半环扁尾海蛇神经毒素的融合表达及活性检测  

Fusional Expression of Erabutoxin in E.coli and its Activity Analysis

作　　者：吴方晖[1] 董玉轩[1] 应天翼[1] 张华东[1] 张金平[1] 王惠芳[1] 李艳军[1] 

机构地区：[1]防化研究院第四研究所,北京102205

出　　处：《生物技术通讯》2006年第5期713-715,共3页Letters in Biotechnology

摘　　要：目的:将海蛇神经毒素基因克隆到融合表达载体pET32a中,诱导表达海蛇神经毒素,鉴定融合表达蛋白的生物功能,为大量合成海蛇神经毒素奠定基础。方法:利用融合表达载体将海蛇神经毒素与硫氧还蛋白融合表达,使其在胞内可溶;采用亲和层析与G50凝胶过滤纯化融合蛋白;利用豚鼠直肠纵肌电刺激检验融合蛋白的神经信号阻断功能。结果:得到电泳纯的融合蛋白,该蛋白对豚鼠直肠纵肌电刺激有明显的阻断作用。结论:融合表达能促进海蛇神经毒素的可溶性表达,表达产物能阻断神经信号的传递。Objective： To obtain high level expression of erabutoxin in E.coli, the study was carried out by the fusional expression through pET32a vector, the purification and the detection of activity were completed. It provided a base for a large scale production of erabutoxin. Methods： The gene of erabutoxin was amplified by PCR and was cloned into pET32a vector, the recombinant vector was transformed into E.coli BL21 （DE3）, then the expression was induced by 0.5 mmol/L IPTG. The recombinant fusion protein was analyzed by SDS-PAGE and purified by affinity chromatograph and G50 gel filtration. Results： The fusional protein was efficiently expressed, the purity achieved the electrophoretic grade and it could blockd transmission of electrical signal of rectal longitudinal muscle of cavia cobaya. Conclusion： The fusional expression could favor soluble expression of erabutoxin, the pure fusion protein had similar potency as the native neurotoxin in blocking transmission of electrical signal.

关 键 词：半环扁尾海蛇 海蛇神经毒素 融合表达 亲和层析 凝胶过滤 直肠纵肌 

分 类 号：Q786[生物学—分子生物学] R996.3[医药卫生—毒理学]