[Rescue of minireplicon by using the cell line stably expressing the T7 RNA polymerase].
To improve the rescue efficiency of measles virus cDNA clone, the cell line that stably expressed the T7 RNA polymerase was established. Firstly, the T7 RNA polymerase gene was amplified by PCR and then the PCR product was inserted into pcDNA3 to obtain plasmid pcDNA3-T7. Vero cell was transfected with the plasmid and G418 was added to the cell 24h later to kill the cells without the plasmid. Western blotting analysis showed that the Vero/pcDNA3-T7 cell could express T7 RNA polymerase. To analyze the gene function of T7 RNA polymerase, the pT7IP-EGFP plasmid was transfected into the Vero/pcDNA3 T7 cell and EGFP was analized by fluorescence. The result suggested that T7 RNA polymerase expressed in the Vero/pcDNA3-T7 cell could transcribe the gene under control of the T7 promoter. Moreover, the minigenome PminiEGFP inserted reversely with report gene EGFP was established. After trans fection with the plasmid and infection with measles virus, EGFP was expressed, indicating the Vero/pcDNA3-T7 cell could rescue the minigenome.
Xiu
Mei-hong
MH
State Key Laboratory for Infectious Diseases Control and Prevention, National Institute for Viral Disease Control and Prevention, China CDC, Beijing 100052, China.
Wang
Qin
Q
Tang
Li-hua
LH
Cao
Shou-chun
SC
Li
Wei-hong
WH
Wei
Yan
Y
Lu
Peng
P
Liang
Mi-fang
MF
Li
De-xin
DX
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002522:Chlorocebus aethiops; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008459:Measles virus / Q000235:genetics; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases