结核分枝杆菌休眠相关抗原Rv1733c DNA疫苗的构建及免疫学特性研究

Construction and immunogenicity of the DNA vaccine of dormancy related antigen Rv1733c of Mycobacterium tuberculosis

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 DNA vaccine of dormancy related antigen Rv1733c of Mycobacterium tu- berculosis （Mtb）, and to evaluate its immunogenicity in mice. Methods Rv1733c gene was cloned into the eukar- yotic expression vector pcDNA 3.1 （--） from the plamid pMD-18T-Rv1733c, which was constructed previously and preserved in our lab. The constructed plasmid was named pcDNA-Rv1733c. P815 cells were stably transfected with the plasmid pcDNA-Rv1733c, and were detected the protein expression of Rv1733c by indirect immunofluorescence （IFT）. The mice BALB/c were divided randomly into three groups named pcDNA-Rv1733c DNA, saline and BCG, 10 mice per group. The mice were immunized with pcDNA-Rv1733c DNA or saline intramuscularly 3 times at an in- terval of 2 weeks. BCG was immunized subcutaneously only once. The antigen specific antibody level and IgG2a/ IgG1 subtype ratio in immunized mice were detected by ELISA every 2 weeks, At 8 weeks after the first immuniza- tion, the specific proliferation of spleno-lymphocytes of mice was detected by MTS I-3-（4,5-diethylthiazol-2-yl）-5- （3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-etrazolium, inner salt] method, and IFN-T secreted by spleno- lymphocytes was detected by ELISPOT. The percentages of CD4＋ and： CD8＋ T cells in spleno-lymphocytes were an- alyzed by flow cytometry, and the function of cytotoxicity T lymphocyte （CTL） was detected by lactate dehydrogen-ase（LDH） assay. Restlits The eukaryotic expression plasmid of Rv1733c gene was successfully constructed, named peDNA-Rv1733c. In P815 cells stably transfected with peDNA-Rv1733c plasmid, the expression protein of Rv1733c could be stably detected by IFT. In immunized mice, the pcDNA-Rv1733c plasmid could stimulate the pro- duction of the antigen specific antibody, and the antibody subtype biased to IgG2a,but with the extension of immuni- zation time, the ratio of IgG2a/IgG1 was tended to balance. In pcDNA-Rv1733e DNA group, the stimulation index of spleno-lymphocytes was （2.00±0. 36）, and the cell number of secreted IFN-± was （41.48±5.30） SFC/106 , which were both statistically higher than those in saline group （t=3. 096, P〈0.05）. However, the percentages of CD4＋ and CD8＋ T ceils [（18.15±2.30）% and （7.68±1.34）%], and the activity of CTL [（29.52±1.96）%] were not significantly different with those in saline group（t=0. 571,P〉0. 05）. Conclusion The eukaryotic ex- pression plasmid of Rv1733c was constructed successfully. The pcDNA-Rv1733c plasmid DNA could induce specific humoral and cellular immunity in mice. It may be used as the new TB vaccine against latent Mtb infection.