Cloning And Construction Of Recombinant Adenovirus Vector Of Nm23-H1

Objective Nm23-HlcDNA gene was cloned from human liver tissue with the correct sequences and recombinant adenovirus vector was constructed . The results will promote the farther research in molecular biology ofnm23-Hl gene, and the p AdShuttle-CMV-nm23-H1 can be potentially used in tumor gene therapy.Methods A pair of DNA primers were designed and synthesized(23,26 bases respective).From human liver, antimetastic nm23-H1cDNA fragment was successfully amplified by reverse transcription-polymerase chain reaction(RT-PCR), and cloned into pMD18-T plasmid. PCR site-directed mutagenesis was done after sequencing. Mutagenic nm23-H1 gene was cloned in plasmid . Then pAd-Shuttle-CMV vector of nm23-H1 was constructed.Results Analysis of the sequence results showed that the fragment cloned pMD18-T was nm23-H1 gene with two nucleotides different from nm23-Hl gene in Genbank (213C-->>T, 217C-->>T), which reflected that two amino acids were changed (71G-->>A, 73V-->>I). After PCR site-directed mutagenesis, the sequencing results showed that fragment cloned in pMD18-T was nm23-H1. Then a pAd-Shuttle -CMV vector containing nm23-Hl gene was successfully constructed with correct order. Conclusion Succeed in mutating two bases. Succeed in constructingpMD18-nm23 and recombined adenovirus pAdShuttle-CMV-nm23-H1. The results suggest the feasibility of gene therapy for oral cancer. The results can prompt the research of nm23-H1 mutagenesis.