摘要
目的获得鸡白介素-10(chIL-10)的原核表达产物,制备鼠抗血清,分析与真核系统表达的chIL-10的反应原性。方法采用RT-PCR从经脂多糖(LPS)体外刺激的鸡外周血淋巴细胞中扩增编码成熟IL-10的基因,克隆至原核表达载体pET-30a(+),成功构建原核重组质粒pET-chIL-10;转化至大肠杆菌DH5α中IPTG诱导表达,切下含有重组蛋白的SDS-PAGE胶条,免疫制备鼠抗血清;分别与包含信号肽和去除信号肽的chIL-10真核表达细胞系CHO-euchIL-10F和CHO-euchIL-10M进行间接免疫荧光抗体试验(IFA)和蛋白免疫印迹试验(Western blot),验证有无反应性。结果成功构建chIL-10的原核表达载体pET-chIL-10,获得了高效价的鼠抗血清,能与表达chIL-10的真核细胞系反应。结论原核表达的chIL-10特异性鼠抗血清将能用于体内研究的检测。
Objective To preliminarily analyze the immunogenicity of E. coli expressed chicken Interleukin-10 （chlL-10） antiserum in stably chIL-lO-expressing cell line transfected by chIL-10 gene with signal peptide （CHO- chIL-10F） and that without signal peptide （CHO-chIL-10M）. Method Whole chIL-10 gene was amplified by PCR from chicken peripheral blood lymphocytes and cloned into pET-30a （ ＋ ） vector to obtain recombinant plasmid of pET-chIL-10, followed by induce express in E. coli BL21 （DE3）. The SDS-PAGE gel containing foreign protein was immunized BALB/c mice to prepare antisera. Indirect immunofluorescent assay （IFA） and Western blot assay were performed. Result The mice anti-pET-chIL-10 specific serum could react with eukaryotic expressed chIL-10 in IFA and Western blot assay. Conclusion Antiserum to prokaryotic expressed chicken IL-10 could be used in vivo research study.
出处
《实验动物科学》
2017年第3期1-5,共5页
Laboratory Animal Science
基金
国家科技支撑计划子课题(No.2015BAI07B02-02)
关键词
鸡白介素-10
原核表达
鼠抗血清
体内实验
Chicken interleukin-10
prokaryotic expression
mice antiserum
in vivo assay
