SPF鸡肝脏细胞cDNA文库的构建及鉴定
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摘要
旨在构建SPF鸡肝脏组织细胞的cDNA文库并且对文库质量进行鉴定。用TRIzol方法从SPF鸡肝脏组织细胞中提取总RNA,用紫外分光光度仪测定其含量后,应用SMART技术经过LD-PCR合成双链cDNA。利用CHROMA SPIN-400纯化柱纯化得到双链cDNA,并与线性pGADT7-Rec共转化进酵母Y187感受态,以同源重组的方式在酵母细胞内构建鸡肝脏酵母双杂交cDNA表达文库。结果显示,成功构建含有1.70×10~7个重组子的SPF鸡肝脏细胞cDNA文库,插入片段多数在0.4~2.0 kb之间,重组率达100%,重组子中平均插入的片段长度为1.0 kb,文库滴度为1.30×10~7 cfu/mL。结果表明,该文库达到了高质量文库所应具备的条件,为进一步筛选此文库与ARV相互作用的宿主蛋白以及研究其功能提供了数据依据。
In oder to construct the SPF chicken liver cells cDNA library and appraise the quality of library, total RNA from SPF chicken liver cells was extract by TRIzol, the concentration of RNA was determine by ultraviolet spectrophotometric. The double-stranded c DNAs were amplified by using SMART technique and Long-distance-PCR.The double-stranded cDNA was purified by CHROMA SPINTMTE-400 Column. And the linearized pGADT7-Rec vector were co-transformed into yeast Y187 competent cells. The SPF chicken liver cells cDNA library was constructed by using homologous recombination method. The cDNA library contained 1.7×10~7 independent clones with DNA inserts of 0.4~2.0 kb, the recombination rate was 100% and the average length of inserts was approximately 1.0 kb. The amplified library had a titer of 1.30×10~7 colony formation cfu/m L. The results showed that, the library met the necessary conditions of high quality library, which provided a data basis for further screening the host protein interacting with ARV and studying its function.
In oder to construct the SPF chicken liver cells cDNA library and appraise the quality of library, total RNA from SPF chicken liver cells was extract by TRIzol, the concentration of RNA was determine by ultraviolet spectrophotometric. The double-stranded c DNAs were amplified by using SMART technique and Long-distance-PCR.The double-stranded cDNA was purified by CHROMA SPINTMTE-400 Column. And the linearized pGADT7-Rec vector were co-transformed into yeast Y187 competent cells. The SPF chicken liver cells cDNA library was constructed by using homologous recombination method. The cDNA library contained 1.7×10~7 independent clones with DNA inserts of 0.4~2.0 kb, the recombination rate was 100% and the average length of inserts was approximately 1.0 kb. The amplified library had a titer of 1.30×10~7 colony formation cfu/m L. The results showed that, the library met the necessary conditions of high quality library, which provided a data basis for further screening the host protein interacting with ARV and studying its function.
引文
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Carninci P.,Shibata Y.,Hayatsu N.,Sugahara Y.,Shibata K.,Itoh M.,Konno H.,Okazaki Y.,Muramatsu M.,and Hayashizaki Y.,2000,Normalization and subtraction of cap-trapper-selected cDNAs to prepare full length cDNA libraries for rapid discovery of new genes,Genome Res.,10(10):1617-1630
Chien C.T.,Bartel P.L.,Sternglanz R.,and Fields S.,1991,The two-hybrid system:a method to identify and clone genes for proteins that interact with a protein of interest,Proc.Natl.,Acad.Sci.USA,88(21):9578-9582
Coates P.J.,and Hall P.A.,2003,The yeast two-hybrid system for identifying protein-protein interactions,Pathol.,199(1):4-7
Dandár E.,Bálint A.,Kecskeméti S.,Szentpáli-Gavallér K.,Kisfali P.,Melegh B.,Farkas S.L.,and Bányai K.,2013,Detection and c haracterization of a divergent avian reovirus strain from a broiler chicken with central nervous system disease,Arch Virol.158(12):2583-2588
Diatchenko L.,Lau Y.F.,Campbell A.P.,Chenchik A.,Moqadam F.,Huang B.,Lukyanov S.,Lukyanov K.,Gurskaya N.,Sverdlov ED.,and Siebert P.D.,1996,Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries,Proc.Natl.Acad.Sci.USA,93(12):6025-6030
Fields S.,and Song O.,1989,A novel genetic system to detect protein-protein interactions,Nature,340(6230):245-246
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Huang L.,Xie Z.X.,Xie L.J.,Deng X.X.,Xie Z.Q.,Fan Q.,Luo S.S.,Huang J.L.,Zeng T.T.,Zhang Y.F,and Wang S.,2016,Proteomic analysisi of vero cells infected by avian reovirus virulent strain and attenuated vaccine strain,Xumu Shouyi Xuebao(Chinese Journal of Animal and Veterinary Sciences),47(2):331-339(黄莉,谢芝勋,谢丽基,邓显文,谢志勤,范晴,罗思思,黄娇玲,曾婷婷,张艳芳,王盛,2016,禽呼肠孤病毒强毒株和弱毒一秒株感染Vero细胞的蛋白质组学研究,畜牧兽医学报,47(2):331-339)
Liao M.,Xie Z.,Liu J.,Pang Y.,Deng X.,Xie Z.,and Tang X.,2002,Isolation and identification of avian reovirus,Zhongguo Jiaqin(Chinese Poultry),24(1):12-14(廖敏,谢芝勋,刘加波,庞耀珊,邓显文,谢志勤,唐小飞,2002,鸡呼肠孤病毒分离与鉴定,中国家禽,24(1):12-14)
Lin P.,Liu H.,Lai M.,Yu F.,Hsu H.,Lee J.,and Shih W.,2006,Avian Reovirus activates a novel proapoptotic signal by linking Src to p53,Apoptosis,11(12):2179-2193
Sambrook J.,and Russell D.W.,2001,Molecular cloning:A laboratory manual,New York:Cold Spring Harbour Laboratory Press,pp.11857-11891
Van de Zande S.,and Kuhn E.M.,2007,Central nervous system signs in chickens caused by a new avian reovirus strain:Apathogenesis study,J.Vet.Micro.,120(1):42-49
Wellenreut H.R.,Schupp I.,and Stka A.P.,2004,SMART amplification combined with cDNA size factonation in order to obtainfull length clones,BMC Genomics,5(36):1-8
Yang F.,He Z.M.,Zhan X.Q.,Chen Z.C.,Yan B.,Huang H.K.,and Li T.B.,2004,Construction and identification of direc tional c DNA library from Chinese giant salamandersl Andrias davidianus liver,Acta Zool Sin.,50(3):4775-4781
Yao Z.,Tian J.,and Liao H.,2014,Comparative characterization of Gm SPX members reveals that Gm SPX3 is involved in phosphate homeostasis in soybean,Annals of Botany,114(3):477-488
Zhang Z.,2015,The molecular mechanism underlysing ARV sigma-C-induced apoptosisi in host cells,Dissertation for Ph.D.,China Agricultural University,Supervisor:Zheng S.J.,pp.1-3(张志强,2015,禽呼肠孤病毒Sigma-C蛋白引起宿主细胞凋亡的分子机理研究,博士学位论文,中国农业大学,导师:郑世军,pp.1-3)
Carninci P.,Shibata Y.,Hayatsu N.,Sugahara Y.,Shibata K.,Itoh M.,Konno H.,Okazaki Y.,Muramatsu M.,and Hayashizaki Y.,2000,Normalization and subtraction of cap-trapper-selected cDNAs to prepare full length cDNA libraries for rapid discovery of new genes,Genome Res.,10(10):1617-1630
Chien C.T.,Bartel P.L.,Sternglanz R.,and Fields S.,1991,The two-hybrid system:a method to identify and clone genes for proteins that interact with a protein of interest,Proc.Natl.,Acad.Sci.USA,88(21):9578-9582
Coates P.J.,and Hall P.A.,2003,The yeast two-hybrid system for identifying protein-protein interactions,Pathol.,199(1):4-7
Dandár E.,Bálint A.,Kecskeméti S.,Szentpáli-Gavallér K.,Kisfali P.,Melegh B.,Farkas S.L.,and Bányai K.,2013,Detection and c haracterization of a divergent avian reovirus strain from a broiler chicken with central nervous system disease,Arch Virol.158(12):2583-2588
Diatchenko L.,Lau Y.F.,Campbell A.P.,Chenchik A.,Moqadam F.,Huang B.,Lukyanov S.,Lukyanov K.,Gurskaya N.,Sverdlov ED.,and Siebert P.D.,1996,Suppression subtractive hybridization:a method for generating differentially regulated or tissue-specific cDNA probes and libraries,Proc.Natl.Acad.Sci.USA,93(12):6025-6030
Fields S.,and Song O.,1989,A novel genetic system to detect protein-protein interactions,Nature,340(6230):245-246
Gietz R.D.,Schiestl R.H.,Willems A.R.,and Woods R.A.,1995,Study on the transformation of intact yeast cells by Li Ac/SS-DNA/PEG procedure,Yeast,11(4):355-336
González-López C.,Martínez-Costas J.,Esteban M.,and Benavente J.,2003,Evidence that avian reovirus sigma A protein is an inhibitor of the double-stranded RNA-dependent protein kinase,Journal of General Virology,84(6):1629-1639
Huang L.,Xie Z.X.,Xie L.J.,Deng X.X.,Xie Z.Q.,Fan Q.,Luo S.S.,Huang J.L.,Zeng T.T.,Zhang Y.F,and Wang S.,2016,Proteomic analysisi of vero cells infected by avian reovirus virulent strain and attenuated vaccine strain,Xumu Shouyi Xuebao(Chinese Journal of Animal and Veterinary Sciences),47(2):331-339(黄莉,谢芝勋,谢丽基,邓显文,谢志勤,范晴,罗思思,黄娇玲,曾婷婷,张艳芳,王盛,2016,禽呼肠孤病毒强毒株和弱毒一秒株感染Vero细胞的蛋白质组学研究,畜牧兽医学报,47(2):331-339)
Liao M.,Xie Z.,Liu J.,Pang Y.,Deng X.,Xie Z.,and Tang X.,2002,Isolation and identification of avian reovirus,Zhongguo Jiaqin(Chinese Poultry),24(1):12-14(廖敏,谢芝勋,刘加波,庞耀珊,邓显文,谢志勤,唐小飞,2002,鸡呼肠孤病毒分离与鉴定,中国家禽,24(1):12-14)
Lin P.,Liu H.,Lai M.,Yu F.,Hsu H.,Lee J.,and Shih W.,2006,Avian Reovirus activates a novel proapoptotic signal by linking Src to p53,Apoptosis,11(12):2179-2193
Sambrook J.,and Russell D.W.,2001,Molecular cloning:A laboratory manual,New York:Cold Spring Harbour Laboratory Press,pp.11857-11891
Van de Zande S.,and Kuhn E.M.,2007,Central nervous system signs in chickens caused by a new avian reovirus strain:Apathogenesis study,J.Vet.Micro.,120(1):42-49
Wellenreut H.R.,Schupp I.,and Stka A.P.,2004,SMART amplification combined with cDNA size factonation in order to obtainfull length clones,BMC Genomics,5(36):1-8
Yang F.,He Z.M.,Zhan X.Q.,Chen Z.C.,Yan B.,Huang H.K.,and Li T.B.,2004,Construction and identification of direc tional c DNA library from Chinese giant salamandersl Andrias davidianus liver,Acta Zool Sin.,50(3):4775-4781
Yao Z.,Tian J.,and Liao H.,2014,Comparative characterization of Gm SPX members reveals that Gm SPX3 is involved in phosphate homeostasis in soybean,Annals of Botany,114(3):477-488
Zhang Z.,2015,The molecular mechanism underlysing ARV sigma-C-induced apoptosisi in host cells,Dissertation for Ph.D.,China Agricultural University,Supervisor:Zheng S.J.,pp.1-3(张志强,2015,禽呼肠孤病毒Sigma-C蛋白引起宿主细胞凋亡的分子机理研究,博士学位论文,中国农业大学,导师:郑世军,pp.1-3)