抗CD58蛋白单克隆抗体的制备及鉴定
发布时间：2018-06-21 04:23
本文选题：CD58蛋白 + 单克隆抗体 ； 参考：《西北农林科技大学》2009年硕士论文
【摘要】： CD58又名淋巴细胞功能相关抗原-3(LAF-3),在机体免疫中发挥重要作用,对动物妊娠早期子宫局部细胞因子网络的调节也存在着影响。本试验利用酶消化法结合柱层析法制备了绵羊红细胞表面活性蛋白CD58;通过小鼠免疫,细胞融合以及有限稀释法筛选阳性克隆,制备了抗CD58蛋白单克隆抗体,并进行了鉴定;建立了检测山羊血清中CD58的间接竞争抑制性ELISA方法,检测了未发情,发情和怀孕山羊血清中CD58的含量。获得以下结果: 1.酶消化法结合柱层析法制备了绵羊红细胞表面活性蛋白CD58,SDS-PAGE结果表明制备了高纯度的CD58蛋白。玫瑰花环抑制试验检测结果抑制率为64.68±3.93%。 2.建立了抗CD58蛋白抗体测定的间接ELISA方法。方阵滴定法确定包被原最适工作浓度为10.0μg/mL,最佳的包被温度和时间为37℃1h后4℃过夜,1.0%明胶封闭,辣根过氧化物酶标记山羊抗小鼠IgG(酶标二抗)最适稀释倍数为l∶3 2 000,作用时间为30min。 3.用45%聚乙二醇(PEG)4000融合骨髓瘤细胞和免疫脾细胞,用间接ELISA法筛选阳性孔,克隆率为81.94%,阳性克隆率45.15%。用有限稀释法筛选得到四株阳性杂交瘤细胞,分别命名为A6,B9,F2,G6。 4.四株杂交瘤细胞经冻存和传代培养,证明均具有稳定分泌抗体的能力。玫瑰花环抑制试验和Western-blotting鉴定,所分泌的是针对CD58蛋白的单克隆抗体。单抗的类型为IgG1型,亲和力常数分别为3.050×10-8,4.724×10-9,6.768×10-10和3.801×10-9。 5.建立了测定山羊血清中CD58的间接竞争抑制性ELISA方法,此方法的线性范围为10.0～1 000 000.0ng/mL,回归方程y=-0.1543X+1.0695,R2=0.9644,该方法灵敏度为3.087ng/mL。通过对不同生理时期山羊血清中CD58含量检测表明,怀孕山羊血清中CD58含量显著高于未发情和发情山羊(P㩳0.05)。 本试验成功制备了抗CD58蛋白单克隆抗体,建立了检测山羊血清中CD58含量的间接竞争ELISA方法,对不同生理时期山羊血清中的CD58含量进行了测定,为进一步研究CD58在山羊不同生理时期的作用提供了理论依据。
[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 卤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，
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