【摘要】 目的构建人重组TNFα-Tumstatin45-132融合蛋白的表达载体,预测接头的合理性和可行性,并对融合蛋白活性进行验证。方法采用基因融合序列重叠延伸(SOE)策略,构建新型TNFα-Tumstatin45-132表达载体;应用核酸和蛋白质序列软件Antheprot和PepTool对融合基因及接头部位翻译后在二级结构水平上的柔性、抗原性、亲水性等生物特性加以预测;用细胞毒试验和内皮细胞增殖试验测定表达的融合蛋白活性。结果构建新型重组TNFα-Tumstatin45-132融合基因,经DNA测序证实与设计完全一致;TNFα-Tumstatin45-132融合基因的氨基酸序列经软件分析,并与TNFα和Tumstatin45-132单独分析的结果比较,未出现新的抗原性,亲水性没有改变,接头部位具有很低的抗原性,接头部位呈中性;表达的融合蛋白经证实具有杀伤L929细胞和抑制ECV304细胞增殖的作用。结论通过计算机软件对TNFα-Tumstatin45-132融合蛋白的预测,并与TNFα和Tumstatin45-132分别对比分析,有利于合理设计融合蛋白,最大程度地保留TNFα和Tumstatin45-132各自的生物活性和功能;生物活性分析证实设计具有一定的科学性和合理性。更多还原

【Abstract】 Objective To construct the expressed vector which express recombinant TNFα-Tumstatin45-132and to predict the rationality and feasibility of the linker,and to certify the bioactivity.Methods To fuse TNFα and Tumstatin45-132encoding gene through sequence overlapping extension(SOE)technique and to analyze the flexibility,antigenicity and hydrophilicity of the recombinant TNFα-Tumstatin45-132 by sequence analysis software.Cell toxicity and proliferation assays were used.Results Sequence analysis showed that splicing order,the direction and the sequence in TNFα-Tumstatin45-132 fusion gene were completely correct.The linker has low antigenicity and high flexibility.The fusion protein killed L929 and inhibited ECV304.Conclusion The results of computer analysis could help us to design the recombinant TNFα-Tumstatin45-132rationally and keep it in maxium biological activity.And bioactivity analysis certified the rationality and feasibility.更多还原