三疣梭子蟹PtToll-1受体的原核表达及组织、细胞分布研究

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PtToll-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。SDS-PAGE结果表明, 体外诱导表达重组蛋白rPtToll-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 kD; Western-Blot分析表明, 小鼠抗血清能与rPtToll-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PtToll-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PtToll-1完整蛋白的真核表达载体pEGFP-N2-PtToll-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, PtToll-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PtToll-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, pEGFP-PtToll-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。

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

Toll like receptors known as pathogen-associated molecular pattern (PAMPs) recognize microbe-specific immune elicitors and play fundamental roles in pathogen recognition and the activation of innate immunity. To better understand the functions of PtToll-1 in Portunus trituberculatus, the ectodomain of PtToll-1 was sequenced and subcloned into the expression vector, and the expressed recombinant ectodomain of PtToll-1 was obtained. The polyclonal antibody against rPtToll-1 was obtained from immunized mice using a conventional method. Results of SDS-PAGE showed that the recombinant protein had a molecular mass of 87.18 kD and it was predominantly expressed in the insoluble fraction. Moreover, western-blot analysis revealed that antiserum could specifically react with the rPtToll-1. The PtToll-1 were expressed in the tissues including intestine, hepatopancreas, gills, heart and muscle with high expression in gills and intestine. The PtToll-1 was mainly located in the connective tissue of digestive tract and gland, epithelial cells of the gill and hepatopancreatic tuble. Cell immunofluorescence and immunohistochemistry staining showed that PtToll-1 was mainly located in the membrane of the crab hemoctyes. Furthermore, the green fluorescence of the PtToll-1-GFP fusion protein was also located in the membrane of the HEK293 cells. These findings will help to better understand potential immune functions of the PtToll-1.