Rapid Construction of homologous recombination in bacteria and preparation of expression hSDF.doc

 PAGE \* MERGEFORMAT 10 Rapid Construction of homologous recombination in bacteria and preparation of expression hSDF Authors: Jun-Ming Tang, GUO Ling-Yun, Kong Xia, Jian-Ye Yang, Pan Guodong, Chen, Huang Zhang, Jia-Ning [Keywords:] homologous recombination; human stromal cell-derived factor-source Construction of recombinant adenoviral plasmid bearing hSDF1α cDNA by homologous recombination in bacteria and preparation of recombinant adenovirus expressing hSDF1α [Abstract] AIM: To construct recombinant adenoviral plasmid containing hSDF1α cDNA using homologous recombination in bacteria and to prepare recombinant adenovirus expressing hSDF1α . METHODS: Adenoviral backbone plasmid was transformed into competent BJ5183 and the competent BJ5183 transformed with pAdEasy1 was prepared. The linearized pShuttleEGFPhSDF1α plasmid with Pme I digestion and CIAP dephosphorylation was transformed into the competent cells BJ5183 transformed with pAdEasy1. The identified recombinant adenoviral plasmid pAdEGFPhSDF1α was digested with Pac I and transfected into AD293 cells with cationic liposome LipoVec to package recombinant adenovirus AdEGFPhSDF1. AdEGFPhSDF1α was propagated by repeated rounds of infection of AD293 cells with supernatant of the recombinant adenovirus. AdEGFPhSDF1α was purified with CsCl density gradient ultracentrifugation. RESULTS: pShuttleEGFPhSDF1α was successfully transformed into competent BJ5183 transformed with pAdEasy1 and homologous recombination between pAdEasy1 and pShuttleEGFPhSDF1α took place within BJ5183 bacteria. Liposomemediated transfection of pAdEGFPhSDF1α digested with Pac I into AD293 cells was performed. The packaging of recombinant adenovirus AdEGFPhSDF1α within AD293 cells was confirmed by fluorescent microscopy. The viral titer was 4.1 * 1015 pfu / L. CONCLUSION: The recombinant adenovirus expressing hSDF1α was prepared successfully by a simple and rapid homologous recombination in bacteria. This study provides a basis