文章摘要
杨宁,周素明,王国良,刘顺,李猛.三疣梭子蟹B型清道夫受体蛋白的原核表达及细胞、组织分布研究[J].水产学报,2018,42(1):39~47
三疣梭子蟹B型清道夫受体蛋白的原核表达及细胞、组织分布研究
Study on prokaryotic expression, cell and tissue distribution of the class B scavenger receptor in Portunustrituberculatus
投稿时间:2016-11-07  修订日期:2017-03-23
DOI:10.11964/jfc.20161110605
中文关键词: 三疣梭子蟹  B型清道夫受体  原核表达  组织分布
英文关键词: Portunustrituberculatus  Class B scavenger receptor  prokaryotic expression  tissue distribution
基金项目:宁波市海洋蟹类产业科技创新团队项目(2011B81003);宁波大学“水产”浙江省重中之重开放基金(xkzsc1404)
作者单位E-mail
杨宁 宁波大学应用海洋生物技术教育部重点实验室 yangning04@163.com 
周素明 宁波大学应用海洋生物技术教育部重点实验室  
王国良 宁波大学应用海洋生物技术教育部重点实验室 wangguoliang@nbu.edu.cn 
刘顺 宁波大学应用海洋生物技术教育部重点实验室  
李猛 宁波大学应用海洋生物技术教育部重点实验室  
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中文摘要:
      B型清道夫受体是清道夫受体超家族成员,可识别多种配体,在机体脂类转运及免疫防御过程中发挥重要作用。为深入研究三疣梭子蟹B型清道夫受体(Pt-SRB)的功能,本研究在前期工作基础上构建了Pt-SRB胞外结构域原核表达载体,并成功获得了重组蛋白rPt-SRB。利用亲和层析方法获得纯化的rPt-SRB,并将纯化rPt-SRB蛋白免疫大鼠获得抗rPt-SRB重组蛋白免疫抗血清以用于后续研究。SDS-PAGE检测发现,体外诱导表达重组rPt-SRB以包涵体的形式出现在大肠杆菌BL21裂解液的沉淀中,分子量大小约为49.2kD。Western-Blot分析表明,大鼠抗rPt-SRB血清能与rPt-SRB特异性结合。本研究还利用免疫荧光技术,对Pt-SRB在三疣梭子蟹血淋巴细胞的定位及不同组织中分布进行研究。研究结果表明Pt-SRB在三疣梭子蟹消化道、肝胰腺、鳃、心脏、肌肉组织中均有分布,但不同组织中Pt-SRB的分布不同。免疫荧光结果显示绿色荧光信号在消化道及腺体结缔组织中较强,另外在肝小管上皮、鳃丝上皮细胞亦有较强的阳性信号,表明Pt-SRB蛋白在上述组织结构中分布较广;在三疣梭子蟹血淋巴细胞中,绿色荧光信号主要分布于细胞质和细胞膜,在细胞核没有明显荧光信号,提示Pt-SRB在三疣梭子蟹血淋巴细胞的细胞质和细胞膜上表达。本结果将为三疣梭子蟹B型清道夫受体蛋白的生理及免疫学功能的研究奠定基础。
英文摘要:
      Class B scavenger receptors are a subclass of scavenger receptors family that recognize a large repertoire of ligands, and they have been shown to have a wide range of functions in lipids metabolism and immune defence. To better understand thefunctions of class B scavenger receptors inPortunustrituberculatus (Pt-SRB), the extracellular loop of Pt-SRB was sequenced and subcloned into the expression vector, and the expressed recombinant rPt-SRB protein was obtained in present study. Then the rPt-SRB was purified by nickel-nitrilotriacetic acid chromatography, and the polyclonal antibody against rPt-SRB was obtained from an immunized rat using a conventional method. Results of SDS-PAGE showed that the recombinant protein had a molecular mass of 49.2kDa and was predominantly expressed in the insoluble fraction. Moreover, western-blot analysis revealed that antiserum could specifically react with the rPt-SRB. Furthermore, immunofluorescent staining technique was used to determine and tissue distribution the cellular localization of Pt-SRB in this study. The results showed that the Pt-SRBwere expressedinthe tissues including intestine, hepatopancreas, gills, heart and muscle, but the Pt-SRB located in differentStissueSstructures. The results displayed that the green fluorescent signals were mainly located in the connective tissue of digestive tract and gland, epithelial cells of the gill and hepatopancreatictuble. In the crab hemocytes, the green fluorescent signalswere located in the membrane and cytoplasm but not in the nucleus which suggested that thePt-SRB was localized both in membrane and cytoplasm in the crab hemocytes. Our findings will help us to better understand potential functions of the Pt-SRB.
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