Feng Zhao1, Qinghua Zhou, Yang Qin, Zhiling Sun, Zefang Sun. 1. Department of Thoracocardiac Surgery, West China Hopsital, Sichuan University (Former The First University Hospital, West China University of Medical Sciences), Chengdu, Sichuan 610041, P.R.China.
Abstract
BACKGROUND: To construct the recombinant adenovirus of mutant k-ras by using the method of homogenous recombination in bacteria. METHODS: Mutant k-ras gene was liberated from the vector of pcDNA3-k-ras 12(Val) via KpnI+XhoI digestion, and subcloned into shuttle vector of pAdTrack-CMV, forming transfer vector of pAdTrack CMV/k-ras 12(Val). Then it was linearized with PmeI and cotransformed into BJ5183 cells with adenovirual geonomis plasmid of pAdEasy-1. The DNA of identified recombinant plasmid was digested with PacI and transfected to 293 cells to package adenovirus. The PCR technique was used to detect target gene. The titre and its infection rate of the Ad-k-ras 12(Val) was measured with the aid of GFP expression. RESULTS: There were over 25% positive recombinant bacterial clones after co transformation of BJ5183 bacterial cells with pAdTrack-CMV/k-ras 12 (Val) and pAdEasy-1 by method of CaCl₂. PCR test indicated each the recombinant adenovirus contained the insert of k-ras 12(Val). The titre of purified recombinant adenovirus was 1.2×10¹² pfu/ml. CONCLUSIONS: The method of homologous recombination in bacteria is convenient and efficient, which compared with that of in cell and the pepared Ad-k-ras 12(Val) paves a sound foundation for further study.
BACKGROUND: To construct the recombinant adenovirus of mutant k-ras by using the method of homogenous recombination in bacteria. METHODS: Mutant k-ras gene was liberated from the vector of pcDNA3-k-ras 12(Val) via KpnI+XhoI digestion, and subcloned into shuttle vector of pAdTrack-CMV, forming transfer vector of pAdTrack CMV/k-ras 12(Val). Then it was linearized with PmeI and cotransformed into BJ5183 cells with adenovirual geonomis plasmid of pAdEasy-1. The DNA of identified recombinant plasmid was digested with PacI and transfected to 293 cells to package adenovirus. The PCR technique was used to detect target gene. The titre and its infection rate of the Ad-k-ras 12(Val) was measured with the aid of GFP expression. RESULTS: There were over 25% positive recombinant bacterial clones after co transformation of BJ5183 bacterial cells with pAdTrack-CMV/k-ras 12 (Val) and pAdEasy-1 by method of CaCl₂. PCR test indicated each the recombinant adenovirus contained the insert of k-ras 12(Val). The titre of purified recombinant adenovirus was 1.2×10¹² pfu/ml. CONCLUSIONS: The method of homologous recombination in bacteria is convenient and efficient, which compared with that of in cell and the pepared Ad-k-ras 12(Val) paves a sound foundation for further study.