摘要
目的建立稳定高效表达人重组大麻受体2(CB2)的中国仓鼠卵巢(CHO-K1)细胞株,为体外高通量筛选CB2激动剂和拮抗剂奠定基础。方法通过脂质体介导的方法将构建的表达载体pcDNA3.1(+)-CB2转染入CHO-K1细胞中,然后用含G418的选择性培养液进行筛选,挑取耐药克隆;培养并收集耐药克隆细胞,用RT-PCR方法做进一步筛选;序列测定鉴定整合基因的序列;筛选的阳性克隆用放射性配体-受体结合实验进行进一步的鉴定和受体活性分析。结果转染细胞在含G418的选择性培养基中生长出28个耐药单克隆,用RT-PCR方法检测出17个CB2mRNA表达量较高的阳性克隆;RT-PCR扩增片段测定鉴定正确;挑选其中最优的克隆进行放射性配体-受体结合实验,结果显示,表达受体具有与CB2激动剂WIN55212-2特异结合的活性,其Kd和Bmax值分别(1.21±0.47)nmol.L-1和(3.12±0.7)nmol.g-1蛋白,这一结果与天然CB2的特性相似。结论建立了稳定高效表达人重组CB2的CHO-K1细胞株。
AIM To establish Chinese hamster ovary（CHO） cell strains stably expressing human cannabinoid receptor 2（CB2）.METHODS Plasmid vector pcDNA3.1（＋）-CB2 that contains a cDNA fragment coding for human CB2 was transfected into CHO cells by a Lipofectamine based method,and transfected CHO cells were selected in culture medium containing G418.Positive cell clones were measured by RT-PCR.The DNA fragment was sequenced.The activity of CB2 was analyzed by radioligand receptor binding assays.RESULTS Through screening by G418,twenty-eight CHO cell monoclones were obtained.Seventeen positive CHO cell clones expressed CB2 mRNA analyzed by RT-PCR,and the sequence of the PCR fragment of CB2 was right.The saturation analysis indicated that the CB2 receptor of the recombinants CHO cell strain should be similar to the natural CB2.The value of Kd and Bmax was（1.21±0.47）nmol·L^-1 and（3.12±0.7）nmol·g-1 protein,respectively.CONCLUSION CHO cell stains stably expressing human CB2 with activity are established.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2009年第2期140-145,共6页
Chinese Journal of Pharmacology and Toxicology
基金
国家自然基金资助项目(30600761)~~
关键词
大麻受体2
基因转染
CHO细胞
cannabinoid receptor ，gene transfection
CHO cells
