蛋白重组技术在制备视黄醇结合蛋白质控品和候选参考物质中的方法研究  

Method and application of high efficient preparation of procalcitonin standby reference material based on protein recombination technology

作　　者：张超 周伟燕[1] 张江涛[1] 汪静[1] 张传宝[1] Zhang Chao;Zhou Weiyan;Zhang Jiangtao;Wang Jing;Zhang Chuanbao(National Center for Clinical Laboratories,Institute of Geriatric Medicine,Chinese Academy of Medical Sciences,Beijing Hospital/National Center of Gerontology,Beijing100730,China)

机构地区：[1]北京医院国家老年医学中心,国家卫生健康委临床检验中心,中国医学科学院老年医学研究院,北京100730

出　　处：《中华检验医学杂志》2022年第11期1131-1135,共5页Chinese Journal of Laboratory Medicine

基　　金：北京医院“科技新星”(BJ-2020-087);国家自然科学基金(82003809)。

摘　　要：目的通过蛋白重组技术低成本、快速制备高浓度人视黄醇结合蛋白4(hRBP4)参考物质,解决临床hRBP4高值参考物质难以获取的需求。方法在美国国立生物技术信息中心NCB查找hRBP4的互补DNA序列,经大肠埃希菌密码子优化后人工合成,然后将DNA片段克隆至原核表达质粒pET-28a(+)中,以构建重组hRBP4表达载体。将重组质粒转化至E.coli BL21感受肽中,优化诱导表达条件(如温度、转速和异丙基硫代半乳糖苷IPTG浓度等)、诱导质粒表达并对重组蛋白进行His融合标签纯化,获取纯化后的hRBP4重组蛋白。结果DNAMAN软件对比显示编码hRBP4蛋白的碱基序列完全正确,成功构建重组hRBP4原核表达质粒;SDS-聚丙烯酰胺凝胶电泳结果显示亲和纯化产物中可清晰可见约相对分子质量26000电泳条带;临床尿液和血清hRBP4常规检测系统均可检测到纯化的hRBP4蛋白,且稀释倍数与检测浓度间呈现良好线性关系。结论成功构建重组hRBP4高水平表达系统,重组hRBP4蛋白纯度大、浓度高、生产周期短,有望在室间质量评价质控品和有证参考物质研制中发挥重要作用。Objective To construct a prokaryotic expression vector for human retinol binding protein 4(hRBP4)that allows technicians to obtain hRBP4 purified protein with low cost,high efficiency,high concentration and high purity.Methods The hRBP4 coding sequence provided by National Center for Biotechnology Information was optimized by E.coli codons,and a synthetic DNA fragment was cloned into the PET-28A(+)prokaryotic expression vector to construct a recombinant hRBP4 expression plasmid.The recombinant protein was transformed into E.coli BL21,and the induced expression conditions(temperature,rotate speed and isopropylβ-d-thiogalactoside concentration)were optimized.The recombinant protein was purified by His fusion tag.Results The recombinant hRBP4 prokaryotic expression plasmid was successfully constructed,and the expression concentration and induction temperature of the recombinant protein were optimized.The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that a band with a relative molecular weight of 26000 daltons was clearly visible in the purified product.The purified hRBP4 protein could be detected clinically,and there was a good linear relationship between the dilution ratio and the detection concentration.Conclusions The recombinant hRBP4 protein has high purity,high concentration,and short production cycle.It has the potential to become a candidate for reference materials for laboratory quality evaluations.

关 键 词：视黄醇结合蛋白质类 重组蛋白 参考物质 质控品 室间质量评价 

分 类 号：Q78[生物学—分子生物学] R318[医药卫生—生物医学工程]