gp130结合分子融合蛋白的表达、纯化及其mAb的制备

Expression and purification of gp130 associated molecule fusion protein and preparation of its mAbs

ｇｐ１３０结合分子（ＧＡＭ）是一种新发现的可与ｇｐ１３０胞浆内近膜区结合的蛋白质，其在ｇｐ１３０信号传导中的作用尚待深入研究。我们在原核表达ＧＡＭｔｈｉｏｒｅｄｏｘｉｎ（Ｔｈｉｏ）融合蛋白的基础上，制备并纯化了抗Ｔｈｉｏ的多克隆抗体，将其与Ｓｅｐｈａｒｏｓｅ４Ｂ偶联，经亲和层析提纯该融合蛋白，以此为免疫原，通过杂交瘤技术获得了３株抗ＧＡＭｍＡｂ。其中两株为ＩｇＧ１，１株为ＩｇＧ２ａ，与Ｔｈｉｏ及空载体转染的工程菌裂解液均无交叉反应。３株ｍＡｂ腹水抗体效价为（１．２４．８）×１０－７，同变性与非变性、单体与聚合体形式的ＧＡＭＴｈｉｏ融合蛋白都能起反应，用于Ｗｅｓｔｅｒｎｂｌｏｔ的效果满意，为ＧＡＭ的功能研究提供了重要的手段。

gp130 associated molecule(GAM) is a recently cloned M r24 000 protein which binds to gp130 at its cytoplasmic memembrane proximal region. Although the function of GAM is still unknown, it seems to be an important factor in the regulation of transcription. We produced rabbit anti thioredoxin(Thio) polyclonal antibody(pAb), and purified it with Thio coulped Sepharose 4B. After expressing GAM Thio fusion protein in E.coli, we coulped the anti Thio pAb to sepharose 4B and purified GAM Thio fusion protein from E.coli lysates with it. By immunizing Balb/c mice with GAM Thio, three hybridoma cell lines secreting mAbs against GAM were raised by conventional hybridoma technique.Two of the mAbs were IgG1, the other was IgG2a.The titre of mAbs from mouse ascites were (1.2￣4.8)×10 -7 . All three mAbs could recognize nature and denatured GAM Thio, without any cross reaction with Thio or components of E.coli.