γ-干扰素诱导蛋白-10融合蛋白的表达纯化及其生物学活性测定  

Expression and purification of IP-10 fusion protein and identification of its biology activity

作　　者：代吕霞[1] 李明远[2] 蒋中华[2] 张林[3] 李虹[2] 

机构地区：[1]成都医学院临床技能中心,四川成都610083 [2]四川大学基础医学与法医学院微生物实验室,四川成都610041 [3]四川大学华西人类疾病生物治疗教育部重点实验室,四川成都610041

出　　处：《中国现代医学杂志》2009年第23期3521-3524,3528,共5页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30470613);教育部博士点专项基金(No:20040610053)

摘　　要：目的表达大鼠γ-干扰素诱导蛋白-10(IP-10)融合蛋白,并测定其生物学活性。方法从大鼠脾细胞中提取总RNA,用RT-PCR方法得到IP-10cDNA,双酶切将其插入pET-32(a)+载体中。重组质粒pET-32a(+)-IP10经测序证实。将pET-32a(+)-IP10转化大肠杆菌BL21(DE3)中进行表达,目的蛋白经镍离子亲和层析柱纯化。微室跨膜迁移实验测定其生物学活性。结果测序表明,所构建的重组质粒pET-32a(+)-IP10序列完全正确,诱导表达的蛋白的相对分子量同预期分子量相同,微室跨膜迁移实验测定出IP-10融合蛋白对激活的T淋巴细胞具有良好的趋化活性。结论成功在大肠杆菌中表达了IP-10融合蛋白,并具有生物学活性。【Objective】To express rat IFN-γ-inducible protein-10 （IP-10） fusion protein in E. coli. and analyze its biological activity.【Methods】Total RNA was isolated from rat spleen cells, and reverse transcription- PCR was performed based on the IP-10 nucleotide sequence, PCR products were cloned into the pET-32（a）＋ vector after being digested by two restrictive enzymes of Kpn I and EcoR I. The sequence of recombinant plasmid pET-32a （＋）-IP10 was verified by DNA sequencing, and then transformed into BL21 （DE3） cells.The expression of IP-IO fusion protein was induced with IPTG and purified using the Ni2＋ affinity chromatography. The bioaetivity of IP-10 fusion protein was detected by transwell migration assay. [ Results ] The constructed recombinant plasmid pET-32a （＋）- IP10 was correct, which was identified by DNA sequencing. The relative molecular mass （Mr） of the expressed prod- ucts was 37 000 with expected molecular weight. The satisfactory chemotactic activity of IP-10 was detected by transwell migration assay on activated T cells. [ Conclusion ] The fusion protein of pET-32a（＋）-IP10 with bioactivity is expressed successfully in E. coli.

关 键 词：γ-干扰素诱导蛋白-10 融合蛋白 生物学活性 

分 类 号：Q78[生物学—分子生物学]