敖汉细毛羊Notch1基因真核表达载体的构建及在成纤维细胞中表达量的研究
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摘要
本研究旨在构建敖汉细毛羊Notch1基因表达载体及转染成纤维细胞后基因表达量变化。试验以敖汉细毛羊40日龄胎儿为研究对象,提取RNA反转录并参照Gen Bank中Notch1基因序列信息设计1对引物,通过PCR反应扩增获得Notch1基因片段,将得到的Notch1基因片段连接至pGM-T载体,构建pGM-T-Notch1克隆载体并转化大肠杆菌(E.coli) DH5α感受态细胞,提取质粒进行酶切鉴定后构建pcDNA3.1-Notch1重组表达载体,转化大肠杆菌(E.coli) DH5α感受态细胞。将敖汉细毛羊的成纤维细胞进行分离培养,并将构建的pcDNA3.1-Notch1瞬时转染成纤维细胞,利用荧光定量PCR技术检测Notch1基因在成纤维细胞中表达量的变化。结果显示:经酶切、测序鉴定质粒pcDNA3.1-Notch1构建成功,并且转染成纤维细胞后Notch1基因的表达量升高,且转染组的表达量极显著地高于对照组(P<0.01)。结论:成功构建了敖汉细毛羊Notch1基因的表达载体,并且成功转染成纤维细胞,基因表达量明显升高,结果可为进一步研究其功能奠定基础。
This study was aimed to identify the expression of the Notch1 plasmid and the expression of the fibroblast gene.Firstly,we extracted RNA reverse transcription and referred to the Notch1 gene sequence in Gen Bank to design a pair of primers,and amplified the Notch1 fragment using PCR.We then ligated the obtained Notch1 gene into the pGM-T vector to construct pGM-T-Notch1 recombinant plasmids and transformed them into the E.coli DH5α competent cells.We identified the plasmids by restriction enzyme digestion.We constructed the recombinant plasmid pcDNA3.1-Notch1 and transformed it into the E.coli DH5α competent cell.Next,we transferred the pcDNA3.1-Notch1 plasmids into fibroblasts,and used qRT-PCR to detect the expression level of Notch1.The results showed that we successfully constructed the pcDNA3.1-Notch1 plasmid and identified them by enzyme and sequencing.The expression level of the Notch1 gene in the fibroblasts was much higher than in the control group(P<0.01).In conclusion:we constructed the plasmids successfully and transfected them into the fibroblasts.The gene expression was significantly increased.These results would lay a foundation for the further research on the function of the gene.
This study was aimed to identify the expression of the Notch1 plasmid and the expression of the fibroblast gene.Firstly,we extracted RNA reverse transcription and referred to the Notch1 gene sequence in Gen Bank to design a pair of primers,and amplified the Notch1 fragment using PCR.We then ligated the obtained Notch1 gene into the pGM-T vector to construct pGM-T-Notch1 recombinant plasmids and transformed them into the E.coli DH5α competent cells.We identified the plasmids by restriction enzyme digestion.We constructed the recombinant plasmid pcDNA3.1-Notch1 and transformed it into the E.coli DH5α competent cell.Next,we transferred the pcDNA3.1-Notch1 plasmids into fibroblasts,and used qRT-PCR to detect the expression level of Notch1.The results showed that we successfully constructed the pcDNA3.1-Notch1 plasmid and identified them by enzyme and sequencing.The expression level of the Notch1 gene in the fibroblasts was much higher than in the control group(P<0.01).In conclusion:we constructed the plasmids successfully and transfected them into the fibroblasts.The gene expression was significantly increased.These results would lay a foundation for the further research on the function of the gene.
引文
[1]刘海山,李恒荣,于洪杰,等.敖汉细毛羊品种形成与发展前景[J].当代畜禽养殖业,2011(08):33-35.
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[3]苏蕊,张文广,常子丽,等.内蒙古绒山羊毛囊不同发育时期BMP2、Noggin及SHH基因在皮肤中的表达[J].扬州大学学报(农业与生命科学版),2009,30(01):54-57.
[4]Kolly C,Suter M M,Muller E J.Proliferation cell cycle exit and onset of terminal differentiation in cultured keratinoey tes:Pre-Programmed Pathways in Control of C-Myc and Notchl Prevail over extracellular caleium signals[J].Invest Dermatol,2005,124(5):1014-1025.
[5]武晓英,任有蛇,张春香,等.毛囊周期发育相关信号通路的研究进展[J].中国兽医杂志,2010,46(06):69-71.
[6]柳楠,王春亮,贺建宁,等.敖汉细毛羊不同部位皮肤毛囊发育及形态结构研究[J].中国畜牧杂志,2015,51(17):1-5.
[7]Mohr OL.Character changes caused by mutation of an entire region of a chromosome in Drosophila[J].Genetics,1919,4(3):275-282.
[8]付亚娟,叶枫,吕卫国,等.Notch信号通路的研究现状[J].医学分子生物学杂志,2007(05):447-450.
[9]吴萍.Notch信号通路与绒山羊绒毛生长的研究[D].呼和浩特:内蒙古农业大学,2010.
[10]张梦瑶,柳楠,贺建宁,等.敖汉细毛羊DKK1基因重组质粒的构建及其在成纤维细胞中表达量的研究[J].中国畜牧兽医,2018,45(01):131-139.
[11]Chen CW,Jung HS,Jiang TX et al.Asymmetric expression of Notch/Delta/Serrate is associated with the anterior-posterior axis of feather buds.[J]Dev Biol,1997,188(1):181-187.
[12]Espinoza I,Pochampally R,Xing F,et al.Notch signaling:targeting cancer stem cells and epithelial-to-mesenchymal transition[J].Onco Targets Ther,2013,6:1249-1259.
[13]Mullendore ME,Koorstra JB,Li YM,et al.Ligand-dependent Notch signaling is involved in tumor initiation and tumormaintenance in pancreatic cancer[J].Clin Cancer Res,2009,15(7):2291-2301.
[14]Wang Z,Kong D,Banerjee S,et al.Down-regulation of plateletderived growth factor-D inhibits cell growth and angiogenesis through inactivation of Notch-1 and nuclear factor-kappaB signaling[J].Cancer Res,2007,67(23):11377-11385.
[15]罗深秋,姜勇.细胞信号转导的分子基础与功能调控[M].北京:科学出版社,2005:179-180.
[16]鲁茁壮,王立生,吴祖泽.Notch信号通路研究进展[J].生理科学进展,2004(02):135-138.
[17]Nyhan KC,Faherty N,Murray G,et al.Jagged/Notch signaling for a subset of TGF-β1 responses in human kidney epithelial cells[J].Biochim Biophys Acta,2010,1803:1386-1395.
[2]荣威恒,田春英,王峰.内蒙古细毛羊产业状况和发展前景探析[J].畜牧与饲料科学,2010,31(Z1):86-89.
[3]苏蕊,张文广,常子丽,等.内蒙古绒山羊毛囊不同发育时期BMP2、Noggin及SHH基因在皮肤中的表达[J].扬州大学学报(农业与生命科学版),2009,30(01):54-57.
[4]Kolly C,Suter M M,Muller E J.Proliferation cell cycle exit and onset of terminal differentiation in cultured keratinoey tes:Pre-Programmed Pathways in Control of C-Myc and Notchl Prevail over extracellular caleium signals[J].Invest Dermatol,2005,124(5):1014-1025.
[5]武晓英,任有蛇,张春香,等.毛囊周期发育相关信号通路的研究进展[J].中国兽医杂志,2010,46(06):69-71.
[6]柳楠,王春亮,贺建宁,等.敖汉细毛羊不同部位皮肤毛囊发育及形态结构研究[J].中国畜牧杂志,2015,51(17):1-5.
[7]Mohr OL.Character changes caused by mutation of an entire region of a chromosome in Drosophila[J].Genetics,1919,4(3):275-282.
[8]付亚娟,叶枫,吕卫国,等.Notch信号通路的研究现状[J].医学分子生物学杂志,2007(05):447-450.
[9]吴萍.Notch信号通路与绒山羊绒毛生长的研究[D].呼和浩特:内蒙古农业大学,2010.
[10]张梦瑶,柳楠,贺建宁,等.敖汉细毛羊DKK1基因重组质粒的构建及其在成纤维细胞中表达量的研究[J].中国畜牧兽医,2018,45(01):131-139.
[11]Chen CW,Jung HS,Jiang TX et al.Asymmetric expression of Notch/Delta/Serrate is associated with the anterior-posterior axis of feather buds.[J]Dev Biol,1997,188(1):181-187.
[12]Espinoza I,Pochampally R,Xing F,et al.Notch signaling:targeting cancer stem cells and epithelial-to-mesenchymal transition[J].Onco Targets Ther,2013,6:1249-1259.
[13]Mullendore ME,Koorstra JB,Li YM,et al.Ligand-dependent Notch signaling is involved in tumor initiation and tumormaintenance in pancreatic cancer[J].Clin Cancer Res,2009,15(7):2291-2301.
[14]Wang Z,Kong D,Banerjee S,et al.Down-regulation of plateletderived growth factor-D inhibits cell growth and angiogenesis through inactivation of Notch-1 and nuclear factor-kappaB signaling[J].Cancer Res,2007,67(23):11377-11385.
[15]罗深秋,姜勇.细胞信号转导的分子基础与功能调控[M].北京:科学出版社,2005:179-180.
[16]鲁茁壮,王立生,吴祖泽.Notch信号通路研究进展[J].生理科学进展,2004(02):135-138.
[17]Nyhan KC,Faherty N,Murray G,et al.Jagged/Notch signaling for a subset of TGF-β1 responses in human kidney epithelial cells[J].Biochim Biophys Acta,2010,1803:1386-1395.