三疣梭子蟹PtToll-1受体的原核表达及组织、细胞分布研究  

PROKARYOTIC EXPRESSION OF THE TOLL RECEPTOR PTTOLL-1 AND ITS CELLULAR DISTRIBUTION IN PORTUNUS TRITUBERCULATUS

作　　者：张丹枫[1] 王国良[1] 杨宁 张鑫[1] 周素明[1] 刘顺[1] ZHANG Dan-Feng;WANG Guo-Liang;YANG Ning;ZHANG Xin;ZHOU Su-Ming;LIU Shun(Key Laboratory of Applied Marine Biotechnology, Ministry of Education, School of Marine Science, Ningbo University, Ningbo Key Laboratory of Applied Marine Biotechnology, Ministry of Education, School of Marine Science, Ningbo University, Ningbo;Marine Science and Engineering College, Qingdao Agricultural University, Qingdao 266109, China)

机构地区：[1]宁波大学应用海洋生物技术教育部重点实验室,宁波315211 [2]青岛农业大学海洋科学与工程学院,青岛266109

出　　处：《水生生物学报》2018年第2期300-306,共7页Acta Hydrobiologica Sinica

基　　金：国家自然科学基金(41476124);浙江省自然科学基金(LY17C190005);宁波市科技计划项目(2016C10037);宁波大学"水产"浙江省重中之重开放基金(xkzsc1404);浙江省大学生科技创新活动计划暨新苗人才计划(2016R405043)资助~~

摘　　要：为深入研究三疣梭子蟹Toll受体的功能,在前期工作的基础上构建了Pt Toll-1胞外结构域原核表达载体,并成功获得其重组蛋白。将获得的重组蛋白免疫小鼠获得抗此重组蛋白免疫抗血清用于后续研究。SDSPAGE结果表明,体外诱导表达重组蛋白r Pt Toll-1以包涵体的形式出现在大肠杆菌裂解液的沉淀中,分子量大小约为87.18 k D;Western-Blot分析表明,小鼠抗血清能与r Pt Toll-1特异性结合。利用免疫荧光及细胞免疫化学方法对三疣梭子蟹Pt Toll-1在消化道、肝胰腺、鳃、心脏和肌肉等组织及血淋巴细胞中的分布进行研究。此外成功构建Pt Toll-1完整蛋白的真核表达载体p EGFP-N2-Pt Toll-1,并转染HEK293T细胞研究其在哺乳动物细胞HEK293中的表达。组织免疫荧光结果显示,Pt Toll-1在三疣梭子蟹消化道、肝胰腺、鳃、心脏及肌肉等组织中均有分布,但在鳃及消化道的组织结构中表达较强;细胞免疫荧光及免疫化学结果均表明Pt Toll-1主要分布在血淋巴的细胞膜上;激光共聚焦显微技术观察发现,p EGFP-Pt Toll-1融合蛋白也主要在HEK-293T细胞膜上表达。研究结果将为三疣梭子蟹Toll受体蛋白的免疫学功能的研究奠定基础。Toll like receptors known as pathogen-associated molecular pattern(PAMPs)recognize microbe-specific immuneelicitors and play fundamental roles in pathogen recognition and the activation of innate immunity.To better understandthe functions of PtToll-1in Portunus trituberculatus,the ectodomain of PtToll-1was sequenced and subclonedinto the expression vector,and the expressed recombinant ectodomain of PtToll-1was obtained.The polyclonalantibody against rPtToll-1was obtained from immunized mice using a conventional method.Results of SDS-PAGEshowed that the recombinant protein had a molecular mass of87.18kD and it was predominantly expressed in the insolublefraction.Moreover,western-blot analysis revealed that antiserum could specifically react with the rPtToll-1.The PtToll-1were expressed in the tissues including intestine,hepatopancreas,gills,heart and muscle with high expressionin gills and intestine.The PtToll-1was mainly located in the connective tissue of digestive tract and gland,epithelialcells of the gill and hepatopancreatic tuble.Cell immunofluorescence and immunohistochemistry stainingshowed that PtToll-1was mainly located in the membrane of the crab hemoctyes.Furthermore,the green fluorescenceof the PtToll-1-GFP fusion protein was also located in the membrane of the HEK293cells.These findings will help to better understand potential immune functions of the PtToll-1.

关 键 词：三疣梭子蟹 TOLL受体 胞外结构域 原核表达 组织分布 真核表达 细胞定位 

分 类 号：Q344.1[生物学—遗传学]