有双重活性的重组胸腺素α1与复合α干扰素融合蛋白的分离纯化(英文)

Separation and purification of recombinant thymosin alpha 1 and interferon alfacon-1 fusion protein

背景:研究表明,将干扰素与胸腺素α1联合应用可增强干扰素的抗病毒作用。目的:获得既具有干扰素抗病毒活性又有胸腺素α1增强免疫功能的双重活性重组融合蛋白。设计、时间及地点:体外对比试验,于2003-03/2004-12在重庆康尔威药业股份有限公司药物研究所完成。材料:融合基因片断由上海生工合成,人羊膜细胞、水泡性口炎病毒购自上海生化与细胞生物所,对照品IFNα1b、IFNα2a和胸腺素α1为市售药品。方法:选择大肠杆菌偏爱的密码子,将合成的胸腺素α1与复合干扰素编码序列构成的融和基因克隆至大肠杆菌表达载体pET-22b(+)、在宿主菌BL21(DE3)-Codonplus-RP-X中表达融合蛋白。通过硫酸铵沉淀、疏水层析、阴离子交换层析、阳离子交换层析、分子筛层析等方法进行分离纯化。采用细胞病变抑制法测定融合蛋白的抗病毒活性,采用细胞增殖实验检测融合蛋白对小鼠脾淋巴细胞增殖的影响。主要观察指标:重组融合蛋白抗病毒的活性和促小鼠脾淋巴细胞增殖活性。结果:①在大肠杆菌中表达的融合蛋白呈胞内可溶性表达,表达量占细菌总蛋白的20%以上。②纯化后,融合蛋白的纯度达96%以上。③融合蛋白的抗病毒活性优于市售的IFNα1b、IFNα2a。④融合蛋白的促淋巴细胞增殖活性与市售胸腺素α1相似。结论:在大肠杆菌中表达的胸腺素α1与复合干扰素融合蛋白既具有干扰素抗病毒活性又有胸腺素α1的增强免疫功能。

BACKGROUND： It demonstrated that combined application of thymosin a 1 （TM- α 1） and interferon （IFN） can enhance the antiviral activity of IFN. OBJECTIVE： To obtain recombinant fusion protein of TM-α 1 and consensus IFN α （IFN α -con） with double activity of antiviral activity and immunity enhancement. DESIGN, TIME AND SETTING： The in vitro contrast experiment was conducted in the Biochemical Laboratory of Research Institute of Medicine, Chongqing K.E.W Pharmaceutical Co., Ltd. from March 2003 to December 2004. MATERIALS： The fusion gene fragment （TM- α 1＋IFN α -con） was synthesized by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, WISH cell, and vesicular stomatitis virus （VSV） was purchased from Institute of Biochemistry and Cell Biology, commercial products of IFN α 1b, IFN α 2α and TM- α 1 was served as reference substance. METHODS： The preference for E. coil of fusion sequence coding TM- α 1 and IFN α -con were cloned into expression vector of pET-22b（＋） and expressed in BL21（DE3）-Codon plus-RP-X, which was purified by precipitation of （NH4）2SO4, hydrophobic interaction chromatography, anion-exchange chromatography, cation-exchange chromatography and molecular exclusion chromatography. The antiviral activity of fusion protein was tested by cytopathic-effect inhibition assay, and effect of fusion protein on lymphocyte proliferation was tested by cell proliferative assay. MAIN OUTCOME MEASURES： The specific activity of fusion protein and its biological activity in promoting lymphocyte proliferation. RESULTS： The fusion protein was expressed as a soluble form, accounting for over 20% of the total cell protein in E. coil, which approached 96% after purification. The antiviral activity of fusion protein was superior to IFN a lb and IFN α 2α. However, the activity of fusion protein for promoting lymphocyte proliferation was similar to TM- α 1. CONCLUSION： The fusion protein of TM- α 1 and IFN α -con expressed in EL coil has both effects on anti-virus and promoting lymphocyte proliferation.