类弹性蛋白展示NT-proBNP双抗原表位重组蛋白的制备和纯化

doi:10.13481/j.1671-587x.20200305

摘要/Abstract

摘要： 目的：利用大肠杆菌（E.coli）高效制备类弹性蛋白（ELP）展示N端脑钠肽前体（NT-proBNP）双抗原表位的重组蛋白，为低成本、高效地制备NT-proBNP检测校准品提供依据。方法：分别设计NT-proBNP第13~20位和第63~71位氨基酸残基表位，通过柔链连接上述2个抗原表位与ELP融合，获得3种融合蛋白。利用基因工程技术合成编码以上3种融合蛋白的基因并克隆到pET-28a （+）载体上，转入E.coli BL21（DE3）自动诱导表达，利用多次可逆相变循环（ITC）纯化重组蛋白，并应用Western blotting和ELISA法检测其抗体特异结合表位的能力。结果：成功构建了3种ELP展示NT-proBNP抗原表位的载体，并在E.coli中表达获得相应重组蛋白。Western blotting和直接ELISA检测，ELP展示的特异抗原表位均具有良好的与相应抗体结合的能力。夹心ELISA法检测，ELP展示NT-proBNP双抗原表位重组蛋白的蛋白浓度与450 nm处吸光度（A）值呈双对数线性剂量依赖关系（r=0.9197，P<0.01）。结论：成功利用ELP展示NF-proBNP双抗原表位，为低成本、高效地制备出与NT-proBNP检测校准品奠定了基础。

关键词: N-端脑钠肽前体, 类弹性蛋白, 抗原表位, 大肠杆菌, 心力衰竭

Abstract: Objective: To use Escherichia coli (E. coli)to prepare the recombinant proteins of elastin-like polypeptide(ELP) for displaying the two epitopes of N-terminal pro-brain natriuretic peptide(NT-proBNP), and to provide the basis for the low-cost and high-efficiency preparation of NT-proBNP detection calibrator. Methods: The epitopes of 13-20 and 63-71 amino acid residues of NT-proBNP were designed and fused with ELP by flexible chain;three kinds of fusion proteins were obtained. The genes encoding the three fusion proteins mentioned above were synthesized by genetic engineering technique and cloned into the pET-28a (+) vector; the recombinant proteins were induced and expressed automatically in E.coli BL21(DE3),the recombinant proteins were purified with inverse transition cycling(ITC),and the abilities of their antibodies specificly binding epitopes were detected by Western blotting and ELISA methods. Results: Three kinds of vectors of ELP for displaying NT-proBNP epitopes were successfully constructed, and the corresponding recombinant proteins were expressed in the E.coli. The western blotting and direct ELISA results showed that the specific epitopes displayed by ELP had the better binding ability with the relative antibodies.The results of Sandwich ELISA showed that the protein concentrations of recombinant proteins of ELP for displaying two epitopes of NT-proBNP had a double logarithmic linear dose-dependent relationship with the absorbance (A) value at 450 nm (r=0.9197, P<0.01). Conclusion: The ELP is successfully used to display the two epitopes of NT-proBNP, and lay a foundation for the low-cost and high-efficiency preparation of the NT-proBNP detection calibrator.

Key words: amino-terminal pro-brain natriuretic peptide, elastic-like polypeptide, epitope, Escherichia coli, heart failure

中图分类号:

付鑫, 龚福梅, 丁宁, 朱静, 杨春光, 胡学军. 类弹性蛋白展示NT-proBNP双抗原表位重组蛋白的制备和纯化[J]. 吉林大学学报(医学版), 2020, 46(03): 458-463.

FU Xin, GONG Fumei, DING Ning, ZHU Jing, YANG Chunguang, HU Xuejun. Preparation and purification of recombinant proteins of elastin-like polypeptide for displaying two epitopes of NT-proBNP[J]. Journal of Jilin University(Medicine Edition), 2020, 46(03): 458-463.

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