重组蓖麻毒素A链蛋白的可溶性表达、纯化与抗原性分析  

Soluble Expression of Recombinant Ricin Toxin A Chain Protein in Escherichia coli and its Purification and Antigenicity

作　　者：孙嗣梅[1] 王利春[2] 康琳[1] 王景林[1] 

机构地区：[1]中国人民解放军军事医学科学院微生物流行病研究所病原微生物与生物安全国家重点实验室,北京100071 [2]内蒙古大学生物工程中心,呼和浩特010020

出　　处：《中国生物工程杂志》2005年第4期47-51,共5页China Biotechnology

基　　金：中国人民解放军军事医学科学院创新基金资助项目

摘　　要：用PCR方法从克隆质粒pUC19-RTA中扩增出蓖麻毒素A(RTA)链基因,序列分析正确后, 亚克隆到原核表达质粒pET-His中,构建重组表达质粒pET-HisRTA,再转化到E.coli BL21(DE3) plysS中获得表达工程菌株BL21/pET-HisRTA。该工程菌在30℃经0.4mmol／L IPTG诱导4h后获 得可溶性表达的目的蛋白,约占菌体总蛋白的18.45％,SDS-PAGE分析显示表达的蛋白区带与 RTA相对分子量相符,约32kDa左右。表达产物经Ni-NTA亲和层析法一步纯化,蛋白纯度约达 97.53％,并可得到约18mg/L重组RTA蛋白。Western印迹和间接ELISA结果证明,重组RTA蛋 白与抗天然蓖麻毒素多抗可发生特异性的抗原抗体反应,具有良好的抗原性,这为制备RT特异 性抗体及建立RT的检测方法奠定了基础。An 825 bp gene fragment encoding ricin toxin A chain was amplified by PCR from cloning plasmid pUC19-RTA. As confirmed by sequencing, the RTA gene fragment was subcloned into the expression vector pET-His to construct recombinant expression vector pET-HisRTA. The resulting expression vector pET-HisRTA was transformed into E. coli BL21(DE3)plysS competent cells and induced at 301 for 4 hours by adding IPTG to a final concentration of 0.4 mmol/L. A specific expression band with a relative molecular mass 32kDa was detected by SDS-PAGE and both in soluble form and inclusion body, and the soluble protein accounted for 18.45% of total cell protein. The expressed protein was one-step purified to 97.53% using Ni-NTA affinity chromatography method under native condition, and with a yield of 18mg/L of induced culture. Both Western blot and indirect ELISA showed that antiserum against native ricin had a specific affinity for the rRTA protein. It laid the foundation of raising specific antibodies against RTA and establishing method to detect ricin.

关 键 词：可溶性表达 蓖麻毒素 抗原性 纯化 WESTERN印迹 间接ELISA A链 原核表达质粒 重组表达质粒 E.coli 抗原抗体反应 PCR方法 相对分子量 亲和层析法 特异性抗体 pET 序列分析 工程菌株 目的蛋白 显示表达 PAGE 表达产物 检测方法 

分 类 号：Q786[生物学—分子生物学] TQ460.4[化学工程—制药化工]