GST-Ccd1融合蛋白的表达、纯化及多克隆抗体制备

EXPRESSION,PURIFICATION AND POLYCLONAL ANTIBODY PREPARATION OF GST-Ccd1 FUSION PROTEIN

目的:利用大肠杆菌DH5α表达GST—Ccd1融合蛋白,并用亲和层析分离纯化,进行动物免疫制备多克隆抗体。方法:利用本室构建好的pGEX-5X-1-Ccd1-N原核表达重组质粒,转化大肠杆菌DH5α,经IPTG诱导表达,在大肠杆菌表达系统中获得可溶性表达。经谷胱甘肽Sepharose 4B介质填充的层析柱分离纯化蛋白,制备抗原免疫动物,得到Ccd1的兔源多克隆抗体。结果:ELISA结果显示血清抗体效价可以达到1∶40 000。免疫组化分析表明自制的抗体能特异性与Ccd1蛋白相互作用,可以用于实验分析。结论:制备了效价高特异性良好的抗Ccd1多克隆抗体,经实验验证获得的抗体能够满足针对Ccd1的免疫印迹和免疫组化检测的实验要求,为今后深入研究Ccd1表达的组织分布、细胞内定位及其生物学功能提供了有用的实验工具。

Aim： By using of Escherichia coli DH5α to express GST-Ccdl fusion protein and which was purified by affinity chromatographic separation. The purified target protein was used to immunize rabbits to prepare polyclonal antibody. Methods： The previously con- structed recombinant prokaryotic expression vector pGEX-SX-1-Ccdl was transformed into Escherichia coli DHSα and was induced to expression by IPTG. The recombinant target protein was expressed with soluble state in Escherichia coli expression system which was separated and purified by chromatographic column stuffed with glutathione Sepharose 4B. The prepared antigen was used to immunize rabbits to get anti-Ccdl specific rabbit original polyclonal antibody. Results： ELISA data demonstrated that the antibody titer of the serurn was up to 1：40 000. Immunohistochemistry analysis indicated that the ＂home-made＂ antibody had a specific interaction with Ccdl protein and which could be used for extended experimental research. Conclusion： The anti-Ccdl polyclonal antibody we had prepared had a high quality of potency and specificity. The antibody was demonstrated by experiments that which could totally fulfill the requirement of immunoblotting and immunohistochemistry study of Ccdl. The antibody provided an useful experimental tool to profoundly research the tissue expression profile, intercellular location and biological function of Ccdl .