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目的构建结核分枝杆菌(Mtb)休眠相关抗原Rv1733c的真核表达载体,并评价其作为DNA疫苗的免疫学特性。方法利用限制性酶切的方法从本室前期保存的pMD-18T-Rv1733c质粒中构建Rv1733c的真核表达载体pcDNA-Rv1733c。将pcDNA-Rv1733c重组质粒稳定转染P815细胞,并用间接免疫荧光法检测Rv1733c的表达。采用数字表法随机将BALB/c小鼠分为3组,每组10只,即pcDNA-Rv1733c质粒DNA组、生理盐水组和BCG组。pcDNA-Rv1733c质粒DNA组和生理盐水组采用肌内注射方式免疫,间隔2周免疫1次,共免疫3次。BCG组采用皮下免疫一次。各组小鼠每2周采血,ELISA检测血清中特异性抗体水平和IgG2a/IgG1抗体亚类比率与比值。初次免疫8周后,MTS[3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Hetrazolium,inner salt](四氮唑蓝盐化合物)法检测小鼠脾淋巴细胞特异性增殖、ELISPOT检测脾淋巴细胞分泌IFN-γ的水平。流式细胞法检测脾淋巴细胞中CD4+和CD8+T细胞所占比率;LDH法检测CTL(cytotoxic T lymphocytes;细胞毒性T淋巴细胞)活性。结果成功构建Rv1733c的真核表达载体pcDNA-Rv1733c。间接免疫荧光实验表明pcDNA-Rv1733c质粒稳定转染的P815细胞中能够稳定表达Rv1733c蛋白。pcDNA-Rv1733c质粒DNA免疫小鼠后能诱导小鼠产生特异性抗体,抗体亚类以IgG2a为主,随着免疫时间的延长,IgG2a/IgG1的比值趋于平衡;脾淋巴细胞增殖指数(2.00±0.36)高于BCG组(1.1±0.06)(t=3.096,P<0.05);特异性分泌IFN-γ的脾淋巴细胞频数(41.48±5.30)SFC/106高于生理盐水组(2.75±1.37)SFC/106(t=4.752,P<0.05);然而脾淋巴细胞中CD4+、CD8+T细胞所占比率[分别为(18.15±2.30)%、(7.68±1.34)%]、CTL杀伤活性[(29.52±1.96)%]都与生理盐水组相当[(16.43±2.02)%(t=0.571,P>0.05)、(7.32±0.42)%(t=0.234,P>0.05)、(25.28±2.51)%(t=0.726,P>0.05)]。结论成功构建Rv1733c真核表达载体pcDNA-Rv1733c;并能够诱导小鼠机体产生特异性的体液和细胞免疫应答,提示用于结核病新型疫苗的研究具有一定的意义。
Objective To construct an eukaryotic expression vector for Rv1733c, an hibernant-associated antigen of Mtb, and evaluate its immunological properties as a DNA vaccine. Methods The eukaryotic expression vector pcDNA-Rv1733c of Rv1733c was constructed by restriction enzyme digestion from pMD-18T-Rv1733c plasmids stored in our laboratory. The pcDNA-Rv1733c recombinant plasmid was stably transfected into P815 cells and the expression of Rv1733c was detected by indirect immunofluorescence assay. BALB / c mice were randomly divided into 3 groups of 10, pcDNA-Rv1733c plasmid DNA group, saline group and BCG group. The pcDNA-Rv1733c plasmid DNA group and the saline group were immunized with intramuscular injection and immunized once every 2 weeks for 3 times. The BCG group was immunized subcutaneously once. Blood samples were taken every 2 weeks in each group. Serum specific antibodies and IgG2a / IgG1 antibody subclass ratios and ratios were determined by ELISA. MTS [3- (4,5-diethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2Hetrazolium, inner salt] ) Method was used to detect the specific proliferation of splenic lymphocytes in mice. ELISPOT was used to detect the level of IFN-γ secreted by splenic lymphocytes. The proportion of CD4 + and CD8 + T cells in splenic lymphocytes was detected by flow cytometry. The cytotoxic T lymphocytes (CTLs) activity was detected by LDH assay. Results The eukaryotic expression vector pcDNA-Rv1733c of Rv1733c was successfully constructed. Indirect immunofluorescence assay showed that Rv1733c protein was stably expressed in P815 cells stably transfected with pcDNA-Rv1733c plasmid. After immunization with pcDNA-Rv1733c plasmid DNA, mice were induced to produce specific antibodies. The antibody subtypes were mainly IgG2a. The ratio of IgG2a / IgG1 tended to balance with the prolongation of immunization time. The spleen lymphocyte proliferation index (2.00 ± (41.48 ± 5.30) SFC / 106 was higher than that of saline group (2.75 ± 1.37) (t = 3.096, P <0.05) However, the percentage of CD4 + and CD8 + T cells in splenic lymphocytes were (18.15 ± 2.30)% and (7.68 ± 1.34)%, respectively. CTL activity [( 29.52 ± 1.96)%] were significantly higher than those in the saline group [(16.43 ± 2.02)% vs (7.32 ± 0.42)% vs (t = 0.234, P> 0.05) % (t = 0.726, P> 0.05)]. Conclusion The eukaryotic expression vector pcDNA-Rv1733c of Rv1733c was successfully constructed and could induce specific humoral and cellular immune responses in mice, suggesting that it is of great significance to study the novel vaccine against tuberculosis.