论文部分内容阅读
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: In order to obtain the high expression of single chain antibody, we studied the expression, renaturation and purification of human colon cancer single chain antibody CL-3 in Escherichia coli. Methods: The recombinant plasmid pJW2-CL-3 was transformed into E. coli DH5α. The recombinant clones were induced by temperature control in culture medium and single-chain antibodies were expressed as inclusion bodies. The inclusion bodies were dissolved, renatured and purified. The expressed proteins were identified by SDS-PAGE and Western Blot. The immunological activity of single-chain antibodies was identified by ELISA. Results: The expression of protein at 42 ℃ for 5 h accounted for 30% of the bacterial protein. 8 mol / L urea can dissolve most of the inclusion body, diluted in renaturation buffer for more than 48 h at 10 ℃ can make inclusion body protein renaturation. Purified peak was identified. The protein expressed at 27 KD was the anti-human colon cancer single-chain antibody CL-3 with E-tag and was proved to have the specific binding to the antigen CEA. The immunocompetence of the purified antibody was the same as that of the parental antibody CL-3 similar. CONCLUSION: CL-3, an anti-human colon cancer single-chain antibody expressed in E. coli with inclusion bodies, has similar immunocompetence to that of the parental antibody and is highly efficient for the expression of single-chain antibodies for the diagnosis and treatment of tumors Provided the basis.