利用Gateway技术构建重组腺病毒pAd-NK4

Gateway technical supported construction of a recombinant adenovirus vector pAd-NK4

目的利用Gateway技术构建含人肝细胞生长因子(HGF)NK4基因的重组腺病毒载体。方法以含有HGF/NK4基因的质粒为模板PCR扩增NK4基因,将回收的NK4PCR产物片段克隆至腺病毒穿梭载体pYr-adshuttle-6获得重组质粒pYr-adshuttle-6-NK4。从该重组质粒上,利用LR同源重组将NK4-IRES-EGPF表达框转移至腺病毒骨架质粒pAD/BL-DEST,获得重组腺病毒质粒pAd-NK4-IRES-EGFP。该质粒经pacI线性化后转染293包装细胞,获得重组腺病毒rAd-NK4-IRES-EGFP。采用TCID 50(tissue cell infectiousdosage 50,TCID 50)法测定重组腺病毒滴度。重组腺病毒分别经酶切和PCR等方法进行鉴定。荧光显微镜观察转染效果。结果证实腺病毒载体中含有NK4基因的目的片段;病毒滴度为6.3×1011 PFU.L-1;荧光显微镜发现该重组腺病毒能在HEK293细胞中高效的表达。结论利用Gateway技术可以快速地构建同时表达NK4蛋白和绿色荧光的重组腺病毒,为下一步开展关于NK4基因的肿瘤治疗提供了基础。

Aim To construct a recombinant adenovirus vector pAd-NK4 with Gateway technology.Methods The NK4 gene was amplified by PCR with plasmid con-taining hepatocyte growth factor（HGF）/NK4 as tem-plate,and then cloned into adenovirus adshuttle vector pYr-adshuttle-6 to acquire recombinant plasmid pYr-adshuttle-6-NK4,from which,the expression cassette of NK4-IRES-EGPF was transfered to pAD/BL-DEST with LR recombinant reaction to acquire recombinant adenovirus plasmid pAd-NK4-IRES-EGFP.The correct clone was linearized and transfected into 293A cells for packing and amplified adenovirus pAd-NK4 was obtained and identified.The titer was determined by TCID50.The transfection efficiency was observed under the fluorescent microscope.Results The recombinant adenovirus vector containing human NK4 gene was constructed successfully with the titer 6.3×1011 PFU·L-1.This vector was found to infect HEK293 cells efficiently.Conclusion The recombinant adenovirus expressing NK4 gene and green fluorescence can be successfully constructed with Gateway technology,which lays the foundation for further study.