[Construction of a eukaryotic expression vector of the gene encoding rat interferon-gamma-inducible protein and its expression in NIH 3T3 cells].
OBJECTIVE
To construct an expression vector of the gene encoding rat interferon-gamma-inducible protein (IP-10) and identify its expression in NIH 3T3 cells.


METHODS
IP-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.


RESULTS
PCR, 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.


CONCLUSIONS
A 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.
Zhao
Yu-jie
YJ
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Lin
Yuan
Y
Li
Ming-yuan
MY
Li
Hong
H
Jiang
Zhong-hua
ZH
D000818:Animals; D054357:Chemokine CXCL10 / Q000235:genetics* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids / Q000235:genetics; D051381:Rats; D014162:Transfection / Q000379:methods*
D054357:Chemokine CXCL10; D004262:DNA Restriction Enzymes