Construction of recombinant adenoviral vector overexpressing PTG
- Author:
Chenxi Wang
1
;
Xia Deng
1
;
Zhicong Zhao
1
;
Zhensheng Cai
1
;
Panpan Zhang
1
;
Lian Li
1
;
Haoxiang Li
1
;
Li Zhao
1
;
Dong Wang
1
;
Ling Yang
1
;
Guoyue Yuan
1
Author Information
- Publication Type:Journal Article
- Keywords: PTG; recombinant adenovirus vector; homologous recombination; type 2 diabetes mellitus
- From: Acta Universitatis Medicinalis Anhui 2022;57(4):558-563
- CountryChina
- Language:Chinese
-
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. - Full text:2025021809193746433PTG重组腺病毒过表达载体的构建_王晨玺.pdf
