Construction of recombinant adenoª²viral plasmid bearing hSDFª²1¦Á cDNA by homologous recombination in bacª²teria and preparation of recombinant adenovirus expressing hSDFª²1¦Á

    TANG Junª²Ming1,2, GUO Lingª²Yun1, KONG Xia1, YANG Jianª²Ye1, PAN Guoª²Dong1, CHEN Long1, HUANG Yongª²Zhang1, WANG Jiaª²Ning1

    1Institute of Clinical Medicine, People¬ðs Hospital, 2Department of Physiology, Yunyang Medical College, Shiyan 442000, China

    ¡¾Abstract¡¿ AIM£º To construct recombinant adenoviral plasmid containing hSDFª²1¦Á cDNA using homologous recombination in bacteria and to prepare recombinant adenovirus expressing hSDFª²1¦Á. METHODS: Adenoviral backbone plasmid was transformed into competent BJ5183 and the competent BJ5183 transformed with pAdEasyª²1 was prepared. The linearized pShuttleª²EGFPª²hSDFª²1¦Á plasmid with Pme I digestion and CIAP dephosphorylation was transformed into the competent cells BJ5183 transformed with pAdEasyª²1. The identified recombinant adenoviral plasmid pAdª²EGFPª²hSDFª²1¦Á was digested with Pac I and transfected into AD293 cells with cationic liposome LipoVec to package recombinant adenovirus Adª²EGFPª²hSDFª²1. Adª²EGFPª²hSDFª²1¦Á was proª²pagated by repeated rounds of infection of AD293 cells with supernatant of the recombinant adenovirus. Adª²EGFPª²hSDFª²1¦Á was purified with CsCl density gradient ultracentrifugation. RESULTS: pShuttleª²EGFPª²hSDFª²1¦Á was successfully transformed into competent BJ5183 transformed with pAdEasyª²1 and homologous recombination between pAdEasyª²1 and pShuttleª²EGFPª²hSDFª²1¦Á took place within BJ5183 bacteria. Liposomeª²mediated transfection of pAdª²EGFPª²hSDFª²1¦Á digested with Pac I into AD293 cells was performed. The packaging of recombinant adenovirus Adª²EGFPª²hSDFª²1¦Á within AD293 cells was confirmed by fluorescent microscopy. The viral titer was 4.1¡Á1015 pfu/L. CONCLUSION: The recombinant adenovirus expressing hSDFª²1¦Á was prepared successfully by a simple and rapid homologous recombination in bacteria. This study provides a basis for exploring the migration mechanism of bone marrowª²derived stem cells into injured tissue such as infarcted myocardium. ......