脑钠肽的重组表达、纯化及其在临床检验质控中的应用  

Recombinant expression and purification of brain natriuretic peptide(BNP)and its application to quality control in clinical laboratories

作　　者：李贺鑫[1,4] 汤小琨 孙高远 黄薇 王璐瑶[1,4] 张俊华 王萌 邹丽辉[2,4] LI He-xin;TANG Xiao-kun;SUN Gao-yuan;HUANG Wei;WANG Lu-yao;ZHANG Jun-hua;WANG Meng;ZOU Li-hui(Clinical Biobank,Beijing Hospital,Beijing 100730;Beijing Institute of Geriatrics of National Health Commission,Key Laboratory of Geriatrics of National Health Commission,Beijing Hospital,Beijing 100730;Department of Laboratory,Beijing Hospital,Beijing 100730;National Center of Gerontology,Institute of Geriatric Medicine,Chinese Academy of Medical Sciences,Beijing 100730,China)

机构地区：[1]北京医院临床生物样本管理中心,北京100730 [2]北京医院,国家卫生健康委北京老年医学研究所,国家卫生健康委老年医学重点实验室,北京100730 [3]北京医院检验科,北京100730 [4]国家老年医学中心,中国医学科学院老年医学研究院,北京100730

出　　处：《生物技术》2023年第5期558-562,共5页Biotechnology

基　　金：国家自然科学基金项目(81871107)。

摘　　要：[目的]制备脑钠肽(brain natriuretic peptide,BNP)重组蛋白,为临床实验室检测BNP提供稳定可靠、方便快捷的质控品或参考品原料。[方法]优化BNP编码密码子,与麦芽糖结合蛋白(maltose-binding protein,MBP)编码基因及FXa识别序列一并克隆至表达载体pRSFDuet-1,在大肠杆菌工程菌BL21(DE3)中诱导表达,可溶性表达产物MBP-BNP经纯化、丝氨酸蛋白酶FXa切除MBP后,获得具有生物活性的BNP蛋白。[结果]将NPPB(natriuretic peptide precursor B,NPPB)基因参考序列内26%的罕见密码子全部替换成了大肠杆菌常用密码子,成功构建了重组表达质粒MBP-BNP-pRSFDuet-1,酶切、测序鉴定正确后转入BL21(DE3)获得可溶性高表达MBP-BNP融合蛋白的工程菌株。用镍柱和交联葡聚糖G50分子筛纯化后获得250 mg/L的MBP-BNP融合蛋白,经FXa切除MBP后得到1.65 mg/L的BNP蛋白,比临床实验室常规BNP检测区间上限高出约50倍。[结论]与传统的参考品制备方法相比,运用基因工程技术可在大肠杆菌中稳定可溶性表达浓度较高的BNP蛋白,可为临床实验室提供更方便快捷、质优价廉的质控品或标准品原材料,提供了理论基础和技术保障。[Objective]To prepare recombinant brain natriuretic peptide,and provide a stable,reliable and convenient reference material for the detection of BNP in clinical laboratories.[Method]The codons in the BNP coding were optimized,along with maltose binding protein(MBP)coding gene and FXa recognition site coding sequence were cloned into expression vector pRSFDuet-1,and the expression was induced in E.coli BL21(DE3),and the soluble expression product MBP-BNP was purified,and after excision of MBP by serine protease FXa,the biologically active BNP protein was obtained.[Result]The 26%of rare codons in NPPB reference sequence were entirely replaced with common codons used in E.coli,the recombinant plasmid MBP-BNP-pRSFDuet-1 was successfully constructed,and its sequence was verified by restriction enzymes digestion and Sanger sequencing,an engineered strain in which MBP-BNP fusion protein could be soluble expressed was obtained by transforming recombinant plasmid into E.coli BL21(DE3).250 mg/L MBP-BNP fusion protein was obtained after purification from nickel column and sephadex G50 molecular sieve,1.65 mg/L BNP protein was detected after excision of MBP by FXa resection,which was about 50 times higher than the upper limit of BNP detection in clinical laboratories.[Conclusion]Compared with traditional methods of BNP preparation,a higher concentration of soluble BNP could be steadily expressed in E.coli by the gene recombinant technologies,which could provide a theoretical basis and technical guarantee for clinical laboratories to provide more convenient,higher quality and inexpensive reference materials.

关 键 词：脑钠肽 基因重组 蛋白质表达纯化 标准品 质控 

分 类 号：R34[医药卫生—基础医学] R394