幽门螺杆菌重组中性粒细胞激活蛋白蛋黄抗体的制备  

Preparation of egg yolk immunoglobulin against recombinant neutrophil activating protein in Helicobacter pylori

作　　者：邓颖[1] 杨致邦[1] 黄伟[2] 林珊珊[1] 黄进[1] 叶翠莲[1] 

机构地区：[1]重庆医科大学微生物学与免疫学教研究室,重庆市400016 [2]西南大学蚕学与生物技术学院生物技术系,重庆市402460

出　　处：《世界华人消化杂志》2007年第5期471-475,共5页World Chinese Journal of Digestology

摘　　要：目的:制备高效价的抗Nap蛋黄抗体(IgY).方法:大量诱导、培养重组菌pQE30-NapA-DH5α获得重组蛋白Nap,经Ni2+-NTA树脂纯化后,Bradford法测定蛋白浓度.用纯化的Nap蛋白免疫鸡,水稀释结合氯仿有机沉淀法提取IgY.ELISA法测定抗体产生的时间-效价变化.将效价高的Nap-IgY用硫酸铵沉淀法纯化浓缩,间接ELISA法检测效价,Bradford法测定蛋白含量.Western blot检测制备的Nap-IgY的抗体活性.结果:Nap重组蛋白主要以包涵体形式表达,蛋白含量为0.37g/L.免疫鸡的蛋黄提取物可与Nap发生特异性反应,IgY效价随免疫时间增加而升高,在110d达最高效价.经纯化浓缩后,Nap-IgY的效价为1∶12800,蛋白浓度为23.67g/L.结论:成功制备了高浓度、高效价的Nap特异性IgY.AIM： To prepare a highly specific and efficient egg yolk immunoglobulin （IgY） against recombinant neutrophil activating protein （Nap） of Helicobacter pylori from the yolk of hen＇s eggs. METHODS： Recombinant bacteria of pQE30- NapA-DH5α were cultured in large numbers to collect Nap protein. The recombinant protein was purified by Ni^2＋-NAT chromatography, and its protein concentration was detected by Bradford method. After laying hens were immunized with recombinant Nap, IgY was isolated by water-dilution （WD） combined chloroform precipitation method. The relationship between IgY titer and immune time was assessed by enzyme-linked immunosorbent assay （ELISA）,High-titer IgY was purified and concentrated by ammonium sulphate precipitation. The Nap-IgY titer and protein concentration were measured by indirect ELISA and Bradford method, respevtively, and the activity of Nap-IgY was identified by Western blot. RESULTS： SDS-PAGE result showed that the recombinant protein was mainly expressed as inclusion bodies. After purification, the protein concentration was 0.37 g/L. The recombinant protein could be recognized by egg yolk extraction. The antibody titer became higher as the increase of immune time, and reached the highest value on the 110th day. The titer and protein concentration were 1 ： 12800 and 23.67 g/L, respectively. CONCLUSION： Nap-IgY with high concentration, purity and specificity is successfully prepared.

关 键 词：幽门螺杆菌 重组蛋白 中性粒细胞激活蛋白 蛋黄抗体 

分 类 号：R392[医药卫生—免疫学]