重组蓖麻毒素A链的原核表达和单克隆抗体的制备、鉴定及应用  

Expression and purification of recombination ricin toxin A and its monoclonal antibody based sensitive ELISA

作　　者：郭建巍[1] 王顺涛[2] 郭黎明[2] 冯健男[2] 马骢[1] 沈倍奋[2] 

机构地区：[1]海军总医院检验科,北京100037 [2]军事医学科学院基础医学研究所分子免疫学实验室,北京100850

出　　处：《免疫学杂志》2007年第4期424-428,共5页Immunological Journal

基　　金：国家"863"国家高技术研究发展计划项目(2004AA002020);中国博士后基金项目(2004035289)资助

摘　　要：目的 实现重组蓖麻毒素A链（rRTA）的高效表达,获得rRTA的单克隆抗体,建立基于单抗的蓖麻毒素检测方法.方法 用计算机辅助设计的RNA二级结构预测和偏性密码子等手段设计引物 用PCR将rRTA基因克隆至载体pE132a（＋） 用双酶切和DNA测序技术对构建的载体进行鉴定 IFTG诱导rRTA原核表达载体pET32a（＋）/rRA/BL21,用镍离子亲和层析纯化,ELISA及Western blot鉴定rRTA蛋白 rRTA免疫Balb/c小鼠,建立杂交瘤细胞株,获得抗rRTA的单克隆抗体,用Westem blot鉴定.结果 构建了rRTA原核表达载体pET32a（＋）/rRA/BL21,实现了rRTA在大肠杆菌BL21中的高效可溶性表达,获得了高纯度约10～20 mg/L rRTA 获得抗rRTA的单克隆抗体 建立的ricin EIJSA检测方法检出ricin的最低浓度为3.125 ng/mL,目视比色对蓖麻毒素的最低检出浓度为6.25 ng/mL.结论 本研究表达的rRTA及其单克隆抗体和基于单抗的ricin检测方法,将在肿瘤生物治疗、ricin生物恐怖袭击的侦检、治疗和预防中发挥重要作用.Objective To construct a prokaryotic expression vector in which the maximum level of rRTA-Trx fusion protein was attained with homogeneous, soluble, and full bioactive, and to prepare anti-rRTA monoclonal antibody （mAb） and establish sandwich ELISA for ricin detection. Methods A pair of RTA special primer was designed based on computer-aided design, RNA secondary structure, and also Escherichia coli pardalism cedon. RTA gene was cloned from PUC19/RA to pET32a （ ＋ ） by PCR. Prokaryotic expression vector pET32a （ ＋ ）/ rRA/JM109 was eatablished and identified by double enzyme digestion and DNA sequencing. After induction with IPTG, the rRTA protein was obtained, and then purified by Ni^2＋ -NTA resin column and evaluated by ELISA and Western blot assays. An anti-RTA mAb was obtained by using hybridoma technique and identified by Western blot assay. A sandwich ELISA for ricin detection was also established. Results a prokaryotic expression vector pET32a （ ＋ ）/rRA/BL21 was constructed. Recombinant RTA proteins with high biological activity were successfully produced in the pET32a vector expression system. After purification with Ni^2＋ -NTA resin column, the extraction showed a yield of approximately 10-20 mg/L. An anti-RTA mAb was obtained with hybridoma technique and specific ELISA screening. Western blotting showed that the mAb had specific binding ability to RTA in reduced SDS-PAGE. A sandwich ELISA for detects ricin was established with a ricin detection limit of 3. 125 ng/mL.Conclusion rRTA, anti- RTA mAb, and a sandwich ELISA for ricin detection have practical use in biotherapy against carcinoma and in prophylaxis, diagnosis and therapeutics against bioterror attack of in the future.

关 键 词：蓖麻毒素 蓖麻毒素A链 融合蛋白 夹心EIJSA 

分 类 号：R392.11[医药卫生—免疫学]