半环扁尾海蛇神经毒素的融合表达及活性检测

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。

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

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.