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

中国防痨杂志 ›› 2013, Vol. 35 ›› Issue (9): 679-685.

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

段安虎张薇柏银兰康健王瑞徐志凯王丽梅

710048 西安，陕西省结核病防治研究所（段安虎、王瑞）；第四军医大学唐都医院儿科（张薇）；第四军医大学微生物学与病原生物学教研室（柏银兰、康健、徐志凯、王丽梅）

收稿日期:2013-07-08 出版日期:2013-09-10 发布日期:2013-09-08
通信作者: 王丽梅 E-mail:limwang@tom.com
基金资助:
“十二五”国家科技重大专项（2012ZX10003008-007）；国家自然科学基金（30801055）

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

DUAN An-hu， ZHANG Wei， BAI Yin-lan， KANG Jian， WANG Rui，XU Zhi-kai， WANG Li-mei

Shaanxi Research Institute for Tuberculosis Prevention and Control，Xi’an 710048，China

Received:2013-07-08 Online:2013-09-10 Published:2013-09-08
Contact: WANG Li-mei E-mail:limwang@tom.com

摘要/Abstract

摘要： 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)-2H-etrazolium,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。

关键词: 分枝杆菌, 结核, 抗原, 细菌, 疫苗, DNA, 抗体生成, 免疫, 细胞

Abstract: Objective To construct DNA vaccine of dormancy related antigen Rv1733c of Mycobacterium tuberculosis (Mtb), and to evaluate its immunogenicity in mice. Methods Rv1733c gene was cloned into the eukaryotic 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 interval 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 immunization, the specific proliferation of spleno-lymphocytes of mice was detected by MTS ［3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-etrazolium, inner salt］ method, and IFN-γ secreted by spleno-lymphocytes was detected by ELISPOT. The percentages of CD4⁺ and CD8⁺ T cells in spleno-lymphocytes were analyzed by flow cytometry, and the function of cytotoxicity T lymphocyte (CTL) was detected by lactate dehydrogenase(LDH) assay． Results The eukaryotic expression plasmid of Rv1733c gene was successfully constructed, named pcDNA-Rv1733c. In P815 cells stably transfected with pcDNA-Rv1733c plasmid，the expression protein of Rv1733c could be stably detected by IFT. In immunized mice, the pcDNA-Rv1733c plasmid could stimulate the production of the antigen specific antibody, and the antibody subtype biased to IgG2a，but with the extension of immunization time，the ratio of IgG2a/IgG1 was tended to balance. In pcDNA-Rv1733c DNA group, the stimulation index of spleno-lymphocytes was (2.00±0.36), and the cell number of secreted IFN-γ 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 cells ［(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 expression 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.

Key words: Mycobacterium tuberculosis, Antigens, bacterial, Vaccines, DNA, Antibody formation, Immunity, cellular

段安虎张薇柏银兰康健王瑞徐志凯王丽梅. 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[J]. 中国防痨杂志, 2013, 35(9): 679-685.

DUAN An-hu， ZHANG Wei， BAI Yin-lan， KANG Jian， WANG Rui，XU Zhi-kai， WANG Li-mei. Construction and immunogenicity of the DNA vaccine of dormancy related antigen Rv1733c of Mycobacterium tuberculosis[J]. Chinese Journal of Antituberculosis, 2013, 35(9): 679-685.

参考文献

［1］World Health Organization. Tuberculosis［EB/OL］. Geneva:WHO,2013［2013-04-18］. http://www.who.int/mediacentre/factsheets/fs104/en/index.html.
［2］Voskuil MI. Mycobacterium tuberculosis gene expression during environmental conditions associated with latency. Tuberculosis (Edinb)，2004，84(3/4)：138-143.
［3］Muttucumaru DG，Roberts G，Hinds J，et al. Gene expression profile of Mycobacterium tuberculosis in a non-replicating state. Tuberculosis (Edinb)，2004， 84(3/4)：239-246.
［4］Leyten EM，Lin MY，Franken KL,et al. Human T-cell responses to 25 novel antigens encoded by genes of the dormancy regulon of Mycobacterium tuberculosis. Microbes Infect，2006, 8(8)：2052-2060.
［5］Lin MY，Geluk A，Smith SG，et al. Lack of immune responses to Mycobacterium tuberculosis DosR regulon proteins following Mycobacterium bovis BCG vaccination. Infect Immun，2007，75(7)：3523-3530.
［6］Black GF, Thiel BA, Ota MO,et al. Immunogenicity of novel DosR regulon-encoded candidate antigens of Mycobacterium tuberculosis in three high-burden populations in Africa. Clin Vaccine Immunol，2009，16(8)：1203-1212.
［7］张薇，柏银兰，康健，等.结核分枝杆菌持续感染期抗原Rv1733c的表达、纯化和鉴定. 现代生物医学进展，2012，12(10)：1868-1871.
［8］吴雪琼.新型结核病疫苗的研究现状与发展趋势.中国防痨杂志，2012，34(3)：133-137.
［9］Huygen K. On the use of DNA vaccines for the prophylaxis of Mycobacterial diseases.Infect Immun，2003，71(4)：1613-1621.
［10］Roupie V，Romano M，Zhang L. Immunogenicity of eight dormancy regulon-encoded proteins of Mycobacterium tuberculosis in DNA-vaccinated and tuberculosis-infected mice. Infect Immun，2007，75 (2)：941-949.
［11］Bivas-Benita M, Lin MY, Bal SM,et al. Pulmonary delivery of DNA encoding Mycobacterium tuberculosis latency antigen Rv1733c associated to PLGA-PEI nanoparticles enhances T cell responses in a DNA prime/protein boost vaccination regimen in mice. Vaccine，2009，27(30)：4010-4017.