[Construction of eukaryotic expression plasmid containing rat interleukin-10 gene and its expression in BRL cells in vitro].
OBJECTIVE
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.


CONCLUSIONS
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.
Chen
Yun-xin
YX
Department of Gastroenterology, Union Hospital, Fujian Medical University, Fuzhou 350001, China. drcyx@sohu.com
Huang
Yue-hong
YH
Chen
Zhi-xin
ZX
Zheng
Wei-da
WD
Wang
Xiao-zhong
XZ
D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism*; D008081:Liposomes / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction; D051381:Rats; D014162:Transfection / Q000379:methods*
D008081:Liposomes; D016753:Interleukin-10