兔抗人MIG7多克隆抗体的制备和纯化

The construction and purification of the rabbit polyclonal antibody of MIG7

目的:构建、表达迁移诱导蛋白7(migration-inducing protein 7,MIG7),制备该融合蛋白的兔多克隆抗体,纯化并鉴定该多克隆抗体。方法:设计并合成MIG7蛋白特异性多肽片段,合成的MIG7多肽与马来酰胺活化匙孔血蓝蛋白(KLH)偶联,构建半抗原-载体偶联物。以MIG7特异性多肽-KLH为抗原免疫大白兔,制备抗MIG7多克隆抗体。40%硫酸铵沉淀、甘氨酸溶液洗脱纯化抗体,Dot blot检测抗体血清效价。免疫组化与qRT-PCR验证MIG7抗体。结果:免疫组织化学染色与qRT-PCR检测结果一致,证明该抗体可特异性结合MIG蛋白,获得MIG7多克隆抗体。结论:本研究成功制备并纯化了特异性较高的抗MIG7多克隆抗体,为进一步研究MIG7在血管生成拟态(vasculogenic mimicry,VM)形成和肝癌侵袭转移中的机制奠定了基础。

Objective： To construct, purify and identify the rabbit polyclonal antibody of MIG7. Methods： The MIG7 protein specific polypeptide fragment was designed and synthesized. The MIG7 polypeptides were coupled with the key pore blood blue protein （KLH）. Conjugate semi antigen carrier were constructed. The MIG7 - KLH were in-jected into white rabbit as an antigen to produce the polyclonal antibody of MIG7. The antibody were purified by 40% ammonium sulfate precipitation and glycine solution. The title was tested by Dot blot. The rabbit polyclonal antibody of MIG7 were verified by qRT - PCR and immunohistochemical staining. Results ： The qRT - PCR and immunohisto- chemical staining showed the same result. We got the rabbit polyclonal antibody of MIG7 successfully. Conclusion ： The antibody of MIG7 were produced,it was a premise to the further study of the vasculogenic mimicry （ VM） forma-tion and metastasis in liver cancer.