Construction of a Novel Recombineant Adnovirus Vector pAdBM5-GFP-mAFP

Objective 　To const ruct 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 rest rict enzyme digestion to const ruct pAdBM5-GFP-mAFP recombinant vector. mAFP gene was expressed in adenovirus by homologous recombinant in HEK-93A cells af ter pAdBM5-GFP-mAFP vector and virus skeleton plasmid cotransfected HEK-93A cells by standard calcium phosphate coprecipitation method. Virus titer was detected by TCID50 method. Results 　pAdBM5-GFP-mAFP recombinant vector was successfully constructed and high titer recombinant adenovirus carrying mAFP gene was harvested. Virus titer reached to 3. 2 10⁸ PFU/ ml. Conclusion 　pAdBM5-GFP-mAFP recombinant adenoviral vector carrying mAFP gene was successfully constructed and high titer recombinant adenovirus was harvested. it will lay down the basis for further developing gene therapy of primary liver cancer.