2型大麻受体的克隆及真核表达体系的构建

Clone and eukaryotic expression of human cannabinoid receptor 2 gene

目的克隆人2型大麻受体(hCB2)基因,并构建相应的稳定表达细胞株。方法利用人T淋巴细胞株HPB-ALL的总RNA,以RT-PCR及酶切、连接等分子生物学方法克隆hCB2基因于质粒载体pFlag-CMV-3内,以HEK293细胞构建稳定表达细胞株,以Western blot方法利用蛋白标记Flag的单克隆抗体(Anti-Flag)和CB2多克隆抗体(Anti-CB2)同时检测质粒的表达。结果酶切及测序结果表明hCB2基因克隆成功,Western blot检测结果表明Anti-Flag和Anti-CB2都能检测Flag-hCB2蛋白。结论成功克隆hCB2基因,并获得稳定表达细胞株,同时证实Anti-CB2多克隆抗体有效。

Objective To clone human cannabinoid receptor 2 gene and establish stable cell line which constantly expresses hCB2 protein. Methods Full length of hCB2 cDNA was obtained by RT-PCR with total RNA isolated from human T cell line HPB-ALL, and then constructed into plasmid vector pFlag-CMV-3. The stable cell line was established with HEK293 cells. The expression of Flag-CB2 was analyzed by Western blot- ting with monoclonal Anti-Flag and multiclonal Anti-CB2. Results pFLag-hCB2 was confirmed containing proper hCB2 gene with diagnostic digestion and sequencing, and the expression products were detected by Anti- Flag and Anti-CB2 parallelly. Conclusion Gene of hCB2 has been cloned and constantly expressed in HEK293 cells. The multiclonal Anti-CB2 is proved efficient in detecting CB2 protein epitope.