Construction Of Recombinant Adenovirus Vector Of CGRP

AIM: To develop a gene therapy vector of Calcitonin gene-related peptide (CGRP) using recombinant adenovirus. METHODS: RT-PCR was performed to get full-length cDNA of CGRP gene. The gene was then cloned into the PUC₅₇ and pshuttle-CMV shuttle vector, respectively. After linearization with Pme I, the recombinant plasmid (pshuttle-CMV-CGRP) was transformed into E.coli BJ5183 cells by electroporation to construct the homologous recombination of AdEasy-pShuttle-CGRP. The linearized recombinant plasmids were used to transform XL10-Gold ultracompetent cells and the recombinant adenovirus plasmid DNA can be amplified. Then the recombinant plasmid was transfected into 293 cells. RESULTS: The RT-PCR product was confirmed a full-length cDNA of CGRP in PUC₅₇ by sequencing. The corresponding double endonuclease and PCR analysis certified the successful cloning of the gene into the pshuttle-CMV. The recombinants of AdEasy-pShuttle-CGRP were selected for kanamycin resistance and digested by Pac I endonuclease to form the typical DNA segments, whose length was about 3kb and 30kb. The transfected cells show evidence of a cytopathic effect (CPE). Restriction endonuclease and PCR analyses confirmed that the CGRP gene was successfully inserted into the adenovirus vector with very strong power of transfection. The titer of the recombinant adenovirus was 5×10⁸ PFU /ml. CONCLUSION: Therecombinant adenovirus vector of CGRP gene was successfully constructed by the method of homogenous recombination in bacteria.