本文關(guān)鍵詞： 結(jié)核分枝桿菌 亞單位疫苗 融合蛋白 佐劑　出處：《蘭州大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：1.結(jié)核分枝桿菌ESAT6-RpfE亞單位疫苗的構(gòu)建與疫苗免疫原性研究 目的：構(gòu)建結(jié)核分枝桿菌ESAT6-RpfE(早期分泌抗原靶抗原-復(fù)活促進(jìn)因子E)的原核表達(dá)載體,表達(dá)和純化融合蛋白,并對(duì)其免疫原性進(jìn)行研究。 方法：采用PCR方法分別以結(jié)核分枝桿菌H37Rv及臨床株的基因組DNA為模板,擴(kuò)增esat6、rpfe基因,依次克隆入載體pET30a中,將測(cè)序正確的原核表達(dá)載體轉(zhuǎn)化到E. coli BL21(DE3)宿主菌,通過(guò)IPTG誘導(dǎo)表達(dá)蛋白。經(jīng)過(guò)處理后的ESAT6-RpfE蛋白進(jìn)行IEX (DEAE)陰離子交換柱層析,HIC (Octyl FF)疏水層析兩步純化。將蛋白樣品與佐劑二甲基三十六烷基胺(dimethyl-dioctyldecyl ammonium bromide, DDA)進(jìn)行混合制備亞單位疫苗。對(duì)C57BL/6小鼠,進(jìn)行腹股溝皮下免疫,間隔三周,免疫三次,末次免疫8周后,小鼠眼球取血處死后,無(wú)菌取脾,分離脾淋巴細(xì)胞后,通過(guò)ELISA方法檢測(cè)脾淋巴細(xì)胞中分泌抗原特異性IFN-γ的水平；檢測(cè)血清中針對(duì)蛋白的特異性IgG2b. IgG1水平。 結(jié)果：PCR擴(kuò)增的esat6、rpfe基因序列與GenBank報(bào)道一致；融合蛋白主要以包涵體形式表達(dá)；ESAT6-RpfE組,體外脾細(xì)胞受RpfE刺激時(shí)分泌IFN-γ分泌水平顯著高于BCG組(p0.05)；亦明顯高于PBS組(p0.01)。同時(shí)ESAT6-RpfE免疫組能誘導(dǎo)產(chǎn)生特異性抗體。 結(jié)論：成功構(gòu)建并表達(dá)ESAT6-RpfE融合蛋白。此融合蛋白可在C57BL/6小鼠中誘導(dǎo)抗原特異性的免疫應(yīng)答,可作為新型結(jié)核亞單位疫苗的候選疫苗以進(jìn)一步研究。 2.新型結(jié)核亞單位疫苗的有效性評(píng)價(jià) 目的：對(duì)已有的四種融合蛋白ESAT6-Ag85B、TB10.4-Ag85B、 ESAT6-TB8.4、ESAT6-RpfE聯(lián)合佐劑進(jìn)行免疫原性及保護(hù)力評(píng)價(jià)。 方法：將融合蛋白ESAT6-Ag85B、TB10.4-Ag85B、ESAT6-TB8.4、 ESAT6-RpfE與佐劑DPG[DDA+poly(I:C)+明膠]混合,制備成亞單位疫苗。設(shè)立BCG和磷酸緩沖液(PBS)免疫組為對(duì)照。不同融合蛋白構(gòu)成的亞單位疫苗強(qiáng)化免疫小鼠三次,間隔三周。末次加強(qiáng)免疫10周后,通過(guò)ELISPOT方法檢測(cè)脾細(xì)胞分泌抗原特異性IFN-γ的水平。同時(shí)以H37Rv毒株攻擊被免疫小鼠。疫苗免疫小鼠感染6周后,檢測(cè)小鼠體內(nèi)結(jié)核菌載量,并分析肺組織病理切片。分析ESAT6-Ag85B、TB10.4-Ag85B、ESAT6-TB8.4、ESAT6-RpfE聯(lián)合佐劑亞單位疫苗的免疫保護(hù)效應(yīng)。 結(jié)果：四種亞單位疫苗免疫小鼠三次,均可產(chǎn)生較強(qiáng)的抗原特異性IFN-γ;肺部菌落計(jì)數(shù)表明四個(gè)亞單位疫苗免疫小鼠后,肺部荷菌量都較PBS組低；肺組織損傷顯示除ESAT6-Ag85B外,其他各疫苗組肺組織病理?yè)p傷均較PBS組輕。 結(jié)論：本實(shí)驗(yàn)室已構(gòu)建的四種融合蛋白(ESAT6-Ag85B、TB10.4-Ag85B、 ESAT6-TB8.4、ESAT6-RpffE)具有較強(qiáng)的免疫原性,并對(duì)小鼠感染結(jié)核分枝桿菌有一定的免疫保護(hù)效應(yīng)。
[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- 緯 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- 緯 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- 緯 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- 緯. 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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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相關(guān)期刊論文 前1條

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本文編號(hào)：1480210