Construction of recombinant adenovirus vectors expressing two different subtypes of ndrg2 gene
AIM: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 recom-binanted with the expression vector pAd-CMV/V5-DEST using Invitrogen's LR Clonase? Ⅱ Enzyme Mix to obtain the expression clones,pAd-CMV/V5-DEST-NDRG2L and pAd-CMV/V5-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 × 1012 and 6. 3 × 1012 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 successful- ly constructed recombinant adenovirus Ad-NDRG2L and Ad-NDRG2S,which can be used in further research on tumor gene therapy.
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