Zhao Yu-jie, Lin Yuan, Li Ming-yuan, Li Hong, Jiang Zhong-hua
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2009 Apr;29(4):615-8.
To construct an expression vector of the gene encoding rat interferon-gamma-inducible protein (IP-10) and identify its expression in NIH 3T3 cells.
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.
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.
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.
构建大鼠干扰素γ诱导蛋白（IP - 10）编码基因的表达载体，并鉴定其在NIH 3T3细胞中的表达。
通过聚合酶链反应（PCR）扩增IP - 10基因，并将其插入真核表达载体pcDNA3.1(+)。经PCR、限制性内切酶消化及序列分析鉴定后，将重组表达载体pcDNA3.1(+)-IP - 10通过脂质体转染至NIH 3T3细胞。采用免疫荧光法证实pcDNA3.1(+)-IP - 10在转染的NIH 3T3细胞中的表达。通过蛋白质印迹法检测转染细胞上清液中IP - 10蛋白的表达。
PCR、限制性内切酶消化及序列分析证实重组载体pcDNA3.1(+)-IP - 10构建成功。免疫荧光测定鉴定了pcDNA3.1(+)-IP - 10在NIH 3T3细胞中的表达，蛋白质印迹法在转染细胞的上清液中检测到了IP - 10蛋白的表达。
成功构建了真核表达载体pcDNA3.1(+)-IP - 10，为研究IP - 10对Th1型自身免疫性疾病的治疗作用提供了依据。
