Construction And Identification Of Recombinant Adenoviral Vector RhAdEasy-nm23-H1 And Research Of Expression In Vitro

[Objective ] :To construct the recombination vector of replication defective adenovirus and nm23-H1 cDNA and further to examine the expression of nm23-H1 which was deliverd by the recombination vector in tumor cell cultivated in vitro.[ Methods ] : A pair of DN A primers with Kpn I ,Xho I restriction Enzyme cutting site were designed and synthesized.With the primers,nm23-H1 cDNA was cloned from pMD18-nm23 plasmid by PCR technology and cloned into pShuttle-CMV in correct direction. The product was pShuttle-nm23. PShuttle-nm23 had been identified by double digestion with Kpn I and Xho I restriction emzymes and DNA sequensing. Homologous recombination occurred between pShuttle-nm23 and advenoviral backbone plasmid pAdEasy-1 in BJ5183 competent bacteria and the recombinant advenovirus was called rhAdEasy-nm23-H1. The recombinant vector was selected by Kanamycin.Identified by digestion with Pac I and PCR, it was amplified broadly in XL10-Gold competent bacteria and purified by CsCL density gradient centrifugation and was packaged and amplified in AD-293 cell.The infectious titer TCIDso of the recombinant advenovirus was tested. A series of assays including immunofluorescence .immunohistochemistry and Western Blot were carried out to monitor the expression of nm23 protein in lung cancer cell lines:YTMLC and GLC.[Results] :Digestion with Kpn I and Xho I got a fragment size being 460bp.The result of DNA sequensing showed that the sequense of gene combined with pShuttle-CMV was identical to the coding region of nm23-H1 gene in Genebank. It demonstrated that nm23-H1 cDNA had been linked with pShuttle-CMV vector with...