人IL-6重组蛋白及IL-6荧光免疫层析检测试剂卡的研制  

Expression of human IL-6 recombinant protein and development of IL-6 fluorescence immuno-chromatography testing card

作　　者：裴晓萌 王静 魏治静 吴萌[1] PEI Xiao-meng;WANG Jing;WEI Zhi-jing;WU Meng(Cell and Biochemistry Laboratory,Institute of Biology,Hebei Academy of Sciences,Shijiazhuang 050081,China)

机构地区：[1]河北省科学院生物研究所细胞生化室,石家庄050081

出　　处：《现代免疫学》2023年第5期406-412,共7页Current Immunology

基　　金：河北省科学院科技计划项目(2021G03)。

摘　　要：研制一种人IL-6荧光免疫层析检测试剂卡,用于IL-6分子的临床快速检测,并且研制一种重组表达人IL-6蛋白作为IL-6检测试剂卡的标准品。首先获得人IL-6基因序列,经密码子优化后连接到表达载体pET30a,然后转化到大肠杆菌BL21感受态细胞诱导表达。经异丙基硫代-β-D-半乳糖苷(isopropylthio-β-D-galactoside, IPTG)诱导后,得到可溶性表达的IL-6蛋白。经镍柱亲和层析纯化和超滤,得到重组人IL-6蛋白。基于荧光免疫层析技术及双抗体夹心法原理,应用时间分辨荧光微球制备IL-6荧光免疫层析检测试剂卡。经性能评估,试剂卡的空白限为5.2 pg/mL,变异系数在7.34%~11.11%之间,重复性较好;回收率在96.3%~107.2%之间;试剂卡37℃放置6周稳定性较好,与常见竞争物无交叉反应,参考值范围0~9.0 pg/mL;与电化学发光免疫分析(electrochemiluminescence immunoassay, ECLIA)比较临床样本检测结果相关性较好,相关系数r值为0.965。综上,研制的IL-6重组蛋白具有较高的纯度及浓度,可作为试剂盒检测的标准品;制备的IL-6定量检测试剂卡性能可满足临床应用的需求,且具有简便、快捷等优点。In this study,a type of fluorescence immuno-chromatography reagent card that may be applied for the quick quantitative detection of IL-6 was developed.A recombinant human IL-6 protein was expressed in E.coli as a standard for the IL-6 testing card.First,the human IL-6 gene was cloned to the expression vector pET30a after codon optimization,which was subsequently transformed into E.coli BL21 competent cells for inducible expression.By isopropylthio-β-D-galactoside(IPTG)induction,the soluble form of IL-6 protein was obtained.After Ni2+column affinity chromatography purification and ultrafiltration,the IL-6 protein standard sample was acquired.The fluorescence immuno-chromatography reagent card was prepared by time-resolved fluorescent microspheres,based on the fluorescence immuno-chromatography technology and sandwich ELISA.By property evaluation,the blank limit was 5.2 pg/mL.The coefficient of variation was between 7.34%to 11.11%,exhibiting good repeatability.The recovery rate was between 96.3%and 107.2%.The reagent card showed good stability after six weeks storage at 37℃.There was no cross-reaction with common competitors,with the reference value ranging from 0 to 9.0 pg/mL.The outcome of our method on the clinical samples testing was comparable to that of the electrochemi-luminescence immunoassay(ECLIA)(r=0.965).In conclusion,the recombinant IL-6 protein prepared in this study is of high purity and concentration,which is qualified as kit testing standard.And the developed reagent card is convenient to use and could meet the needs of clinical applications.

关 键 词：人白介素6 重组表达 荧光免疫层析 

分 类 号：R446.62[医药卫生—诊断学]