Zhang Heng, Li Guangxing, Ren Xiaofeng
College of Veterinary Medicine, Northeast Agricultural University, Xiangfang District, Harbin, China.
Hybridoma (Larchmt). 2010 Dec;29(6):525-9. doi: 10.1089/hyb.2010.0063. Epub 2010 Nov 18.
The gene encoding mature chicken interleukin-18 (ChIL-18) was cloned into prokaryotic expression vector pET-30a(+), resulting in a recombinant plasmid pET-30a-ChIL-18. After pET-30a-ChIL-18 was transformed into Escherichia coli Rosseta, the expression of ChIL-18 induced by 1 mM IPTG at 37°C was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The expressed fusion protein of 26 kDa was purified with a Ni-NTA affinity column and used to generate a hyperimmune antiserum in a rabbit. The specificity and titer of anti-ChIL-18 serum were analyzed by the enzyme-linked immunosorbent assay. Western blot and immunofluorescence assays indicated that the anti-ChIL-18 antibody specifically reacted with the ChIL-18 expressed from E. coli or ChIL-18-transfected eukaryotic cells. Moreover, the renatured ChIL-18 stimulated the production of nitric oxide (NO) from macrophages via eliciting the secreting of IFN-γ from lymphocytes.
将编码成熟鸡白细胞介素-18（ChIL-18）的基因克隆到原核表达载体pET-30a(+)中，构建重组质粒pET-30a-ChIL-18。将pET-30a-ChIL-18转化至大肠杆菌Rosetta中后，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析1 mM异丙基-β-D-硫代半乳糖苷（IPTG）在37°C诱导下ChIL-18的表达情况。用镍-亚氨基二乙酸（Ni-NTA）亲和柱纯化表达的26 kDa融合蛋白，并用于在兔体内制备超免疫抗血清。通过酶联免疫吸附测定分析抗ChIL-18血清的特异性和效价。蛋白质印迹法和免疫荧光测定表明，抗ChIL-18抗体与大肠杆菌表达的ChIL-18或ChIL-18转染的真核细胞特异性反应。此外，复性的ChIL-18通过诱导淋巴细胞分泌γ干扰素（IFN-γ）刺激巨噬细胞产生一氧化氮（NO）。
