共表达人PUMA和HGF／NK4基因的重组腺病毒的制备及在胰腺癌细胞中的表达  

Construction and identity of recombinant adenovirus co-expressing NK4 and PUMA gene

作　　者：黄柏强 李二毛 孔冠群 黄志平 

机构地区：[1]广州市番禺区石楼人民医院,511447 [2]广州医科大学实验医学研究中心,510182 [3]广州市番禺区石楼镇社区卫生服务中心,511447

出　　处：《国际医药卫生导报》2013年第16期2474-2478,共5页International Medicine and Health Guidance News

基　　金：2012广东省科技厅科技计划项目（01656900179474038）

摘　　要：目的制备同步表达HGF／NK4基因和PUMA基因的重组腺病毒，感染胰腺癌细胞后检测基因的有效表达。方法将PUMA基因和HGF／NK4基因以串联方式依次克隆至腺病毒穿梭载体，构建重组穿梭载体pAdTrack-PUMA-NK4，转化大肠杆菌BJ5183并在细菌内与腺病毒载体同源重组制备重组腺病毒载体pAd-PUMA-NK4。PacⅠ酶切后转染至HEK293细胞包装得到重组腺病毒。一定滴度的腺病毒感染人胰腺癌细胞株，荧光定量RT-PCR检测细胞中NK4和PUMA基因mRNA表达水平。结果双酶切重组pad-PUMA-NK4穿梭载体鉴定证实目的基因PUMA和HGF／NK4正确克隆；重组腺病毒载体经PacⅠ酶切得到特异的酶切产物转染HEK293细胞后成功包装出重组腺病毒；病毒感染胰腺癌细胞后3-9d内PUMA和NK4基因的表达水平较高，整体持续表达可维持2w。结论成功构建了同时表达NK4和PUMA基因的重组腺病毒载体并包装出相应的重组腺病毒颗粒，该腺病毒能有效感染胰腺癌细胞并高表达NK4和PUMA基因，这为进一步确定NK4和PUMA基因的功能及指导双基因联合治疗胰腺癌提供了理论依据和必要的材料。Objective To construct replication deficient recombinant adenovirus co-expressing PUMA and NK4 genes using AdEasy-1 system, observe the expression of PUMA and NK4 genes in pancreatic cancer cell after andenovirus infection. Methods PUMA and HGF/NK4 gene were cloned to adenovirus shutter plasmid for the construction of recombinant plasmid pAdTrack-PUMA/NK4. The recombinant plasmid digested by PmeI was transformed into bacteria BJ5183 containing backbone vector pAdEasy-t for homologous recombination to obtain the recombinant adenoviral vector pAd- PUMA- NK4.The recombinant adenoviral vector was then transfected into HEK293 package cells to produce virus particles. The expression of NK4 and puma gene in cells were detected in human pancreatic cancer cells by qRT-PCR after adenovirus infection. Results The recombinant adenoviral vector was identified by PCR and restriction endonuclease digestion. High level expression of NK4 and PUMA mRNA was maintained for 6 days in SW1990 cells after adenovius infection. Conclusion The infection of Recombinant adenovirus Ad-PUMA-NK4 can mediate high level expression of NK4 and PUMA, it provides an important tool for further gene therapy of pancreatic cancer with them.

关 键 词：腺病毒 NK4 PUMA 胰腺癌 

分 类 号：R735.9[医药卫生—肿瘤]