He Yan, Zhu Wentao, Xu Tianbing, Liao Zhiwei, Su Jianguo
Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, 430070, China; Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology, Qingdao, 266237, China; Hubei Hongshan Laboratory, Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt, Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China.
Department of Aquatic Animal Medicine, College of Fisheries, Huazhong Agricultural University, Wuhan, 430070, China.
Fish Shellfish Immunol. 2022 Apr;123:314-323. doi: 10.1016/j.fsi.2022.03.009. Epub 2022 Mar 16.
Thrombocytes are an important component in peripheral blood cells and play a crucial role in immune regulation. CD41 is one of the biomarkers of thrombocytes. In this study, grass carp (Ctenopharyngodon idella) CD41 protein was expressed in Escherichia coli and purified by affinity chromatography. Subsequently, New Zealand rabbits were immunized with this protein via subcutaneous injection. The antibody titer examined by enzyme linked immunosorbent assay was 1:12800. The concentration of rabbit polyclonal antibody purified by HiTrap-rprotein-AFF affinity chromatography column was 1.9 mg/mL. The specificity was identified by SDS-PAGE, Western blot, flow cytometry, and indirect immunofluorescence assays. The purified antibody was used to screen grass carp thrombocytes, and CD41 cells were 14.13%. CD41 cells were further verified by Giemsa staining, transmission electron microscopy and RT-PCR. mRNA expression of CD41 in thrombocytes was not affected by viral or bacterial challenge in vitro, while CD41 transcripts were remarkably induced post pathogenic infections in vivo, which results from the immature hematopoietic stem cells and thrombocytes. Indirect immunofluorescence assay revealed that grass carp reovirus (GCRV) could not invade thrombocytes; however, mRNA expressions of some representative innate immune genes (IFN1, IL-1β, TNFα and Mx2) were significantly up-regulated post GCRV challenge. Meanwhile, the transcripts of some innate immune genes (IL-6 and TNFα) were swiftly increased post bacterial infection. These results indicated that the rabbit anti-CD41 polyclonal antibody possesses good specificity and can effectively bind to the CD41 protein on the surface of grass carp thrombocytes. Grass carp thrombocytes participate in immune regulation in viral and bacterial infections.
血小板是外周血细胞的重要组成部分，在免疫调节中发挥关键作用。CD41是血小板的生物标志物之一。在本研究中，草鱼（Ctenopharyngodon idella）CD41蛋白在大肠杆菌中表达并通过亲和层析纯化。随后，通过皮下注射用该蛋白免疫新西兰兔。用酶联免疫吸附测定法检测的抗体效价为1:12800。通过HiTrap-rprotein-AFF亲和层析柱纯化的兔多克隆抗体浓度为1.9 mg/mL。通过SDS-PAGE、蛋白质免疫印迹、流式细胞术和间接免疫荧光测定法鉴定其特异性。纯化的抗体用于筛选草鱼血小板，CD41阳性细胞为14.13%。通过吉姆萨染色、透射电子显微镜和RT-PCR进一步验证CD41阳性细胞。体外病毒或细菌攻击不影响血小板中CD41的mRNA表达，而在体内致病性感染后CD41转录本显著诱导，这是由于造血干细胞和血小板不成熟所致。间接免疫荧光测定显示草鱼呼肠孤病毒（GCRV）不能侵入血小板；然而，在GCRV攻击后一些代表性先天免疫基因（IFN1、IL-1β、TNFα和Mx2）的mRNA表达显著上调。同时，在细菌感染后一些先天免疫基因（IL-6和TNFα）的转录本迅速增加。这些结果表明兔抗CD41多克隆抗体具有良好的特异性，能有效结合草鱼血小板表面的CD41蛋白。草鱼血小板参与病毒和细菌感染中的免疫调节。
