摘要
为了获得有免疫原性的人脂皮素 -1 ( lipocortin-1 ,LC-1 )蛋白 ,以便疫动物获得抗体 ,建立了免疫分析方法 ,而且更深入地探讨 LC-1抗炎机制及其在临床上的应用。采用基因工程的方法进行重组人 LC-1的原核表达并鉴定 ,主要结果如下 :( 1 )从 U93 7细胞中提取总 RNA,以此为模板行反转录 PCR,成功地扩增了 1 0 38bp的人 LC-1 c DNA基因 ,并将此基因全长克隆入 p UC1 8载体 ,经测序分析证实克隆序列与文献报道的序列基本一致 ;( 2 )成功地构建了原核表达质粒 p BV2 2 0 / LC-1 ,并在大肠杆菌中实现表达。经 SDS-PAGE电泳和 West-ern免疫印迹分析 ,证实重组菌产生 37k D的表达产物 ,且此蛋白可与 LC-1抗体特异结合。实现了人 LC-1基因的克隆及原核表达 ,获得了有免疫学活性的 LC-1 ,为
In order to study the mechanism of action of lipocortin-1(LC-1)and its foreground of clinical application, this LC-1 protein was highly effectively expressed in Escherichia coli by gene engineering methods, for getting enough immunogen to raise antibody by immunizing rabbits, then to establish highly sensitive immunoassay methods of LC-1. Main results are as follows: (1) The total RNA was isolated from the human U 937 cells and cDNA of human lipocortin-1 was cloned by the reverse transcription-polymerase chain reactions (RT-PCR)method. The fragment was sequenced after it had been inserted into clone vector pUC18. The DNA sequencing analysis showed that the cloned gene was in agreement with the reported sequence; (2) The fragment was subcloned into expression vector pBV220 and the recombinant plasmid was transformed into Escherichia coli strains. The construction of pBV220-LC was verified by restriction analysis. When it was induced at 42℃, the engineering bacteria expressed a 37 kD protein which was demonstrated by SDS-PAGE. Western blotting assay showed that the protein could react with anti-LC-1 antibodies specifically. The successful expression of lipocortin-1 in Escherichia coli will provide great amounts of lipocortin-1 for its clinical and basic research.
出处
《标记免疫分析与临床》
CAS
2001年第2期84-89,共6页
Labeled Immunoassays and Clinical Medicine
