抗人结肠癌单链抗体CL-3在大肠杆菌中的表达、复性和纯化  

Expression, renatureation and purification of anti human colon cancer scFv CL 3 in E.Coli

作　　者：贾宝庆[1] 袁玫[2] 闫锡蕴 余征[2] 李力[2] 费丽华[2] 方永鑫[2] 林星石[2] 张晓平[2] 蒋彦永[1] 

机构地区：[1]解放军总医院普通外科,北京100853 [2]解放军总医院肿瘤生物研究室 [3]中国科学院微生物研究所,北京100080

出　　处：《军医进修学院学报》1999年第4期267-269,共3页Academic Journal of Pla Postgraduate Medical School

基　　金：国家自然科学基金

摘　　要：目的：为得到单链抗体高效表达，我们对抗人结肠癌单链抗体ＣＬ－３ 在大肠杆菌中的表达、复性和纯化进行了研究。方法：以重组质粒表达载体ｐＪＷ２－ＣＬ－３ 转化大肠杆菌ＤＨ５α，重组克隆在培基中温控诱导，以包涵体形式表达单链抗体。包涵体经溶解、复性、纯化后，以ＳＤＳ－ＰＡＧＥ及Ｗｅｓｔｅｒｎ Ｂｌｏｔ鉴定表达蛋白，ＥＬＩＳＡ 法鉴定单链抗体的免疫活性。结果：４２ ℃诱导５ ｈ 蛋白表达量占菌体蛋白的３０％ 。８ ｍ ｏｌ／Ｌ尿素可将包涵体大部分溶解，稀释于复性缓冲液中静置１０ ℃４８ ｈ 以上可使包涵体蛋白复性。纯化峰经鉴定， ２７ ＫＤ 处表达之蛋白为带有Ｅ－ｔａｇ的抗人结肠癌单链抗体ＣＬ－３，并证明其具有与抗原ＣＥＡ 特异性结合的功能，免疫活性与亲代抗体ＣＬ－３ 相似。结论：本研究获得的以包涵体形式在大肠杆菌中表达的抗人结肠癌单链抗体ＣＬ－３，具有与亲代抗体相似的免疫活性，为高效表达单链抗体，用于肿瘤的诊断和治疗提供了依据。Objective:To expect more efficient expression, we expressed scFv CL 3, which specifically targets to human colon cancer membrane antigen, as inclusion bodies in E.Coli. Methods:Recombinant plasmid pJW2 CL 3 were transformed into E.Coli DH5 α. Induced by temperature control, scFv CL 3 were expressed as inclusion bodies in the plasms of bacteria when the positive clone has been cultured in LB till OD600=0.6. After being digested and sonicated, inclusion bodies were dissolved in 8M urea and then diluted 100 fold into renaturation buffer at 10℃. The scFv were purified on ion exchange Blot. Immunoactivity of the scFv was appraised by ELISA. Results:After 5 hour inducing, the inclusion bodies turn out to account for about 30% of total bacterial proteins. Inclusion bodies were dissolved in 8M urea and have been refolded in renaturation buffer after 48 hours at 10℃. When eluted with 0.3 mol/L NaCl/20 nmol/L TE, scFv were obtained from ion exchange column elution. SDS PAGE and Western Blot proved that the 27KD protein expressed in E.Coli is scFv with E tag fused to its Cterminus. The renatured scFv CL 3 has similar specificity and Immunoactivity to its parent antibody as evaluated by ELISA. Conclusion:Expressed scFv CL 3 as inclusion bodies in E.Coli. After renaturation and purification, the scFv were showed could bind specifically to human colon cancer antigen.

关 键 词：单链抗体 包涵体 表达 大肠杆菌 结肠癌 

分 类 号：Q78[生物学—分子生物学] R392－33[医药卫生—免疫学]