抗chTLR3单克隆抗体制备及其初步应用  

Preparation of Monoclonal Antibody against chTLR3 and Its Preliminary Application

作　　者：唐泽群 罗海燕[1,2] 葛铭 郭荣 许丹蕾 张瑞莉 TANG Zequn;LUO Haiyan;GE Ming;GUO Rong;XU Danlei;ZHANG Ruili(College of Veterinary Medicine,Northeast Agriculture University,Harbin 150030,China;Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine,Harbin 150030,China)

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030 [2]黑龙江省实验动物与比较医学重点实验室,哈尔滨150030

出　　处：《畜牧兽医学报》2019年第6期1292-1300,共9页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(31272533)

摘　　要：Toll样受体3(TLR3)是激发机体天然免疫的模式识别受体,为了制备能与天然结构的鸡TLR3(chTLR3)蛋白结合的单克隆抗体,将前期构建的可以在细胞内表达的真核重组质粒pVAX1-Igk-chTLR3免疫BALB/c小鼠,采用杂交瘤技术制备抗chTLR3单克隆抗体;并应用该抗体采用Western blot、流式细胞术及间接免疫荧光技术检测镉中毒雏鸡外周血淋巴细胞与脾内chTLR3的表达变化。结果发现,获得1株阳性杂交瘤细胞,该细胞株单个细胞染色体数量为(94±2)条,细胞上清效价为1∶25,抗体亚类鉴定为IgG3,细胞上清能够与原核表达的chTLR3胞外区蛋白及鸡外周血淋巴细胞中的chTLR3特异性结合。应用此抗体采用Western blot、流式细胞术及间接免疫荧光技术均能够在对照组和染镉组雏鸡外周血淋巴细胞及脾内检测到chTLR3的蛋白表达。结果表明,成功制备了具有天然活性的chTLR3单克隆抗体,该抗体可用于Western blot、间接免疫荧光技术及流式细胞术研究chTLR3在鸡组织细胞的分布定位、表达变化,为示踪chTLR3蛋白分布、表达及研究其在鸡发病机制中的作用提供了重要的物质基础。Toll-like receptor 3 (TLR3) is a pattern recognition receptor that stimulates innate immunity. In order to prepare a monoclonal antibody that can bind to chicken TLR3 (chTLR3) protein with natural structure,BALB/c mice were immunized with recombinant plasmid pVAX1-Igk-chTLR3,which could be expressed in cells,and monoclonal antibodies against chTLR3 were prepared by hybridoma technique. The expression of chTLR3 expression of chTLR3 in peripheral blood lymphocytes and spleen of cadmium poisoned chickens were detected by Western blot,flow cytometry and indirect immunofluorescence. The results showed that a positive hybridoma cell line was obtained. The number of chromosomes of the cell line was 94±2,the titer of the cell supernatant was 1∶2 5,and the antibody subclass was identified as IgG3. The cell supernatant not only specifically bound to the prokaryotic expression protein of chTLR3,but also bound to chTLR3 protein in chicken peripheral blood lymphocytes. Western blot,flow cytometry and indirect immunofluorescence were used to detect the expression of chTLR3 in peripheral blood lymphocyte and spleen of chickens in control group and cadmium-exposed group. The results indicated that the monoclonal antibody against chTLR3 with natural activity was successfully prepared. The antibody can be used to study the distribution,localization and expression of chTLR3 in chicken tissues and cells by Western blot,indirect immunofluorescence and flow cytometry. It provides an important material basis for tracing the distribution and expression of chTLR3 protein and its role in the pathogenesis of chicken diseases.

关 键 词：chTLR3 单克隆抗体 WESTERN BLOT 间接免疫荧光 流式细胞术 

分 类 号：Q813[生物学—生物工程]