高原鼠兔瘦素蛋白乳腺特异表达载体的构建及细胞表达
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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504 bp,改造并构建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。本研究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。
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 504 bp open-reading frame of obese( ob) gene was amplified from the full-length pika leptin c DNA 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.
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 504 bp open-reading frame of obese( ob) gene was amplified from the full-length pika leptin c DNA 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.
引文
Chung J H,Bell A C,Felsenfeld G.1997.Characterization of the chickenβ-globin insulator.Proc Natl Acad Sci USA,94:575-580.
Chung J H,Whiteley M,Felsenfeld G.1993.A 5’element of the chickenβ-globin domain serves as an insulator in human erythroid cells and protects against posit-ioneffect in drosophila.Cell,74:505-514.
Denman J,Hayes M,O’Day C,Edmunds T,Bartlett C,Hirani S,Ebert K M,Gordon K,Mc Pherson J M.1991.Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk:purification and characterization of the recombinant enzyme.BioTechnology,9:839-843.
Du J Z,Li Q F.1982.Effects of simulated hypoxic acclimation on organism,organ and hematology in Ochotona curzoniae and rats.Acta Theriologica Sinica,2(1):35-42.(in Chinese)
Edmunds D,Patten S M V,Pollock J,Hanson E,Bernasconi R,Higgins E,Manavalan P,Ziomek C,Meade H,Mc Pherson J M,Cole E S.1998.Transgenically produced human antithrombin:structural and functional comparison to human plasmaderived antithrombin.Blood,91:4561-4571.
Li Q F,Sun R Y,Huang C X,Wang Z K,Li N.2001.Cold adaptive thermogenesis in small mammals from different geographical zones of China.Comp Biochem Phys A,129(4):949-961.
Liang Z X,Yin F Q,Liu Q Y,Li L.2015.Progress and technology in mammary gland bioreactor of transgenic animals.China Biotechnology,35(2):92-98.(in Chinese)
Persuy M A,Stinnakre M G,Printz C,Mahe M F,Mercier J C.1992.High expression of the caprineβ-casein gene in transgenic mice.Eur J Biochem,205:887-893.
Roberts B,Di Tullio P,Vitale J,Hehir K,Gordon K.1992.Cloning of the goatβ-case in-encodinggene and expression in transgenic mice.Gene,121:255-262.
Schmittgen T D,Livak K J.2008.Analyzing real-time PCR data by the comparative CT method.Nature Protocols,3(6):1101-1108.
Wang Q H,Liu J F,Ding Y P,Wang J L,Shao B P.2017.Cloning of the CDS of yak Endothelin-1 gene and construction of its eukaryotic expression plasmid.Acta Theriologica Sinica,37(3):293-299.(in Chinese)
Yang J,Zhao X Q,Guo S C,Li H G,Qi D L,Wang D P,Cao J H.2006.Leptin c DNA cloning and its mRNA expression in plateau pikas(Ochotona curzoniae)from different altitudes on Qinghai-Tibet Plateau.Biochemical and Biophysical Research Communications,345(4):1405-1413.
Yang J,Wang Z L,Zhao X Q,Wang D P,Qi D L,Xu B H,Ren Y H,Tian H F.2008.Natural selection and adaptive evolution of leptin in the Ochotona family driven by the cold environmental stress.PLoS ONE,3(1):e1472.
Yang J,Bromage T G,Zhao Q,Xu B H,Gao W L,Tian H F,Tang HJ,Liu D W,Zhao X Q.2011.Functional evolution of leptin of Ochotona curzoniae in adaptive thermogenesis driven by cold environmental stress.PLoS ONE,6(6):e19833.
Young M W,Okita W B,Brown E M,Curling J M.1997.Production of biopharmaceutical proteins in the milk of transgenic dairy animals.BioPharm,10:34-38.
Zhang Y Y,Barone M,Friedman J M,Leopold L,Maffei M,Proenca R.1994.Positional cloning of the mouse obese gene and its human homologue.Nature,374(6521):425-432.
Zhu X F,Qu J F,Wu X J,Fan S T,Ma Q M,Gao R.2007.Progress in mammary gland bioreacror of transgenic animals.Journal of Animal Science and Veterinary Medicine,26(3):50-52.(in Chinese)
朱小甫,渠敬峰,吴旭锦,樊松涛,马秋明,高睿.2007.转基因动物乳腺生物反应器研究进展.畜牧兽医杂志,26(3):50-52.
杜继增,李庆芬.1982.模拟高原低氧对高原鼠兔和大鼠器官与血液若干指标的影响.兽类学报,2(1):35-42.
汪迁昊,刘健锋,丁艳平,王建林,邵宝平,2017.牦牛Endothelin-1基因CDS序列克隆及其真核表达质粒构建.兽类学报,37(3):293-299.
梁振鑫,尹富强,刘庆友,李力.2015.转基因动物乳腺生物反应器相关技术及研究进展.中国生物工程杂志,35(2):92-98.
Chung J H,Whiteley M,Felsenfeld G.1993.A 5’element of the chickenβ-globin domain serves as an insulator in human erythroid cells and protects against posit-ioneffect in drosophila.Cell,74:505-514.
Denman J,Hayes M,O’Day C,Edmunds T,Bartlett C,Hirani S,Ebert K M,Gordon K,Mc Pherson J M.1991.Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk:purification and characterization of the recombinant enzyme.BioTechnology,9:839-843.
Du J Z,Li Q F.1982.Effects of simulated hypoxic acclimation on organism,organ and hematology in Ochotona curzoniae and rats.Acta Theriologica Sinica,2(1):35-42.(in Chinese)
Edmunds D,Patten S M V,Pollock J,Hanson E,Bernasconi R,Higgins E,Manavalan P,Ziomek C,Meade H,Mc Pherson J M,Cole E S.1998.Transgenically produced human antithrombin:structural and functional comparison to human plasmaderived antithrombin.Blood,91:4561-4571.
Li Q F,Sun R Y,Huang C X,Wang Z K,Li N.2001.Cold adaptive thermogenesis in small mammals from different geographical zones of China.Comp Biochem Phys A,129(4):949-961.
Liang Z X,Yin F Q,Liu Q Y,Li L.2015.Progress and technology in mammary gland bioreactor of transgenic animals.China Biotechnology,35(2):92-98.(in Chinese)
Persuy M A,Stinnakre M G,Printz C,Mahe M F,Mercier J C.1992.High expression of the caprineβ-casein gene in transgenic mice.Eur J Biochem,205:887-893.
Roberts B,Di Tullio P,Vitale J,Hehir K,Gordon K.1992.Cloning of the goatβ-case in-encodinggene and expression in transgenic mice.Gene,121:255-262.
Schmittgen T D,Livak K J.2008.Analyzing real-time PCR data by the comparative CT method.Nature Protocols,3(6):1101-1108.
Wang Q H,Liu J F,Ding Y P,Wang J L,Shao B P.2017.Cloning of the CDS of yak Endothelin-1 gene and construction of its eukaryotic expression plasmid.Acta Theriologica Sinica,37(3):293-299.(in Chinese)
Yang J,Zhao X Q,Guo S C,Li H G,Qi D L,Wang D P,Cao J H.2006.Leptin c DNA cloning and its mRNA expression in plateau pikas(Ochotona curzoniae)from different altitudes on Qinghai-Tibet Plateau.Biochemical and Biophysical Research Communications,345(4):1405-1413.
Yang J,Wang Z L,Zhao X Q,Wang D P,Qi D L,Xu B H,Ren Y H,Tian H F.2008.Natural selection and adaptive evolution of leptin in the Ochotona family driven by the cold environmental stress.PLoS ONE,3(1):e1472.
Yang J,Bromage T G,Zhao Q,Xu B H,Gao W L,Tian H F,Tang HJ,Liu D W,Zhao X Q.2011.Functional evolution of leptin of Ochotona curzoniae in adaptive thermogenesis driven by cold environmental stress.PLoS ONE,6(6):e19833.
Young M W,Okita W B,Brown E M,Curling J M.1997.Production of biopharmaceutical proteins in the milk of transgenic dairy animals.BioPharm,10:34-38.
Zhang Y Y,Barone M,Friedman J M,Leopold L,Maffei M,Proenca R.1994.Positional cloning of the mouse obese gene and its human homologue.Nature,374(6521):425-432.
Zhu X F,Qu J F,Wu X J,Fan S T,Ma Q M,Gao R.2007.Progress in mammary gland bioreacror of transgenic animals.Journal of Animal Science and Veterinary Medicine,26(3):50-52.(in Chinese)
朱小甫,渠敬峰,吴旭锦,樊松涛,马秋明,高睿.2007.转基因动物乳腺生物反应器研究进展.畜牧兽医杂志,26(3):50-52.
杜继增,李庆芬.1982.模拟高原低氧对高原鼠兔和大鼠器官与血液若干指标的影响.兽类学报,2(1):35-42.
汪迁昊,刘健锋,丁艳平,王建林,邵宝平,2017.牦牛Endothelin-1基因CDS序列克隆及其真核表达质粒构建.兽类学报,37(3):293-299.
梁振鑫,尹富强,刘庆友,李力.2015.转基因动物乳腺生物反应器相关技术及研究进展.中国生物工程杂志,35(2):92-98.