The Construction Of PKC ε Recombinant Adenovirus Vector And Its Expression In The Eukaryotic Cells

Objective:In this study,pAdEasy system was used to generate recombinant adenovirus vector carrying PKC epsilon protein adenovirus expression vector.We construct recombinant adenovirus infection of primary rat cardiac cells,use western-blot technique to detect the protein levels of PKC epsilon expression,and through the MTT colorimetric assay for testing the cell survival rate to make sure that the recombinant vector is successfully constructed.Methods:Amplificate the full-length DNA of PKC epsilon gene from wild-type plasmid PKC ε by using PCR method,and double enzyme the purified cDNA and pShuttle-CMV respectively by Kpn Ⅰ and Xho Ⅰ. Then recover fragments with low melting agarose gel electrophoresis,and connects the two parts to transform Escherichia coli. Then extract the recombinant plasmid pShuttle-CMV-PKC epsilon, which was identified by enzyme digestion and sequence identification.Using a shuttle plasmid pShuttle-CMV-PKC epsilon in Escherichia coli BJ5183and backbone plasmid PAdEasy-lto do some chemical conversion,homologous recombination to form PKCe/PAdEasy-1recombinant plasmid,extraction and purification of plasmid,linearization by Pac Ⅰ digestion,transfecting HEK293cells by Calcium phosphate,packaging intact virus,collecting virus. Infected HEK293cells again,amplification of collecting and purifing virus. Infection in primary cultured rat myocardial cells with PKC epsilon adenovirus.Detection of PKC epsilon with high expression at the protein level through Western-blot technology,and through the MTT colorimetric assay for testing the cell survival rate,thus confirming the recombinant adenovirus vector was constructed successfully.Result:Demonstrated by enzyme digestion and DNA sequencing,plasmid construction of shuttle plasmid pShuttle-CMV-PKC epsilon was successful. Plasmid with target gene and backbone plasmid were homologous recombinated in BJ5183, and then packaged to complete virus in HEK293cells,then amplified to get high titers of virus,detected PKC epsilon protein expression using Western blot method,and through the MTT colorimetric assay for testing the cell survival,through the MTT colorimetric assay for testing the cell survival rate,then confirmed by high expression of adenovirus vector.Conclusion:Building on the success of the PKC epsilon protein adenovirus to expression vector,and the success of primary rat myocardial cells with infection, through Western-blot technology at the protein level to detect the PKC epsilon expression,and the MTT colorimetric assay for testing the cell survival rate, to make sure that the PKC epsilon protein adenovirus expression vector was constructed successfully, and to provide tools for getting on with the study of PKC epsilon myocardial protection.