[Eukaryonization of T7 RNA polymerase prokaryotic expression system and development of its couple expression system].
To make transcription of the target gene be driven by T7 RNA polymerase (T7 RNAP) in the eukaryotic cells, and the transcripts be CAP-independent translated. Firstly, the T7 RNAP was introduced into eukaryotic cells by two methods: (1) the BHK-21 cells were contransfected by the plasmid expressing T7 RNAP and pIERS-EGFP-ET vector; (2) by transfection of the cell line stably expressing T7 RNAP. The internal ribosome entry site (IRES) element from FMDV was cloned into the downstream of the T7 promoter sequence of the prokaryotic expressing vector pET-40a-c (+), resulted in the plasmid would express the transcripts carrying the IERS element at its 5' end. The enhanced green fluorescent protein (EGFP) gene was cloned into the downstream of the IERS element, resulted in plasmid pIERS-EGFP-ET. Then, the two kinds of cells expressing T7 RANP were transfected by pIERS-EGFP-ET. The green fluorescence in the transfected cells was observed under a fluorescence microscope equipped with a video documentation system. And the expressional efficiency was analyzed with flow cytometry (FCM). The results show that the IRES element from FMDV has the role of initiating CAP-independent translation, and lay foundation for researching function of the element and interrelated proteins. It would be potential for expressing target gene by the T7 RNAP couple expression system.
Zheng
Hai-Xue
HX
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China.
Jin
Ye
Y
Yin
Shuang-Hui
SH
Guo
Hui-Chen
HC
Shang
You-Jun
YJ
Bai
Xing-Wen
XW
Liu
Xiang-Tao
XT
Xie
Qing-Ge
QG
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis / Q000235:genetics*; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics*; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*
D014764:Viral Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases