Construction of recombinant adenovirus vector containing rat insulin-like growth factor 1 gene and its expression in islet beta-cells.
- Author:
Zhihong CHEN
1
;
Tang LI
;
Sujuan HU
;
Guiqin WANG
Author Information
1. Department of Pediatrics, The Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China. chchch96@sina.com
- Publication Type:Journal Article
- MeSH:
Adenoviridae;
genetics;
metabolism;
Animals;
Cell Line;
Diabetes Mellitus, Type 1;
therapy;
Genetic Therapy;
Genetic Vectors;
genetics;
Insulin-Like Growth Factor I;
biosynthesis;
genetics;
Islets of Langerhans;
metabolism;
Rats;
Recombinant Proteins;
biosynthesis;
genetics;
Transfection
- From:
Journal of Biomedical Engineering
2009;26(5):1067-1071
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to construct the recombinant adenovirus containing rat insulin-like growth factor 1(rIGF-1), and then to investigate its expression in islet beta-cells. RNA was extracted using Trizol from rat livers. rIGF-1 cDNA was obtained using RT-PCR. The purified RT-PCR products and pAdTrack-CMV were digested using Bg1 II and EcoR V and religated by T4 DNA ligase, then transformed into electro-competent JM109 bacteria and selected on Kanamycin LB plates. This plasmid pAd-CMV-rIGF-1 was linearized by PmeI and co-transformed into electro-competent BJ5183 bacteria with pAdEasy-1 and selected on Kanamycin LB plates. After having been screened, the extracted plasmid of positive bacteria was transfected into HEK 293 cells with liposome and was identified by the green fluorescence protein (GFP) expression. The recombinant adenovirus encoding rIGF-1 was named Ad-rIGF-1, and the viral particles were further amplified, purified, and its titer was about 4.0 x 10(8)pfu/ml. Ad-rIGF-1 was transfected into rat pancreatic beta cell lines- RINm5F cells, RT-PCR was carried out to detect the transfer genes, rIGF-1 protein in cells culture supernatants was detected by ELISA method, and its concentration was 91.6 +/- 26.8 ng/ml. rIGF-1 was present in Ad-rIGF-1-infected RINm5F cells as measured by Western blotting. The recombinant adenovirus vector containing rIGF-1 was constructed successfully, and the rIGF-1 protein was expressed by RINm5F cells. This method provided the mechanism of rLGF-1 to prevent beta cell from impairmentand to treat the case of type 1 diabetes.
