重组腺病毒Ad5-hSH2-EGFP及其突变体的构建与鉴定  

Construction and Identification of Recombinant Adenovirus Ad5-hSH2-EGFP and Its Mutant

作　　者：史静[1] 彭智[1] 罗红伟[1] 曹唯希[1] 陶崑[1] 李亚娟[1] 王海霞[1] 冯文莉[1] 

机构地区：[1]重庆医科大学临床血液学教研室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》2011年第5期507-512,共6页Chinese Journal of Biologicals

基　　金：国家自然科学基金(30871102)

摘　　要：目的构建携带增强型绿色荧光蛋白(EGFP)和HA标签的SH2基因重组腺病毒Ad5-hSH2-EGFP及其突变体Ad5-hSH2mt-EGFP,并观察其对K562细胞增殖的影响。方法设计并化学合成引物,采用RT-PCR法扩增hSH2基因,重叠PCR法扩增hSH2mt基因,分别克隆至pAdTrack-CMV,构建穿梭质粒,经PmeⅠ酶切,转化感受态pAdEasy-BJ5183,在细菌内同源重组产生复制缺陷型重组腺病毒质粒pAd5-hSH2-EGPF和pAd5-hSH2mt-EGPF,PacⅠ酶切后转染AD293细胞进行包装,获得重组腺病毒Ad5-hSH2-EGFP和Ad5-hSH2mt-EGFP,扩增病毒,测定滴度并经PCR和Western blot鉴定。重组腺病毒感染K562细胞,MTT法检测病毒对K562细胞增殖活性的影响。结果重组腺病毒质粒pAd5-hSH2-EGFP和pAd5-hSH2mt-EGFP经酶切鉴定构建正确;重组腺病毒Ad5-hSH2-EGFP和Ad5-hSH2mt-EGFP的滴度均可达1.6×1012;PCR结果表明,2种重组腺病毒包装和扩增成功,Western blot结果表明,重组腺病毒携带的目的基因能够在AD293细胞中表达;MTT检测证实,Ad5-hSH2-EGFP对K562细胞的增殖具有明显的抑制作用。结论已成功构建重组腺病毒Ad5-hSH2-EGFP和Ad5-hSH2mt-EGFP,Ad5-hSH2-EGFP能在体外明显抑制K562细胞的增殖,为进一步探讨SH2基因对慢性粒细胞白血病的治疗作用及其机制奠定了基础。Objective To construct recombinant adenovirus Ad5-hSH2-EGFP for SH2 gene,containing enhance green fluorescent protein（EGFP） gene and HA tag,as well as its mutant Ad5-hSH2mt,and observe its effect on proliferation of K562 cells.Methods Primers were designed and synthesized chemically,with which hSH2 gene was amplified by RT-PCR,while hSH2mt gene by overlapping PCR,and cloned into vector pAdTrack-CMV separately.The constructed shuttle plasmids were digested with PmeⅠ and transformed to competent pAdEasy-BJ5183,based on which replication-defective recombinant adenovirus plasmids pAd5-hSH2-EGFP and pAd5-hSH2mt-EGFP were obtained by homologous recombination in bacteria,digested with PacⅠ,and transfected to AD293 cells for packaging.The obtained recombinant adenoviruses Ad5-hSH2-EGFP and Ad5-hSH2mt-EGFP were amplified and determined for titers,then identified by PCR and Western blot and infected to K562 cells.The proliferative activity of K562 cells was determined by MTT method.Results Restriction analysis proved that both recombinant adenovirus plasmids pAd5-hSH2-EGFP and pAd5-hSH2mt-EGFP were constructed correctly.Both the titers of recombinant adenoviruses Ad5-hSH2-EGFP and Ad5-hSH2mt-EGFP reached 1.6 × 1012.PCR proved that the both the recombinant adenoviruses were successfully packaged and amplified.Western blot showed that the target genes in recombinant adenoviruses were expressed in AD293 cells.MTT method proved significantly inhibitory effect of Ad5-hSH2-EGFP on proliferation of K562 cells.Conclusion Recombinant adenoviruses Ad5-hSH2-EGFP and Ad5-hSH2mt-EGFP were successfully constructed,which laid a foundation of further study on curative effect of SH2 gene on chronic myeloid leukemia.

关 键 词：重组腺病毒 含SH2域蛋白质酪氨酸磷酸酶类 K562细胞 细胞增殖 

分 类 号：Q782[生物学—分子生物学]