基于基因工程蛋白重组技术高效制备降钙素原参考物质的方法及应用  

Methodology and application of high-efficiency preparation of procalcitonin reference substance based on genetically-engineered protein recombination

作　　者：张超 张江涛[1] 汪静[1] 周伟燕[1] 张传宝[1] Zhang Chao;Zhang Jiangtao;Wang Jing;Zhou Weiyan;Zhang Chuanbao(National Geriatrics Center of Beijing Hospital,Clinical Laboratory Center of National Health Commission,Institute of Geriatrics,Chinese Academy of Medical Sciences,Beijing 100730,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：《中华生物医学工程杂志》2022年第4期419-423,共5页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金青年项目(82003809);北京医院"科技新星"项目(BJ-2020-087)。

摘　　要：目的构建人降钙素原(hPCT)原核表达载体, 低成本、快速、高浓度、高纯度获取hPCT纯化蛋白, 为临床实验室检测制备质控品和参考物质奠定基础。方法将GenBank提供的hPCT编码序列经大肠杆菌密码子优化后, 人工合成DNA片段克隆至pET-28a(+)原核表达载体以构建重组hPCT表达质粒。转化至大肠杆菌E. coli BL21感受态中, 优化诱导表达条件、诱导质粒表达并对重组蛋白进行His融合标签纯化。结果成功构建重组hPCT原核表达质粒, 优化异丙基硫代半乳糖苷(IPTG)诱导重组蛋白表达浓度及诱导温度, 最适IPTG浓度为0.2 mmol/L, 最佳诱导温度为37 ℃。纯化后hPCT蛋白可通过临床检测, 且稀释倍数与检测浓度间呈现良好线性关系, 回归方程为y=-0.0085x+112.63, R2值为0.9975。结论成功构建重组hPCT的高效表达系统, 重组hPCT蛋白纯度大、浓度高、生产周期短, 有成为候选室间质量评价参考物质的潜力。Objective To construct a prokaryotic expression vector for human procalcitonin(hPCT),obtain the purified hPCT protein with low cost,rapidity,high concentration and high purity,and to lay a foundation for preparation of quality control and reference substances used in clinical laboratory testing.Methods The hPCT coding sequence provided by GenBank was optimized by E.coli codon and artificially synthesized into a DNA fragment which was cloned into the PET-28A(+)prokaryotic expression vector to construct the recombinant hPCT expression plasmid.The recombinant plasmid was then transformed into BL21 competent E.coli.The expression conditions with induction of plasmid expression were optimized.the recombinant protein was purified by His-tagging.Results A recombinant hPCT prokaryotic expression plasmid was successfully constructed,and the expression concentration and induction temperature of the recombinant protein induced by isopropylthiogalactoside(IPTG)were optimized.The optimal IPTG concentration was 0.2 mmol/L,and the optimal induction temperature was 37℃.The purified hPCT protein was detectable in clinical setting,with a good linear relationship between the dilution factor and detected concentration that followed the regression equation y=-0.0085x+112.63(R2=0.9975).Conclusion We successfully constructed a high-efficiency recombinant hPCT expression system.The recombinant hPCT protein yielded from this system shows high purity,high concentration and a short production cycle,and thereby is promising as a candidate reference substance for inter-laboratory quality assessment.

关 键 词：降钙素 重组蛋白质类 质粒 质控品 

分 类 号：R392[医药卫生—免疫学]