[Construction of hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 and identification of its expression]
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Published on: March 22, 2011
Shao-yan Si1, Yan-fang Sui, Pei-zhen Hu
1Department of Pathology, Fourth Military Medical University, Xi'an 710032, China. sishy306@yahoo.com.cn
Aim:
To construct hepatoma-targeting recombinant co-expression adenovirus vector of Staphylococcal enterotoxin A (SEA) and CD80 gene.
Methods:
Us-ing the adenovirus transfer plasmids pShuttle and pShuttle-CMV, we constructed a new transfer plasmid pShuttle2 with polyA signal sequence instead of CMV enhancer/promoter. AFP enhancer, promoter, SEA or CD80 gene was subcloned into pShuttle2 from the vectors pKS-EP or pMD18-T-BIS respectively, and then the constructed plasmid pShuttle2-BIS containing AFP enhancer, promoter, SEA or CD80 gene was cotransformed into E.coli BJ5183 with backbone vector pAdEasy-1 to obtain recombinant adenovirus DNA. The recombinant adenovirus DNA was transfected into 293 cells to prepare adenovirus. After AFP-producing cell line Hepa1-6 and AFP-nonproducing cell lines B16 and NIH3T3 were infected by recombinant adenovirus, the expression of SEA and CD80 on the surface of cells was detected by indirect immunofluorescent staining, laser confocal microscope and flow cytometry (FCM).
Results:
SEA and CD80 was specifically co-expressed on the surface of infected Hepa1-6 cells but not on B16 and NIH3T3 cells.
Conclusion:
Hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 gene was successfully constructed, which lays the foundation for further research on application of SEA and CD80 in targeted gene therapy for hepatoma and the underlying immunological mechanisms.
