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

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.