蓖麻毒素A链与绿色荧光蛋白基因的融合表达和活性测定

目的:表达蓖麻毒素A链(RTA)与绿色荧光蛋白(GFP)的融合蛋白 (GFP-RTA),用于蓖麻毒素A链在细胞内的转运机理研究.方法:采用PCR法从重组质粒pKK233.2-RTA中扩增出蓖麻毒素A链基因,然而将其插入真核表达质粒pEGFPC1,构成GFP-RTA融合基因;GFP-RTA经PCR扩增,与T载体连接,用内切酶从T-GFP-RTA中切下GFP-RTA片段,插入原核表达质粒pET-28a( );在BL21(DE3)中表达GFP-RTA融合蛋白,通过金属螯合和层析纯化,MTT法检测融合蛋白的细胞毒性.结果:测序发现插入的融合基因全长序列完全正确,SDS-PAGE鉴定表达产物分子量正确;经诱导表达后,菌体产生绿色荧光,MTT法表明该融合蛋白呈现与RTA相似的细胞毒性.结论:从大肠杆菌表达的蓖麻毒素A链与绿色荧光蛋白的融合蛋白,具有蓖麻毒素A链和绿色荧光蛋白的生物学活性,可用于蓖麻毒素A链在细胞内的转运途径研究.

Construction and expression of ricin A chain and green fluorescent protein fusion gene in E.coli

Objective: To study the expression and purificationof a fusion protein of ricin A chain (RTA) and green fluorescent protein (GFP).Methods: The DNA sequence encoding ricin A chain was inserted into pEGFPC1 first to make the template sequence of the fusion protein.The fusiongene was amplified from the plasmid pEGFP-RTA by PCR,and directly subcloned into T vector.The fusion gene then was cloned into expression vector pET-28a(+),and the sequence was confirmed by sequencing.Expression was induced by IPTG in E.coli BL21(DE3).The fusion protein was purified by metal chelated affinity chromatography.The cytotoxicity of fusion protein was analyzed by the MTTassay in HepG2 and Hela cells. Results: The fusion protein of ricin A chain and GFP could be produced in E.coli transformed with the expression plasmid of pET-28a(+)-GFP-RTA.The molecular weight of the recombinant protein was measured by SDS-PAGE.The fusion protein showed a green fluorescence andhad a similar cytotoxicity of RTA. Conclusion: A recombinant fusion protein of RTA and GFP expressed in E.coli is possessed of similar biological activity of individual GFP and RTA,which could be used in study of the intracellular trafficking and translocation of RTA.