重组蓖麻毒素A链的表达及半乳糖修饰  

Expression and galactose modification of recombinant ricin toxin A chain

作　　者：边红 王燕 孙成彪 许娜 朱文赫 刘文森 王秀然[1] BIAN Hong;WANG Yan;SUN Cheng-biao;XU Na;ZHU Wen-he;LIU Wen-sen;WANG Xiu-ran(College of Life Sciences,Jilin Agricultural University,Changchun 130118,Jilin Province,China)

机构地区：[1]吉林农业大学生命科学学院,吉林长春130118 [2]军事医学研究院军事兽医研究所吉林省人兽共患病预防与控制重点实验室,吉林长春130122

出　　处：《中国生物制品学杂志》2019年第5期565-569,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金面上项目(81773630);吉林省科技厅重点科技攻关计划(20180201004YY)

摘　　要：目的表达重组蓖麻毒素A链(ricin toxin A,RTA),并进行半乳糖糖基化修饰。方法将重组质粒pET-28a-RTA转化E. coli BL21(DE3),经IPTG诱导表达重组蛋白RTA,通过镍离子亲和层析柱纯化包涵体,并复性。采用1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride,EDC]/N,N-羟基琥珀酰亚胺(N-hydroxysuccinimide,NHS)法对重组RTA进行半乳糖糖基化修饰,并对偶联反应温度(20、25、30和37℃)和半乳糖与RTA的投料比(100∶1、500∶1、1 000∶1、1 500∶1和2 000∶1)进行优化。结果 RTA蛋白相对分子质量约34 000,主要以包涵体形式表达,纯化复性后纯度大于90%。最适偶联反应温度为25℃,半乳糖与RTA最适投料比为1 000∶1。结论成功表达了RTA蛋白,并通过EDC/NHS法实现了半乳糖与RTA的偶联。Objective To express recombinant ricin toxin A chain(RTA) and modify by glycosylation with galactose.Methods Recombinant plasmid pET-28 a-RTA was transformed to E. coli BL21(DE3)and induced by IPTG. The expressed RTA was purified by nickel ion affinity chromatography and refolded,then modified by galactose glycosylation using 1-(3-dimethylaminoprophyl)-3-ethylcarbodiimide hydrochloride(EDC)/N-hydroxysuccinimide(NHS),and the temperature for coupling reaction(20,25,30 and 37 ℃) and the ratio of galactose to RTA(100 ∶ 1,500 ∶ 1,1 000 ∶ 1,1 500 ∶ 1 and 2 000 ∶ 1)were optimized. Results The expressed RTA with a relative molecular mass of about 34 000 was mainly in a form of inclusion body,of which the purity was more than 90%. The optimal temperature for coupling reaction was25 ℃,while the optimal ratio of galactose to RTA was 1 000 ∶ 1. Conclusion RTA was expressed successfully and coupled to galactose by EDC/NHS method.

关 键 词：蓖麻毒素A链 糖基化修饰 工艺优化 

分 类 号：Q816[生物学—生物工程]