人NT-proBNP的高效原核表达及免疫学检测标准品的研究  

Construction and preparation of prokaryotic high-level expression vector of human NT-proBNP

作　　者：易维京[1] 李鹏[1] 李淑慧[1] 智强[1] 胡川闽[1] 

机构地区：[1]第三军医大学医学检验系临床生物化学教研室,重庆400038

出　　处：《第三军医大学学报》2008年第6期525-529,共5页Journal of Third Military Medical University

摘　　要：目的构建人NT-proBNP(氨基末端脑钠肽,amino-terminal pro-brain natriuretic peptide)的高效原核表达载体,建立重组NT-proBNP纯化工艺,制备NT-proBNP免疫学检测工作标准品。方法采用人工合成和套叠(GeneSOEing)PCR技术相结合的方式对NT-proBNP基因序列进行密码子进行偏嗜性改造后克隆入原核表达质粒pET32a和pET42a构建不同形式的NT-proBNP原核表达重组质粒;采用离子交换、HIS和GST亲和层析柱以及肠激酶(EK酶)酶切等方法建立NT-proBNP重组蛋白的纯化工艺;用Roche公司NT-proBNP检测试剂盒测定所制备的标准品的免疫活性和稳定性。结果pET32a和pET42a构建的不同形式NT-proBNP原核表达质粒在BL21(DE3)均实现了高效可溶性表达,通过多种纯化方案制备了纯度达98.3%的NT-proBNP-83重组蛋白,经测定具有良好免疫活性和稳定性,适合用于免疫学检测工作标准品。结论成功构建了人NT-proBN的高效原核表达载体,筛选出较适合用于免疫学检测工作标准品的NT-proBNP重组蛋白。Objective To construct a prokaryotic high-level expression system for human NT-proBNP （amino-terminal pro-brain natriuretic peptide ） and establish the working standard for immunoassay. Methods According to the frequency of E. coli codons, an E. coli favorite DNA fragment, encoding amino acids sequence identical to that of the wild type NT-proBNP was cloned into plasmid pET32a and pET42a by synthesized oligoes and GeneSOEing PCR techniques. The NT-proBNP protein was isolated by cation exchange, his-tag affinity column, GST affinity chromatograph and digested by enterokinase. Immunoreactivity and stability of the protein were identified by using NT-proBNP test kit （ Roche. Ltd）. Results After induced by IPTG, the NT-proBNP was expressed efficiently as soluble protein in E. coil cells. The purified NT-proBNP （98.3%） was immunoreactive and stable. Conclusion The prokaryotic high-level expression vector of human NT-proB- NP was successfully constructed. A recombinant protein NT-proBNP suitable for standards of immunodetection was obtained.

关 键 词：NT—proBNP高效 套叠PCR肠激酶 蛋白纯化 工作标准品 

分 类 号：R341[医药卫生—基础医学] R392-32