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

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.