人NP9基因蛋白重组腺病毒的制备

目的：制备表达人NP9蛋白的复制缺陷型重组腺病毒。方法：酶切pMD18T-NP9质粒获得NP9基因编码区序列并克隆至芽梭载体构建重组pAcTrack-CMV-NP9载体，线性化后与pAdEasy-1共转化BJ5183，通过同源重组得到重组的pAD-NP9病毒载体将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒。结果：酶切鉴定得到阳性pAD-NP9重组腺病毒载体，该载体能有效转染HEK293细胞并在细胞内成功包装，转染3d后可以观察到绿色荧光蛋白(CFP)表达并逐渐增多、增强：结论：成功构建了NP9基因的重组腺病毒载体并制备重组腺病毒颗粒，为进一步研究NP9基因的功能及应用NP9进行基因治疗奠定基础。

Construction of Replication Deficient Recombinant Adenovirus of NP9 Protein

Objective:To construct replication deficient recombinant adenovirus vector of human NP9 protein by homologous recombination in bacteria using AdEasy system,and produce adenoviral particles.Methods: The CDS of NP9 gene was obtained from pMD18T-NP9 plasmid by restriction endonuclease digestion, and was inserted into the downstream of CMV promoter of a shutter plasmid pAdTrack-CMV. The recombinant transfer plasmid pAdTrack-CMV-MCP-1 was linearized by Pme I and co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for homologous recombination to obtain the recombinant adenoviral plasmid. The recombinant adenoviral plasmid was linearized and was then transfected into HEK293 package cells to produce virus particles. The detection of recombinant adenovirus was characterized by using PCR.Results:The recombinant adenoviral plasmid was successfully established and confirmed by restriction endonuclease digestion. GFP expression was observed on the 5th day after transfection. Recombinant adenovirus was identified by PCR.Conclusion:The achievement of recombinant adenoviral plasmid and recombinant adenovirus of NP9 laid a foundation for further investigation of its function and application.