大鼠干扰素诱导蛋白-10的体外表达和鉴定  

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

作　　者：赵玉杰[1] 林媛[1] 李明远[1] 李虹[1] 蒋忠华[1] 

机构地区：[1]四川大学华西医学中心基础医学与法医学院微生物学教研室,四川成都610041

出　　处：《南方医科大学学报》2009年第4期615-618,共4页Journal of Southern Medical University

基　　金：国家自然科学基金(30470613)

摘　　要：目的构建大鼠干扰素诱导蛋白-10(IP-10)真核表达载体,在NIH3T3细胞中表达重组载体pcDNA3.1(+)-IP-10,并对其表达产物进行鉴定。方法通过PCR扩增IP-10基因片段,通过酶切和连接反应,构建IP-10的真核表达载体pcDNA3.1(+)-IP-10,重组载体经限制性内切酶、PCR及DNA序列测定等证实构建成功后,用PolyFect脂质体转染NIH3T3细胞,免疫荧光技术检测pcDNA3.1(+)-IP-10在NIH3T3细胞的表达情况。转染的NIH3T3细胞通过G418筛选出稳定表达的细胞克隆,Western Blotting鉴定IP-10蛋白在细胞培养上清的表达情况。结果酶切分析、PCR及DNA序列测定证实,IP-10基因被成功克隆入真核表达载体pcDNA3.1(+),免疫荧光技术检测证实重组载体可在NIH3T3细胞表达。Western Blotting结果显示细胞培养上清有IP-10蛋白表达。结论成功构建了真核表达载体pcDNA3.1(+)-IP-10,为进一步研究其对Th1型自身免疫性疾病的治疗效果奠定基础.Objective To construct an expression vector of the gene encoding rat interferon-γ-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.

关 键 词：干扰素-Γ 诱导蛋白10 真核表达 自身免疫性疾病 

分 类 号：R346[医药卫生—基础医学]