重组腺病毒Ad5-hNK4-EGFP的构建与鉴定  

Construction and identification of recombinant adenovirus Ad5-hNK4-EGFP

作　　者：匡文斌[1] 成凤[1] 秦晓林[1] 范晓卿[1] 王秦[1] 董晋豫[1] 梁勤东[1] 涂植光[1] 

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《中国生物制品学杂志》2012年第6期676-681,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30872417)

摘　　要：目的构建携带绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)标签和hNK4基因的重组腺病毒Ad5-hNK4-EGFP,并检测其对乳腺癌MDA-MB-231细胞增殖的影响。方法以pcDNA3/hNK4质粒为模板,采用高保真DNA聚合酶扩增hNK4基因,克隆至腺病毒穿梭载体pAdTrack-CMV中,构建重组穿梭质粒pAdTrack-hNK4,穿梭质粒经PmeⅠ酶切线性化,与腺病毒骨架质粒pAdEasy-1在大肠杆菌BJ5183中进行同源重组,获得重组腺病毒质粒pAd5-hNK4-EGFP,经PacⅠ酶切线性化后转染AD-293细胞进行包装,获得重组腺病毒Ad5-hNK4-EGFP,经4轮扩增后,测定病毒滴度;将重组腺病毒感染AD-293细胞,采用RT-PCR及Western blot法检测感染细胞中NK4基因的转录及蛋白的表达;MTT法检测Ad5-hNK4-EGFP对肝细胞生长因子(Hepatocyte growth factor,HGF)诱导的MDA-MB-231细胞增殖的影响。结果重组腺病毒质粒pAd5-hNK4-EGFP经PacⅠ酶切鉴定证明构建正确;经包装及4轮扩增后,重组腺病毒Ad5-hNK4-EGFP的滴度可达1.5×1011PFU/ml;经RT-PCR及Western blot鉴定表明,重组腺病毒携带的NK4基因能够在AD-293细胞中表达;HGF可促进MDA-MB-231细胞增殖,而Ad5-hNK4-EGFP可拮抗HGF诱导的MDA-MB-231细胞增殖。结论成功构建了表达hNK4基因的重组腺病毒Ad5-hNK4-EGFP,其可拮抗HGF诱导的MDA-MB-231细胞的增殖,为进一步深入研究HGF和NK4的功能、相互作用及作用机制奠定了基础,也为乳腺癌的靶向基因治疗提供了一个新的靶点。Objective To construct recombinant adenovirus Ad5-hNK4-EGFP with enhance green fluorescent protein（EGFP） tag and NK4 gene and observe its effect on the proliferation of breast cancer MDA-MB-231 cells.Methods The hNK4 gene was amplified with high fidelity DNA polymerase using plasmid pcDNA3 / hNK4 as a template,and cloned into adenovirus shuttle vector pAdTrack-CMV.The constructed recombinant shuttle plasmid pAdTrack-hNK4 was linearized with PmeⅠ and inserted into adenovirus backbone plasmid pAdEasy-1 for homologous recombination in E.coli BL5183.The obtained recombinant adenovirus plasmid pAd-hNK4-EGFP was linearized with PacⅠ,then transfected to AD-293 cells for packaging.The obtained recombinant adenovirus Ad5-hNK4-EGFP was subjected to four cycles of propagation and determined for titer.AD-293 cells were infected with the recombinant adenovirus and determined for transcription of NK4 gene and expression of NK4 protein by RT-PCR and Western blot respectively.The effect of Ad5-hNK4-EGFP on proliferation of MDA-MB-231 cells induced by hepatocyte growth factor（HGF） was determined by MTT method.Results Digestion with PacⅠ proved that recombinant adenovirus plasmid pAd5-hNK4-EGFP was constructed correctly.After packaging and four cycles of propagation,recombinant adenovirus Ad5-hNK4-EGFP reached a titer of 1.5 × 1011 PFU / ml.RT-PCR and Western blot showed that the NK4 gene in Ad5-hNK4-EGFP was expressed in AD-293 cells.HGF promoted the proliferation of MDA-MB-231 cells,while Ad5-hNK4-EGFP antagonized the proliferation induced by HGF.Conclusion Recombinant adenovirus Ad5-hNK4-EGFP for expression of NK4 gene was successfully constructed,which antagonized the proliferation of MDA-MB-231 cells induced by HGF.It laid a foundation of further study on the functions,interaction and action mechanisms of HGF and NK4 and provided a novel target for gene therapy of breast cancer.

关 键 词：腺病毒 肝细胞生长因子 NK4 乳腺癌 基因治疗 

分 类 号：Q782[生物学—分子生物学]