Found programs:
Authors:Wang Chenxi; Deng Xia; Zhao Zhicong; Cai Zhensheng; Zhang Panpan; Li Lian; Li Haoxiang; Zhao Li; Wang Dong; Yang Ling; Yuan Guoyue
Keywords:PTG;recombinant adenovirus vector;homologous recombination;type 2 diabetes mellitus
DOI:10.19405/j.cnki.issn1000-1492.2022.04.010
〔Abstract〕 Objective To construct and identify an overexpression recombinant adenovirus vector carrying the mouse PTG gene(NM_016854), and to lay a foundation for in-depth study of the function of PTG. Methods The coding sequence of the mouse PTG gene was chemically synthesized, amplified by polymerase chain reaction(PCR), digested with restriction enzymes, and inserted into the GV314 vector(CMV-MCS-3 FLAG-SV40-EGFP) to obtain the recombinant shuttle plasmid pGV314-PTG. BamHⅠ/AgeⅠ double enzyme digestion was further carried out, and the product was transferred into linearized expression vector pDC315 to construct recombinant adenovirus Ad-PTG, which was transfected into HEK293 T cells and packaged into recombinant virus particles. After repeated amplification of several generations of HEK293 T cells, the recombinant adenovirus was purified and titer detected. Finally, PCR, Western blot and sequencing were used to verify the recombinant adenovirus. Results After PCR, Western blot and sequencing, the results showed that the pGV314-CMV-MCS-3 FLAG-SV40-EGFP-PTG overexpression adenovirus vector(Ad-PTG) was successfully constructed, and the virus titer measured by end-point dilution method was 4×1010PFU/ml, Western blot and RT-qPCR showed that the protein and mRNA expression levels of PTG increased significantly. Conclusion The recombinant adenovirus vector carrying mouse PTG gene is successfully constructed, and the expression of PTG gene in hepatocytes is effectively up regulated.