人粒细胞集落刺激因子受体膜外区在昆虫细胞中的表达

Expression of the CRH Region of G CSF Receptor in Insect Cell

以胎盘组织提取的ｍ Ｒ Ｎ Ａ 为模板， Ｒ Ｔ Ｐ Ｃ Ｒ 扩增人粒细胞集落刺激因子（ Ｇ Ｃ Ｓ Ｆ） 受体膜外区 Ｃ Ｒ Ｈ 的ｃ Ｄ Ｎ Ａ 片段，克隆于供体质粒ｐ Ｆ Ａ Ｓ Ｔ Ｂ Ａ Ｃ１ ，与杆状病毒表达载体 Ｂａｃｍｉｄ 同源重组后，转染昆虫细胞 Ｓ Ｆ９ ，获得重组杆状病毒并证明了 Ｃ Ｒ Ｈ 的高效表达。表达产物经 Ｇ Ｃ Ｓ Ｆ 亲和层析进一步纯化，纯度可达９０ ％ 以上。受体竞争性结合实验结果表明，该表达产物能特异性结合 Ｇ Ｃ Ｓ Ｆ，具有较高的亲和力（ Ｋｄ ＝ ３ ．８ｎ ｍｏｌ／ Ｌ） 。

The CRH cDNA of G CSF receptor gene was cloned into the plasmid pFASTBAC. After transformed the E.coli DH10BAC, recombinant baculovirus genome which contained CRH cDNA was obtained. By transfecting the insect cell SF 9 with the recombinant baculavirus genome, the recombinant baculavirus was obtained. SDS PAGE and Western blot analysis of the infected cell lysit showed that CRH was expressed in the SF 9 cell. The result of the receptor binding experiment also proved that the expression product purfied by the G CSF affinity column can specifically bind the 125 IG CSF with the Kd of 3.8nmol/L.