家蚕BmCIB基因的原核表达及其亚细胞定位
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摘要
钙整合素结合蛋白(Calcium and Integrin Binding protein,CIB)是含有EF-hand结构域的钙离子结合蛋白,可与血小板integrinαⅡbβ3等许多效应蛋白结合,并依赖Ca~(2+)的调控而调节它们的功能。它在各组织中广泛表达,作为一个重要的中间分子,在机体中执行多种功能。目前,国内外对钙整合素结合蛋白的研究多数集中在脊椎动物上,而对无脊椎动物的报道很少。
我们从本实验室构建的家蚕蛹cDNA文库中获得一条cDNA序列,通过同源序列比对后发现其为家蚕钙整合素结合蛋白(Bombyx mori Calcium and Integrin Binding protein,BmCIB)基因。该基因全长为939 bp,ORF为558 bp,编码185个氨基酸残基。通过扩增BmCIB基因完整的ORF序列,克隆到表达载体pET-28a(+)相应的酶切位点上,构建了重组表达载体pET28a(+)-BmCIB。经PCR、酶切鉴定以及测序证明了该重组子的正确性后,将其转化大肠杆菌BL21(DE3)。以终浓度为1 mM的IPTG诱导,并收集菌体裂解,经SDS-PAGE分析,在26 kD左右的位置出现了一条特异性蛋白条带,与预期值(融合标签3.56 kD,BmCIB 21.16 kD)大体相符。重组蛋白以包涵体形式存在,超声裂解菌体后12000 rpm离心,收集沉淀经包涵体洗涤,溶解在含8 M尿素的溶解液中,再以亲和层析的方法进一步纯化。经透析除盐后,进行了质谱分析,测定该重组蛋白的精确分子量为24877.0508 Da,与其理论值24720.99 Da非常接近,可以确定该蛋白为重组的BmCIB。以纯化所得重组蛋白为抗原免疫雄性新西兰兔,制备了多克隆抗体,ELISA检测该抗血清的效价在抗原浓度为10μg/mL时达到1:12800以上。用Protein A凝胶柱纯化了多克隆抗体。利用荧光定量PCR的方法,对家蚕四个发育时期以及五龄幼虫各组织中的靶mRNA的量进行比较,结果显示其mRNA转录水平分别以蛹期和生殖腺中最高,同时我们提取家蚕各发育时期及五龄幼虫的各组织蛋白,用于Western blotting实验。实验结果表明,BmCIB蛋白的表达量在家蚕各发育时期中差异不大;在五龄幼虫各组织中以生殖腺中最多。通过免疫荧光法分析BmCIB蛋白在家蚕BmN细胞的亚细胞定位,发现其定位于细胞质中。以上工作为进一步研究BmCIB的功能奠定了基础。
Calcium and integrin binding protein(CIB),which contains EF-hand motifs, is one member of the Ca~(2+) binding protein superfamily. It binds many effector proteins including the plateletαⅡbβ3 integrin and potentially regulates their functions in a Ca~(2+) related manner. CIB are widely distributed in a variety of tissues, as a intermediate molecular it plays an important role in many cell possess. To date, most researches of CIB were focused on rather vertebrate than invertebrate.
Here we screened a cDNA sequence from a silkworm pupa cDNA database constructed by our laboratory. After blasting, we found that it is Bombyx mori calcium and integrin binding protein gene (BmCIB). The CIB cDNA is 939 bp in size, containing a 558 bp open reading frame that encodes 185 amino acids.We obtained His-tagged fusion protein by transforming pET28a(+)-CIB recombinant plasmid into E.coli BL21 (DE3). The fusion protein which was purified with liquid solution of inclusion bodies and metal-chelating affinity chromatography. The molecular weight of this fusion protein was 24877.0508 Da (His tag was weight of 3.56 kD, and BmCIB was weight of 21.16 kD), characterized by mass-spectrum after dialysis. This result proved that His-tag BmCIB was expressed in the right form. The purified fusion protein was used to immunize a male New Zealand rabbit. Then, antiserum was harvested, and IgG was prepared by affinity chromatography using immobilized protein A. According to real-time Quantitative PCR and Western blotting analyses, we found that BmCIB was widely expressed in a variety of tissues and different developmental stages of silkworm. The transcriptional level of BmCIB mRNA is highest in gonad and pupa. The expression of protein can be detected in all the developmental stages and is highest in gonad among different tissues. Moreover, after being stained with immunofluorescence, the subcellular localization of the BmCIB was analyzed. The result show that BmCIB proteins are all accumulated in cytoplasm. The protein of BmCIB has been cloned and expressed successfully. The specific polyclonal antibody against the protein of BmCIB has been obtained which can be used for further research. Additionally we have completed the related expression and subcellular localization analysis of BmCIB which can be used for further research into the function of BmCIB.
我们从本实验室构建的家蚕蛹cDNA文库中获得一条cDNA序列,通过同源序列比对后发现其为家蚕钙整合素结合蛋白(Bombyx mori Calcium and Integrin Binding protein,BmCIB)基因。该基因全长为939 bp,ORF为558 bp,编码185个氨基酸残基。通过扩增BmCIB基因完整的ORF序列,克隆到表达载体pET-28a(+)相应的酶切位点上,构建了重组表达载体pET28a(+)-BmCIB。经PCR、酶切鉴定以及测序证明了该重组子的正确性后,将其转化大肠杆菌BL21(DE3)。以终浓度为1 mM的IPTG诱导,并收集菌体裂解,经SDS-PAGE分析,在26 kD左右的位置出现了一条特异性蛋白条带,与预期值(融合标签3.56 kD,BmCIB 21.16 kD)大体相符。重组蛋白以包涵体形式存在,超声裂解菌体后12000 rpm离心,收集沉淀经包涵体洗涤,溶解在含8 M尿素的溶解液中,再以亲和层析的方法进一步纯化。经透析除盐后,进行了质谱分析,测定该重组蛋白的精确分子量为24877.0508 Da,与其理论值24720.99 Da非常接近,可以确定该蛋白为重组的BmCIB。以纯化所得重组蛋白为抗原免疫雄性新西兰兔,制备了多克隆抗体,ELISA检测该抗血清的效价在抗原浓度为10μg/mL时达到1:12800以上。用Protein A凝胶柱纯化了多克隆抗体。利用荧光定量PCR的方法,对家蚕四个发育时期以及五龄幼虫各组织中的靶mRNA的量进行比较,结果显示其mRNA转录水平分别以蛹期和生殖腺中最高,同时我们提取家蚕各发育时期及五龄幼虫的各组织蛋白,用于Western blotting实验。实验结果表明,BmCIB蛋白的表达量在家蚕各发育时期中差异不大;在五龄幼虫各组织中以生殖腺中最多。通过免疫荧光法分析BmCIB蛋白在家蚕BmN细胞的亚细胞定位,发现其定位于细胞质中。以上工作为进一步研究BmCIB的功能奠定了基础。
Calcium and integrin binding protein(CIB),which contains EF-hand motifs, is one member of the Ca~(2+) binding protein superfamily. It binds many effector proteins including the plateletαⅡbβ3 integrin and potentially regulates their functions in a Ca~(2+) related manner. CIB are widely distributed in a variety of tissues, as a intermediate molecular it plays an important role in many cell possess. To date, most researches of CIB were focused on rather vertebrate than invertebrate.
Here we screened a cDNA sequence from a silkworm pupa cDNA database constructed by our laboratory. After blasting, we found that it is Bombyx mori calcium and integrin binding protein gene (BmCIB). The CIB cDNA is 939 bp in size, containing a 558 bp open reading frame that encodes 185 amino acids.We obtained His-tagged fusion protein by transforming pET28a(+)-CIB recombinant plasmid into E.coli BL21 (DE3). The fusion protein which was purified with liquid solution of inclusion bodies and metal-chelating affinity chromatography. The molecular weight of this fusion protein was 24877.0508 Da (His tag was weight of 3.56 kD, and BmCIB was weight of 21.16 kD), characterized by mass-spectrum after dialysis. This result proved that His-tag BmCIB was expressed in the right form. The purified fusion protein was used to immunize a male New Zealand rabbit. Then, antiserum was harvested, and IgG was prepared by affinity chromatography using immobilized protein A. According to real-time Quantitative PCR and Western blotting analyses, we found that BmCIB was widely expressed in a variety of tissues and different developmental stages of silkworm. The transcriptional level of BmCIB mRNA is highest in gonad and pupa. The expression of protein can be detected in all the developmental stages and is highest in gonad among different tissues. Moreover, after being stained with immunofluorescence, the subcellular localization of the BmCIB was analyzed. The result show that BmCIB proteins are all accumulated in cytoplasm. The protein of BmCIB has been cloned and expressed successfully. The specific polyclonal antibody against the protein of BmCIB has been obtained which can be used for further research. Additionally we have completed the related expression and subcellular localization analysis of BmCIB which can be used for further research into the function of BmCIB.
引文
[1] Barry WT, Boudignon-Proudhon C, Shock DD, McFadden A, Weiss JM, Sondek J, Parise LV. Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain[J]. J Biol Chem 2002, 277 (32):28877-83.
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[3] Fang X, Chen C, Wang Q, Gu J, Chi C. The interaction of the calcium- and integrin-binding protein (CIBP) with the coagulation factor VIII[J]. Thromb Res 2001, 102 (2):177-85.
[4] Grabarek Z. Structural basis for diversity of the EF-hand calcium-binding proteins[J]. Mol Biol 2006, 359 (3):509-25.
[5] Ikura M. Calcium binding and conformational response in EF-hand proteins[J]. Trends Biochem Sci 1996, 21 (1):14-7.
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[2] Kirchhofer D, Grzesiak J, Pierschbacher MD. Calcium as a potential physiological regulator of integrin-mediated cell adhesion[J]. J Biol Chem 1991, 266 (7):4471-7.
[3] Fang X, Chen C, Wang Q, Gu J, Chi C. The interaction of the calcium- and integrin-binding protein (CIBP) with the coagulation factor VIII[J]. Thromb Res 2001, 102 (2):177-85.
[4] Grabarek Z. Structural basis for diversity of the EF-hand calcium-binding proteins[J]. Mol Biol 2006, 359 (3):509-25.
[5] Ikura M. Calcium binding and conformational response in EF-hand proteins[J]. Trends Biochem Sci 1996, 21 (1):14-7.
[6] Vijay-Kumar S, Kumar VD. Crystal structure of recombinant bovine neurocalcin[J]. Nat Struct Biol 1999, 6 (1):80-8.
[7] Berridge MJ, Bootman MD, Lipp P. Calcium--a life and death signal[J]. Nature 1998, 395 (6703):645-8.
[8] Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling[J]. Nat Rev Mol Cell Biol 2000, 1 (1):11-21.
[9] Carafoli E, Santella L, Branca D, Brini M. Generation, control, and processing of cellular calcium signals[J]. Crit Rev Biochem Mol Biol 2001, 36 (2):107-260.
[10] Kahl CR, Means AR. Regulation of cell cycle progression by calcium/calmodulin-dependent pathways[J]. Endocr Rev 2003, 24 (6):719-36.
[11] Adriana Isvoran LP, Constantin T. Craescu , Adrian Chiriac Fractal aspects of calcium binding protein structures[J]. Solitons and Fractals 2008, 35 (960–966).
[12] Barroso MR, Bernd KK, DeWitt ND, Chang A, Mills K, Sztul ES. A novel Ca2+-binding protein, p22, is required for constitutive membrane traffic[J]. J Biol Chem 1996, 271 (17):10183-7.
[13] Lin X, Barber DL. A calcineurin homologous protein inhibits GTPase-stimulated Na-H exchange[J]. Proc Natl Acad Sci U S A 1996, 93 (22):12631-6.
[14] Naik UP, Patel PM, Parise LV. Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain[J]. J Biol Chem 1997, 272 (8):4651-4.
[15] Kretsinger RH. Structure and evolution of calcium-modulated proteins[J]. CRC Crit Rev Biochem 1980, 8 (2):119-74.
[16] Persechini A, Moncrief ND, Kretsinger RH. The EF-hand family of calcium-modulated proteins[J]. Trends Neurosci 1989, 12 (11):462-7.
[17] Stabler SM, Ostrowski LL, Janicki SM, Monteiro MJ. A myristoylated calcium-binding protein that preferentially interacts with the Alzheimer's disease presenilin 2 protein[J]. J Cell Biol 1999, 145 (6):1277-92.
[18] Bockaert J, Fagni L, Dumuis A, Marin P. GPCR interacting proteins (GIP) [J]. Pharmacol Ther 2004, 103 (3):203-21.
[19] Bockaert J, Marin P, Dumuis A, Fagni L. The 'magic tail' of G protein-coupled receptors: an anchorage for functional protein networks[J]. FEBS Lett 2003, 546 (1):65-72.
[20] Hwang PM, Vogel HJ. Structures of the platelet calcium- and integrin-binding protein and thealphaIIb-integrin cytoplasmic domain suggest a mechanism for calcium-regulated recognition; homology modelling and NMR studies[J]. J Mol Recognit 2000, 13 (2):83-92.
[21] Ames JB, Ishima R, Tanaka T, Gordon JI, Stryer L, Ikura M. Molecular mechanics of calcium-myristoyl switches[J]. Nature 1997, 389 (6647):198-202.
[22] Kauselmann G, Weiler M, Wulff P, Jessberger S, Konietzko U, Scafidi J, Staubli U, Bereiter-Hahn J, Strebhardt K, Kuhl D. The polo-like protein kinases Fnk and Snk associate with a Ca(2~+)- and integrin-binding protein and are regulated dynamically with synaptic plasticity[J]. EMBO J 1999, 18 (20):5528-39.
[23] Zhu J, Stabler SM, Ames JB, Baskakov I, Monteiro MJ. Calcium binding sequences in calmyrin regulates interaction with presenilin-2[J]. Exp Cell Res 2004, 300 (2):440-54.
[24] Gentry HR, Singer AU, Betts L, Yang C, Ferrara JD, Sondek J, Parise LV. Structural and biochemical characterization of CIB1 delineates a new family of EF-hand-containing proteins[J]. J Biol Chem 2005, 280 (9):8407-15.
[25] Yamniuk AP, Anderson KL, Fraser ME, Vogel HJ. Auxiliary Ca2+ binding sites can influence the structure of CIB1[J]. Protein Sci 2009, 18 (5):1128-34.
[26] Blamey CJ, Ceccarelli C, Naik UP, Bahnson BJ. The crystal structure of calcium- and integrin-binding protein 1: insights into redox regulated functions[J]. Protein Sci 2005, 14 (5):1214-21.
[27] Shock DD, Naik UP, Brittain JE, Alahari SK, Sondek J, Parise LV. Calcium-dependent properties of CIB binding to the integrin alphaIIb cytoplasmic domain and translocation to the platelet cytoskeleton[J]. Biochem J 1999, 342 Pt 3:729-35.
[28] Tsuboi S. Calcium integrin-binding protein activates platelet integrin alpha IIbbeta 3[J]. J Biol Chem 2002, 277 (3):1919-23.
[29] Ma S, Liu MA, Yuan YL, Erikson RL. The serum-inducible protein kinase Snk is a G1 phase polo-like kinase that is inhibited by the calcium- and integrin-binding protein CIB[J]. Mol Cancer Res 2003, 1 (5):376-84.
[30] Yuan W, Leisner TM, McFadden AW, Clark S, Hiller S, Maeda N, O'Brien DA, Parise LV. CIB1 is essential for mouse spermatogenesis[J]. Mol Cell Biol 2006, 26 (22):8507-14.
[31] Leisner TM, Liu M, Jaffer ZM, Chernoff J, Parise LV. Essential role of CIB1 in regulating PAK1 activation and cell migration[J]. J Cell Biol 2005, 170 (3):465-76.
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