Construction And Identification Of Recombinant Adenovirus Eukaryotic Vector Carrying Mouse Gene Brcc3

Objective:This study aims to construct recombinant adenovirus eukaryotic expression vector carrying Brcc3gene, namely pAV.Exld-CMV>Brcc3/GST/IRES/EGFP, which can express obviously in the eucaryotic cells and be used to study the function of Brcc36in mouse cardiac fibroblast.Methods:1. Amplifying of target gene fragmentWe used the upstream primer (Ndel-Sacl-Brcc3-F) and downstream primer (Brcc3-R) to amplify Sacl-Brcc3, and used Brcc3-GST-F and Xhol-Spel-GST-R to amplify GST-Xhol. And then, using Ndel-Sacl-Brcc3-F and Xhol-Spel-GST-R as the primers, Sacl-Brcc3and GST-Xhol acts as the templates to amplify Sacl-Brcc3-GST-Xhol.2. Using restriction enzyme digestion and ligase technology to construct pDown-Brcc3/GST/IRES/EGFPWe used Sacl/Xhol double enzyme to digest backbone plasmid pDown-MCS-IRES/EGFP and recycle PCR product Sacl-Brcc3-GST-Xhol. Next, the digested skeleton fragments of pDown-MCS-IRES/EGFP and Brcc3-GST gene were connected with ligases and then we transformed the connected product into competent cells stbl3of Escherichia coli for amplification. Finally, we picked up the single clone and identify using PCR methods and sequencing. The identified positive plasmid (pDown-Brcc3/GST/IRES/EGFP) were amplified and extracted. 3. Construction of pAV.Ex1d-CMV>Brcc3/GST/IRES/EGFP by Gateway technologyAfter LR reaction of pDown-Brcc3/GST/IRES/EGFP and pAV.Des1d were done, the target gene was cloned into adenovirus vector pAV.Des1d, and the reaction product was transformed into Escherichia coli competent cells stbl3for next amplification, then picking single clone and extracting the target plasmid and sequencing.4. Transecting the recombinant adenovirus into293A cells for packaging and amplifying adenovirus carrying Brcc3gene. At last, we used the adenovirus to transfect the adult mouse cardiac fibroblasts to detect Brcc36protein expression level by Western-blot analysis.Result:1. It has shown by agarose electrophoresis that amplification of Scal-Brcc3-GST-Xhol was successful.2. It has shown by PCR detecting, agarose electrophoresis and gene sequencing that construction of pDown-Brcc3/GST/IRES/EGFP was successful.3. It has shown by PCR detecting, agarose electrophoresis and gene sequencing that construction of pAV.Ex1d-CMV>Brcc3/GST/IRES/EGFP was successful.4. The result of western blot shows that the construction of adenovirus is well and Brcc36protein could effectively express in the transfected cardiac fibroblasts.Conclusion:A recombinant adenovirus eukaryotic expression vector carrying mouse Brcc3gene (namely pAV.Exld-CMV>Brcc3/GST/IRES/EGFP) was constructed successfully. It can successfully over-express Brcc3gene in the eukaryotic cells.