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

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.