SCIRR 10截短体真核表达载体的构建及其在COS-7细胞中的表达

Construction of three truncated SCIRR 10 protein eukaryotic expression vectors and their expressions in COS-7 cells

目的:构建SCIRR 10蛋白3个截短体的哺乳动物细胞表达系统,探讨SCIRR 10蛋白的功能位点。方法:PCR扩增出SCIRR 10蛋白的3个功能截短体表达DNA序列,构建3种真核表达质粒载体pcDNA3.1/Myc-His-SCIRR 10(1-145aa)、pcDNA3.1/Myc-His-SCIRR 10(1-90aa)和pcDNA3.1/Myc-His-SCIRR 10(1-70aa)。将3个表达载体质粒转染到COS-7细胞中,应用Western blotting方法检测3个蛋白截短体的表达情况。结果:3个被截短的SCIRR 10蛋白相对分子质量分别为18 800、12 800和10 700。在COS-7细胞中可以成功表达3个SCIRR 10蛋白截短体。相对3个SCIRR 10截短体蛋白的表达量,β-tubulin蛋白无明显变化。结论:成功构建SCIRR 10蛋白3个截短体的哺乳动物细胞表达系统,通过此系统获得有生物活性的3个SCIRR10截短体蛋白。

Objective To construct three truncated SCIRR 10 protein mammalian cells systems,and discuss the protein function sites of SCIRR 10.Methods Three truncated SCIRR 10 genes were amplified by PCR,and three kinds of recombinant eukaryotic expression vectors were constructed as follows：pcDNA3.1/Myc-His-SCIRR 10（1-145aa）,pcDNA3.1/ Myc-His-SCIRR 10（1-90aa） and pcDNA3.1/Myc-His-SCIRR 10（1-70aa）.Then they were transiently transfercted into COS-7 cells at the same time.After 48 h,the recombinant proteins were detected by Western blotting.Results The molecular weights of three truncated SCIRR 10 proteins were 18 800,12 800 and 10 700.Three SCIRR 10 truncated proteins were successfully expressed in COS-7 cells.β-tubulin protein had no change.Conclusion The three truncated SCIRR 10 protein mammalian cell systems are constructed successfully,and the proteins are obtained by this system.