LanCL1融合蛋白表达纯化及其多克隆抗体制备鉴定

Expression,Purification of LanCL1 Fusion Protein and Preparation,Identification of Its Polyclonal Antibody

目的:获得LanCL1原核和真核表达融合蛋白,制备兔抗LanCL1的多克隆抗体以用于LanCL1基因功能研究。方法:PCR扩增LanCL1 CDS编码区序列,分别亚克隆至原核和真核表达载体上。将真核表达重组质粒用脂质体转染HEK293细胞成功获得了pRK5-LanCL1真核表达融合蛋白。同时将原核表达重组质粒化学转化BL21(DE3)感受态细胞,IPTG诱导GST-LanCL1原核表达融合蛋白,谷胱甘肽琼脂糖珠亲和纯化,透析浓缩获得高纯度GST-LanCL1融合蛋白。将纯化的融合蛋白免疫新西兰大白兔制备多克隆抗体,并用Western Blot检测抗体。结果:获得了LanCL1真核和原核表达重组融合蛋白。经亲和纯化后的GST-LanCL1融合蛋白纯度达到90%,BCA蛋白定量浓度约0.44mg/mL。获得了高效价的特异性兔抗LanCL1多克隆抗体。结论:成功进行了LanCL1融合蛋白的原核和真核表达,并获得抗LanCL1的高灵敏度的多克隆抗体,为LanCL1进一步功能研究奠定了基础。

Objective：To express the fusion protein of LanCL1 in prokaryotic and eukaryotic cells,and prepare the anti-LanCL1 polyclonal antibody.Methods：The CDS of LanCL1 was amplified by PCR from normal mice,and then subcloned into the GST-fused protein expression vector pGEX-6p-1,After transformed with the vector,the BL21 strains were induced by IPTG.To get high purity protein,we treated the lysates with immobilized Glutathione Sepharose,and got rid of ions by dialysis.The purified protein was analyzed by SDS-PAGE,and then immuned the New Zealand white rabbits to prepare antibody.The antibody titer and specification were indentified by western bolt.Results：The GST-fusion protein of LanCL1 had 90% purity,0.44 mg/mL concentration after series procedures and polyclonal antibody from the rabbit could detect LanCL1 both from the brain and from the eukaryotic cell lysates at a high titer.Conclusion：We had successfully obtained eukaryotic and prokaryotic fusion protein of LanCL1,got high sensitivity and specification anti-LanCL1 polyclonal antibody.That lays a solid foundation for the further studies of LanCL1 function.