禽源HSP70、HSP40和RPL4基因的克隆和表达

Cloning and Expression of Avian-origin Genes of HSP70,HSP40 and RPL4

本研究参照GenBank中编码禽源热休克蛋白HSP70、HSP40和核糖体蛋白RL4等的基因序列,分别设计特异性引物,采用RT-PCR扩增HSP70、HSP40和RPL4基因,经双酶切后克隆到真核表达载体pEF1α-Myc,得到重组质粒pEF1α-Myc-HSP70、pEF1α-Myc-HSP40和pEF1α-Myc-RPL4;将构建好的重组质粒,转染HEK293T细胞后,采用间接免疫荧光和Western-blot技术,对目的蛋白进行验证。结果表明,Myc-HSP70、Myc-HSP40和Myc-RPL4融合蛋白在HEK293T细胞内得到了正确表达。本研究为后续禽源HSP70、HSP40和RPL4基因的功能研究奠定了基础。

In this study,speci c primers were respectively designed according to the gene sequences of the heat-shock protein(HSP70 and HSP40)and the ribosomal protein(RPL4)registered in GenBank,then the genes of HSP70,HSP40 and RPL4 were ampli ed by RT-PCR,and after double enzyme digestion,the quali ed genes were cloned into the eukaryotic expression plasmid,pEF1α-Myc,to obtain the recombinant plasmids including pEF1α-Myc-HSP70,pEF1α-Myc-HSP40 and pEF1α-Myc-RPL4;after the recombinant plasmids were transfected into HEK293T cells,the expressed protein was veri ed by indirect immuno uorescence and Western-blot.The results showed that the recombinant proteins of Myc-HSP70,Myc-HSP40 and Myc-RPL4 were correctly expressed in HEK293T cells.Therefore,the foundation was laid for further study on the genes of HSP70,HSP40 and RPL4.