水牛睾丸曲精细管蛋白质组双向电泳方法的建立及优化
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摘要
建立并优化了沼泽型水牛睾丸曲精细管蛋白质分离的双向电泳体系,为后续水牛睾丸曲精细管蛋白质表达谱的鉴定和研究奠定基础。比较不同IPG胶条(线性与非线性)、胶条pH范围和蛋白质上样量这三个参数对双向电泳结果的影响。结果显示,采用上样量350μg的曲精细管总蛋白、24 cm且pH值4～7的线性IPG胶条能够更好地分离水牛睾丸曲精细管蛋白质,获得质量较好且分辨率较高的双向电泳图谱,得到大约486个蛋白点。双向凝胶电泳技术能够对水牛睾丸曲精细管蛋白质进行有效分离,并通过对双向电泳体系的优化可获得蛋白质点清晰且分辨率更高的双向电泳图谱。
A two-dimensional electrophoresis system for separating proteins of swamp buffalo testicular seminiferous tubules was established and optimized, which laid a good foundation for further identification and research of protein expression profiling in the testicular seminiferous tubules of buffalo. The effects of different IPG strips(linear or nonlinear), different pH range of strips and protein sample loading quantity on the results of two-dimensional electrophoresis were compared. The results showed that using 350 μg total protein of seminiferous tubules and 24 cm linear IPG strip with pH 4～7 could better separate the protein of buffalo testicular seminiferous tubules, obtain a two-dimensional electrophoresis map with good quality and high resolution, and obtain about 486 protein spots.The two-dimensional gel electrophoresis technology could effectively separate the protein of buffalo testicular seminiferous tubules, and by two-dimensional electrophoresis system optimization, two dimensional electrophoresis map with clear protein spots and higher resolution also could be obtained.
A two-dimensional electrophoresis system for separating proteins of swamp buffalo testicular seminiferous tubules was established and optimized, which laid a good foundation for further identification and research of protein expression profiling in the testicular seminiferous tubules of buffalo. The effects of different IPG strips(linear or nonlinear), different pH range of strips and protein sample loading quantity on the results of two-dimensional electrophoresis were compared. The results showed that using 350 μg total protein of seminiferous tubules and 24 cm linear IPG strip with pH 4～7 could better separate the protein of buffalo testicular seminiferous tubules, obtain a two-dimensional electrophoresis map with good quality and high resolution, and obtain about 486 protein spots.The two-dimensional gel electrophoresis technology could effectively separate the protein of buffalo testicular seminiferous tubules, and by two-dimensional electrophoresis system optimization, two dimensional electrophoresis map with clear protein spots and higher resolution also could be obtained.
引文
Amy A.K.,and Margaret T.F.,2001,Male germ-line stem cells,Stem Cell Biology,Cold Spring Harbor Laboratory Press,pp.149-181
Bjellqvist B.,Ek K.,Giorgio Righetti P.,Gianazza E.,G觟rg A.,Westermeier R.,and Postel W.,1982,Isoelectric focusing in immobilized p H gradients:principle,methodology and some applications,Journal of Biochemical&Biophysical Methods,6(4):317-39
Farrell P.H.,1975,High resolution two-dimensional electrophoresis of proteins,Journal of Biological Chemistry,250(10):4007-4021
Fu W.,Li C.X.,Liu W.J.,Ma X.Q.,Ren L.,Huang L.,Jin S.Y.,and Zheng Y.C.,2014,Identification of the differential-ly expressed testis proteins between yaks and cattle-yaks by two-dimensional electrophoresis combined with mass spectrometry,Xinan Minzu Daxue Xuebao(Ziran Kexue Ban)(Journal of Southwest University for Nationalities(Natural Science Edition)),40(1):11-15(付伟,李彩霞,刘文静,马晓琴,任亮,黄林,金素钰,郑玉才,2014,利用双向电泳-质谱技术鉴定牦牛和犏牛睾丸中的差异表达蛋白质,西南民族大学学报(自然科学版),40(1):11-15)
Gorg A.,Weiss W.,and Dunn M.J.,2004,Current two-dimensional electrophoresis technology for proteomics,Proteomics,4(12):3665-3685
Gould K.L.,Ren L.,Feoktistova A.S.,Jennings J.L.,and Link A.J.,2004,Tandem affinity purification and identification of protein complex components,Methods,33(3):239-244
Guo X.J.,Zhao C.,Wang F.Q.,Zhu Y.F.,Cui Y.F.,Zhou Z.M.,Huo R.,and Sha J.H.,2010,Investigation of human testis protein heterogeneity using 2-dimensional electrophoresis,Journal of Andrology,31(4):419-429
Liang G.M.,Pang H.Y.,Wang Y.L.,Sun L.J.,Liu Y.,Xu D.F.,Chen J.J.,and Li J.R.,2015,Establishment and optimization of two-dimensional electrophoresis(2-DE)technique in muscle proteome of Litopenaeus vannamei,Zhongguo Shipin Xuebao(Journal of Chinese Institute of Food Science and Technology),15(10):240-246(梁光明,庞欢瑛,王雅玲,孙力军,刘阳,徐德峰,陈进军,励建荣,2015,凡纳滨对虾肌肉蛋白质双向电泳分离技术的建立及优化,中国食品学报,15(10):240-246)
Li J.Y.,Liu F.J.,Liu X.,Liu J.,Zhu P.,Wan F.C.,Jin S.H.,Wang W.T.,Li N.,Liu J.,and Wang H.Y.,2011,Mapping of the human testicular proteome and its relationship with that of the epididymis and spermatozoa,Molecular&Cellular Proteomics Mcp,10(3):318-324
Li F.,Zhou Y.G.,Zheng P.,Zhao L.,Li T.Q.,Zhou Q.Z.,Li J.M.,and Mao X.M.,2009,Establishment and optimization of two-dimensional polyacrylamide gel electrophoresis for proteome analysis of the human testicular tissue,Zhongguo Nankexue Zazhi(Chinese Journal of Andrology),23(4):3-6(李飞,邹亚光,郑平,赵亮,李铁求,周其赵,李建明,毛向明,2009,人睾丸组织蛋白质组双向凝胶电泳技术的建立及其优化,中国男科学杂志,23(4):3-6)
Lu Z.K.,Zhang Q.W.,Ma Y.J.,Zhao X.X.,and Ruan C.M.,2015,Establishment and optimization of two-dimensional gel electrophoresis system in testis of sheep(Ovis aries),Nongye Shengwujishu Xuebao(Journol of Agricultural Biotechnology),23(12):1660-1666(卢曾奎,张全伟,马友记,赵兴绪,阮崇美,2015,绵羊睾丸蛋白质双向电泳体系的建立及优化,农业生物技术学报,23(12):1660-1666)
Paz M.,Morín M.,and Mazo J.D.,2006,Proteome profile changes during mouse testis development,Comparative Biochemistry&Phys iology Part D Genomics&Proteomics,1(4):404-415
Poon T.C.W.,and Johnson P.J.,2001,Proteome analysis and its impact on the discovery of serological tumor markers,Clinica Chimica Acta,313(1-2):231-239
Huang S.Y.,Lin J.H.,Teng S.H.,Sun H.S.,Chen Y.H.,Chen H.H.,Liao J.Y.,Chung M.T.,Chen M.Y.,Chuang C.K.,Lin E.C.,and Huang M.C.,2011,Differential expression of porcine testis proteins during postnatal development,Animal Reproduction Science,123(3):221-233
Huang Y.L.,Fu Q.,Yang L.,Guan J.L.,Pan H.,Chen F.M.,Lu K.H.,and Zhang M.,2015,Differences between high-and low-motility buffalo sperm identified by comparative proteomics,Reproduction in Domestic Animals,50(3):443-451
Johnson L.,1996,Efficiency of spermatogenesis,Microscopy Research&Technique,32(5):385-422
Molloy M.P.,Herbert B.R.,Walsh B.J.,Tyler M.I.,Traini M.,Sanchez J.C.,Hochstrasser D.F.,Williams K.L.,and Gooley A.A.,1998,Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis,Electrophoresis,19(5):837-844
Nakata H.,Wakayama T.,Sonomura T.,Honma S.,Hatta T.,Iseki S.,2015,Three-dimensional structure of seminiferous tubules in the adult mouse,Journal of Anatomy,227(5):686-694
Organizer J.M.,and Goode J.,1994,Somatic cell-germ cell relationships in mammalian testes during development and spermatogenesis,Ciba Foundation Symposium,182:271-281
Xue W.,Yang L.,Fu Q.,Liu Z.F.,Zhang M.,and Lu K.H.,2012,Establishment and optimization of a two-dimensional electrophoresis technique for proteome of buffalo sperm,Shengwujishu Tongbao(Biotechnology Bulletin),(1):151-155(薛雯,杨丽,付强,刘振方,张明,卢克焕,2012,水牛精子蛋白质组双向电泳体系的建立和优化,生物技术通报,(1):151-155)
Zhu J.J.,Shen X.S.,Fu Q.,Zhou Y.,Tan Y.,and Gan X.,2013,Establishment and optimization analysis of two-dimensional electrophoresis system on GIFT tilapia liver,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),26(5):2122-2126(朱佳杰,沈夏霜,付强,周宇,谭芸,甘西,2013,吉富罗非鱼肝脏蛋白质组双向电泳技术的建立与优化,西南农业学报,26(5):2122-2126)
Bjellqvist B.,Ek K.,Giorgio Righetti P.,Gianazza E.,G觟rg A.,Westermeier R.,and Postel W.,1982,Isoelectric focusing in immobilized p H gradients:principle,methodology and some applications,Journal of Biochemical&Biophysical Methods,6(4):317-39
Farrell P.H.,1975,High resolution two-dimensional electrophoresis of proteins,Journal of Biological Chemistry,250(10):4007-4021
Fu W.,Li C.X.,Liu W.J.,Ma X.Q.,Ren L.,Huang L.,Jin S.Y.,and Zheng Y.C.,2014,Identification of the differential-ly expressed testis proteins between yaks and cattle-yaks by two-dimensional electrophoresis combined with mass spectrometry,Xinan Minzu Daxue Xuebao(Ziran Kexue Ban)(Journal of Southwest University for Nationalities(Natural Science Edition)),40(1):11-15(付伟,李彩霞,刘文静,马晓琴,任亮,黄林,金素钰,郑玉才,2014,利用双向电泳-质谱技术鉴定牦牛和犏牛睾丸中的差异表达蛋白质,西南民族大学学报(自然科学版),40(1):11-15)
Gorg A.,Weiss W.,and Dunn M.J.,2004,Current two-dimensional electrophoresis technology for proteomics,Proteomics,4(12):3665-3685
Gould K.L.,Ren L.,Feoktistova A.S.,Jennings J.L.,and Link A.J.,2004,Tandem affinity purification and identification of protein complex components,Methods,33(3):239-244
Guo X.J.,Zhao C.,Wang F.Q.,Zhu Y.F.,Cui Y.F.,Zhou Z.M.,Huo R.,and Sha J.H.,2010,Investigation of human testis protein heterogeneity using 2-dimensional electrophoresis,Journal of Andrology,31(4):419-429
Liang G.M.,Pang H.Y.,Wang Y.L.,Sun L.J.,Liu Y.,Xu D.F.,Chen J.J.,and Li J.R.,2015,Establishment and optimization of two-dimensional electrophoresis(2-DE)technique in muscle proteome of Litopenaeus vannamei,Zhongguo Shipin Xuebao(Journal of Chinese Institute of Food Science and Technology),15(10):240-246(梁光明,庞欢瑛,王雅玲,孙力军,刘阳,徐德峰,陈进军,励建荣,2015,凡纳滨对虾肌肉蛋白质双向电泳分离技术的建立及优化,中国食品学报,15(10):240-246)
Li J.Y.,Liu F.J.,Liu X.,Liu J.,Zhu P.,Wan F.C.,Jin S.H.,Wang W.T.,Li N.,Liu J.,and Wang H.Y.,2011,Mapping of the human testicular proteome and its relationship with that of the epididymis and spermatozoa,Molecular&Cellular Proteomics Mcp,10(3):318-324
Li F.,Zhou Y.G.,Zheng P.,Zhao L.,Li T.Q.,Zhou Q.Z.,Li J.M.,and Mao X.M.,2009,Establishment and optimization of two-dimensional polyacrylamide gel electrophoresis for proteome analysis of the human testicular tissue,Zhongguo Nankexue Zazhi(Chinese Journal of Andrology),23(4):3-6(李飞,邹亚光,郑平,赵亮,李铁求,周其赵,李建明,毛向明,2009,人睾丸组织蛋白质组双向凝胶电泳技术的建立及其优化,中国男科学杂志,23(4):3-6)
Lu Z.K.,Zhang Q.W.,Ma Y.J.,Zhao X.X.,and Ruan C.M.,2015,Establishment and optimization of two-dimensional gel electrophoresis system in testis of sheep(Ovis aries),Nongye Shengwujishu Xuebao(Journol of Agricultural Biotechnology),23(12):1660-1666(卢曾奎,张全伟,马友记,赵兴绪,阮崇美,2015,绵羊睾丸蛋白质双向电泳体系的建立及优化,农业生物技术学报,23(12):1660-1666)
Paz M.,Morín M.,and Mazo J.D.,2006,Proteome profile changes during mouse testis development,Comparative Biochemistry&Phys iology Part D Genomics&Proteomics,1(4):404-415
Poon T.C.W.,and Johnson P.J.,2001,Proteome analysis and its impact on the discovery of serological tumor markers,Clinica Chimica Acta,313(1-2):231-239
Huang S.Y.,Lin J.H.,Teng S.H.,Sun H.S.,Chen Y.H.,Chen H.H.,Liao J.Y.,Chung M.T.,Chen M.Y.,Chuang C.K.,Lin E.C.,and Huang M.C.,2011,Differential expression of porcine testis proteins during postnatal development,Animal Reproduction Science,123(3):221-233
Huang Y.L.,Fu Q.,Yang L.,Guan J.L.,Pan H.,Chen F.M.,Lu K.H.,and Zhang M.,2015,Differences between high-and low-motility buffalo sperm identified by comparative proteomics,Reproduction in Domestic Animals,50(3):443-451
Johnson L.,1996,Efficiency of spermatogenesis,Microscopy Research&Technique,32(5):385-422
Molloy M.P.,Herbert B.R.,Walsh B.J.,Tyler M.I.,Traini M.,Sanchez J.C.,Hochstrasser D.F.,Williams K.L.,and Gooley A.A.,1998,Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis,Electrophoresis,19(5):837-844
Nakata H.,Wakayama T.,Sonomura T.,Honma S.,Hatta T.,Iseki S.,2015,Three-dimensional structure of seminiferous tubules in the adult mouse,Journal of Anatomy,227(5):686-694
Organizer J.M.,and Goode J.,1994,Somatic cell-germ cell relationships in mammalian testes during development and spermatogenesis,Ciba Foundation Symposium,182:271-281
Xue W.,Yang L.,Fu Q.,Liu Z.F.,Zhang M.,and Lu K.H.,2012,Establishment and optimization of a two-dimensional electrophoresis technique for proteome of buffalo sperm,Shengwujishu Tongbao(Biotechnology Bulletin),(1):151-155(薛雯,杨丽,付强,刘振方,张明,卢克焕,2012,水牛精子蛋白质组双向电泳体系的建立和优化,生物技术通报,(1):151-155)
Zhu J.J.,Shen X.S.,Fu Q.,Zhou Y.,Tan Y.,and Gan X.,2013,Establishment and optimization analysis of two-dimensional electrophoresis system on GIFT tilapia liver,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),26(5):2122-2126(朱佳杰,沈夏霜,付强,周宇,谭芸,甘西,2013,吉富罗非鱼肝脏蛋白质组双向电泳技术的建立与优化,西南农业学报,26(5):2122-2126)