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

作　　者：井申荣[1] 邹全明[1] 洪愉[1] 毛旭虎[1] 

机构地区：[1]第三军医大学医学检验系临床微生物学及免疫学教研室重庆市生物制药工程技术研究中心,重庆400038

出　　处：《免疫学杂志》2006年第4期399-401,405,共4页Immunological Journal

摘　　要：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.

关 键 词：重组人白细胞介素10 融合蛋白 可溶性 生物学活性 

分 类 号：R392[医药卫生—免疫学]