粒细胞巨噬细胞集落刺激因子受体α链（GMCSFRα）的克隆与表达

Cloning and Expression of Granulocyte macrophage Colony stimulating Factor Receptor α Subunit(GM CSFR α)

ＧＭＣＳＦ高亲和力受体至少由两条链组成，低亲和力的α链及无结合力活性的β链。本文采用ＲＴＰＣＲ的方法，从ＧＭＣＳＦ依赖细胞株ＴＦ１中克隆了全长的ＧＭＣＳＦＲα链ｃＤＮＡ（包括信号肽部分），经酶切及全基因测序鉴定，并分别在原核及真核细胞表达系统中表达。在大肠杆菌中ＧＭＣＳＦＲα以ＧＳＴ融合蛋白形式表达，谷胱甘肽Ｓｅｐｈａｒｏｓｅ４Ｂ亲和层析纯化融合蛋白。ＧＳＴＣＳＦＲαＣ无受体活性，推测主要与缺乏翻译后修饰有关。将全基因克隆入真核表达载体ｐＳＶＬ中，转染ＣＯＳ７细胞瞬时表达以重建ＧＭＣＳＦ的低亲和力受体，１２５ＩＧＭＣＳＦ平衡结合实验证明转染后的ＣＯＳ７细胞膜上有受体的表达，Ｃｒｏｓｓｌｉｎｋｉｎｇ显示表达的受体α链分子量略低于ＴＦ１细胞。胞外区基因（ＣＳＦＲαＢ）克隆入ｐＳＶＬ构建的ｐＳＶＬ／ＣＳＦＲαＢ表达载体，转染ＣＯＳ７细胞后，Ｗｅｓｔｅｒｎｂｌｏｔ检测表达细胞上清，有单一的反应带，表达上清有ＧＭＣＳＦ的受体活性。

The high affinity receptor for GM CSF is consisted of two subunits,α chain and β chain.We have cloned whole length of GM CSFR α cDNA from TF 1 cells by RT PCR method and verified by restriction analysis and DNA sequencing. E.coli and COS 7 cell expression system are used to express the GM CSFR α cDNA.PCR is first used to modify the GM CSFR α cDNA and expressed in E.coli by fusing to GST.The fusion protein can be purified by Glutathione Sepharose 4B.GM CSFR α fusion protein can't bind with GM CSF.The reason exists in lack of post translation modification.GM CSFR α cDNA is then cloned into eukaryotic expression vector pSVL and transfect the COS 7 cell. 125 I GM CSF binding experiment indicate GM CSFR α is expressed on the COS 7 cell membrane.But MW of expressed GM CSFR is a little lower compared with that of TF 1 cells.Then we subcloned extracellular domain of GM CSFR α(CSFR α B) into the pSVL and transfected COS 7 cells.The supernatant of the transfected cells has a reaction band with specific antisera using Western blot.It also has GM CSFR activity.