鸡Toll样受体21单克隆抗体的研制及其初步应用

Preparation and preliminary application of monoclonal antibodies against chicken Toll-like receptor 21

摘要:

【目的】研制鸡Toll样受体21(chTLR21)单克隆抗体，考察禽致病性大肠杆菌(APEC)及高致病性禽流感病毒(HPAIV)感染鸡组织中的chTLR21蛋白的表达情况。【方法】以人工合成的多肽免疫原chTLR21(203–225 aa)与KLH偶联蛋白免疫6周龄BALB/c小鼠，以多肽免疫原chTLR21(203–225 aa)与BSA偶联蛋白作为检测抗原，通过间接ELISA技术筛选阳性杂交瘤细胞株，同时，采用间接免疫荧光技术(IFA)检测单克隆抗体的荧光反应性；上述单克隆抗体用于chTLR21在鸡巨噬细胞(HD11)中的定位；在APEC O1血清型E516菌株、HPAIV H5N6亚型毒株感染35日龄SPF鸡模型中，应用获得的单克隆抗体以免疫印迹(Western blotting)和免疫组化(IHC)方法检测感染鸡组织中chTLR21的表达情况。【结果】建立了间接ELISA检测方法，确定最适抗原包被浓度为2.5 μg/mL，最适血清稀释度为1:6 400。经ELISA、IFA筛选，获得4株阳性杂交瘤细胞株，分别命名为1G3、2C10、3B6和4F11。上述单克隆抗体亚类分别为IgG2b、IgG1、IgG2b和IgG2a型，其中3B6具有较强的荧光反应性；制备单克隆抗体3B6腹水，其ELISA效价为1:102 400；IFA结果表明，chTLR21存在于HD11细胞质内；APEC感染鸡组织中chTLR21 Western blotting和IHC检测结果表明，感染鸡肝、脾、肺、肾、法氏囊中chTLR21表达量显著上升，而胰腺中表达量显著下降；HPAIV H5N6感染鸡上述组织中chTLR21的表达量均未见明显变化。【结论】本研究成功研制出针对鸡chTLR21受体的单克隆抗体，单克隆抗体3B6可用于检测鸡体内chTLR21表达情况，为chTLR21在病原体感染及天然免疫应答中的机制、功能研究提供了重要生物材料。

关键词:chTLR21;单克隆抗体;细胞定位;免疫印迹;免疫组化

Abstract:

[Objective] To develop monoclonal antibodies (MAbs) against chicken Toll-like receptor 21 (chTLR21) and determine the expression of chTLR21 in chicken tissues challenged by avian pathogenic Escherichia coli (APEC) or highly pathogenic avian influenza virus (HPAIV). [Methods] The 6-week-old BALB/c mice were immunized with the synthetic polypeptide chTLR21 (203-225 aa) conjugated with keyhole limpet hemocyanin (KLH). The chTLR21 (203-225 aa) conjugated with bovine serum albumin (BSA) was employed as the coating protein in indirect enzyme-linked immunosorbent assay (ELISA). The positive hybridoma cells were screened by indirect ELISA. The MAbs selected by ELISA were detected by indirect immunofluorescence assay (IFA) and then used to trace the localization of chTLR21 in chicken macrophages (HD11) and determine the expression of chTLR21 in tissues of the chickens infected with APEC O1 serotype E516 strain and HPAIV H5N6 strain. [Results] An indirect ELISA method was established, with the optimal antigen coating concentration of 2.5 μg/mL and serum dilution of 1:6 400. Four positive hybridoma cell strains were obtained and named 1G3, 2C10, 3B6, and 4F11, which belonged to the IgG2b, IgG1, IgG2b, and IgG2a subclasses, respectively. The MAb 3B6 had a robust fluorescence reactivity. The results of IFA demonstrated that chTLR21 was localized in the cytoplasm of HD11 cells. Compared with that in mock birds, the expression of chTLR21 was up-regulated in liver, spleen, lung, kidney, and bursa of Fabricius while down-regulated in pancreas of the 35-day-old specific pathogen-free (SPF) chickens infected with APEC E516 strain while exhibited no changes in the tissues of the chickens challenged with HPAIV H5N6 strain. [Conclusion] We developed the MAbs against chTLR21, among which the MAb 3B6 can be used to probe the localization and expression of chTLR21 in both chickens and macrophages, providing a tool for studying the effect of chTLR21 on the pathogenesis of bacterial or viral infections in chicken.

Key words:chTLR21;monoclonal antibody;subcellular localization;Western blotting;immunohistochemistry