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高原鼠兔瘦素蛋白乳腺特异表达载体的构建及细胞表达

庞礴 于鸿浩 张湑泽 谢玲  付林 赵新全 郭松长   

  1. (1 中国科学院西北高原生物研究所,高原生物适应与进化重点实验室,西宁 810001)
    (2 中国科学院大学,北京 100049)(3 桂林医学院生物技术学院,桂林 541004)
    (4 青海民族大学生态环境与资源学院,西宁 810007)
    (5 温州医科大学药学院分子药理学研究中心,温州 325035)
    (6 湖南农业大学动物科学技术学院,长沙 410128)
  • 出版日期:2019-01-30 发布日期:2019-01-18
  • 通讯作者: 郭松长E-mail: scguo_as@126.com; 赵新全 E-mail: xqzhao@nwipb.cac.cn
  • 基金资助:
    中国科学院知识创新工程项目(KSCX2-EW-J-26)

Construction of mammary gland-specific expression vector for plateau pika(Ochotona curzoniae)Leptin gene and its expression in cells

PANG Bo, YU Honghao, ZHANG Xuze, XIE Ling, FU Lin, ZHAO Xinquan, GUO Songchang   

  1. (1 Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining 810001, Qinghai, China)
    (2 University of Chinese Academy of Sciences, Beijing 100049, China)
    (3 College of Biotechnology, Guilin Medical University, Guilin 541004, Guangxi, China)
    (4 College of Eco-Environment & Resources, Qinghai University For Nationalities, Xining 810007, Qinghai, China)
    (5 Molecular Pharmacology Research Center, School of Pharmaceutical Science, Wenzhou Medical University, Wenzhou 325035, Zhejiang, China)
    (6 College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, Hunan, China)
  • Online:2019-01-30 Published:2019-01-18

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

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