口蹄疫病毒WFL株ORF基因真核表达质粒的构建及病毒的拯救

Rescue of FMDV from Contransfecting Full Length Infectious RNA of WFL Strain and Eukaryotic Expression Plasmid of FMDV ORF Gene

构建了FMDVWFL株ORF(open reading frame)基因真核表达质粒pEGFP-C1-A3I3,并进行了表达研究和共转染研究。结果发现,该质粒可以在BHK-21细胞中表达。将其与体外转录获得的FMDV RNA共转染BHK-21细胞后,用夹心ELISA、RT-PCR法以及电镜观察证明共转染后的细胞培养液中有病毒粒子,且病毒量高于单独转染RNA所得病毒量。证明用FMDV基因组真核表达质粒与其基因组体外转录RNA共转染敏感细胞可提高拯救病毒的数量。

An eukaryotic expression plasmids pEGFP-C1-A313, with open reading frame （ORF） of FMDV WFL strain, was constructed based on the pEGFP-C1. The results of transient expression showed that the eukaryotic expression plasmids can be expressed in BHK-21. When cotransfected the plasmid with RNA transcribed from linearized cDNA clone, FMDV antigen and nucleotide acids was detected in cell culture fluid by sandwich ELISA and RT-PCR,respectively. And the virus particles were also found by electron microscope. The results proved that cotransfection of eukaryotic expression plasmid with RNA can improve virus rescuing.