[Construction of eukaryotic expression plasmid containing rat interleukin-10 gene and its expression in BRL cells in
Yun-xin Chen1, Yue-hong Huang, Zhi-xin Chen
1Department of Gastroenterology, Union Hospital, Fujian Medical University, Fuzhou 350001, China. drcyx@sohu.com
Aim:
To construct eukaryotic expression vector of rat IL-10 gene and observe its expression in hepatocyte cell line BRL.
Methods:
Total RNA was extracted from rat peripheral blood mononuclear cells. The full length coding region of IL-10 was amplified by RT- nested PCR and cloned into eukaryotic expression vector pcDNA3.0. The recombinant plasmid was transfected into BRL cells with either liposome Transfast or asialoglycoprotein receptor mediated liposome PEIjet-gal respectively. The expression of IL-10 mRNA was detected with PCR and that of IL-10 secreted from BRL cells transfected by liposome PEIjet-gal was detected with ELISA.
Results:
The recombinant plasmid was identified and confirmed with digestion of restriction endonuclease and DNA sequencing. Receptor mediated liposome PEIjet-gal exhibited significantly higher transfection efficiency than liposome Transfast and higher level secretory IL-10 expressed in BRL cells.
Conclusion:
The eukaryotic expression vector of IL-10 gene was successfully constructed. Asialoglycoprotein receptor-mediated liposome had high transfection efficiency on hepatocytes, suggesting that it could be a potential hepatocyte-targeting delivery system for IL-10 gene therapy.
