抗猪免疫球蛋白单克隆抗体制备与鉴定.pdf

中文摘要猪传染性疾病给我国的养殖业带来巨大的危害。传统的血清学检测方法难以了解猪群的早期发病情况,同时也无法区分抗体性质,影响了诊断传染病的准确性。随着我国规模化养猪业的快速发展,深入研究这些疾病免疫特点,提高免疫监测和诊断能力,对预防该类传染性疾病已迫在眉睫。研制猪免疫球蛋白的单克隆抗体,对于进一步研究猪传染性疾病具有十分重要的现实意义。本文主要研究了抗猪免疫球蛋白单克隆抗体的制备和鉴定。首先,采用饱和硫酸铵盐析和Sephadex G一200柱层析法从猪血清中获得IgM和IgG,经SDS-- PAGE电泳鉴定,纯化后的IgM和IgG达到了电泳纯。Folin--酚法测定IgM和 IgG浓度,分别为O,7mg/mL和2mg/mL。其次,采用间接ELISA法筛选阳性杂交瘤细胞,两组方法分别建立并优化后得出以下最佳反应条件。猪IgM包被的间接ELISA筛选方法:抗原包被量为 ,阳性和阴性对照血清稀释度为1:2000, 的稀释度为l:20000:猪IgG包被的间接ELISA筛选方法:抗原包被量为 ,阳性和阴性对照血清稀释度为1:2000, 的稀释度为1:20000。最后,用纯化后的猪IgG,采用常规免疫法免疫6-8周龄雌性BABL/c小鼠。取三次免疫后的小鼠脾细胞与SP2/0骨髓瘤细胞按10:1混合,用lmL 50% PEG3000进行细胞融合,。试验中共融合两次,%和75%,初检阳性率分别为13%%。经两次亚克隆后,共获得两株可稳定分泌单克隆抗体的杂交瘤细胞株,分别命名为c11和G3。两株杂交瘤细胞经过体外多次传代培养后,仍能分泌高效价的抗体,且细胞上清效价均稳定在1:64~128之间。用12周龄以上的BABL/c小鼠大量制备单抗腹水, G3株小鼠腹水单抗ELISA效价为1:12800以上。经HBT Mouse Monoclonal AntibodyIsotypingKit鉴定,cll细胞株为IgM类,K型。分别用IgM和IgG抗原包被的间接ELISA法检测G3株细胞上清和小鼠腹水效价,两种方法检测结果相近。Weston--Blotting试验证实二株细胞均为分泌针对猪Ig轻链抗体的杂交瘤细胞株,与间接ELISA法的检测结果相符。关键词:猪免疫球蛋白纯化单克隆抗体制备鉴定 Abstract Porcine infectious diseases havethreatened swine industryenormously in China. Traditional serological detection isdifficult tofind outtheporcine diseases earlierand distinguish the antibody’S character,which cause the infection—overspreading thelarge scalefostering ofswine inindustry,it isingreaturgency to research the immune mechanism inthis infection mad enhalice the immune isveryimportant forthefurtherresearch todevelop the monoclonal antibody againstporcine immunoglobulin. Inthisstudy,we obtained andidentifiedthe monoclonal antibody againstporcine immuno’globulin. Fistly,IgM and IgG were isolatedfrom porcine serum by aseriesprocedure of precipitation with saturated ammonium sulfateand chromatography with Sephadex G· SDS—PAGE electrophoreisis,purified IgM and IgG reach the electrophoretic purity,and the concentration were and 2mg/mL respectively determined byLowry method. Secondly,indirect