摘要
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.
出处
《第四军医大学学报》
北大核心
2009年第9期771-774,共4页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30772516)