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

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,本实验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49.18 ku。Western-Blot分析表明,大鼠抗rPt-SRB血清能与rPt-SRB特异性结合。本实验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的分布进行研究。结果显示,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、鳃丝上皮细胞中亦有较强的阳性信号,表明PtSRB蛋白在上述组织结构中分布较广;在血淋巴细胞中,绿色荧光信号主要分布于细胞质和细胞膜,在细胞核中没有明显荧光信号,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。

Study on prokaryotic expression, cell and tissue distribution of the class B scavenger receptor in Portunustrituberculatus

Class B scavenger receptors are a subclass of scavenger receptors family that can 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 the functions of class B scavenger receptors in Portunus trituberculatus (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 49.18 ku 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 the tissue distribution and the cellular localization of Pt-SRB in this study. The results showed that the Pt-SRB were expressed in the tissues including intestine, hepatopancreas, gills, heart and muscle, but the Pt-SRB were located in different tissue structures. 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 hepatopancreatic tubules. In the crab hemocytes, the green fluorescent signals were located in the membrane and cytoplasm, but not in the nucleus, which suggested that Pt-SRB was localized both in membrane and cytoplasm in the crab hemocytes. Our findings will help us to better understand potential functions of Pt-SRB.