骆驼FGF21蛋白的原核表达及其多克隆抗体的制备与鉴定

Prokaryotic expression and polyclonal antibody preparation of camel FGF21 protein

本研究旨在为深入研究骆驼成纤维细胞生长因子21(FGF21)调节糖脂代谢的作用机制提供蛋白和抗体材料。在NCBI数据库中检索骆驼FGF21基因序列(XM_010996368.2),删除信号肽区段后合成携带His标签的FGF21全长基因,通过Eco RⅠ和XhoⅠ双酶切位点嵌入p ET-28a(+)表达载体,获得重组表达质粒p ET-28a(+)-FGF21-His,转入大肠杆菌Rosetta(DE3)中进行重组蛋白FGF21的表达,通过镍离子亲和层析柱纯化重组蛋白,利用SDS-PAGE和Western-blot鉴定纯化的重组蛋白。将重组蛋白FGF21与弗氏佐剂混合乳化后免疫新西兰大白兔,制备家兔源多克隆抗血清,利用protein A亲和层析纯化柱纯化多克隆抗体,利用SDS-PAGE技术分析纯化多克隆抗体的纯度,利用Western-blot和间接ELSIA方法验证纯化多克隆抗体的反应性。结果表明,成功表达并纯化出骆驼重组蛋白FGF21,以其免疫家兔后制备并纯化出抗FGF21的特异性多克隆抗体。本研究为建立ELISA方法检测骆驼FGF21蛋白提供了材料,为阐明FGF21在骆驼糖脂代谢中的功能机制奠定了基础。

The aim of this study was to provide protein and antibody materials for an in-depth study of the mode of action of camel fibroblast growth factor 21(FGF21)in regulating glycolipid metabolism.The camel FGF21 gene sequence(XM_010996368.2)was searched in the NCBI database,and the full-length FGF21gene carrying His tag was synthesized after deleting the signal peptide segment,inserted into p ET-28a(+)expression vector,and the recombinant expression plasmid p ET-28a(+)-FGF21-His was obtained,and was transferred into E.coli Rosetta(DE3)for the expression of recombinant protein FGF21.The recombinant protein was purified by nickel ion affinity chromatography column,and the purified recombinant protein was identified by SDS-PAGE and Western-blot assay.The recombinant protein FGF21 was mixed with Freund's adjuvant and immunized New Zealand large white rabbits to prepare rabbit-derived polyclonal antibody serum.The polyclonal antibody serum was purified by using a protein a affinity chromatography purification column,and the purity of the purified polyclonal antibody was analyzed by using SDS-PAGE,and the reactivity of the purified polyclonal antibody was verified by using Western-blot and indirect ELSIA methods.The results showed that camel recombinant protein FGF21 was successfully expressed and purified,and the specific polyclonal antibody against FGF21 was prepared and purified after immunizing rabbits with it.This study provides materials for the detection of camel FGF21 protein by ELISA method,and lays the foundation for elucidating the functional mechanism of FGF21 in camel glycolipid metabolism.