【摘要】 目的:在大肠杆菌中进行含硒抗病毒多肽的可溶性半胱氨酸缺陷型融合蛋白的表达,并对其纯化蛋白进行生物学活性的初步鉴定。方法:将含硒抗病毒多肽Se-GBVA10基因克隆到GST融合表达载体pGEX-2T中,转化至半胱氨酸缺陷型大肠杆菌BL21cysE51后进行半胱氨酸缺陷型表达,通过谷胱甘肽Sepharose 4B亲和柱纯化目的蛋白质,并对其含硒量、抗病毒活力等性质进行初步鉴定。结果:利用半胱氨酸缺陷型表达法成功地可溶性表达了sjGST-Se-GBVA10和sjGSTGBVA10融合蛋白,并经凝血酶切后获得了纯化的Se-GBVA10和GBVA10抗病毒多肽。测定SeGBVA10的含硒量为0.974 mol/mol peptide,GPX活力为47.52 U/μmol,其EC50为21.73μmol/L,CC50为849.41μmol/L。结论:Se-GBVA10具有与GBVA10相同的抗病毒活性,并具有一定的抗氧化活力。更多还原

【Abstract】 Objective: To express the soluble antiviral peptide,the cysteine auxotrophic expression system was used in Escherichia coli( E. coli). And the biological activities of the purified antiviral peptide were initially identified. Method: The gene sequence of antiviral peptide Se-GBVA10 was chemically synthesized according to the optimized codons of E. coli,and the gene was cloned into GST fusion expression vector pGEX-2T. Then,the plasmid pGEX-2T-GBVA10 was converted to cysteine auxotrophic expression strains( E. coli BL21cysE51). Expressed proteins were purified by glutathione Sepharose 4B affinity column. Finally,the content of selenium, the antiviral activity and the antioxidant activity( the vitality of glutathione peroxidase) of the antiviral peptides were detected. Results: The fusion protein,sjGST-Se-GBVA10 and sjGST-GBVA10,were successfully expressed by means of cysteine auxotrophic expression system. After cutting with thrombin,the purified antiviral peptides, Se-GBVA10 and GBVA10 were obtained. The content of selenium of the Se-GBVA10 was 0. 974 mol / mol peptide,the glutathione peroxidase vitality of Se-GBVA10 was 47. 52 U / μmol,the concentration for 50% of maximal effect( EC50) of Se-GBVA10 was 21. 73μmol/L,and the median cytotoxic concentration( CC50) of SeGBVA10 was 849. 41μmol / L. Conclusion: The antiviral peptide Se-GBVA10 has the same antiviral activity as the GBVA10,but also has some antioxidant activity.更多还原