人S100 A9重组腺病毒载体的构建和鉴定

Construction and identification of recombinant adenovirus vector expressing human S100A9

目的制备人S100A9(hS100A9)重组腺病毒载体,为进一步研究人S100A9蛋白的分子生物学作用奠定基础。方法从pHAHA-hS100A9中PCR扩增hS100A9片段,亚克隆至穿梭质粒pAdTrack-TOX,获得重组穿梭质粒pAdTrack-TOX-hS100A9。酶切、聚合酶链反应(PCR)及测序鉴定正确后经PmeⅠ酶切电转入AdEasier感受态细胞,获得重组腺病毒质粒pAdhS100A9,该质粒经PacⅠ酶切后由脂质体转染至HEK293细胞中包装扩增重组腺病毒并进行滴度测定,最后通过PCR及蛋白印迹法(Western blot)鉴定重组腺病毒。结果正确扩增出hS100A9基因;重组穿梭质粒pAdTrack-TOX-hS100A9均及重组腺病毒质粒pAdhS100A9构建成功,并在HEK293细胞中成功包装且扩增后滴度为1 010 I U/mL;重组腺病毒AdhS100A9在HEK293细胞中成功转录和表达。结论用pAdEasy系统成功构建hS100A9重组腺病毒载体,为探讨其生物学作用奠定了基础。

Objective To construct the recombinant adenovirus vector expressing human S100A9 for further studying its molecular biology effects.Methods Human S100A9 gene was amplified using PCR method and inserted into pAdTrack-TOX vector.The recombinant plasmid pAdTrack-TOX-hS100A9 was confirmed by restriction endonuclease digestion,PCR and gene sequencing,Then the resultant vector was electro-transduted into competent AdEasier cells after linearized by PmeⅠto acquire recombinant adenovirus plasmid pAdhS100A9.Subsequently,the recombinant vector was transfected into HEK293 cells by liposome transfection to acquire recombinant adenovirus（AdhS100A9）.Then AdhS100A9 was amplified and evaluated the titer and identified by PCR and Western blot.Results Human S100A9 gene was amplified accurately.The shuttle plasmid pAdTrack-TOX-hS100A9 and adenovirus plasmid pAdhS100A9 were constructed successfully.AdhS100A9 was successfully packaged in HEK293 cells and the virus titer was 1 010 IU/mL after amplification.The recombinant adenovirus could transcribe human S100A9 gene and express protein.Conclusion A recombinant adenovirus AdhS100A9 was successfully constructed with pAdEasy system.The adenovirus will be used to investigate the biological role of human S100A9.