Construction of rat interleukin-10 adenoviral vector and its expression in bone marrow mesenchymal stem cells.
- Author:
Wen-Ting ZHANG
1
;
Juan TANG
;
Hong-Mei ZHAO
;
Jie-Yu YOU
Author Information
1. Department of Gastroenterology, Hunan Children's Hospital, Changsha 410007, China. yjy660111@sina.com.
- Publication Type:Journal Article
- MeSH:
Adenoviridae;
Animals;
Bone Marrow Cells;
Genetic Vectors;
HEK293 Cells;
Humans;
Interleukin-10;
Mesenchymal Stem Cells;
Rats;
Transfection
- From:
Chinese Journal of Contemporary Pediatrics
2019;21(7):708-712
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To construct the recombinant adenoviral vector carrying the rat interleukin-10 (rIL-10) gene, and to investigate whether it is stably expressed in bone marrow mesenchymal stem cells.
METHODS:The rIL-10 gene was amplified by PCR from template rIL-10 cDNA, and the recovered 656 bp rIL-10 DNA fragment was cloned into pcDNA3.1 to construct pcDNA3.1-IL-10. Then HEK293 cells were transfected with pcDNA3.1-IL-10 and adenoviral vector for homologous recombination, and sequencing and PCR were used to evaluate whether recombination was successful. HEK293 cells were lysed by repeated freeze-thaw cycles, and bone marrow mesenchymal stem cells were infected with the virus solution containing the rIL-10 gene. Western blot was used to measure the expression of rIL-10 in bone marrow mesenchymal stem cells.
RESULTS:Sequencing and PCR verified that the rIL-10 adenoviral vector was successfully constructed, with a virus titer of 4×10 PFU/mL. The expression of IL-10 was detected after bone marrow mesenchymal stem cells were infected by the virus solution containing the rIL-10 gene.
CONCLUSIONS:The constructed rIL-10 recombinant adenovirus can mediate the stable expression of rIL-10 gene in bone marrow mesenchymal stem cells, which provides a basis for gene transplantation therapy of inflammatory bowel disease.
