一种高效制备人β2微球蛋白标准物质的方法和应用  

An efficient method for the preparation of human β2-microglobulin reference material and its application prospects

作　　者：邹丽辉[1] 王萌[2] 黄薇[1] 张恩毅[1] 肖飞[1] 王玉梅[3] ZOU Li-hui;WANG Meng;HUANG Wei;ZHANG En-yi;XIAO Fei;WANG Yu-mei(The MOH Key Laboratory of Geriatrics,Department of Laboratory Medicine,Beijing Hospital,National Center of Gerontology,Beijing 100730,China;Department of Biomaterials,National Institute for Food and Drug Control,Beijing 100050,China)

机构地区：[1]北京医院/国家老年医学中心/卫生部老年医学重点实验室,北京100730 [2]北京医院/国家老年医学中心/检验科,北京100730 [3]中国食品药品检定研究院生物材料室,北京100050

出　　处：《中国医药生物技术》2018年第5期412-417,共6页Chinese Medicinal Biotechnology

基　　金：北京市自然科学基金(7172193);“重大新药创制”国家科技重大专项(2017ZX09304026);国家自然科学基金(81571384)

摘　　要：目的构建重组人β2微球蛋白表达体系,高效快速获取β2微球蛋白,为临床实验室检测参考物质的制备奠定基础。方法优化人β2微球蛋白序列,人工合成优化序列片段并与麦芽糖结合蛋白(MBP)分别克隆至pRSF-Duet载体以构建重组人β2微球蛋白表达质粒。使用大肠杆菌E.coli BL21(DE3)对重组质粒进行诱导表达,TEV蛋白酶切除MBP,全自动生化仪测定β2微球蛋白浓度。对重组蛋白在4℃和–20℃储存条件下的稳定性进行检测,对β2微球蛋白纯品与校准品的互通性进行分析。结果成功构建重组人β2微球蛋白表达质粒,异丙基硫代半乳糖苷(IPTG)成功诱导重组蛋白表达,MBP-β2微球蛋白(MBP-β2-MG)浓度可达100 mg/L,β2微球蛋白浓度可达185 mg/L。在4℃和–20℃储存条件下监测64周,β2微球蛋白稳定存在。β2微球蛋白纯品与RANDOX生化校准品互通性良好。结论通过密码子优化,成功构建了重组β2微球蛋白的高效表达系统,β2微球蛋白可溶性高,稳定性好,与国际通用校准品互通性好,可充分满足该产品诊断试剂制备的需求。Objective To construct optimized human β2-microglobulin prokaryotic expression system, and obtain recombinant β2-microglobulin efficiently and rapidly which is expected to provide basis for the preparation of reference materials and application in quality control in clinical laboratories. Methods The optimized human β2-microglobulin gene was synthesized and cloned into pRSF-Duet vector with maltose binding protein （MBP） to generate MBP-β2-MG pRSF expression vector. Recombinant human β2-MG protein was expressed in E.coli BL21（DE3） and its concentration was analyzed by Hitachi analysis system after MBP was cut by TEV protease. The stability of β2-MG protein was detected during long-term storage at 4℃ and -20 ℃. The compatibility of the purified β2 microglobulin and the β2 microglobulin from RANDOX was analyzed.Results The prokaryotic expression vector MBP-β2-MG pRSF was successfully constructed. When expressed in E.coli BL21（DE3）, the concentration of soluble MBP-β2-MG was about 100 mg/L, and the concentration of β2-MG protein could reach 185 mg/L. β2-MG protein was stable after 64 weeks at 4℃ and -20℃. The purified β2 microglobulin had good compatibility with the β2 microglobulin from RANDOX. Conclusions The recombinant MBP- β2-MG protein is soluble and the concentration of β2-MG can reach 185 mg/L, which can fully meet the requirement of the preparation of diagnostic reagent.

关 键 词：Β2微球蛋白 重组蛋白质类 参考物质 

分 类 号：R392-33[医药卫生—免疫学]