本文選題：CD58蛋白 + 單克隆抗體　； 參考：《西北農林科技大學》2009年碩士論文

【摘要】： CD58又名淋巴細胞功能相關抗原-3(LAF-3),在機體免疫中發(fā)揮重要作用,對動物妊娠早期子宮局部細胞因子網(wǎng)絡的調節(jié)也存在著影響。本試驗利用酶消化法結合柱層析法制備了綿羊紅細胞表面活性蛋白CD58;通過小鼠免疫,細胞融合以及有限稀釋法篩選陽性克隆,制備了抗CD58蛋白單克隆抗體,并進行了鑒定;建立了檢測山羊血清中CD58的間接競爭抑制性ELISA方法,檢測了未發(fā)情,發(fā)情和懷孕山羊血清中CD58的含量。獲得以下結果: 1.酶消化法結合柱層析法制備了綿羊紅細胞表面活性蛋白CD58,SDS-PAGE結果表明制備了高純度的CD58蛋白。玫瑰花環(huán)抑制試驗檢測結果抑制率為64.68±3.93%。 2.建立了抗CD58蛋白抗體測定的間接ELISA方法。方陣滴定法確定包被原最適工作濃度為10.0μg/mL,最佳的包被溫度和時間為37℃1h后4℃過夜,1.0%明膠封閉,辣根過氧化物酶標記山羊抗小鼠IgG(酶標二抗)最適稀釋倍數(shù)為l∶3 2 000,作用時間為30min。 3.用45%聚乙二醇(PEG)4000融合骨髓瘤細胞和免疫脾細胞,用間接ELISA法篩選陽性孔,克隆率為81.94%,陽性克隆率45.15%。用有限稀釋法篩選得到四株陽性雜交瘤細胞,分別命名為A6,B9,F2,G6。 4.四株雜交瘤細胞經凍存和傳代培養(yǎng),證明均具有穩(wěn)定分泌抗體的能力。玫瑰花環(huán)抑制試驗和Western-blotting鑒定,所分泌的是針對CD58蛋白的單克隆抗體。單抗的類型為IgG1型,親和力常數(shù)分別為3.050×10-8,4.724×10-9,6.768×10-10和3.801×10-9。 5.建立了測定山羊血清中CD58的間接競爭抑制性ELISA方法,此方法的線性范圍為10.0～1 000 000.0ng/mL,回歸方程y=-0.1543X+1.0695,R2=0.9644,該方法靈敏度為3.087ng/mL。通過對不同生理時期山羊血清中CD58含量檢測表明,懷孕山羊血清中CD58含量顯著高于未發(fā)情和發(fā)情山羊(P㩳0.05)。 本試驗成功制備了抗CD58蛋白單克隆抗體,建立了檢測山羊血清中CD58含量的間接競爭ELISA方法,對不同生理時期山羊血清中的CD58含量進行了測定,為進一步研究CD58在山羊不同生理時期的作用提供了理論依據(jù)。
[Abstract]:CD58, also known as lymphocyte function-associated antigen -3laf-3, plays an important role in the body immunity, and also has an effect on the regulation of local cytokine network in the early stage of pregnancy. In this experiment, sheep erythrocyte surface protein CD58 was prepared by enzyme digestion and column chromatography, and monoclonal antibody against CD58 protein was prepared and identified by mouse immunity, cell fusion and limited dilution method. An indirect competitive inhibitory Elisa was established for the detection of CD58 in goat serum, and the content of CD58 in the serum of unestrus, estrous and pregnant goats was determined. The following results were obtained: 1. Sheep erythrocyte surface protein CD58 / SDS-PAGE was prepared by enzyme digestion combined with column chromatography. The results showed that high purity CD58 protein was prepared. The inhibition rate of rosette inhibition test was 64.68 鹵3.93.2. An indirect Elisa method for the determination of anti-CD 58 protein antibody was established. The optimum working concentration of the coating was 10.0 渭 g / mL by square array titration, and the best coating temperature and time was 37 鈩，

本文編號：2047199