应用AdEasy-1腺病毒系统构建含人nm23-H1基因的重组腺病毒质粒

Construction of Recombinant Adenovirus Plasmid Encoding Human nm23-H1 Gene by AdEasy-1 System

目的应用AdEasy-1腺病毒载体系统制备含人nm23-H1基因的重组腺病毒载体。方法设计带有限制性内切酶KpnⅠ和XhoⅠ双酶切位点的引物,利用PCR法从质粒pMD18-T-nm23-H1上克隆出nm23-H1 cDNA并定向连接至穿梭载体pShuttle-CMV上,行KpnⅠ和XhoⅠ双酶切鉴定和测序。将pAdEasy-1腺病毒骨架载体电转化B J5183感受态细菌,Pm eⅠ酶线性化并去磷酸化处理重组质粒pShuttle-CMV-nm23-H1,并电转化含pAdEasy-1的B J5183感受态细菌,利用卡那霉素筛选,PacⅠ酶切鉴定,重组腺病毒质粒在XLGold-10感受态细菌中大量扩增,产物行PCR鉴定。结果经KpnⅠ和XhoⅠ双酶切pShuttle-CMV-nm23质粒得到460bp的片段,该片段经测序与nm23-H1基因序列一致,重组腺病毒质粒pAdEasy-nm23-H1经PacⅠ酶切后可以得到3.0kb和4.5kb的片段,大量扩增重组腺病毒质粒后行PCR仍然得到460bp的片段。结论利用AdEasy-1腺病毒系统成功构建含人nm23-H1基因重组腺病毒质粒。

Objective To construct the recombinant adenovirus vector encoding human nm23-H1 gene by AdEasy-1 adenovirus vector system. Methods A pair of DNA primers with Kpn Ⅰ and Xho Ⅰ restriction enzyme site were designed. Nm23-H1 eDNA was cloned from pMD18-T-nm23 plasmid by PCR technology and cloned into pShuttle-CMV in correct direction and the recombinant plasmid was called pShuttle-CMV-nm23-H1. It was identified by double digestion with KpnⅠ and Xho Ⅰ restriction enzymes and DNA sequencing. The replication-defective adenovirus type 5 backbone plasmid pAdEasy-Ⅰ was transformed into competent bacteria BJ5183 by eleetroporation, pShuttle-CMV-nm23-H1 was linearized by Pme Ⅰ and dephosphorylated and transformed into competent bacteria BJ5183 which contained pAdEasy-1 backbone plasmid. Then the product was selected by kanamyein and identified by digestion with Pae Ⅰ. The recombinant adenovirus vector plasmid was amplified broadly in XL10-Gold competent bacteria and identified by PCR. Results A fragment size about 460bp was got by digestion with Kpn Ⅰ and Xho Ⅰ. Its sequence was identical to that of gene nm23-Hl by DNA sequencing, The recombinant advenoviral plasmid could produce a fragment whose size was 3.0kb or 4.5kb when digested with PaeⅠ. The 460bp fragment was appeared after PCR identification. Conclusion The recombinant adenovirus plasmid encoding human nm23-H1 gene by AdEasy-1 adenovirus vector system was constructed successfully.