四种结核亚单位疫苗的制备和有效性评价

发布时间：2018-01-31 22:56

本文关键词： 结核分枝杆菌亚单位疫苗融合蛋白佐剂　出处：《兰州大学》2012年硕士论文　论文类型：学位论文

【摘要】：1.结核分枝杆菌ESAT6-RpfE亚单位疫苗的构建与疫苗免疫原性研究目的：构建结核分枝杆菌ESAT6-RpfE(早期分泌抗原靶抗原-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：采用PCR方法分别以结核分枝杆菌H37Rv及临床株的基因组DNA为模板,扩增esat6、rpfe基因,依次克隆入载体pET30a中,将测序正确的原核表达载体转化到E. coli BL21(DE3)宿主菌,通过IPTG诱导表达蛋白。经过处理后的ESAT6-RpfE蛋白进行IEX (DEAE)阴离子交换柱层析,HIC (Octyl FF)疏水层析两步纯化。将蛋白样品与佐剂二甲基三十六烷基胺(dimethyl-dioctyldecyl ammonium bromide, DDA)进行混合制备亚单位疫苗。对C57BL/6小鼠,进行腹股沟皮下免疫,间隔三周,免疫三次,末次免疫8周后,小鼠眼球取血处死后,无菌取脾,分离脾淋巴细胞后,通过ELISA方法检测脾淋巴细胞中分泌抗原特异性IFN-γ的水平；检测血清中针对蛋白的特异性IgG2b. IgG1水平。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；ESAT6-RpfE组,体外脾细胞受RpfE刺激时分泌IFN-γ分泌水平显著高于BCG组(p0.05)；亦明显高于PBS组(p0.01)。同时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以进一步研究。 2.新型结核亚单位疫苗的有效性评价目的：对已有的四种融合蛋白ESAT6-Ag85B、TB10.4-Ag85B、 ESAT6-TB8.4、ESAT6-RpfE联合佐剂进行免疫原性及保护力评价。方法：将融合蛋白ESAT6-Ag85B、TB10.4-Ag85B、ESAT6-TB8.4、 ESAT6-RpfE与佐剂DPG[DDA+poly(I:C)+明胶]混合,制备成亚单位疫苗。设立BCG和磷酸缓冲液(PBS)免疫组为对照。不同融合蛋白构成的亚单位疫苗强化免疫小鼠三次,间隔三周。末次加强免疫10周后,通过ELISPOT方法检测脾细胞分泌抗原特异性IFN-γ的水平。同时以H37Rv毒株攻击被免疫小鼠。疫苗免疫小鼠感染6周后,检测小鼠体内结核菌载量,并分析肺组织病理切片。分析ESAT6-Ag85B、TB10.4-Ag85B、ESAT6-TB8.4、ESAT6-RpfE联合佐剂亚单位疫苗的免疫保护效应。结果：四种亚单位疫苗免疫小鼠三次,均可产生较强的抗原特异性IFN-γ;肺部菌落计数表明四个亚单位疫苗免疫小鼠后,肺部荷菌量都较PBS组低；肺组织损伤显示除ESAT6-Ag85B外,其他各疫苗组肺组织病理损伤均较PBS组轻。结论：本实验室已构建的四种融合蛋白(ESAT6-Ag85B、TB10.4-Ag85B、 ESAT6-TB8.4、ESAT6-RpffE)具有较强的免疫原性,并对小鼠感染结核分枝杆菌有一定的免疫保护效应。
[Abstract]:1. Construction and immunogenicity of Mycobacterium tuberculosis ESAT6-RpfE subunit vaccine Objective: to construct the prokaryotic expression vector of Mycobacterium tuberculosis ESAT6-RpfE (early secretory antigen target antigen-resurrection promoting factor E) and to express and purify the fusion protein. Its immunogenicity was studied. Methods: using the genomic DNA of Mycobacterium tuberculosis H37Rv and clinical strains as templates, the EST 6 rpfe gene was amplified by PCR and cloned into the vector pET30a in turn. The correctly sequenced prokaryotic expression vector was transformed into E. coli BL21 (DE3) host strain. The expressed protein was induced by IPTG. The treated ESAT6-RpfE protein was subjected to IEX DEAE anion exchange column chromatography. HIC Octyl FFF was purified by hydrophobic chromatography. The protein was purified with the adjuvant dimethyl 36 alkylamine (Dimethyl 36 alkylamine). Dimethyl-dioctyldecyl ammonium bromide. C57BL / 6 mice were immunized subcutaneously in groin for three weeks, three times after the last immunization, and the mice were killed after 8 weeks of last immunization. The level of antigen-specific IFN- 纬 in splenic lymphocytes was detected by ELISA method. The specific IgG 2 b. IgG1 level in serum was detected. Results the sequence of esat6 rpfe gene amplified by PCR was consistent with that reported by GenBank. The fusion protein was mainly expressed in the form of inclusion body. In ESAT6-RpfE group, the level of IFN- 纬 secreted by splenocytes stimulated by RpfE in vitro was significantly higher than that in BCG group (P 0.05). It was also significantly higher than that of PBS group (P 0.01), and ESAT6-RpfE immunized group could induce specific antibody. Conclusion: ESAT6-RpfE fusion protein was successfully constructed and expressed, which can induce antigen-specific immune response in C57BL / 6 mice. It can be used as a candidate vaccine for new TB subunit vaccine for further study. 2. Evaluation of the effectiveness of new TB subunit vaccines Objective: to identify four fusion proteins, TB10.4-Ag85B, ESAT6-TB8.4. The immunogenicity and protective power of ESAT6-RpfE combined with adjuvant were evaluated. Methods: the fusion protein ESAT6-Ag85BH4, ESAT6-TB8.4, ESAT6-RpfE and adjuvant DPG were prepared. [DDA polyI: C) gelatin. The subunit vaccine was prepared. The mice were immunized with BCG and phosphoric acid buffer. The mice were immunized with subunit vaccine consisting of different fusion proteins for three times. The interval was 3 weeks. 10 weeks after the last booster immunization. The levels of antigen-specific IFN- 纬 secreted by splenocytes were detected by ELISPOT method. The mice were immunized with H37Rv strain. The mice were inoculated with the vaccine for 6 weeks. The amount of tuberculous bacilli in mice was measured, and the pathological sections of lung tissue were analyzed, and the ESAT6-TB8.4 of TB10.4-Ag85B-1 in ESAT6-Ag85B was analyzed. Immune protective effect of ESAT6-RpfE combined with adjuvant subunit vaccine. Results: four subunit vaccines were immunized for three times, all of them produced strong antigen-specific IFN- 纬. The count of pulmonary colony showed that after immunization with the four subunit vaccines, the amount of pulmonary mycorrhizal bacteria in mice was lower than that in PBS group. Lung tissue injury showed that the pathological injury of lung tissue in all vaccine groups except ESAT6-Ag85B was lighter than that in PBS group. Conclusion: four fusion proteins, TB10.4-Ag85B, ESAT6-TB8.4, have been constructed in our laboratory. ESAT6-RpffE has strong immunogenicity and has a protective effect on Mycobacterium tuberculosis infection in mice.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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