ABSTRACT
OBJECTIVE: To construct a recombinant adenovirus of survivin vector and provid valuable reference for gene therapy of laryngeal cancer. METHODS: The survivin gene was cloned by PCR. After confirmation by enzyme restriction analysis and sequencing, the gene and the adenovirus vector were recombined together to construct the recombinant adenovirus vector. The recombinant adenovirus vector was confirmed via both sequencing and digestion restriction analysis, and then linearized and transfected into the HEK 293 cell line to generate recombinant adenovirus. RESULTS: The sequence analysis demonstrated that the survivin gene sequence was the same as published in the literature, suggesting that a recombinant adenovirus vector has been successfully constructed. CONCLUSIONS: A survivin recombinant adenovirus has been successfully constructed.
Subject(s)
Adenoviridae/genetics , Genetic Vectors , Inhibitor of Apoptosis Proteins/genetics , Recombinant Fusion Proteins/genetics , HEK293 Cells , Humans , Inhibitor of Apoptosis Proteins/metabolism , Plasmids , Polymerase Chain Reaction , Recombinant Fusion Proteins/metabolism , Survivin , TransfectionABSTRACT
This study was purposed to investigate the role of integrin beta3 cytoplasmic domain in signal transduction mediated by integrin alphaIIbbeta3 and to explore the effect of integrin beta3 on signal transduction and specificity in condition without alphaIIb subunit. The fusion protein (Tac/beta3) was stably expressed in CHO cell line expressing GPIbIX, integrin alphaIIbbeta3 (IbIX/IIbIIIa-CHO cell line) by combining extracellular and transmembrane domains (Tac) of IL-2 receptor with integrin beta3 cytoplasmic domain (beta3) for formation of fusion protein (Tac/beta3). Then a series of tests were performed, including spreading and stable adhesion of IbIX/IIbIIIa-CHO cell line in solid phase fibrinogen (Fg), fibrin clot restriction and soluble fibrinogen binding, which represent outside-in and inside-out signal transduction events. The results showed that the bidirectional signal transduction mediated by alphaIIbbeta3 in IbIX/IIbIIIa-CHO/Tac-762 cells stably expressing Tac/beta3 was seriously inhibited. It is concluded that the Tac/beta3 can play a significant role in IbIX/IIbIIIa-CHO/Tac-762 cells through a dominant negative mode, the independent presence of beta3 subunit cytoplasmic domain can regulate the bidirectional signal transduction mediated by integrin alphaIIbbeta3.