue of ULBP in mouse, to elucidate the mechanism of γδT cells recognizing ULBP. ULBP can be upregulated on the surface of other relative cells infected by mycobacterium tuberculosis. Given the chonic characteristic of mycobacterium tuberculosis infection and the fact that γδT cells can not be cultured for a relatively long time in vitro, we tried to immortalize human γδT cells for the better understanding of the longterm effect of γδT cells against mycobacterium tuberculosis.So our research was composed of the following three parts:the prokaryotic expression of mycobacterium tuberculosis recombined protein, the propagation and genotype identification of transgenic mice and the immortalization of human γδT cells. Scientifically, we obtained the conserved protein Rv1194c of mycobacterium tuberculosis which could induce the amplification ofγδT cells in vitro, the positive offsprings of transgenic mice, and human yγδT cells which had a relatively long lifespan. So we laid a foundation for the study of the mechanism ofγδT against mycobacterium tuberculosis.In order to elucidate the recognition mechanism of γδTCR towards proteins of mycobacterium tuberculosis, we expressed the conserved protein Rv1194c of mycobacterium tuberculosis H37Rv. After obtaining the gene of Rv1194c through PCR (Polymerase chain reaction), we inserted the gene into the expressing-plasmid, and then transformed into the prokaryotic expressing E.coli BL21(DE3). And we used IPTG (Isopropyl β-D-1-thiogalactopyranoside) to induce the expression of Rvl194c. When the protein was extracted and purified under native and denaturing conditions respectively, we found that it might induce the amplification of human γδT cells.In order to elucidate the recognition mechanism of NKG2D (Natural killer group2member D) towards ULBP molecule, we established Mult1/Mult1S transgenic mouse model (MULT1is human analogue of ULBP in mouse) previously. In this part of study, we set up the breeding of the male transgenic mice with wildtype female mice to get the offsprings of them. Then we identified and obtained the positive transgenic mice.The characteristic of mycobacterium tuberculosis infection is chronic. During the long course of infection, the longterm role of y8T cells had not been clearly elucidated yet. The study of human γδT cell against mycobacterium tuberculosis was limited partly for y8T cells can not be cultured for a relatively long time in vitro. In order to resolve this obstacle, we immortalized human γδT cells through electric shock and retrovirus infection.After reconstructing the plasmid of pRevTRE-hTERT successfully, we immortalized human γδT cells with high voltage electricity and retrovirus infection. During the process of transfection, we optimized some factors that might have influence on the results and we obtained positive retrovirus-producing cell colonies and γδT cells which have a relatively long lifespan.To sum up, the results of our study are as follows:1. We expressed the recombinant protein of Rv1194c of mycobacterium tuberculosis H37Rv, and we found that this protein might induce the amplification of human γδT cells.2. We obtained16Mult IS and5Multl positive transgenic mice after genotype identification of the offsprings, and we provided the indemnity for next research steps.3. We obtained human γδT cells which had a partly altered growth condition through the insertion of exogenous gene of hTERT with two different methods, and we gained the experiences of immortalization of human γδT cells in our lab.We tried to elucidate the mechanism of γδTCR recognizing antigens of mycobacterium tuberculosis through the expression of Rv1194c, and we intended to understand the process of NKG2D coupling ULBP by establishing Multl transgenic mice, and we showed that insertion of exogenous hTERT could extend the lifespan of human γδT cells. Thus we laid a foundation for the research of the mechanism of γδT cells against mycobacterium tuberculosis... |
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