CSF－1R部分基因在大肠杆菌中表达、抗融合蛋白血清制备及丝氨酸／苏氨酸磷酸化的初步研究

鼠巨噬细胞集落刺激因子－１受体（ｍＣＳＦ－１Ｒ）部分序列与质粒ｐＧＥＸ－２Ｔ谷胱苷肽转移酶（ＧＳＴ）融合，融合蛋白ＧＳＴ－ＣＤ－Ｐｓｔ（胞浆区），ＧＳＴ－ＣＴｅｒｍ（Ｃ－末端）和ＧＳＴ－ＫＩ（激酶插入区）成功地在大肠杆菌ＪＭ１０９株表达。初步结果指出：（１）ＧＳＴ－融合蛋白在体外激酶分析中可以作为底物；（２）由ＰＫＡ导致的磷酸化可能具有生理学意义；（３）ｍＣＳＦ－１Ｒ被ＣＫＩＩ磷酸化。３２Ｐ标记ＧＳＴ－ＣＤ－Ｐｓｔ的磷酸氨基酸分析证实，ｍＣＳＦ－１Ｒ的胞浆区丝氨酸上被磷酸化，已制备抗ＧＳＴ－ＣＴｅｒｍ，ＧＳＴ－ＫＩ和ＧＳＴ－ＣＤ－Ｐｓｔ兔抗体。抗血清的筛选通过野生型３２Ｄ－ＣＳＦ－１Ｒ转染子免疫沉淀进行。

Expression of CSF-IR Partial Genes in E. Coli and Preparation of Antiserum for Fusion Proteins and Investigation on Serine/Threoprine Phosphorylation

The bacterial expressed proteins containing parts of murine CSF-1R (mCSF-1R) were fused in frameto glutathione S-transferase(GSF) in the vector, pGEX-2T. The fusion protein, GST-CD-Pst (cytoplasmicdomain), GST-CTerm (C-terminus) and GST-KI (kinase insert)have been successfully expressed in E. Coltstrain JM109. To determine whether mCSF-1R can be phosphorylated in vitro by pruified Ser/Thr kinases.GST-CD-Pst was utilized as the substrate in in vitro kinase assay. The preliminary results presented thusindicate that (1). GST-fusion protsins can function as substrates in in vitro kinals assay; (2)phosphorylation by PKA is likely to be physiologically significant; (3) mCSF-1R is phosphorylated byCKII. Phosphoamino acid analysis of 32P labeled GST-CD-Pst demonstrated that cytoplasmic domain ofmCSF-1R is phosphorylated on serines. Rabbit antibodies have been raised against GST-CTerm, GST-KIand GSF-CD-Pst. Sera was screened by immunoprecipitution of 32D-CSF-1R (wlid-type) transfectunt.