CAJ | 학술논문

背景:研究表明,将干扰素与胸腺素α 1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α 1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α 1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IFN α 1b、IFN α 2a.④融合蛋白的促淋巴细胞增殖活性与市售胸腺素α 1相似.结论:在大肠杆菌中表达的胸腺素α 1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α 1的增强免疫功能.
배경:연구표명,장간우소여흉선소α 1연합응용가증강간우소적항병독작용.목적:획득기구유간우소항병독활성우유흉선소α 1증강면역공능적쌍중활성중조융합단백.설계、시간급지점:체외대비시험,우2003-03/2004-12재중경강이위약업고빈유한공사약물연구소완성.재료:융합기인편단유상해생공합성,인양막세포、수포성구염병독구자상해생화여세포생물소,대조품IFNα1b、IFNα2a화흉선소α1위시수약품.방법:선택대장간균편애적밀마자,장합성적흉선소α 1여복합간우소편마서렬구성적융화기인극륭지대장간균표체재체pET-22b(+)、재숙주균BL21(DE3)-Codonplus-RP-X중표체융합단백.통과류산안침정、소수층석、음리자교환층석、양리자교환층석、분자사층석등방법진행분리순화.채용세포병변억제법측정융합단백적항병독활성,채용세포증식실험검측융합단백대소서비림파세포증식적영향.주요관찰지표:중조융합단백항병독적활성화촉소서비림파세포증식활성.결과:①재대장간균중표체적융합단백정포내가용성표체,표체량점세균총단백적20%이상.②순화후,융합단백적순도체96%이상.③융합단백적항병독활성우우시수적IFN α 1b、IFN α 2a.④융합단백적촉림파세포증식활성여시수흉선소α 1상사.결론:재대장간균중표체적흉선소α 1여복합간우소융합단백기구유간우소항병독활성우유흉선소α 1적증강면역공능.
BACKGROUND: It demonstrated that combined application of thymosin α 1 (TM-α1) and interferon (IFN) can enhance the antiviral activity of IFN.OBJECTIVE: To obtain recombinant fusion protein of TM- α1 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 2a and TM- α 1 was served as reference substance.METHODS: The preference for E. coli 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. coli, which approached 96% after purification. The antiviral activity of fusion protein was superior to IFN α 1b and IFN α 2a. 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 E. coli has both effects on anti-virus and promoting lymphocyte proliferation.