本文關(guān)鍵詞： 旋毛蟲 重組蛋白 單克隆抗體 排泄分泌抗原　出處：《東北農(nóng)業(yè)大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 旋毛蟲病是由旋毛形線蟲(Trichinella spiralis)引起的一種人獸共患寄生蟲病,主要寄生于豬、野豬、鼠、熊等150多種動物及人體。人體主要因生食或半生食含有旋毛蟲肌幼蟲囊包的豬肉或其他動物肉類而感染。由于其病原傳播的復(fù)雜性,以致該病發(fā)現(xiàn)一百多年來,不僅沒有得到完全有效的控制,而且發(fā)生范圍反而逐漸擴大。要迅速、有效地控制和預(yù)防旋毛蟲病的流行,必須有快速準(zhǔn)確的檢測技術(shù)。本研究選用旋毛蟲P53ES基因重組蛋白免疫小鼠制備McAb,為進一步研制旋毛蟲病診斷試劑盒提供理想、必備的試驗材料。 以旋毛蟲P53ES基因的重組蛋白免疫BALB/c鼠,取小鼠脾細胞和骨髓瘤細胞SP2/0融合,經(jīng)間接ELISA篩選和有限稀釋法克隆,篩選分泌高滴度McAb雜交瘤細胞株,制備腹水。Southern Biotechnology Associates,Inc的SBA ClonotypingTMSystem/AP對單克隆抗體進行亞類鑒定;采用ELISA間接法測定雜交瘤細胞上清液和小鼠腹水效價;Western-blot檢測單克隆抗體的特異性;測定雜交瘤細胞分泌抗體的穩(wěn)定性;間接ELISA檢測單克隆抗體與隱孢子蟲、豬鞭蟲、豬囊尾蚴囊液抗原、多頭蚴囊液、日本血吸蟲抗原是否存在交叉反應(yīng);間接免疫熒光方法檢測單克隆抗體與肌幼蟲的免疫學(xué)反應(yīng)。結(jié)果表明:得到兩株能穩(wěn)定分泌單克隆抗體的雜交瘤細胞株,分別命名為2H5和3D2;試劑盒鑒定兩株McAb均屬IgM亞類,其輕鏈均為κ鏈;腹水效價分別為1:24000和1:12000;Western-blot證實兩株McAb均能與約53ku處的ES抗原反應(yīng),出現(xiàn)特異性條帶;雜交瘤細胞株連續(xù)傳20代,細胞生長良好,效價穩(wěn)定;所得單抗與隱孢子蟲、豬鞭蟲、豬囊尾蚴囊液抗原、多頭蚴囊液、日本血吸蟲抗原均無交叉反應(yīng);間接免疫熒光試驗結(jié)果表明單克隆抗體與肌幼蟲發(fā)生免疫學(xué)反應(yīng)。本研究成功制備了兩株抗旋毛蟲P53ES重組蛋白的單克隆抗體,為旋毛蟲病的研究和研制旋毛蟲病診斷試劑盒奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

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