摘要
1型1-磷酸鞘氨醇受体(S1P1)是淋巴细胞表面的重要受体。为研究S1P1中ERY保守基序与FTY720诱导其内化的关系,以HA-S1P1-Myc-EGFP-N1为模板,应用重叠PCR的方法,将S1P1第142位的Arg(R)突变为Asn(N),连同N端HA标签一起,克隆入pcDNA3.1(+)真核表达载体。同时PCR扩增野生型S1P1,克隆入pcDNA3.1(+)中。限制性酶切消化、PCR和测序鉴定后,经Polyfect转染入HEK293细胞。G418筛选出稳定细胞株。100 nmol/L FTY720处理12 h后,抗HA-PE抗体染色,用流式细胞仪检测S1P1在HEK293细胞上的表达情况。结果显示HA-S1P1(WT)-pcDNA3.1(+)和HA-S1P1(R142N)-pcDNA3.1(+)载体被成功构建,HA-S1P1(WT)和HA-S1P1(R142N)蛋白表达在HEK293稳定细胞株的表面。FTY720能诱导HA-S1P1(WT)内化,但不能诱导HA-S1P1(R142N)内化。提示FTY720诱导S1P1内化与ERY保守基序有关。
Sphingosine 1-phosphate receptor type 1(S1P1) is the dominant receptor on lymphocytes.In order to study the relation between the conserved ERY motif of S1P1 and FTY720-induced internalization,the gene encoding R142N mutant S1P1 was amplified from HA-S1P1-Myc-EGFP-N1 by overlap-PCR and the wild type S1P1 gene was amplified by PCR.These genes including N-terminal hemagglutinin(HA)-tag were cloned into pcDNA3.1(+) vector.The recombinant vectors were confirmed by restriction enzyme digestion,PCR and sequencing,...
出处
《现代免疫学》
CAS
CSCD
北大核心
2008年第3期214-218,共5页
Current Immunology
