Construction of recombinant adenovirus vector of human wnt5a

Objective: To construct the recombinant adenovirus vector containing human wnt5a gene by homogenous recombination in E.coli for later research of the mechanism of Wnt signal pathway in tooth development. Methods: The cDNA of human wnt5a was derived from MCF-7 cell, then was cloned into pMD18-T vector, reproduced in E. coli JM109, identified by digestion, and the positive clone was sequenced. Then the reproduced pMD18-wnt5a plasmid was cut by two endonucleases and directional subcloned into pAdtrack-CMV shuttle vector. The recombinant was linearized by PmeI, following co- transformation with the backbone vector pAdEasy-1 in E.coli BJ5183. The homologous recombinant pAd-wnt5a was linearized by PacI, then transfected into AD293 cell line to pack the adenovirus. The expression of green fluorescence protein was observed under fluorescence microscope. For further amplification and purification, the titer of adenovirus was determined. Results: Through PCR, endonuclease cutting and gene sequencing, the target gene was verified to be correctly cloned in adenovirus vector. The high expression of green fluorescence protein in AD293 cell line was confirmed under fluorescent microscope. Conclusion: The recombinant adenovirus vector containing human wnt5a gene was successfully constructed using AdEasy system, and the adenovirus can be packaged in AD293 cell line. These results provide the basis for further study on the role of human wnt5a gene in dental development.