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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 SH5 recombinant adenovirus Ad5-hSH2-EGFP and its mutant Ad5-hSH2mt-EGFP carrying enhanced green fluorescent protein (EGFP) and HA tag and observe its effect on the proliferation of K562 cells. Methods The hSH2 gene was amplified by RT-PCR. The hSH2mt gene was amplified by overlap PCR and cloned into pAdTrack-CMV respectively. The shuttle plasmid was constructed and digested with PmeⅠ. The recombinant plasmid pAdEasy-BJ5183 was transformed into E. coli BL21 Recombinant adenoviral vector pAd5-hSH2-EGPF and pAd5-hSH2mt-EGFP were generated by homologous recombination in E.coli and were digested with PacⅠ and then transfected into AD293 cells for packaging. Recombinant adenovirus Ad5-hSH2-EGFP and Ad5-hSH2mt-EGFP , Amplify the virus, determine the titer and identify by PCR and Western blot. K562 cells were infected with recombinant adenovirus and the effect of virus on the proliferation of K562 cells was detected by MTT assay. Results The recombinant adenovirus plasmids pAd5-hSH2-EGFP and pAd5-hSH2mt-EGFP were correctly constructed by restriction enzyme digestion. The titer of recombinant adenovirus Ad5-hSH2-EGFP and Ad5-hSH2mt-EGFP reached 1.6 × 1012. , Two kinds of recombinant adenovirus were successfully packaged and amplified. The results of Western blot showed that the recombinant adenovirus carrying the target gene can be expressed in AD293 cells; MTT assay showed that Ad5-hSH2-EGFP significantly inhibited the proliferation of K562 cells . Conclusions The recombinant adenovirus Ad5-hSH2-EGFP and Ad5-hSH2mt-EGFP have been successfully constructed and Ad5-hSH2-EGFP can inhibit the proliferation of K562 cells in vitro. To further investigate the therapeutic effect and mechanism of SH2 gene on chronic myeloid leukemia Foundation.