家蚕BmIBP基因的原核表达及其亚细胞定位研究
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摘要
胰岛素(insilin)和类胰岛素多肽(insulin-related peptides)是调节生长和代谢过程中主要的激素,在脊椎动物和非脊椎动物中均发现了类胰岛素多肽,包括哺乳动物、原索动物、线虫、软体动物和昆虫。这些激素通过类似胰岛素受体(insulin receptor)的异源四聚体跨膜受体酪氨酸蛋白激酶相互作用而发挥其功能。目前,国内外对胰岛素结合蛋白(insulin-related binding proteins, IBP)的研究多数集中在脊椎动物上,而对非脊椎动物的研究很少。
我们从本实验室构建的家蚕蛹cDNA文库中发现了一条编码类胰岛素结合蛋白(insulin-related binding protein)的基因,我们将其命名为BmIBP (Bombyx mori insulin-related binding protein), GenBank登录号为DQ443372.1。该基因的ORF为768bp,编码255个氨基酸残基,是由六个外显子和五个内含子剪接而成。将推测的氨基酸序列进行同源性(BlastP)比对,发现此蛋白序列含有两个保守的IG domains,生物信息学预测其分子量为27.98kD,等电点为7.36。
我们利用大肠杆菌表达系统成功表达了含有His标签的BmIBP融合蛋白,主要以包涵体形式存在。镍柱纯化后获得较纯的融合蛋白,将其为抗原免疫新西兰白兔,制备该融合蛋白的多克隆抗体。ELISA检测该抗血清(多克隆抗体)的效价可达到1：12800。用制备的多克隆抗体进行Western blotting检测发现,BmIBP蛋白在家蚕五龄幼虫卵巢、精巢、脂肪体、马氏管和头等组织和家蚕卵、蛹、蛾期的发育阶段均有表达。实时荧光定量PCR的结果表明,家蚕BmIBP mRNA在家蚕的各个发育时期及不同组织中广泛存在,在蛹期和五龄幼虫中的卵巢、肠和马氏管中表达量最高。用获得的多克隆抗体进行亚细胞定位,信号显示BmIBP蛋白主要定位在家蚕Bm5细胞的细胞质中。
Insulin and related peptides are key hormones for the regulation of growth and metabolism. Insulin-related peptides have been identified in both vertebrates and invertebrates including mammals, protochordates, nematodes, mollusks, and insects. Many of these hormones have been shown to act through receptors structurally similar to the insulin receptor,being heterotetrameric,membrane-spanning tyrosine kinases. To date, most researches of insulin-related binding protein were focused on rather vertebrate than invertebrate.
A pupae cDNA library of silkworm has been constructed in our Lab. We identified a gene with an open reading frame(ORF) of 768bp which encodes 255 amino acids containing two conserved IG domains. We nominated this gene as BmIBP (Bombyx mori insulin-related binding protein), and its accession number in Genbank is DQ443372.The ORF contains six exons and five introns The predicted protein has a molecular weight of 27.98kD and the theoretical pI lies at 7.36.
The recombinant fusion protein His-tag BmIBP was successfully espressed, purified and then immuned a male New Zealand rabbit to get the antiserum(polyclonal antibodies).The titer of the polyclonal antibodies reaches 1:12800 measured by ELISA.Western blotting revealed the protein was expressed in some fully differentiated tissues of the fifth instar larvae,such as testicle,ovary,fat body, malpighian tubule and head,also in some differentiated growth stages of the silkworm,such as egg,pupa and adult.The result of real-time PCR revealed that BmIBP mRNA is widespread in different tisssues and growh stages of silkworm,which highest expression in the ovary,midgut, malpighian tubule and pupa. The subcellular localization experiment with polyclonal antibodies indicated that the BmIBP protein was distributed mainly in cytoplasm of the Bm5 cells.
我们从本实验室构建的家蚕蛹cDNA文库中发现了一条编码类胰岛素结合蛋白(insulin-related binding protein)的基因,我们将其命名为BmIBP (Bombyx mori insulin-related binding protein), GenBank登录号为DQ443372.1。该基因的ORF为768bp,编码255个氨基酸残基,是由六个外显子和五个内含子剪接而成。将推测的氨基酸序列进行同源性(BlastP)比对,发现此蛋白序列含有两个保守的IG domains,生物信息学预测其分子量为27.98kD,等电点为7.36。
我们利用大肠杆菌表达系统成功表达了含有His标签的BmIBP融合蛋白,主要以包涵体形式存在。镍柱纯化后获得较纯的融合蛋白,将其为抗原免疫新西兰白兔,制备该融合蛋白的多克隆抗体。ELISA检测该抗血清(多克隆抗体)的效价可达到1：12800。用制备的多克隆抗体进行Western blotting检测发现,BmIBP蛋白在家蚕五龄幼虫卵巢、精巢、脂肪体、马氏管和头等组织和家蚕卵、蛹、蛾期的发育阶段均有表达。实时荧光定量PCR的结果表明,家蚕BmIBP mRNA在家蚕的各个发育时期及不同组织中广泛存在,在蛹期和五龄幼虫中的卵巢、肠和马氏管中表达量最高。用获得的多克隆抗体进行亚细胞定位,信号显示BmIBP蛋白主要定位在家蚕Bm5细胞的细胞质中。
Insulin and related peptides are key hormones for the regulation of growth and metabolism. Insulin-related peptides have been identified in both vertebrates and invertebrates including mammals, protochordates, nematodes, mollusks, and insects. Many of these hormones have been shown to act through receptors structurally similar to the insulin receptor,being heterotetrameric,membrane-spanning tyrosine kinases. To date, most researches of insulin-related binding protein were focused on rather vertebrate than invertebrate.
A pupae cDNA library of silkworm has been constructed in our Lab. We identified a gene with an open reading frame(ORF) of 768bp which encodes 255 amino acids containing two conserved IG domains. We nominated this gene as BmIBP (Bombyx mori insulin-related binding protein), and its accession number in Genbank is DQ443372.The ORF contains six exons and five introns The predicted protein has a molecular weight of 27.98kD and the theoretical pI lies at 7.36.
The recombinant fusion protein His-tag BmIBP was successfully espressed, purified and then immuned a male New Zealand rabbit to get the antiserum(polyclonal antibodies).The titer of the polyclonal antibodies reaches 1:12800 measured by ELISA.Western blotting revealed the protein was expressed in some fully differentiated tissues of the fifth instar larvae,such as testicle,ovary,fat body, malpighian tubule and head,also in some differentiated growth stages of the silkworm,such as egg,pupa and adult.The result of real-time PCR revealed that BmIBP mRNA is widespread in different tisssues and growh stages of silkworm,which highest expression in the ovary,midgut, malpighian tubule and pupa. The subcellular localization experiment with polyclonal antibodies indicated that the BmIBP protein was distributed mainly in cytoplasm of the Bm5 cells.
引文
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[2]Gonzalez. J P, Farnes. O C, R. A T, Conservation of key members in the course of the evolution of the insulin signaling pathway[J]. Biosystems,2009,95(1):7-16.
[3]WU Q, ZHAO H Y, WEN T Q, The conserved signaling pathways and physiological functions of insulin-like peptides in insect[J]. Journal of Shanghai University(English Edition),2004,8(2):227-234.
[4]Brogiolo W, Stocker H, Ikeya T, et al., An evolutionarily conserved function of the Drosophila insulin receptor and insulin-like peptides in growth control[J]. Curr Biol,2001,11(4):213-221.
[5]Rulifson E J, Kim S K, Nusse R, Ablation of insulin-producing neurons in flies:growth and diabetic phenotypes[J]. Science,2002,296(5570):1118.
[6]Claeys I, Simonet G, Poels J, et al., Insulin-related peptides and their conserved signal transduction pathway[J]. Peptides,2002,23(4):807-816.
[7]Mattson M P, Duan W, Maswood N, How does the brain control lifespan?[J]. Ageing Research Reviews, 2002,1(2):155-165.
[8]Osterbur D L, Fristrom D K, Natzle J E, et al., Genes expressed during imaginal discs morphogenesis: IMP-L2, a gene expressed during imaginal disc and imaginal histoblast morphogenesis[J]. Developmental Biology,1988,129(2):439.
[9]Sloth Andersen A, Hertz Hansen P, Schaffer L, et al., A new secreted insect protein belonging to the immunoglobulin superfamily binds insulin and related peptides and inhibits their activities[J]. J Biol Chem, 2000,275(22):16948-16953.
[10]Arquier N, Geminard C, Bourouis M, et al., Drosophila ALS regulates growth and metabolism through functional interaction with insulin-like peptides[J]. Cell Metab,2008,7(4):333-338.
[11]Matsunaga T, Evolution of the immunoglobulin superfamily by duplication of complementarity [J]. Immunology Today,1985,6(9):260-263.
[12]Williams A F, A year in the life of the immunoglobulin superfamily[J]. Immunology Today,1987, 8(10):298-303.
[13]Ladendorff N E,Kanost M R, Bacteria-induced protein P 4(hemolin) from Manduca sexta:A member of the immunoglobulin superfamily which can inhibit hemocyte aggregation[J]. Archives of insect biochemistry and Physiology,1991,18(4):285-300.
[14]Andersson K,Steiner H, Structure and properties of protein P4, the major bacteria-inducible protein in pupae of Hyalophora cecropia[J]. Insect Biochemistry,1987,17(1):133-140.
[15]Nelson R E, Fessler L I, Takagi Y, et al., Peroxidasin:a novel enzyme-matrix protein of Drosophila development[J]. The EMBO Journal,1994,13(15):3438.
[16]Feinstein A, Richardson N, Taussig M I, Immunoglobulin flexibility in complement activation[J]. Immunology Today,1986,7(6):169-174.
[17]Poljak R J, Amzel L M, Phizackerley R P, Studies on the three-dimensional structure of immunoglobulins[J]. Progress in Biophysics and Molecular Biology,1978,31:67-93.
[18]Pipott R,Power C, The adhesion molecule:facts book[J].1994.
[19]Williams A F,Barclay A N, The immunoglobulin superfamily-domains for cell surface recognition[J]. Annual Review of Immunology,1988,6(1):381-405.
[20]Lanz Mendoza H,Faye I, Physiological aspects of the immunoglobulin superfamily in invertebrates[J]. Developmental & Comparative Immunology,1999,23(4-5):359-374.
[21]Burden-Gulley S M,Lemmon V, Ig superfamily adhesion molecules in the vertebrate nervous system: binding partners and signal transduction during axon growth[J]. Seminars in Developmental Biology,1995, 6(2):79-87.
[22]Aricescu A R,Jones E Y, Immunoglobulin superfamily cell adhesion molecules:zippers and signals[J]. Current Opinion in Cell Biology,2007,19(5):543-550.
[23]Bateman A, Eddy S R, Chothia C, Members of the immunoglobulin superfamily in bacteria[J]. Protein Science:A Publication of the Protein Society,1996,5(9):1939.
[24]Day M J, Immune System Development in the Dog and Cat[J]. Journal of Comparative Pathology,2007, 137(Supplement 1):S10-S15.
[25]Mokady O, Occam's Razor, invertebrate allorecognition and Ig superfamily evolution[J]. Research in Immunology,1996,147(4):241-246.
[26]Zapf J, Born W, Chang J-Y, et al., Isolation and NH2-terminal amino acid sequences of rat serum carrier proteins for insulin-like growth factors[J]. Biochemical and Biophysical Research Communications,1988, 156(3):1187-1194.
[27]Chan S, Cao Q P, Steiner D F, Evolution of the insulin superfamily:cloning of a hybrid insulin/insulin-like growth factor cDNA from amphioxus[J]. Proc. Nald. Acad. Sci. USA,1990,87:9319-9323.
[28]Nakae J, Kido Y, Accili D, Distinct and overlapping functions of insulin and IGF-Ⅰ receptors[J]. Endocrine Reviews,2001,22(6):818.
[29]Jones J I,Clemmons D R, Insulin-like growth factors and their binding proteins:biological actions[J]. Endocrine Reviews,1995,16(1):3.
[30]Wheatcroft S B,Kearney M T, IGF-dependent and IGF-independent actions of IGF-binding protein-1 and-2:implications for metabolic homeostasis[J]. Trends in Endocrinology & Metabolism,2009, 20(4):153-162.
[31]Mohan S, Nakao Y, Honda Y, et al., Studies on the mechanisms by which insulin-like growth factor (IGF) binding protein-4 (IGFBP-4) and IGFBP-5 modulate IGF actions in bone cells[J]. Journal of Biological Chemistry,1995,270(35):20424.
[32]Rajah R, Valentinis B, Cohen P, Insulin-like growth factor (IGF)-binding protein-3 induces apoptosis and mediates the effects of transforming growth factor-beta1 on programmed cell death through a p53-and IGF-independent mechanism[J]. The Journal of biological chemistry,1997,272(18):12181.
[33]Hwa V, Oh Y, Rosenfeld R G, The insulin-like growth factor-binding protein (IGFBP) superfamily[J]. Endocr Rev,1999,20(6):761-787.
[34]Neumann G M, Marinaro J A, Bach L A, Identification of O-glycosylation sites and partial characterization of carbohydrate structure and disulfide linkages of human insulin-like growth factor binding protein 6[J]. Biochemistry,1998,37(18):6572.
[35]Hashimoto R, Ono M, Fujiwara H, et al., Binding sites and binding properties of binary and ternary complexes of insulin-like growth factor-Ⅱ (IGF-Ⅱ), IGF-binding protein-3, and acid-labile subunit[J]. Journal of Biological Chemistry,1997,272(44):27936.
[36]Forbes B E, Turner D, Hodge S J, et al., Localization of an insulin-like growth factor (IGF) binding site of bovine IGF binding protein-2 using disulfide mapping and deletion mutation analysis of the C-terminal domain[J]. Journal of Biological Chemistry,1998,273(8):4647.
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