中文摘要：

目的构建携带HA标签的重组腺病毒Ad5F35-HF2S。方法体外合成HA标签DNA双链,以K562细胞cDNA为模板,分别扩增FKBP1、FKBP2和SH2基因,依次亚克隆至穿梭质粒pAdTrack-CMV-HA中,构建重组穿梭质粒pAdTrack-CMV-HF2S,穿梭质粒经PmeⅠ酶切线性化,与腺病毒骨架质粒pAd5/F35在Ad5/F35-BJ5183感受态细胞中同源重组,获得重组腺病毒质粒pAd5/F35-HF2S,经PacⅠ酶切线性化后,转染AD-293细胞进行包装、扩增,获得重组腺病毒Ad5/F35-HF2S,经2轮扩增后,测定病毒滴度;采用PCR及Western blot法检测感染细胞中HF2S基因的转录及蛋白的表达。结果重组穿梭质粒pAdTrack-CMV-HF2S及重组腺病毒质粒pAd5/F35-HF2S经鉴定均构建正确;经包装及2轮扩增后,重组腺病毒Ad5/F35-HF2S病毒滴度可达1013IU/ml;经PCR及Western blot鉴定表明,重组腺病毒携带的HF2S基因能够在AD-293细胞中表达。结论已成功构建了表达HF2S基因的重组腺病毒Ad5/F35-HF2S,为研究Grb2-SH2结构域在CML中的基因治疗和作用机制奠定了基础。

英文摘要：

Objective To construct a recombinant adenovirns Ad5/F35-HF2S with HA tag. Methods Double chains of DNA of HA tag were synthesized in vitro. FKBP1, FKBP2 and SH2 gene segments were amplified by PCR using the cDNA of K562 cells as template, and subcloned into shuttle plasmid pAdTrack-CMV-HA in turn. The constructed recombinant shuttle plasmid pAdTrack- CMV-HF2S was linearized with Pme I for homologous recombination with adenoviral backbone vector pAd5 / F35 in competent AdS / F35-BJS183 cells. The obtained recombinant adenovirus plasmid pAd5/F35-HF2S was linearized with Pac I , and transfected to AD- 293 cells for packaging and amplification. The obtained recombinant adenovirus Ad5/F35-HF2S was subjected to two rounds of propagation and determined for titer. The transcription of HF2S mRNA and expression of HF2S protein in infected AD-293 cells were determined by PCR and Western blot respectively. Results Both recombinant shuttle ptasmid pAdTrack-HF2S and recombinant adenovirus vector pAdS/F35-HF2S were constructed correctly. After packaging and two rounds of propagation, recombinant adenovirus AdS/F35-HF2S reached a titer of 1013 IU/ml. PCR and Western blot proved that HF2S gene in recombinant adenovirus was successful expressed in AD-293 cells. Conclusion Recombinant adenovirus AdS/F35-HF2S was successfully constructed, which laid a foundation of further study on role of SH2 domain of Grb2 in gene therapy as well as the relevant mechanism.