摘要
目的：探索亲和纯化人源单克隆抗体片段的简便实用方法。方法：应用自制的抗人免疫球蛋白片段抗体（Ａｎｔｉ－Ｆａｂ）与链球菌蛋白Ｇ亲和胶（Ｇａｍ ｍ ａ Ｂｉｎｄ）交联制备亲和层析柱，用一步法从工程菌培养上清中纯化人源抗ＨＢｓ单克隆Ｆａｂ 片段。经依沙吖啶沉淀、离子交换、分子筛纯化人混合血清提取ＩｇＧ组分，木瓜酶处理后分离、纯化出Ｆａｂ，免疫绵羊制备高效价抗血清。用Ｇａｍ ｍ ａＢｉｎｄ 一步纯化出抗血清中的ＩｇＧ组分，再将该ＩｇＧ经交联剂与Ｇａｍ ｍ ａ Ｂｉｎｄ 交联后制成亲和胶。人源抗ＨＢｓ阳性菌培养上清与亲和胶共育后装柱，ＰＢＳ洗去非特异结合蛋白，再用５ ｍ ｏｌ／Ｌ氯化锂洗脱特异Ｆａｂ。结果：聚丙烯酰胺电泳纯化产物显示，纯化后的抗体片段在电泳中形成单一区带，达到电泳纯；用酶标抗Ｆａｂ 抗体免疫印迹结果证明，电泳显示的单一区带为Ｆａｂ 蛋白区带；抗原特异Ｄｏｔｂｌｏｔ检测表明，该法制备出的Ｆａｂ 保持了抗原结合活性。结论：本实验建立的一步亲和纯化法具操作简便、纯化快速和高效的特点，是人源性单克隆抗体Ｆａｂ
Objective: To prepare and purify human monoclonal antibody fragments from a combinatorial library to hepatitis B virus surface antigens with a self prepared affinity chromatography column. Methods: Human Fab components of IgG obtained from enzyme cut IgG were used to immunize a sheep to obtain high titer anti Fab serum. Affinity column to Fabs was prepared by chemically linking the anti Fab serum to commercial Gamma Bind. One step method was used to purify the supernatants generated from culturing of Anti HBs positive clones, SDS polyacrylamide gel electrophoresis (PAGE) and immune blot were performed to verify the effect of purification. Results: Comparing the PAGE results, after affinity column purification, complex protein components from bacteria were eluted away and only one band remained. Its relative molecular mass is about 5.0×10 4. Immune blot confirmed the results. There were numerous bacterial protein components in the supernatant, purification with ordinary method was difficult. Conclusion: Our results indicate that one step affinity purification is a simple and highly effective method.
出处
《第二军医大学学报》
CAS
CSCD
北大核心
2000年第1期50-52,共3页
Academic Journal of Second Military Medical University
基金
国家自然科学基金!资助项目(39670668)
关键词
乙型肝炎
抗HBS
单克隆抗体
FAB片段
antibodies, monoclone
affinity purification
hepatitis B
hepatitis antibodies
