Abstract:Objective:To identify and construct a recombinant adeno-associated virus (AAV) vector that can secrete Exendin-4 mediated by NT4 leading sequence.Methods: ·L^-1).Conclusion:The recombinant virus pSSHG/NT4 - Exendin - 4 was successfully constructed by molecular cloning. The relatively high levels of recombinant virus were obtained. These results provide the basis for further studies that examine the function of Exendin-4.Exendin-4 cDNA was cloned by complementary primer/template PCR and T carrier cloning method. The Exendin-4 gene was linked to terminal of the signal peptide of NT4 leading sequence. The fusion gene of NT4- Exendin-4 was subcloned into the shuttle plasmid of pSSHG. The recombinant virus was transfected and packaged by human embryo kidney 293 cells and the virus titer was measured by quantitative dot-blot hybridization.Results:Exendin-4 cDNA cloning was confirmed by restriction enzyme digestion and DNA sequencing. The NT4 leading sequence and Exendin-4 cDNA clone were successful constructed in the same ORF and high titer of recombinant AAV was obtained (2.5×10⁹pfu

Key words:plasmid construction,Exendin-4,NT4,adeno-associated virus (AAV)