摘要
目的 :为了获得小鼠催产素受体 (mouseoxytocinreceptor,mOTR)基因的全长编码区和构建mOTR的真核表达载体 ,以及在哺乳动物细胞中真核表达mOTR。方法 :采用RT PCR方法 ,获得有相互重叠序列的mOTR的羧基端和氨基端的cDNA片段并分别将其亚克隆入PMD 18 T载体。通过对这 2个片段加入酶切位点、双酶切和相互连接 ,获得全长的编码序列。然后将mOTR的全长的编码序列克隆入真核表达载体pEGFP N3,再用脂质体转染法将pEGFP N3 mOTR质粒转染到COS 7细胞中并进行了瞬时真核表达。结果 :将mOTR的羧基端和氨基端的cDNA片段亚克隆入了PMD 18 T载体。连接并最终获得了mOTR编码区的全长序列 ,构建了pEGFP N3 mOTR真核表达载体。将pEGFP N3 mOTR载体转染入COS 7细胞 ,并成功地进行了瞬时真核表达。结论 :成功构建了包含mOTR全长编码区序列的pEGFP N3 mOTR真核表达载体 ,并在COS 7细胞中进行了瞬时表达 ,为今后研究mOTR的功能打下了基础。
AIM: To clone the complete coding sequence of mouse oxytocin receptor (mOTR) gene and to express it in mammalian cells. METHODS: Overlapping carboxyl end and amino end cDNAs of mOTR gene were acquired respectively by RT-PCR method and subcloned into PMD 18-T vectors. Through adding the digestion sites, double digestions and ligation, the 2 fragments were linked to form the complete coding sequence which was inserted into the eukaryotic expression vector pEGFP-N3, and the recombinant pEGFP-mOTR plasmid was transfected into COS-7 cells for eukaryotic expression by liposome-mediated gene transfer method. RESULTS: The carboxyl end and amino end cDNAs of mOTR gene were successfully subcloned into PMD 18-T vectors. The complete coding sequence was obtained and cloned into the pEGFP-N3 vector. The recombinant pEGFP-mOTR plasmid was transiently expressed in COS-7 cells successfully. CONCLUSION: The recombinant eukaryotic expression vector pEGFP-N3-mOTR was constructed successfully and transiently expressed in COS-7 cells, which lays a foundation for further functional studies of mOTR.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第4期491-494,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展规划 (973)资助项目 (No .2 0 0 3CB51 50 31 )
