Construction and identification of the recombinant adenovirus AdMxA
- VernacularTitle:人MxA基因重组腺病毒载体的构建及鉴定
- Author:
Guoqiang MAO
- Publication Type:Journal Article
- Keywords:
MxA gene;
RT-PCR;
Gene cloning;
Adenovirus
- From:
Journal of Chongqing Medical University
1986;0(04):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To clone the Chinese MxA gene and to construct the recombinant adenovirus plasmid carring human MxA gene for further study.Methods: The peripheral blood mononuclear cells(PBMCs) were isolated by gradient fractionation,then total RNA was extracted from the PBMCs.The MxA gene was amplified by RT-PCR using the primers based on the published sequence of MxA.The PCR product,double-digested with restrictive endonucleases NotI and XbaI,was cloned into pAdTrack-CMV by molecular cloning technique.The plasimid pAdTrack-MxA was linearized with Pme I,and subsequently co-transformed into electrocompetent BJ5183 cells with an adenoviral backbone plasmid(pAdEasy-1).Recombinants were selected for kanamycin resistence and confirmed by PacI.Finally,the linearized recombinant plasmid was transfected into HEK 293 cells.Recombinant adenoviruses were generated within 7 to 12 days,then viral tite was checked by GFP.Results:Sequencing analysis revealed that the amino acid sequence of MxA gene had only one amino acid residue displace from isoleucine to valine(compared with that of published sequence) although there were five variations in nucleotide sequence.The restrictive endonuclease analysis confirmed that correct recombinant adenovirus plasmid was constructed.24 hours after transfection,the fluorescence was observed in 293 cells,and viral title checked by GFP was 1.59?108(pfu/mL).Conclusion: The MxA gene is cloned from a Chinese donor and a recombinant adenovirus vector containing human MxA gene is constructed successfully.It lays the foundation for further study of gene therapy for HBV.
