Pan Xiaoyi

Construction and identification of recombinant eukaryotic expression vector pCI-VP7 containing GCRV VP7 gene.

A target sequence of 0. 9kb which codes the major capsid protein VP7 of grass carp reovirus (GCRV) was amplified by PCR. The target fragment was inserted into the pMD19-T vector. The positive clone was screened and sequenced. Sequencing result showed the nucleic acid sequence is right. Then the target gene was cloned into eukaryotic expression vector pCI and the positive clone was screened. The recombinant plasmid pCI-VP7 extracted from the positive clone were identified by PCR and digestion. Then the recombinant plasmid pCI-VP-GFP (fusion expression of GFP and partial VP7 gene) was constructed to testify the expression of VP7 gene. The recombinant plasmid pCI-VP7-GFP were transfected into COS-1 and CIK cells by lipofectamine 2000. Fluorescence microscope and RT-PCR was used to detect the transient expression. The results showed that the gene fragment was transfected and expressed in COS-1 and CIK cell successfully. The results indicated that the recombinant plasmid pCI-VP7 can also express successfully. It establishes foundation to research the gene vaccine of GCRV.