[Construction of a eukaryotic expression vector of the gene encoding rat interferon-gamma-inducible protein and its expression in NIH 3T3 cells].

ABSTRACT

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. CONCLUSION: 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.