旋毛虫P53ES基因重组蛋白单克隆抗体的制备和鉴定

发布时间：2018-02-24 09:08

本文关键词： 旋毛虫重组蛋白单克隆抗体排泄分泌抗原　出处：《东北农业大学》2009年硕士论文　论文类型：学位论文

【摘要】： 旋毛虫病是由旋毛形线虫(Trichinella spiralis)引起的一种人兽共患寄生虫病,主要寄生于猪、野猪、鼠、熊等150多种动物及人体。人体主要因生食或半生食含有旋毛虫肌幼虫囊包的猪肉或其他动物肉类而感染。由于其病原传播的复杂性,以致该病发现一百多年来,不仅没有得到完全有效的控制,而且发生范围反而逐渐扩大。要迅速、有效地控制和预防旋毛虫病的流行,必须有快速准确的检测技术。本研究选用旋毛虫P53ES基因重组蛋白免疫小鼠制备McAb,为进一步研制旋毛虫病诊断试剂盒提供理想、必备的试验材料。以旋毛虫P53ES基因的重组蛋白免疫BALB/c鼠,取小鼠脾细胞和骨髓瘤细胞SP2/0融合,经间接ELISA筛选和有限稀释法克隆,筛选分泌高滴度McAb杂交瘤细胞株,制备腹水。Southern Biotechnology Associates,Inc的SBA ClonotypingTMSystem/AP对单克隆抗体进行亚类鉴定;采用ELISA间接法测定杂交瘤细胞上清液和小鼠腹水效价;Western-blot检测单克隆抗体的特异性;测定杂交瘤细胞分泌抗体的稳定性;间接ELISA检测单克隆抗体与隐孢子虫、猪鞭虫、猪囊尾蚴囊液抗原、多头蚴囊液、日本血吸虫抗原是否存在交叉反应;间接免疫荧光方法检测单克隆抗体与肌幼虫的免疫学反应。结果表明:得到两株能稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为2H5和3D2;试剂盒鉴定两株McAb均属IgM亚类,其轻链均为κ链;腹水效价分别为1:24000和1:12000;Western-blot证实两株McAb均能与约53ku处的ES抗原反应,出现特异性条带;杂交瘤细胞株连续传20代,细胞生长良好,效价稳定;所得单抗与隐孢子虫、猪鞭虫、猪囊尾蚴囊液抗原、多头蚴囊液、日本血吸虫抗原均无交叉反应;间接免疫荧光试验结果表明单克隆抗体与肌幼虫发生免疫学反应。本研究成功制备了两株抗旋毛虫P53ES重组蛋白的单克隆抗体,为旋毛虫病的研究和研制旋毛虫病诊断试剂盒奠定了基础。
[Abstract]:Trichinella spiralis is a zoonotic parasitic disease caused by Trichinella spiralis. it is parasitic on pigs, wild boars, and mice. Bears and more than 150 species of animals and human bodies. The human body is mainly infected by raw or semi-raw food containing pork or other animal meat containing trichinella muscle larvae. Due to the complexity of the transmission of its pathogen, the disease has been discovered for more than 100 years. Not only has it not been completely effectively controlled, but the scope of its occurrence has gradually expanded. The epidemic of trichinellosis should be controlled and prevented quickly and effectively. In this study, McAbs were prepared by immunizing mice with recombinant protein of Trichinella spiralis P53ES gene, which provided an ideal and necessary test material for further development of diagnostic kit for trichinellosis. BALB/c mice were immunized with recombinant protein of Trichinella spiralis P53ES gene. Mouse spleen cells and myeloma cells were fused with SP2/0. The hybridoma cell lines secreting high titer McAb were screened by indirect ELISA screening and limited dilution method. The SBA ClonotypingTMSystem/AP of ascites, Southern Biotechnology Associates Inc., was prepared to identify the monoclonal antibody subclass, the specificity of monoclonal antibody was detected by ELISA indirect assay in the supernatant of the hybridoma cell and the mouse ascites titer, and the stability of the antibody secreted by the hybridoma cells was determined by Western-blot. Indirect ELISA was used to detect the cross reaction of monoclonal antibody with Cryptosporidium, Trichuris suis, Cysticercus cellulosae sac fluid, polycaria cyst fluid and Schistosoma japonicum antigen. Indirect immunofluorescence assay was used to detect the immunological reaction between monoclonal antibody and muscle larva. The results showed that two hybridoma cell lines which could secrete monoclonal antibody stably were named 2H5 and 3D2.The two McAb strains were identified as IgM subclasses by the kit. The ascites titers of 1: 24000 and 1: 12000 Western-blot showed that both McAb could react with es antigen of about 53 ku and had specific bands, and the hybridoma cells grew well and their titers were stable. There was no cross reaction between McAb and Cryptosporidium, Trichuris suis, Cysticercus cellulosae sac fluid, polycephalosis cyst fluid, Schistosoma japonicum antigen. The results of indirect immunofluorescence assay showed that monoclonal antibodies reacted with muscle larvae. In this study, two monoclonal antibodies against Trichinella spiralis P53ES recombinant protein were successfully prepared. It lays a foundation for the study of trichinellosis and the development of diagnostic kit for trichinellosis.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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