ndrg2基因2种亚型重组腺病毒载体的构建

Construction of recombinant adenovirus vectors expressing two different subtypes of ndrg2 gene

目的:构建表达N-myc下游调节基因2(ndrg2)2种亚型的重组腺病毒载体,为与ndrg2相关肿瘤的基因治疗奠定基础.方法:采用腺病毒表达系统ViraPowerTM Adenoviral Expression System构建腺病毒载体.首先将ndrg22种亚型(长短2种亚型分别命名为ndrg2L,ndrg2S)利用酶切、连接、转化、扩增后提取质粒等方法克隆入入门载体pENTR2B以获得入门克隆pENTR2B-NDRG2L和pENTR2B-NDRG2S,经双酶切及PCR鉴定正确后,用重组酶LR ClonaseTMⅡ Enzyme Mix进行入门克隆与表达载体pAd-CMV/V5-DEST间的重组反应,以获得表达克隆pAd-CMV/V5-DEST-NDRG2L和pAd-CMV/V5-DEST-NDRG2S的质粒.表达克隆经PCR鉴定后用限制性内切酶PacⅠ线性化后转染HEK293A包装细胞得到重组腺病毒(分别命名为Ad-NDRG2L和Ad-NDRG2S),经过反复的感染扩增后,用半数组织培养感染剂量法(TCID50)检测病毒滴度,用Western Blot法检测重组病毒载体是否能正确表达NDRG2蛋白.结果:证实入门克隆pENTR2B-NDRG2L/S和表达克隆pAd-CMV/V5-DEST-NDRG2L/S经酶切鉴定和PCR鉴定质粒构建正确,转染HEK293A细胞并反复扩增后获得的病毒滴度分别为:Ad-NDRG2L,4.0×1012空斑形成单位(PFU)/L;Ad-NDRG2S,6.3×1012PFU/L.且这2个病毒能正确表达NDRG2蛋白.结论:成功构建了ndrg2基因长短2种亚型的重组腺病毒载体,并包装扩增病毒,可为肿瘤基因治疗的研究提供依据.

AINI： To construct recombinant adenovirus vectors expressing two different subtypes of ndrg2 gene for tumor gene therapy. METHODS： Invitrogen＇s ViraPowerTM Adenoviral Expression System was used to construct recombinant adenovirus vectors. The two subtypes of ndrg2 gene （ long and short subtypes designated respectively as ndrg2L and ndrg2S） were cloned into the entry vector pENTR2B to obtain the entry clones, pENTR2B- NDRG2L and pENTR2B-NDRG2S. After identification by double enzymes digestion and PCR analysis, the entry clones were recombinanted with the expression vector pAd-CMV/VS-DEST using Invitrogen＇s LR Clonase^TM Ⅱ Enzyme Mix to obtain the expression clones, pAd-CMV/VS-DEST-NDRG2L and pAd-CMV/VS-DEST-NDRG2S identified by PCR method. After linearized by Pac I , the expression clones were transfected into HEK293A cells for adenovirus packaging and amplification. Two adenoviruses were designated as Ad-NDRG2L and Ad-NDRG2S. Adenovirus titers were determined by TCID50 method and NDRG2 protein expression was detected by Western blotting. RESULTS： Enzyme digestion and PCR analysis proved that the pAd-CMV/V5-DEST-NDRG2L and pAd-CMV/V5-DEST-NDRG2S expression clones were successfully constructed. High-titer recombinant adenoviruses were acquired after being packaged in HEK293A. The titers of Ad-NDRG2L and Ad-NDRG2S were 4.0 × 10^12 and 6.3 × 10^12 PFU/L respectively and the two virus vectors expressed NDRG2L and NDRG2S correctly. CONCLUSION： Recombinant adenovirus vectors pAd- NDRG2L and pAd-NDRG2S are constructed. We have successfully constructed recombinant adenovirus Ad-NDRG2L and Ad- NDRG2S, which can be used in further research on tumor gene therapy.