mING基因的腺病毒载体的构建与表达

The Construction and Identification of Recombined Adenovirus Vector of mING4

目的构建鼠ING4(肿瘤生长抑制因子4)的腺病毒载体,获得鼠ING4(mING4)重组腺病毒子,为ING4进行肿瘤的基因治疗奠定基础。方法以pcDNA3.0-mING4重组质粒为模板,PCR扩增mING4,酶切连接到带有GFP标记的pAdTrack-CMV质粒上,PmeI线性化重组质粒pAdTrack-CMV-mING4,与腺病毒质粒pAdeasy-1共转化BJ5183细菌,获得重组腺病毒表达载体pAdeasy-1-pAdTrack-CMV-mING4,经PacI线性化后转染293细胞,收获腺病毒重组病毒子,RT-PCR鉴定,并用MTT法检测mING4对肝癌细胞SMMC7721的作用。结果成功构建mING4的重组腺病毒表达载体pAdeasy-1-pAdTrack-CMV-mING4,获得了mING4重组病毒子。结论mING4的重组腺病毒表达载体可抑制SMMC7721细胞的生长。

Objective The recombined adenovirus vector of mING4 was constructed for carcinoma gene therapy. Methods The pAdTrack-CMV-mING4 was constructed with pcDNA3.0-mING4 by PCR recombined plasmid as template and by means of, enzyme digestion and ligation. The pAdTrack- CMV-mING4 lineared by PmeI was co-transformed into BJ5183 with pAdeasy-1. The pAdeasy-1- pAdTrack-CMV-mING4 recombined adenovirus vector was lineared with PacI and then transfected in-to 293 cells. The mING4 recombined adenovirus was obtained and was used to infect SMMC7721 cells. The effect on SMMC7721 cells of mING4 was tested by MTT. Results The pAdeasy-1-pAdTrack-CMV-mING4 vector was constructed and the mING4 recombined adenovirus was obtained successfully. Conclusion mING4 can inhibit the growth of SMMC7721 cells.