A simplified two-step method of homologous bacterial and efficient preparation of recombinant adenovirus vector expressing p53 gene.docVIP

 PAGE \* MERGEFORMAT 19 A simplified two-step method of homologous bacterial and efficient preparation of recombinant adenovirus vector expressing p53 gene Zou Changyong, the whole family flatter Δ, tan, and Yang Jing-sheng, Wu Ji Nam 【Abstract】 Objective homologous recombination in bacteria and efficient preparation of recombinant adenovirus p53 gene. Designed primers p53 gene, respectively, upstream and downstream primers to introduce Xhol and Hind Ⅲ restriction sites will be connected to the p53 gene was subcloned digested by the same shift p shuttle-cmv plasmid transformed into DH5α screening kanamycin Construction of recombinant transfer plasmid resistant colonies p shuttle-cmv-p53 linearized with Pmel digestion of homologous recombination using two-step method: first adenovirus backbone material pAdEasy-1 into BJ5183 calcium chloride prepared by Competent bacteria were screened streptomycin and ampicillin resistance AdBJ5183 transformed bacteria; then linearized p shuttle-cmv-p53 into calcium chloride prepared by AdBJ5183 Competent bacteria, within the homologous recombination in bacteria, selection kanamycin-resistant colonies cultivate and extract the plasmid, agarose gel electrophoresis to select large fragment of recombinant plasmid pAdBJ5183-P53 were digested with restriction enzymes PacI and BstXI and the identification of PCR method. The result successfully constructed recombinant adenovirus p53 expression vector. Conclusion simplified two-step calcium chloride chemical transformation can be replaced electroporation for Constructing recombinant adenovirus expression vector. Keywords: adenovirus vector; homologous recombination; p53 gene 【Abstract】 Objective To construct recombinant replication-defective human adenovirus serotype 5 vector carrying wt-p53 gene by simplified two-step homologous recombination protocol in bacteria.Methods wt-p53 gene was firstly subcloned into a transfer vector p shuttle-cmv and the positive re