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目的:构建活化的激酶C受体1(RACK1)WD1-4真核表达载体,通过细胞转染和Western blot鉴定其是否在细胞中表达。方法:应用PCR法扩增RACK1蛋白中第1～4个WD40区域的片段,将其与PGEM-T载体连接,测序后再将其克隆至pEGFP-N1中。采用脂质体法将重组质粒转染至293T细胞中,观察其在真核细胞中的表达,并用Western blot法进行鉴定。结果:pEGFP-N1-RACK1(WD1-4)重组质粒经酶切鉴定及测序证实,目的基因的序列完全正确。荧光显微镜观察发现,转染了重组质粒的细胞株可见绿色荧光融合蛋白的表达,Western blot结果进一步证实了上述结果。结论:成功构建pEGFP-N1-RACK1(WD1-4)重组质粒且进行了真核表达,为下一步研究RACK1蛋白的功能奠定了基础。
OBJECTIVE: To construct the eukaryotic expression vector of activated RACK1 WD1-4, which was identified by cell transfection and Western blot. METHODS: The first to fourth WD40 regions of RACK1 protein were amplified by PCR. The fragments were ligated with PGEM-T vector and sequenced before being cloned into pEGFP-N1. The recombinant plasmids were transfected into 293T cells by liposome method, and their expression in eukaryotic cells was observed and identified by Western blot. Results: The recombinant plasmid pEGFP-N1-RACK1 (WD1-4) confirmed by restriction enzyme digestion and sequencing showed that the sequence of the target gene was completely correct. Fluorescence microscopy showed that the expression of green fluorescent fusion protein was observed in the cell lines transfected with recombinant plasmids. Western blot results further confirmed the above results. Conclusion: The recombinant plasmid pEGFP-N1-RACK1 (WD1-4) was successfully constructed and eukaryotic expressed, which laid the foundation for the further study on the function of RACK1 protein.