结核分枝杆菌Ag85B-ESAT-6融合蛋白重组耻垢分枝杆菌对小鼠的免疫原性研究

中国防痨杂志 ›› 2012, Vol. 34 ›› Issue (3): 145-149.

结核分枝杆菌Ag85B-ESAT-6融合蛋白重组耻垢分枝杆菌对小鼠的免疫原性研究

王平王丽梅张薇柏银兰康健郝彦斐罗泰来徐志凯

710032 西安，第四军医大学微生物学与病原生物学教研室［王平(研究生)、王丽梅、柏银兰、康健、郝彦斐、罗泰来、徐志凯］；解放军第一五○中心医院病理科（王平）；第四军医大学唐都医院儿科（张薇）

收稿日期:2011-11-26 出版日期:2012-03-10 发布日期:2012-05-03
通信作者: 徐志凯,王丽梅 E-mail:zhikaixu@fmmu.edu.cn, limwang@tom.com
基金资助:
“十二五”传染病重大专项（2012ZX10003008-007）

Immunogenicity of the recombinant M. smegmatis expressing Ag85B-ESAT-6 fusion protein of M. tuberculosis in mice

WANG Ping, WANG Li-mei, ZHANG Wei, BAI Yin-lan, KANG Jian, HAO Yan-fei, LUO Tai-lai, XU Zhi-kai

Department of Microbiology and Pathogenic Biology, the Fourth Military Medical University, Xi’an 710032, China（*Now in：Department of Clinical Laboratory, the 150 Central Hospital, Luoyang 471031, China）

Received:2011-11-26 Online:2012-03-10 Published:2012-05-03
Contact: XU Zhi-kai,WANG Li-mei E-mail:zhikaixu@fmmu.edu.cn, limwang@tom.com

摘要/Abstract

摘要： 目的评价表达Mtb Ag85B-ESAT-6融合蛋白的重组耻垢分枝杆菌（Ag85B-ESAT-6-rM.s）在小鼠中的免疫原性。方法 6周龄BALB/c小鼠经背部皮下注射1×10⁶ CFU（colony-forming unit，菌落形成单位）的Ag85B-ESAT-6-rM.s，同时设M.s免疫组（10只）、BCG免疫组（10只）、Ag85B-ESAT-6融合蛋白免疫组（10只）和生理盐水组（10只）。接种免疫后1～6周，尾静脉采血，检测小鼠外周血CD4⁺和CD8⁺ T细胞所占百分比；免疫后6周，MTS［3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt］法检测小鼠脾淋巴细胞增殖和酶联免疫斑点检测（enzyme-linked immunospot assay，ELISPOT）检测脾淋巴细胞分泌γ干扰素（interferon γ，IFN-γ）、白细胞介素-2（interleukin-2，IL-2）和白细胞介素-4（interleukin-4，IL-4）等细胞因子的水平。结果 Ag85B-ESAT-6-rM.s免疫小鼠后能够明显刺激小鼠外周血CD4⁺和CD8⁺ T细胞的产生，其所占百分比［（59.6±1.5）%］明显高于BCG免疫组［(48.8±2.3)%］（t=12.252，P<0.05）和原始M.s免疫组［(45.7±1.6)%］（t=20.166，P<0.05）。Ag85B-ESAT-6-rM.s免疫小鼠的脾淋巴细胞增殖指数（3.23±0.31）与BCG免疫刺激的增殖指数（2.95±0.36）相当（t=1.864，P>0.05），但其诱生IFN-γ的能力［斑点形成细胞(spot forming cells，SFC) ］［(167.5±36.6)SFC/10.6］明显高于BCG［(98.5±26.9)SFC/10⁶ ］（t=4.804，P<0.05）。结论 Ag85B-ESAT-6融合蛋白在耻垢分枝杆菌中的表达能够明显提高M.s的免疫原性，并能够刺激小鼠机体产生有利于抗Mtb感染的免疫反应，作为TB新型候选疫苗具有一定的研究前景。

关键词: 分枝杆菌, 耻垢, 重组融合蛋白质类, 结核菌苗, 免疫活性

Abstract: Objective To evaluate the immunogenicity of the recombinant M. smegmatis expressing Ag85B-ESAT-6 fusion protein of M. tuberculosis (Ag85B-ESAT-6-rM.s) in mice. Methods Six-week-old BALB/c mice were immunized by subcutaneous injection on the back with 1×10⁶ colony-forming unit (CFU) of Ag85B-ESAT-6-rM.s per mouse. The mice immunized by M. smegmatis (M.s), BCG, Ag85B-ESAT-6 fusion protein and saline used as control. The percentages of CD4⁺ and CD8⁺ T cells in the peripheral blood mononuclear cells (PBMC) of mice were detected once a week for six weeks after immunization. The proliferation of splenic lymphocytes of mice was assayed by MTS［3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt］, and the numbers of splenic lymphocytes secreting interferon γ (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4) were detected by enzyme-linked immunospot assay (ELISPOT) at 6 weeks after immunization. Results The Ag85B-ESAT-6-rM.s could significantly stimulate the generation of CD4⁺ and CD8⁺ T cells in the PBMC from immunized mice, and its percentages of CD4⁺ and CD8⁺ T cells ［(59.6±1.5)%］were significantly higher than that in BCG group ［(48.8±2.3)%］ (t=12.252, P<0.05) and M.s group ［(45.7±1.6)%］(t=20.166, P<0.05). Ag85B-ESAT-6-rM.s could stimulate the proliferation of splenic lymphocytes in immunized mice, and its stimulation index (3.23±0.31) was equivalent to that in BCG group (2.95±0.36). However, the level of IFN-γ in mice immunized by Ag85B-ESAT-6-rM.s ［(167.5±36.6) spot forming cells (SFC)/10⁶］was significantly higher than that in BCG group ［(98.5±26.9)SFC/10⁶ ］(t=4.804, P<0.05), and the level of IFN-γ was also higher than levels of IL-2 and IL-4 (t=6.174 and 6.449, P<0.05) in mice immunized by Ag85B-ESAT-6-rM.s. Conclusion The expression of Ag85B-ESAT-6 fusion protein in M. smegmatis could improve the immunogenicity, and Ag85B-ESAT-6-rM.s could induce immune response preventing M. tuberculosis infection in mice. Ag85B-ESAT-6-rM.s as a TB candidate vaccine is worthy of further study.

王平王丽梅张薇柏银兰康健郝彦斐罗泰来徐志凯. 结核分枝杆菌Ag85B-ESAT-6融合蛋白重组耻垢分枝杆菌对小鼠的免疫原性研究[J]. 中国防痨杂志, 2012, 34(3): 145-149.

WANG Ping, WANG Li-mei, ZHANG Wei, BAI Yin-lan, KANG Jian, HAO Yan-fei, LUO Tai-lai, XU Zhi-kai. Immunogenicity of the recombinant M. smegmatis expressing Ag85B-ESAT-6 fusion protein of M. tuberculosis in mice[J]. Chinese Journal of Antituberculosis, 2012, 34(3): 145-149.

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