SPATA4基因启动子结构功能研究及SPATA4蛋白纯化
摘要
SPATA4基因是本实验室利用巢式PCR扩增和人类基因组草图搜索法,从人的睾丸cDNA文库中克隆出的新基因。前期研究表明,SPATA4在哺乳动物的睾丸组织中特异表达,并呈现了一定的抗凋亡特性。本课题中,我们主要研究了该基因的启动子结构和功能,另外也对SPATA4蛋白进行了初步纯化分析。
首先,在生物信息学预测的基础上,运用SMART RACE技术精确定位了SPATA4基因的转录起始位点,它位于翻译起始密码子上游23bp处。这为我们研究启动子和5'-前导序列的转录调控功能提供了便利。
然后,以小鼠血液基因组DNA为模板,克隆出约2.9 kb的启动子序列;并以它为模板,扩增了7个不同片段长度的5'端截短序列,瞬转到TM4细胞中,检测报告基因的荧光活性。结果显示pGL3-P923具有最高的活性,推测其为SPATA4基因启动子的核心片段。另外,生物信息学比对结果表明,在SPATA4基因转录起始位点上游,存在HSF1、RREB1、ZF5等转录因子的结合位点,推测它们对SPATA4基因的启动子活性具有重要影响。通过以上研究,我们初步明确了SPATA4基因启动子的结构和功能,对于分析SPATA4基因的转录调控机制具有重要的指导意义。
另外,本研究中构建了SPATA4蛋白的原核表达体系。在目的蛋白的N末端加入麦芽糖结合蛋白(Maltose Binding Protein,MBP),增加了表达蛋白的可溶性。表达蛋白通过磁珠亲和层析进行初步纯化后,采用Resource Q柱离子交换层析和Superdex200柱凝胶过滤层析纯化。电泳结果表明蛋白纯度较高(约95 %),不仅达到了做抗体的纯度要求,而且也为研究该蛋白的晶体结构奠定了基础。
Using technique of nested PCR and draft human genome searching, SPATA4 was cloned from a human testis cDNA library. Results of previous research confirmed that SPATA4 was specially expressed in testis of mammalian, and plays an important role in the process of spermatogenic cells apoptosis. Here we study on the structure and function of the SPATA4 gene promoter, and also purification of the SPATA4 protein.
Based on the Bioinformatics forecasting, we have established by SMART RACE analysis the transcription start site (TSS) of SPATA4 gene is located in 23bp upstream ATG that corresponds to the first codon of a 305 amino acid protein. This is foundational for researching functionality of transcriptional regulation of promoters.
Then, we cloned 2838 bp of the SPATA4 5′-flanking region from a mouse genomic DNA. And seven progressively deleted fragments were cloned in the promoter-less luciferase and the resulting plasmid pGL3-P2838 as the template. After transfected in HeLa cells, we analyzed various 5′-deletions of the promoter region by reporter gene assays. The results indicated that pGL3-P923 had the highest activity. So we suspect the P923 has the core fragment of SPATA4 gene promoter. In addition, bioinformatics comparison showed that there are several transcription factor (TF) binding sites exited in upstream of SPATA4 gene, such as HSF1, RREB1, ZF5. Those TFs are likely to significantly impact the activity of SPATA4 gene promoter.
To expression the SPATA4 protein, we cloned the SPATA4 gene to the plasmid pTHM, which added a Maltose Binding Protein (MBP) to the N-terminal of SPATA4. The MBP-tag helps to improve the solubility of SPATA4. The recombination protein, about 80 kD, was preliminary purified by affinity chromatography, and then purified by Ion Exchange Chromatography (IEC) and gel filtration chromatography. Protein harvested last show high purity (about 95%). It could not only be used to produce antibodies, but also helpful for researching on the crystal structure of the protein.
首先,在生物信息学预测的基础上,运用SMART RACE技术精确定位了SPATA4基因的转录起始位点,它位于翻译起始密码子上游23bp处。这为我们研究启动子和5'-前导序列的转录调控功能提供了便利。
然后,以小鼠血液基因组DNA为模板,克隆出约2.9 kb的启动子序列;并以它为模板,扩增了7个不同片段长度的5'端截短序列,瞬转到TM4细胞中,检测报告基因的荧光活性。结果显示pGL3-P923具有最高的活性,推测其为SPATA4基因启动子的核心片段。另外,生物信息学比对结果表明,在SPATA4基因转录起始位点上游,存在HSF1、RREB1、ZF5等转录因子的结合位点,推测它们对SPATA4基因的启动子活性具有重要影响。通过以上研究,我们初步明确了SPATA4基因启动子的结构和功能,对于分析SPATA4基因的转录调控机制具有重要的指导意义。
另外,本研究中构建了SPATA4蛋白的原核表达体系。在目的蛋白的N末端加入麦芽糖结合蛋白(Maltose Binding Protein,MBP),增加了表达蛋白的可溶性。表达蛋白通过磁珠亲和层析进行初步纯化后,采用Resource Q柱离子交换层析和Superdex200柱凝胶过滤层析纯化。电泳结果表明蛋白纯度较高(约95 %),不仅达到了做抗体的纯度要求,而且也为研究该蛋白的晶体结构奠定了基础。
Using technique of nested PCR and draft human genome searching, SPATA4 was cloned from a human testis cDNA library. Results of previous research confirmed that SPATA4 was specially expressed in testis of mammalian, and plays an important role in the process of spermatogenic cells apoptosis. Here we study on the structure and function of the SPATA4 gene promoter, and also purification of the SPATA4 protein.
Based on the Bioinformatics forecasting, we have established by SMART RACE analysis the transcription start site (TSS) of SPATA4 gene is located in 23bp upstream ATG that corresponds to the first codon of a 305 amino acid protein. This is foundational for researching functionality of transcriptional regulation of promoters.
Then, we cloned 2838 bp of the SPATA4 5′-flanking region from a mouse genomic DNA. And seven progressively deleted fragments were cloned in the promoter-less luciferase and the resulting plasmid pGL3-P2838 as the template. After transfected in HeLa cells, we analyzed various 5′-deletions of the promoter region by reporter gene assays. The results indicated that pGL3-P923 had the highest activity. So we suspect the P923 has the core fragment of SPATA4 gene promoter. In addition, bioinformatics comparison showed that there are several transcription factor (TF) binding sites exited in upstream of SPATA4 gene, such as HSF1, RREB1, ZF5. Those TFs are likely to significantly impact the activity of SPATA4 gene promoter.
To expression the SPATA4 protein, we cloned the SPATA4 gene to the plasmid pTHM, which added a Maltose Binding Protein (MBP) to the N-terminal of SPATA4. The MBP-tag helps to improve the solubility of SPATA4. The recombination protein, about 80 kD, was preliminary purified by affinity chromatography, and then purified by Ion Exchange Chromatography (IEC) and gel filtration chromatography. Protein harvested last show high purity (about 95%). It could not only be used to produce antibodies, but also helpful for researching on the crystal structure of the protein.
引文
[1].史小林主编,人类生殖学,科学出版社,2002.
[2]. Ellsworth PI, Ebb RG. The cryptorchid testis. J Med Liban. 2004, 52(4):227-233.
[3].刘上峰,李羃芸,傅俊江等.利用巢式PCR技术和人类基因组草图搜索法快速获得人类睾丸生精细胞凋亡相关基因TSARG2.生物化学与生物物理学报, 2002, 34:378-381.
[4]. Liu SF, Lu GX, Liu G, et al. Cloning of a full-length cDNA of humantestis-specific spermatogenic cell apoptosis inhibitor TSARG2 as a candidate oncogene. Biochemical and Biophysical Research Communications. 2004, 319(1):32-40.
[5]. Liu SF, He S, Liu BW, et al. Cloning and characterization of testis-specific spermatiogensis associated gene homologous to human SPATA4 in rat. Boil. Pharm. Bull. 2004, 27:1867-1870.
[6]. Xie MC, Ai C, Jin XM, et al. Cloning and characterization of chicken SPATA4 gene and analysis of its specific expression. Mol Cell Biochem. 2007, 306:79-85.
[7]. Jiang H, Li LY, Lu GX. Molecular cloning of genes related to apoptosis in spermatogenic cells of mouse. Acta Biochim. Biophys. Sin. 2001, 33:421-425.
[8]. Liu SF, Ai C, Ge ZQ, et al. Molecular Cloning and Bioinformatic Analysis of SPATA4 Gene. Journal of Biochemistry and Molecular Biology. 2005, 38(6):739-747.
[9]. Liu SF, Liu BW, He S, Zhao Y, Wang Z. Cloning and Characterization of Testies-Specific Genes Homologous to SPATA4 in Various Vertebrates. International Symposium on Bioinformatics and Chinese Medicine. 2004, 2:18-27.
[10]. Liu SF, Liu BW, He S, Zhao Y, Wang Z. Cloning and Characterization of Zebra Fish SPATA4 Gene and Analysis of Its Gonad Specific Expression. Biochemistry(Moscow). 2005, 70(6):638-644.
[11]. Yin Y, De Wolf WC, Morgentalar A. Experimental cryptorchidism induces testicular grem cell apoptosis by p53-dependengt and–independent pathways in mice. Biol Reprod. 1998, 58:492-496.
[12]. Yin Y, De Wolf WC, Morgentalar A. p53 is associated with the nuclear envelope in mouse testis. Biochem Biophys Res Commun. 1997, 235:689-694.
[13].郑航,郑新民,李世文等. Bcl-2/Bax基因表达对隐睾生殖细胞凋亡的影响。中国男科学杂志, 2000, 14:81-85.
[14]. Furuchi T, Masuko K, Nishimuine Y, Obinata M, Matsui Y. Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia. Development. 1996, 122(6):1703-1709.
[15]. Lee J, Richburg JH, Younkin SC, Boekelheide K. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology. 1997, 138(5):2081-2088.
[16]. Yin Y, Stahl BC, De Wolf WC, Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis. JAndrol. 2002, 23(1):64-70.
[17]. Nagata S, Golstern P. The Fas death factor. Science. 1995, 267:1449-1456.
[18]. Pesce M, Farrace MG, Piacentini M, Dolci S, De Felici M. Stem cll factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development. 1993, 118(4):1089-1094.
[19]. Mauduit C, Hamamah S, Benahmed M. Stem cell factor/c-kit system in spermatogenesis. Hum Reprod Update. 1999, 5(5):53-545.
[20]. Grigore M, Indrei A. The role of heat shock proteins in reproduction. Rev Med Chir Soc Med Nat Iasi. 2001, 105(4):674-676.
[21]. Werner A, Meinhardt A, Seitz J, Bergmann M. Distribution of heat-shock protein 60 immunoreactivity in testes of infertile men. Cell Tissue Res. 1997, 288(3):539-544.
[22]. Dix DJ, Allen JW, Collins BW, Mori C, Eddy EM. Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. Proc Natl Acad Sci USA. 1996, 93(8):3264-3268.
[23]. Kodaira K, Takahashi R, Hirabayashi M, et al. Overexpression of c-myc induces apoptosis at the prophase of meiosis of rat primary spermatocytes. Mol Reprod Dev. 1996, 45:403-410.
[24]. I. Ovcharenko, G.G. Loots, B.M. Giardine, M. Hou, J. Ma, R.C. Hardison, L. Stubbs, and W. Miller, Mulan: Multiple-sequence local alignment and visualization for studying function and evolution, Genome Research, 15,184-194 (2005)
[2]. Ellsworth PI, Ebb RG. The cryptorchid testis. J Med Liban. 2004, 52(4):227-233.
[3].刘上峰,李羃芸,傅俊江等.利用巢式PCR技术和人类基因组草图搜索法快速获得人类睾丸生精细胞凋亡相关基因TSARG2.生物化学与生物物理学报, 2002, 34:378-381.
[4]. Liu SF, Lu GX, Liu G, et al. Cloning of a full-length cDNA of humantestis-specific spermatogenic cell apoptosis inhibitor TSARG2 as a candidate oncogene. Biochemical and Biophysical Research Communications. 2004, 319(1):32-40.
[5]. Liu SF, He S, Liu BW, et al. Cloning and characterization of testis-specific spermatiogensis associated gene homologous to human SPATA4 in rat. Boil. Pharm. Bull. 2004, 27:1867-1870.
[6]. Xie MC, Ai C, Jin XM, et al. Cloning and characterization of chicken SPATA4 gene and analysis of its specific expression. Mol Cell Biochem. 2007, 306:79-85.
[7]. Jiang H, Li LY, Lu GX. Molecular cloning of genes related to apoptosis in spermatogenic cells of mouse. Acta Biochim. Biophys. Sin. 2001, 33:421-425.
[8]. Liu SF, Ai C, Ge ZQ, et al. Molecular Cloning and Bioinformatic Analysis of SPATA4 Gene. Journal of Biochemistry and Molecular Biology. 2005, 38(6):739-747.
[9]. Liu SF, Liu BW, He S, Zhao Y, Wang Z. Cloning and Characterization of Testies-Specific Genes Homologous to SPATA4 in Various Vertebrates. International Symposium on Bioinformatics and Chinese Medicine. 2004, 2:18-27.
[10]. Liu SF, Liu BW, He S, Zhao Y, Wang Z. Cloning and Characterization of Zebra Fish SPATA4 Gene and Analysis of Its Gonad Specific Expression. Biochemistry(Moscow). 2005, 70(6):638-644.
[11]. Yin Y, De Wolf WC, Morgentalar A. Experimental cryptorchidism induces testicular grem cell apoptosis by p53-dependengt and–independent pathways in mice. Biol Reprod. 1998, 58:492-496.
[12]. Yin Y, De Wolf WC, Morgentalar A. p53 is associated with the nuclear envelope in mouse testis. Biochem Biophys Res Commun. 1997, 235:689-694.
[13].郑航,郑新民,李世文等. Bcl-2/Bax基因表达对隐睾生殖细胞凋亡的影响。中国男科学杂志, 2000, 14:81-85.
[14]. Furuchi T, Masuko K, Nishimuine Y, Obinata M, Matsui Y. Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia. Development. 1996, 122(6):1703-1709.
[15]. Lee J, Richburg JH, Younkin SC, Boekelheide K. The Fas system is a key regulator of germ cell apoptosis in the testis. Endocrinology. 1997, 138(5):2081-2088.
[16]. Yin Y, Stahl BC, De Wolf WC, Morgentaler A. P53 and Fas are sequential mechanisms of testicular germ cell apoptosis. JAndrol. 2002, 23(1):64-70.
[17]. Nagata S, Golstern P. The Fas death factor. Science. 1995, 267:1449-1456.
[18]. Pesce M, Farrace MG, Piacentini M, Dolci S, De Felici M. Stem cll factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development. 1993, 118(4):1089-1094.
[19]. Mauduit C, Hamamah S, Benahmed M. Stem cell factor/c-kit system in spermatogenesis. Hum Reprod Update. 1999, 5(5):53-545.
[20]. Grigore M, Indrei A. The role of heat shock proteins in reproduction. Rev Med Chir Soc Med Nat Iasi. 2001, 105(4):674-676.
[21]. Werner A, Meinhardt A, Seitz J, Bergmann M. Distribution of heat-shock protein 60 immunoreactivity in testes of infertile men. Cell Tissue Res. 1997, 288(3):539-544.
[22]. Dix DJ, Allen JW, Collins BW, Mori C, Eddy EM. Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. Proc Natl Acad Sci USA. 1996, 93(8):3264-3268.
[23]. Kodaira K, Takahashi R, Hirabayashi M, et al. Overexpression of c-myc induces apoptosis at the prophase of meiosis of rat primary spermatocytes. Mol Reprod Dev. 1996, 45:403-410.
[24]. I. Ovcharenko, G.G. Loots, B.M. Giardine, M. Hou, J. Ma, R.C. Hardison, L. Stubbs, and W. Miller, Mulan: Multiple-sequence local alignment and visualization for studying function and evolution, Genome Research, 15,184-194 (2005)