摘要
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 F9 ,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.
出处
《生物工程学报》
CAS
CSCD
北大核心
1999年第4期466-469,共4页
Chinese Journal of Biotechnology