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目的:研究嵌合形式表达的鞭毛蛋白对结核分枝杆菌(MTB)抗原ESAT-6的免疫佐剂效应。方法:用PCR方法扩增鼠伤寒沙门菌鞭毛蛋白基因fliCi及MTB抗原ESAT-6编码序列,通过重叠PCR将ESAT-6编码序列插入fliCi的高变区域,构建嵌合鞭毛基因片段fliC/esa。t将fliC/esat片段分别插入原核表达载体pET,构建pET-fliC/esat质粒。将ESAT-6编码序列插入原核表达载体pBCX的多克隆位点,构建原核表达质粒pBCX-esat。以质粒pET-fliC/esat及pBCX-esat分别转化大肠杆菌BL21(DE3),以异丙基1-1硫代-β呋喃半乳糖苷诱导融合的嵌合蛋白fliC/esat及ESAT-6蛋白的表达。以抗ESAT-6 mAb HYB 076-08为一抗,通过W estern b lot鉴定嵌合蛋白fliC/esat及ESAT-6蛋白。以两种蛋白分别在体外刺激骨髓树突状细胞(BMDCs),通过FACS分析共刺激分子CD40、CD80、CD86和CD54的表达,同时用ELISA检测前炎性因子IL-12p70表达的水平。此外,以两种蛋白分别免疫C57BL/6小鼠,运用ELISPOT法分析ESAT-6特异的IFNγ-及IL-4分泌细胞的产生。结果:嵌合蛋白及ESAT-6蛋白均可溶性表达,相对分子质量(Mr)约为64000和39000。Western blot的结果显示,fliC/esat嵌合蛋白及ESAT-6蛋白均具有良好的反应原性。与ESAT-6蛋白相比较,fliC/esat嵌合蛋白在体外能诱导BMDCs成熟。IL-12p70的检测结果显示,fliC/es-at嵌合蛋白诱导BMDCs分泌的IL-12p70明显高于ESAT-6蛋白诱导分泌的IL-12p70(P<0.01)。体内实验结果表明,嵌合鞭毛蛋白免疫组能够显著增强ESAT-6特异性免疫应答,且免疫应答趋于Th1型。结论:鞭毛嵌合表达ESAT-6抗原,能够有效地上调BMDCs共刺激分子表达,增强ESAT-6特异性细胞的免疫应答,以嵌合形式表达的鞭毛蛋白对ESAT-6抗原具有诱导Th1型免疫应答的佐剂效应。
Objective: To study the immune adjuvant effect of chimeric expressed flagellin on Mycobacterium tuberculosis (MTB) antigen ESAT-6. Methods: FliCi and ESAT-6 coding sequences of Salmonella typhimurium were amplified by PCR. The coding sequence of ESAT-6 was inserted into the hypervariable region of fliCi by overlapping PCR to construct the chimeric flagellin gene fliC / esa. The fliC / esat fragment was inserted into the prokaryotic expression vector pET to construct the plasmid pET-fliC / esat. The ESAT-6 coding sequence was inserted into the multi-cloning site of the prokaryotic expression vector pBCX to construct the prokaryotic expression plasmid pBCX-esat. Escherichia coli BL21 (DE3) was transformed with plasmids pET-fliC / esat and pBCX-esat respectively, and the fusion proteins fliC / esat and ESAT-6 were induced by isopropyl 1-1-thiogalactopyranoside expression. The anti-ESAT-6 mAb HYB 076-08 was used as primary antibody to identify the chimeric proteins fliC / esat and ESAT-6 protein by Western blot. Bone marrow dendritic cells (BMDCs) were stimulated with two proteins in vitro. The expression of co-stimulatory molecules CD40, CD80, CD86 and CD54 was analyzed by FACS. The expression of IL-12p70 was detected by ELISA. In addition, C57BL / 6 mice were separately immunized with two proteins, and the production of ESAT-6-specific IFNγ- and IL-4-secreting cells was analyzed using the ELISPOT method. Results: The chimeric protein and ESAT-6 protein were both soluble in E.coli. The relative molecular mass (Mr) was about 64000 and 39000. The results of Western blot showed that the fliC / esat chimeric protein and ESAT-6 protein all had good reactivity. Compared with ESAT-6 protein, fliC / esat chimeric protein can induce the maturation of BMDCs in vitro. The results of IL-12p70 showed that IL-12p70 induced by fliC / es-at chimeric protein induced BMDCs was significantly higher than IL-12p70 induced by ESAT-6 protein (P <0.01). In vivo experiments showed that the chimeric flagellin immunized group could significantly enhance the ESAT-6-specific immune response, and the immune response tended to Th1 type. Conclusion: Expression of ESAT-6 antigen by flagellar chimerism can effectively up-regulate the expression of costimulatory molecules of BMDCs and enhance the immune response of ESAT-6-specific cells. The flagellin expressed in chimeric form can induce Th1-type immunity to ESAT-6 antigen Adjuvant effect of response.