结核分枝杆菌ESAT6-RpfE亚单位疫苗的构建与免疫原性研究

摘要/Abstract

摘要： 目的构建Mtb ESAT6-RpfE（早期分泌抗原靶6-复活促进因子E）的原核表达载体，表达和纯化融合蛋白，并对其免疫原性进行研究。方法分别以Mtb H37Rv及临床株的基因组为模板，PCR扩增esat6、rpfE基因，依次插入pET30a质粒并在大肠埃希菌BL21中表达纯化ESAT6-RpfE蛋白。采用完全随机的方法将C57BL/6小鼠分组，每组6只，共进行了两次实验，第一次实验分为ESAT6、RpfE、ESAT6RpfE、PBS、BCG五组,佐剂为二甲基三十六烷基胺（dimethyldioctyldecyl ammonium bromide,DDA）；第二次实验有ESAT6-RpfE、PBS、BCG三组，佐剂为DPG［DDA+poly(I:C)+明胶］。分别于0、3、6周皮下免疫C57BL/6小鼠。末次免疫8周后，用特异性抗原ESAT6及RpfE刺激，检测其分泌IFN-γ的水平；ELISA检测血清特异性抗体IgG2b、IgG1。结果 PCR扩增的esat6、rpfE基因序列与GenBank报道一致；融合蛋白主要以包涵体形式表达；亚单位疫苗免疫动物能够分泌RpfE特异性IFN-γ的脾淋巴细胞数量［(427.13±100.46)］显著高于PBS组(26.13±22.34；q=7.924，P＜0.001)；分泌ESAT6刺激特异性IFN-γ的脾淋巴细胞数量［(506.50±140.40)］明显高于PBS组(19.25±14.75；q=11.36，P＜0.001)和 BCG组(125.5±46.41；q=8.882, P＜0.001)。ESAT6-RpfE免疫组能诱导产生特异性抗体。结论成功构建并表达ESAT6-RpfE融合蛋白。此融合蛋白可在C57BL/6小鼠中诱导抗原特异性的免疫应答，可作为新型结核亚单位疫苗的候选疫苗予以进一步研究。

关键词: 疫苗, 亚单位, 重组融合蛋白质类, 细菌蛋白质类, 细胞因子类, 免疫活性

Abstract: Objective To construct and express Mycobacterium tuberculosis esat6-rpfE fused gene, and to purify the fusion protein and study its immunogenicity. Methods The esat6 and rpfE genes were amplified from the genomic DNA of Mycobacterium tuberculosis reference strain H37Rv and clinical stain respectively by PCR, and inserted into the plasmid pET30a. The fusion protein ESAT6-RpfE was expressed in E. coli BL21(DE3) and purified after two successive chromatographic purification steps. In the first experiment, the mice C57BL/6 were divided randomly into five groups named ESAT6, RpfE, ESAT6-RpfE, PBS and BCG, the adjuvant used was dimethyl-dioctyldecyl ammonium bromide (DDA). In the second experiment, the mice were divided randomly into three groups called ESAT6-RpfE, PBS and BCG, the adjuvants used were DPG ［DDA+poly(I:C)+Gelatin］. The mice were immunized subcutaneously at weeks 0, 3 and 6. The level of IFN-γ secreted by spleen lymphocytes stimulated by antigen ESAT6 or RpfE was detected at the 8th week after the last immunization. The antibodies IgG2b and IgG1 in the sera were dectected by ELISA. Results The esat6 and rpfE gene sequences cloned were consistent with GenBank report. The recombinant fusion protein ESAT6-RpfE was expressed in inclusion bodies. The counts of spleen lymphocytes secrecing RpfE-specific IFN-γ in the subunit vaccine group［ (427.13±100.46)］ were significantly higher than those in PBS group (26.13±22.34, q=7.924，P＜0.001), and the counts of spleen lymphocytes secrecing ESAT6-specific IFN-γ in the subunit vaccine group［ (506.50±140.40) ］were significantly higher than those in PBS group (19.25±14.75，q=11.36，P＜0.001) and BCG group (125.5±46.41，q=8.882, P＜0.001). The mice immunized by ESAT6-RpfE protein can induced antigenspcific antibody. Conclusion The recombinant fusion protein ESAT6-RpfE was successfully expressed and obtained. It can induce antigen-specific immune responses in C57BL/6 mice. ESAT6 and RpfE fusion protein can be used as a candidate of the new TB subunit vaccine to further study.

Key words: Vaccines, subunit, Recombinant fusion proteins, Bacterial proteins, Cytokines, Immunocompetence

李志达泽蛟章国平李瑞营刘万波苏娟张颖祝秉东. 结核分枝杆菌ESAT6-RpfE亚单位疫苗的构建与免疫原性研究[J]. 中国防痨杂志, 2012, 34(3): 154-160.

LI Zhi, DA Ze-jiao, ZHANG Guo-ping, LI Rui-ying, LIU Wan-bo, SU Juan, ZHANG Ying, ZHU Bing-dong. Construction and immunogenicity of Mycobacterium tuberculosis subunit vaccine ESAT6-RpfE[J]. Chinese Journal of Antituberculosis, 2012, 34(3): 154-160.

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