A new recombinant adenovirus vector pAdBM5 GFP mAFP Construction.docVIP

 PAGE \* MERGEFORMAT 18 A new recombinant adenovirus vector pAdBM5 GFP mAFP Construction Author: Kuangzhi Peng, XIE Yu-an, LIANG An-min, LUO Xiao-ling, Anmin 【Abstract】 Objective To construct the expression of mouse alpha-fetoprotein (murine alpha fetoprotein, mAFP) gene recombinant adenovirus vector pAdBM5  GFP  mAFP, and determine its titer. Method from Hepa1  6 mouse hepatoma cells to extract total RNA, specific primers by RT  PCR amplified mAFP gene sequencing confirmed, pAdBM5  GFP inserted into the shuttle vector, double digestion of recombinant adeno - viral vector. Recombinant adenovirus vector and virus linear backbone plasmid DNA using calcium phosphate coprecipitation method HEK293A were co-transfected cells by homologous recombination to express mAFP adenovirus genes and HEK293A cells containing a large number of target gene amplification of recombinant adenovirus, recombinant adenovirus with the TCID50 titer determination. The result successfully cloned mice mAFP gene expression mAFP construct a recombinant adenovirus vector pAdBM5  GFP  mAFP, received recombinant adenovirus expressing mAFP, viral titers up to 3.2 × 108PFU/ml. Conclusions This study successfully constructed pAdBM5  GFP  mAFP carrier to obtain a high titer of recombinant adenovirus expressing mAFP for further immuno-gene therapy of liver cancer foundation. Keywords: pAdBM5  GFP; mouse Α-fetoprotein; recombinant adenovirus vector Abstract: Objective To construct a novel recombinant adenovirus vector pAdBM5  GFP  mAFP and detect virus titer. Methods Total RNA were extracted from hepa1  6 murine liver cancer cell line, mAFP (murine alpha fetoprotein, mAFP) gene was cloned by RT  PCR method. The gene was identified by sequencing, then it was inserted into pAdBM5  GFP shuttle vector by Bgl II and Pme I restrict enzyme digestion to construct pAdBM5  GFP  mAFP recombinant vector. mAFP gene was expressed in adenovirus by homologous recombinant in