嵌合鞭毛蛋白fliC/esat佐剂效应的研究

The adjuvant effect of chimeric flagellin fliC/esat

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。

AIM：To determine the role of ESAT-6 chimeric flagellin in TB immunology.METHODS： The coding sequences of flagellin of Salmonella typhimurium and ESAT-6 of Mycobacterium tuberculosis were cloned by PCR and identified by sequencing,respectively.Chimeric flagellin gene fliC/esat was constructed by overlap PCR technique.The ESAT-6 coding fragment was inserted to the hypervariable region of Salmonella flagellin gene fliCi.And then prokaryotic exprssion plasmids of pET-fliC/esat,pET-fliC and pBCX-esat were constructed and transformed into E.coli BL21（DE3）,followed by induction of IPTG.The expressed proteins fliC/esat and ESAT-6 were identified by Western-blot assay using specific monoclonal antibody（mAb） HYB076-08.Bone marrow dendritic cells（BMDCs） were in vitro stimulated by fliC/esat and ESAT-6 proteins,and analyzed for the expression levels of CD40,CD80,CD86 and CD54 molecules.The secreted IL-12p70 was determined by ELISA.Moreover,C57BL/6 mice were immunized intravenously with fliC/esat or ESAT-6 protein.The specific IFN-γ-secreting cells and IL-4-secreting cells from the immunized mice were detected by ELISPOT assay using an ESAT-6 peptide as a stimulus.RESULTS： The results showed that the proteins of fliC/esat and ESAT-6 were expressed solubly,with the sizes of 64 kD and 39 kD respectively.Western blot analysis showed that both proteins reacted with the specific mAb against ESAT-6.BMDCs maturation was triggered by the chimeric flagellin fliC/esat.In contrast,ESAT-6 protein alone didn＇t activate BMDCs.IL-12p70 was also detected in the supernatants of BMDCs.The results showed that the chimeric flagellin fliC/esat induced significantly higher level of the secreted IL-12p70 than that of ESAT-6 protein.Furthermore,the chimeric flagellin fliC/esat significantly enhanced the Th1-biased immune responses against ESAT-6 in the immunized C57BL/6 mice.CONCLUSION： The chimeric flagellin we generated exerts Th1 type adjuvant activity for ESAT-6 protein.