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禽源HSP70、HSP40和RPL4基因的克隆和表达

8494 2020-03-12 14:08:05 任红玉,谢芝勋,谢丽基,等 中国动物检疫
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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。

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

In this study,specific 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 amplified by RT-PCR,and after double enzyme digestion,the qualified 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 verified by indirect immunofluorescence 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.

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