CD11b-BFP融合蛋白在U937细胞株中的表达与鉴定

Expression of CD11b-BFP fusion protein in U937 cell line

目的 :构建并表达巨噬细胞分化抗原 1(Mac- 1)的 α链 CD11b与蓝色荧光蛋白 (BFP)的融合蛋白 CD11b- BFP,为进一步直视研究 Mac- 1在白细胞内的分布、走向及归宿提供物质条件。方法 :首先将 CD11b的全长 c DNA进行酶切并获得 2个片段 ,将其中含有终止密码的片段作为模板 ,通过设计引物进行 PCR,获得不含有终止密码的该片段 ,然后与另一片段进行连接 ,形成不含有终止密码的 Mac- 1全长 c DNA,将其插入到表达载体 p EBFP- N1中 ,构建完成 CD11b- BFP融合基因 ,最后将其转染至 U937细胞株中 ,通过荧光显微镜观察与流式细胞术检测 Mac- 1的表达及其黏附活性来进行鉴定。 结果 :经酶切鉴定 ,p CD11b- BFP构建完全正确 ,转染 U937细胞株后 ,可见 CD11b- BFP融合蛋白发出的蓝色荧光。 结论 :构建完成 CD11b-BFP融合基因并在

Objective:To construct the expressive vector of pCD11b BFP and to express fusion protein CD11b BFP in U937 cell line, providing foundation for advanced researching in intracellular distribution and dynamic trafficking of Mac 1. Methods:CD11b cDNA was digested into 2 fragments by restriction endonuclease. As the terminal codon of CD11b influence the expression of fusion protein CD11b BFP, the fragment with terminal codon should be removed by PCR using designed primer and then be ligated with another fragment into one complete cDNA. After that the full length cDNA was inserted into the MCS of pEBFP N1 vector to construct the expression vector of pCD11b BFP. U937 cell line was transfected with recombinant vector containing fusion gene and expressed fusion proteins CD11b BFP, which were visible with a fluorescence microscope and were measured by flow cytometry and were assayed with ligand. Results:Restriction endonuclease digestive identification was right for recombinant expression vector pCD11b BFP.Blue fluorescence from fusion protein could be seen in U937 cells transfected with plasmid pCD11b BFP. Conclusion:Recombinant expressive vector of pCD11b BFP was constructed and expressed in U937 cell line successfully.