重组人IL-10融合蛋白的表达及其生物学活性测定

Expression of fusion rhIL-10 in E. coli and its bioactivity

目的在大肠杆菌中表达重组人白细胞介素10融合蛋白,获得有生物学活性的IL10。方法限制性内切酶NcoⅠ和BamHⅠ双酶切目的基因IL10,插入到同样双酶切的表达载体pET32a,构建重组载体IL10pET32a,测序正确后转化E.coliBL21(DE3),不同温度、低浓度IPTG诱导工程菌表达目的蛋白,SDSPAGE和Westernblot检测目的蛋白的表达,ELISA和MC9细胞增殖法分别测定IL10的含量及生物学活性。结果构建的表达重组载体IL10pET32a经酶切鉴定和序列测定表明完全正确,诱导工程菌可以表达可溶性的融合蛋白IL10Trx,同时也存在包含体融合蛋白,经Westernblot鉴定存在有目的蛋白IL10,MC9细胞增殖法测定可溶性的融合蛋白具有生物学活性。结论成功表达了具有生物学活性的融合蛋白IL10Trx,有助于下游纯化和制备hIL10基因工程药物。

Objective To express fusion rhIL-10-Trx in E. coli and analyze its biological activity. Methods IL-10 gene was inserted into plasmid pET32a after the genc and the plasmid were digested with endonucleases Nco Ⅰand BamH Ⅰ , respectively. The sequence of recombinant plasmid IL-10/pET32a was confirmed, and then transformed into E. coli BL21 （DE3）. The expression of interest protein in engineering bacteria IL-10/pgET32/BL21 induced by low concentration of IPTG at temperature of 37 ℃ and 25 ℃, respectively. The expressed fusion protein was identified with SDS-PAGE and Western blotting. Amount of IL-10 was assayed with ELISA and its bioactivity was analyzed by MC/9 mast-cell-line assay. Results The constructed recombinant plasmid IL10/pET32a was correct, which was identified by endonucleases digestion and sequence. The proteins expressed by engineering bacteria IL- 10/pET32a/BL21 were soluble and partly in inclusion bodies, provingtobe K.10 protein by Westem blotting. The soluble fusion IL-10-Trx possossed biological activity. Conclusion IL-10 poteinwith hioactivity is expressed in E. coli successfully, which provides a basis for downstream purification and preparation of genetically engineered drug IL-10.