Abstract: Leptin is one of the key factors regulating body energy homeostasis. Previous research showed that the plateau pika（Ochotona curzoniae）leptin gene has undergone adaptive evolution. Functional experiments revealed that pika leptin regulates energy homeostasis via reduced food intake and increased energy expenditure under both warm and cold conditions. Together, these findings suggest that leptin of the plateau pika has a potentially potent role in regulating adaptive thermogenesis. In this study, the 504bp open-reading frame of obese（ob）gene was amplified from the full-length pika leptin cDNA and then cloned into a mammary gland-specific expression vect- or pBC1（pBC1-lep）. Meanwhile, we successfully established dairy goat mammary epithelial cell lines by primary tissue mass cell culture and transfected the modified pBC1-lep plasmid into the cultured cells with lipofectamine, and then screened for and successfully established positive cell lines transfected with leptin. These results provide a probable way for expression of the plateau pika leptin in goat mammary gland specifically by transgenic animal and complete the construction of the eukaryotic mammary gland-specific expression vector.

Key words: Leptin, Mammary epithelial cell, Mammary gland-specific expression vector, pBC1, Plateau pika

摘要： 瘦素（Leptin）蛋白是调节机体能量代谢的关键因子之一。前期研究显示高原鼠兔Leptin蛋白发生了适应性进化。功能实验表明，在温暖或寒冷条件下高原鼠兔Leptin都通过减少食物摄取和增加能量消耗调节能量平衡，显示了其调节适应性产热过程的潜力。本研究以高原鼠兔Leptin cDNA为模板扩增高原鼠兔obese（ob）基因编码区序列504bp，改造并构建了哺乳动物真核细胞乳腺特异表达载体pBC1-lep，同时通过组织块法原代培养建立了奶山羊乳腺上皮细胞系，并通过pBC1-lep质粒脂质体法转染及转基因细胞的筛选，成功获得转染Leptin的阳性细胞。实验为利用转基因动物实现奶山羊乳腺中特异表达高原鼠兔Leptin提供了一条可能的途径，完成了乳腺特异真核表达载体的构建。

关键词: 高原鼠兔, 瘦素蛋白, 乳腺上皮细胞, 乳腺特异表达载体, pBC1