【作者】 张辉； 文科； 黄金林； 胡茂志； 申峻松； 潘志明； 焦新安；

【Author】 ZHANG Hui~1,WEN Ke~1,HUANG Jin-lin~1,HU Mao-zhi~(1,2),SHEN Jun-song~1, PAN Zhi-ming~1,JIAO Xin-an~(1*) (1.Jiangsu Key Laboratory of Zoonosis,Yangzhou University;2.Testing Center of Yangzhou University, Jiangsu 225009,China)

【机构】 扬州大学江苏省人兽共患病学重点实验室； 扬州大学测试中心；

【摘要】 目的:为研究嵌合表达结核分枝杆菌(MTB)抗原ESAT-6的鞭毛蛋白的免疫佐剂效应。方法: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overlap 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/esat。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。将质粒pET-fliC/esat及pBCX-esat分别转化大肠杆菌BL21(DE3),以IPTG诱导融合表达的嵌合蛋白fliC/esat及ESAT-6蛋白。以ESAT-6单克隆抗体HYB 076-08为一抗通过Western-blotting鉴定嵌合蛋白fliC/esat及ESAT-6蛋白。将两种蛋白分别在体外刺激髓系树突状细胞(BMDC),分析共刺激分子CD40、CD80、CD86和CD54表达情况,同时检测前炎性因子IL-12p70的表达水平。此外,将两种蛋白分别免疫C57BL/6小鼠,分析ESAT-6特异的IFN-γ及IL-4分泌细胞产生情况。结果:嵌合蛋白及ESAT-6蛋白均以可溶性表达,分别约为64KD和39kD。Western-blotting结果显示嵌合蛋白及ESAT-6蛋白均具有良好的反应原性。与ESAT-6蛋白相比较,嵌合蛋白在体外能诱导BMDC成熟。IL-12p70检测结果显示,嵌合蛋白诱导分泌的IL-12p70高于ESAT-6蛋白组,差异显著。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,能够有效上调BMDC共刺激分子表达,增强ESAT-6的特异性细胞免疫应答,以嵌合形式表达的鞭毛蛋白对ESAT-6抗原具有Th1型佐剂作用,更多还原

【Abstract】 AIM:To define the role of ESAT-6 chimeric flagellin in TB immunology.METHODS:The coding genes of flagellin of Salmonella typhimurium and ESAT-6 of Mycobacterium tuberculosis were cloned and identified by PCR and sequencing,respectively. Chimeric flagellin gene fliC/esat were 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 plasmid pET-fliC/esat,pET-fliC and pBCX-esat were constructed and transformed into E.coli BL21(DE3) and induced by IPTG.The expressed proteins fliC/esat and ESAT-6 were identified by Western-blotting assay using specific monoclonal antibody(mAb) HYB076-08.Bone marrow dendritic cells(BMDC) were in vitro stimulated by fliC/esat and ESAT-6 protein,analyzed their expression levels of CD40,CD80,CD86 and CD54 molecules,and determined the secreted IL-12p70 of triggered ones using ELISA.Moreover,C57BL/6 mice were immunized intravenously with fliC/esat or ESAT-6 protein and the specific IFN-γ-secreting cells and IL-4-secreting cells from immunized mice were detected by ELISPOT assay using ESAT-6 peptide as stimulus.RESULTS:The results showed that the protein fliC/esat and ESAT-6 were expressed as soluble,with the sizes of 64kD and 39kD respectively.Western-blotting analysis showed that both proteins could react with the specific mAb against ESAT-6.BMDC maturation was triggered by the chimeric flagellin fliC/esat.In contrast, ESAT-6 protein alone didn’t activate BMDC.IL-12p70 was also detected in the superantants of BMDC.The results showed that the chimeric flagellin fliC/esat be able to induce significantly higher level of the secreted IL-12p70 than that of ESAT-6 protein. Furthermore,it was shown that the chimeric flagellin fliC/esat could significantly enhance the Th1-biased immune responses against ESAT-6 in the immunized C57BL/6 mice.Conclusion:The chimeric flagellin can present Thl type adjuvant activity for ESAT-6 protein.更多还原