Construction of a eukaryotic expression vector of the gene encoding rat interferon-gamma-inducible protein and its expression in NIH 3T3 cells.
- Author:
Yu-jie ZHAO
1
;
Yuan LIN
;
Ming-yuan LI
;
Hong LI
;
Zhong-hua JIANG
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Chemokine CXCL10; genetics; metabolism; DNA Restriction Enzymes; metabolism; Fluorescent Antibody Technique; Gene Expression; Genetic Vectors; genetics; Mice; NIH 3T3 Cells; Plasmids; genetics; Rats; Transfection; methods
- From: Journal of Southern Medical University 2009;29(4):615-618
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct an expression vector of the gene encoding rat interferon-gamma-inducible protein (IP-10) and identify its expression in NIH 3T3 cells.
METHODSIP-10 gene was amplified by polymerase chain reaction (PCR) and inserted into the eukaryotic expression vector pcDNA3.1(+). After identification by PCR, restriction endonuclease digestion and sequence analysis, the recombinant expression vector pcDNA3.1(+)-IP-10 was transfected into NIH 3T3 cells via liposome. Immunofluorescence method was used to confirm the expression of pcDNA3.1(+)-IP-10 in the transfected NIH 3T3 cells. The expression of IP-10 protein in the supernatant of the transfected cells was examined by Western blotting.
RESULTSPCR, restriction endonuclease digestion and sequence analyses confirmed successful construction of the recombinant vector pcDNA3.1(+)-IP-10. Immunofluorescence assay identified the expression of pcDNA3.1(+)-IP-10 in NIH 3T3 cells, and the expression of IP-10 protein was detected by Western blotting in the supernatant of the transfected cells.
CONCLUSIONA eukaryotic expression vector pcDNA3.1(+)-IP-10 has been successfully constructed, which provides the basis for investigating the therapeutic effect of IP-10 on Th1 type autoimmune disease.