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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: Studies have shown that the combination of interferon and thymosin α1 enhances the antiviral effect of interferon. OBJECTIVE: To obtain double-active recombinant fusion protein with both interferon antiviral activity and thymosin α1 enhancing immune function. DESIGN, TIME AND SETTING: In vitro comparative tests were performed at the Institute of Materia Medica, Chongqing Kangwei Pharmaceutical Co., Ltd. from March 2003 to December 2004. MATERIALS: Fusion gene fragments were synthesized by Shanghai Institute of Biotechnology. Human amniotic cells and vesicular stomatitis virus were purchased from Shanghai Institute of Biochemistry and Cell Biology. The reference drugs IFNα1b, IFNα2a and thymosin α1 were commercially available drugs. Methods: The fusion gene consisting of the synthetic thymosin α1 and the consensus interferon coding sequence was cloned into E. coli expression vector pET-22b (+) by selecting the codons preferred by E. coli. The fusion gene was expressed in host strain BL21 (DE3) -Codonplus-RP -X expression of the fusion protein. Purification by ammonium sulfate precipitation, hydrophobic chromatography, anion exchange chromatography, cation exchange chromatography, molecular sieve chromatography and other methods. The antiviral activity of the fusion protein was determined by cytopathic effect inhibition. The effect of the fusion protein on the proliferation of mouse splenic lymphocytes was detected by cell proliferation assay. MAIN OUTCOME MEASURES: Antiviral activity of recombinant fusion protein and splenic lymphocyte proliferation activity in mice. Results: ① The fusion protein expressed in Escherichia coli was intracellularly soluble, accounting for more than 20% of total bacterial protein. ② After purification, the purity of the fusion protein was above 96%. ③ The antiviral activity of the fusion protein is better than the commercially available IFNα1b, IFNα2a. ④ fusion protein promoting lymphocyte proliferation activity and commercial thymosin α1 similar. CONCLUSION: The fusion protein of thymosin α1 and IFN-α expressed in Escherichia coli not only has interferon antiviral activity but also thymosin α1 enhances immune function.