摘要
为了构建表达人胰高血糖素样肽-1受体(GLP-1R)基因的BHK细胞株,并利用该重组细胞对GLP-1等相关肽进行活性测定,首先通过酶切、连接方式将人GLP-1R基因克隆至真核表达载体pCDNA3.(1+)中,然后用脂质体转染法将重组质粒转染至BHK-21细胞,转染后的细胞经G418加压筛选、细胞有限稀释等方法获得克隆细胞株。经过该细胞株RT-PCR验证,结果证实目的基因已整合至BHK-21细胞基因组中,并获得成功转录和表达。活性检测实验表明该重组细胞株经过GLP-1的刺激后,其细胞中的cAMP含量得到明显提升。该细胞株的构建为GLP-1及相关肽的活性测定奠定了基础。
In order to establish a recombinant BHK-GLP1R cell line which has a good application to exam biological activity of GLP-1 and its analogues.The human glucagons-like peptide 1 receptor （GLP-1R）gene was cloned into eukaryotic expression vector pCDNA3.1 （＋）.The recombinant expression vector was transfected into BHK-21 cells by mean s of lipofectin procedure.After selected by G418,the positive cell clones were analysed by RT-PCR to confirm the steady expression of GLP-1R. gene in BHK cells.The analysis of RT-PCR show that GLP-1R gene had been integrated into BHK cell genome and could express the GLP-1R protein successfully in BHK cells.The recombinant cells had been applied to exam the quantity of intracellular cAMP after stimulated by GLP-1 or its analogues.
出处
《生物技术通报》
CAS
CSCD
2007年第3期118-121,共4页
Biotechnology Bulletin
