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为获取针对休眠期结核分枝杆菌(MTB)疫苗,本研究以编码MTB休眠相关转录调节蛋白DosR的基因Rv3133c为靶基因构建重组卡介苗,并研究其免疫效应。在本研究中,将从结核分枝杆菌H37Rv基因组获取的Rv3133c插入大肠杆菌-分枝杆菌穿梭表达质粒pMV361构建重组质粒,该重组体以电转化方式导入卡介苗基因组构建重组卡介苗rBCG-Rv3133c。将成功构建的重组卡介苗免疫BALB/c小鼠,分别在免疫后30d及180d,通过检测疫苗诱导的血清抗体滴度水平评价其诱导的体液免疫反应,通过检测免疫小鼠脾淋巴细胞受TB-PPD刺激后脾CD4+和CD8+T细胞的比例变化、细胞因子IFN-γ的表达评价重组疫苗诱导的细胞免疫。结果显示,重组疫苗rB-CG-Rv3133c可诱导产生较BCG更高水平的特异性抗体滴度、更强的CD4+和CD8+T细胞增殖反应及较高水平的IFN-γ表达量,提示该疫苗具有较强的研究应用价值。
In order to obtain a vaccine against M. tuberculosis during dormancy, we constructed a recombinant BCG cell line using the gene Rv3133c, which encodes the transcriptional regulatory protein DosR of MTB, as a target gene and studied its immune effect. In this study, Rv3133c obtained from the Mycobacterium tuberculosis H37Rv genome was inserted into E. coli-mycobacterium shuttle expression plasmid pMV361 to construct a recombinant plasmid which was electroporated into BCG genome to construct recombinant BCG vaccine rBCG-Rv3133c. BALB / c mice were immunized with recombinant BCG vaccine successfully. The induced humoral immune responses were evaluated by detecting the level of serum antibody titers induced by the vaccine at 30 d and 180 d after immunization. The spleen lymphocytes were detected by TB- The ratio of splenic CD4 + and CD8 + T cells after PPD stimulation was changed, and the expression of cytokine IFN-γ was evaluated by recombinant vaccine-induced cellular immunity. The results showed that the recombinant vaccine rB-CG-Rv3133c induced a higher level of specific antibody titers than BCG, stronger CD4 + and CD8 + T cell proliferation responses and higher levels of IFN-γ expression, suggesting that the vaccine Has strong research and application value.