兔抗人MIG7多克隆抗体的制备和纯化

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.

The construction and purification of the rabbit polyclonal antibody of MIG7

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 injected 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 immunohistochemical 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) formation and metastasis in liver cancer.