DI Feng,WU Xiujuan,SHEN Shuijuan, et al. Cloning of the cDNA fragment coding for rat ENPP1 protein and construction of recombinant adeno-associated virus[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2021, 51(1): 13-18.
Abstract：Objective: To clone the full-length cDNA fragment coding of rat ENPP1 and construct the recombinant adeno-associated virus vector of ENPP1. Methods: Using rat vascular smooth muscle cell mRNA as templates, the large fragment of rat ENPP1 gene was obtained through RT-PCR amplification and cloned into pCDH vector. The fragment was then cloned into adeno-associated virus vector (AVV) vector pAAV-MCS by EcoR I/Xho I enzyme digestion, ligation and transformation, after which the positive clone was identified by enzyme digestion and DNA sequencing. Recombinant AAV-ENPP1 was packaged and introduced to the targeted vascular smooth muscle cells. Western blot was used to detect the expression of target ENPP1 protein. Results: The 2 721 bp large fragment of rat ENPP1 gene was successfully cloned and constructed using RT-PCR technique, and finally obtained pAAV/ENPP1 clone with correct sequencing. The results of Western blot testing revealed that the expression of the target protein ENPP1 was increased. Conclusion: The successful construction of recombinant adeno-associated virus vector of rat ENPP1 lays a foundation for the treatment of vascular calcification via ENPP1 gene transfection.
