Efficient generation of human mutant p27 gene recombinant adenovirus by homologous recombination in bacteria

    CHEN Jun, XU Shaoª²Yong, DENG Changª²Sheng, WANG Jiaª²Ning, HUANG Yongª²Zhang

    1Department of Gastroenterology, Affiliated People¡¯s Hospital, Yunyang Medical College, Shiyan 442000, China, 2Department of Gastroenterology, Zhongnan Hospital, Wuhan University, Wuhan 430071, China

    ¡¾Abstract¡¿ AIM: To construct the recombinant adenovirus of human mutant p27 by the method of homogenous recombination in bacteria. METHODS: hp27mt gene was digested from plasmid of pORF9ª²hp27mt and subcloned into the plasmid of pBluescript¢òSK(+) and the plasmid of pBluescriptª²hp27mt was constructed. Then hp27mt gene was liberated from pBluescriptª²hp27mt and subcloned into shuttle vactor and turned into transfer plasmid of pShuttleª²CMVª²hp27mt. Adenovirus genomic DNA plasmid of pAdeasyª²1 was transformed into BJ5183 bacterial cell and prepared ultracompletent BJ5183 containing pAdeasyª²1. pShuttleª²CMVª²hp27mt was linearized with PmeI and transformed into ultracompletent BJ5183 bacterial cell containing pAdeasyª²1 and positive clone of homologous recombination was selected. The cDNA of identified recombinant plasmid was digested with PacI and transferred to Ad293 cells to package Adenovirus particles. PCR technique was used to detect target gene and the titer of the recombinant Ad was determined by measuring the absorbance at 260 nm. RESULTS: There were over 30% positive recombinant bacteria clones after the cotransformation of BJ5183 bacterial cells with pShuttleª²CMVª²hp27mt and Padeasyª²1. The titer of purified recombinant adenovirus was 7.95¡Á1015 pfu/L. CONCLUSION: Homogenous recombination in bacteria can efficiently and conveniently construct high quality recombinant adenovirus. ......