人趋化因子受体CCR6的克隆、表达及其功能分析

目的构建人趋化因子受体6（CCR6）cDNA序列的真核表达载体．并在HEK293细胞中表达．为研究CCR6生物学功能和筛选CCR6拮抗剂奠定基础。方法采用RT-PCR方法从人扁桃体克隆CCR6受体的DNA片段．并将该片段插入真核表达质粒pcDNA3．1（＋）中，构建重组真核表达质粒pcDNA3、1（＋）-CCR6；将该质粒pcDNA3、1（＋）-CCR6转染HEK293细胞．用流式细胞术检测转染pcDNA3．1（＋）-CCR6质粒的HEK293细胞；采用体外趋化实验、钙流实验，验证转染pcDNA3.1（-）．CCR6质粒的HEK293细胞表面表达的CCR6的生物学活性。结果经RT-PCR获得了编码人CCR6受体的DNA片段．构建了重组真核表达质粒pcDNA3．1（＋）-CCR6：转染HEK293细胞．经流式细胞仪检测、趋化实验、钙流实验证实．表达CCR6的HEK293细胞具有趋化因子受体CCR6的生物学活性。结论重组人趋化因子受体CCR6克隆成功．并在HEK293细胞中获得了表达．为研究CCR6的生物学功能及筛选CCR6拮抗剂奠定了基础。

Cloning and expression and function analysis of human chemokine receptor CCR6

Objective To construct an eukaryotic expression vector of human chemokine receptor CCR6 and express on HEK293 cells for further studing its function and making its antagonist.Methods Human CCR6 receptor gene was amplified from the human tonsil by RT-PCR,cloned and constructed into eukaryotic expression vector,pcDNA3.1( ).Then plasmid pcDNA3.1( )-CCR6 was transfected into HEK293 cells.The transfected cells were detected with flow cytometry.The biological activities of CCR6 expressed on HEK293 cells were observed with in vitro chemotaxis test and calcium fluxing assay.Results Human CCR6 gene fragment was obtained with RT-PCR and was inserted into the eukaryotic expression vector,pcDNA3.1( ).The recombination eukaryotic expression vector,pcDNA3.1( )-CCR6 was transfected into HEK293 cells.The HEK293 cells expressing CCR6 is one of biological activities of CCR6 recptor.Conclusion The recombinant human chemokine receptor CCR6 was cloned and expressed on HEK293 cells successfully,laying a foundation for further studing the function of CCR6 and screening its antagonists.