家蚕类胰岛素相关肽结合蛋白2（BmIBP2） 的原核表达与组织特异性分析

doi:10.3864/j.issn.0578-1752.2012.23.019

摘要/Abstract

摘要： 【目的】从体外表达家蚕中新发现的1个免疫球蛋白超家族成员—类胰岛素相关肽结合蛋白2（BmIBP2），并制备其多克隆抗体，为进一步深入研究BmIBP2的生物学功能奠定基础。【方法】以家蚕中肠的总cDNA为模板，利用普通PCR方法，扩增出BmIBP2基因的成熟肽序列；通过构建重组表达质粒pET-28a-BmIBP2对BmIBP2的成熟肽进行重组表达；制备多克隆抗体，通过免疫共沉淀的方法研究BmIBP2蛋白的组织特异性。【结果】SDS-PAGE电泳分析表达产物发现，重组蛋白rBmIBP2主要以包涵体的形式表达，目的蛋白大小约为30 kD；以兔抗His-tag抗体为一抗进行Western blot检测，结果表明rBmIBP2具有良好的免疫反应性；用镍离子亲和层析的方法可获得纯化的rBmIBP2，以纯化的rBmIBP2为抗原免疫新西兰白兔制备多克隆抗体，Western blot结果显示家蚕的中肠组织匀浆液中有1条约28 kD大小的条带，大小与BmIBP2成熟肽的蛋白分子量基本一致，而脂肪体、血淋巴、丝腺、精巢及卵巢的组织匀浆液中没有检测到特异反应条带。【结论】成功地实现了BmIBP2在大肠杆菌中的表达和纯化，并获得其多克隆抗体，Western blot结果证明BmIBP2被正确表达，并明确了该蛋白只在家蚕中肠表达的组织特异性。

Abstract: 【Objective】 The objective of this study is to express insulin-related peptide-binding protein 2 from silkworm (BmIBP2) which belongs to the immunoglobulin (Ig) superfamily in vitro and prepare polyclonal antibody against recombinant BmIBP2 for the further research about its biological function．【Method】 The gene fragment of the mature BmIBP2 peptide was amplified by PCR method with the cDNA synthesized from silkworm midgut. In order to express the protein of mature BmIBP2 peptide, the DNA segment was inserted into the expression plasmid pET-28a(+) to construct a recombinant expression plasmid. Polyclonal antibody was prepared to study the tissue-specificity expression of BmIBP2 protein in silkworm midgut. 【Result】 The SDS-PAGE result showed that the cloned recombinant BmIBP2 (rBmIBP2) was expressed in the form of inclusion bodies in E. coli BL21 with a molecular weight of 30 kD, and Western blot using the antibody against His-tag as the primary antibody indicated that rBmIBP2 had satisfied immunobiological activity. Pure protein was obtained by Ni2+ NTA resin column to prepare the anti-rBmIBP2 polyclonal antibody by immunizing New Zealand white rabbit. Western blot analysis showed that BmIBP2 protein could be bound with the polyclonal antibody in silkworm midgut with a molecular weight of 28 kD, which is consistent with the predicted molecular of BmIBP2 mature peptide．However, no specific bands were found in other tissues including fat body, hemocytes, silk gland, testicle and ovary. 【Conclusion】 Recombinant BmIBP2 protein was successfully expressed in E. coli BL21, and its polyelonal antibody was prepared in this study. Moreover, Western blot showed that BmIBP2 was correctly expressed in vitro and had a tissue-specificity expression in silkworm midgut.

Key words: Bombyx mori , insulin-related peptide-binding protein 2 (IBP2) , prokaryotic expression , purification , antibody

高坤, 邓祥元, 朱峰, 汪生鹏, 钱荷英, 郭锡杰. 家蚕类胰岛素相关肽结合蛋白2（BmIBP2）的原核表达与组织特异性分析[J]. 中国农业科学, 2012, 45(23): 4916-4923.

GAO Kun, DENG Xiang-Yuan, ZHU Feng, WANG Sheng-Peng, QIAN He-Ying, GUO Xi-Jie. Prokaryotic Expression of Bombyx mori Insulin-Related Peptide-Binding Protein 2 (BmIBP2) and Its Tissue-Specificity in Silkworm[J]. Scientia Agricultura Sinica, 2012, 45(23): 4916-4923.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2012.23.019

https://www.chinaagrisci.com/CN/Y2012/V45/I23/4916

参考文献

[1]Gao K, Deng X Y, Qian H Y, Wu P, Qin G X, Liu T, Shen Z Y, Guo X J. Novel protein of IBP from silkworm, Bombyx mori, involved in cytoplasmic polyhedrosis virus infection. Journal of Invertebrate Pathology, 2012, 110(1): 83-91.

[2]Kawakami A, Iwami M, Nagasawa H, Suzuki A, Ishizaki H. Structure and organization of four clustered genes that encode bombyxin, an insulin-related brain secretory peptide of the silkmorm Bombyx mori. Proceedings of the National Academy of Sciences of the United States of America, 1989, 86: 6843-6847.

[3]Gregoire F M, Chomiki N, Kachinskas D, Warden C H. Cloning and developmental regulation of a novel member of the insulin-like gene family in Caenorhabditis elegans. Biochemical and Biophysical Research Communications, 1998, 249: 385-390.

[4]Garofalo R S. Genetic analysis of insulin signaling in Drosophila. Trends in Endocrinology and Metabolism, 2002, 13(4): 156-162.

[5]Wu Q, Brown M R. Signaling and function of insulin-like peptides in insects. Annual Review of Entomology, 2006, 51: 1-24.

[6]Honegger B, Galic M, Kohler K, Wittwer F, Brogiolo W, Hafen E, Stocker H. Imp-L2, a putative homolog of vertebrate IGF-binding protein 7, counteracts insulin signaling in Drosophila and is essential for starvation resistance. Journal of Biology, 2008, 7: 10.

[7]Andersen A S, Hansen P H, Schaffer L, Kristensen C. A new secreted insect protein belonging to the immunoglobulin superfamily binds insulin and related peptides and inhibits their activities. The Journal of Biological Chemistry, 2000, 275(22): 16948-16953.

[8]Yamanaka Y, Wilson E M, Rosenfeld R G, Oh Y. Inhibition of insulin receptor activation by insulin-like growth factor binding proteins. The Journal of Biological Chemistry, 1997, 272(49): 30729-30734.

[9]Claeys I, Simonet G, Poels J, Van Loy T, Vercammen L, De Loof A, Vanden Broeck J. Insulin-related peptides and their conserved signal transduction pathway. Peptides, 2002, 23: 807-816.

[10]Kenyon C. The plasticity of aging: insights from long-lived mutants. Cell, 2005, 120: 449-460.

[11]Grotewiel M S, Martin I, Bhandari P, Cook-Wiens E. Functional senescence in Drosophila melanogaster. Ageing Research Reviews, 2005, 4: 372-397.

[12]Wessells R J, Bodmer R. Age-related cardiac deterioration: insights from Drosophila. Frontiers in Bioscience, 2007, 12: 39-48.

[13]Selman C, Lingard S, Choudhury A I, Batterham R L, Claret M, Clements M, Ramadani F, Okkenhaug K, Schuster E, Blanc E, Piper M D, Al-Qassab H, Speakman J R, Carmignac D, Robinson I C, Thornton J M, Gems D, Partridge L, Withers D J. Evidence for lifespan extension and delayed age-related biomarkers in insulin receptor substrate 1 null mice. The FASEB Journal, 2008, 22(3): 807-818.

[14]Cohen E, Bieschke J, Perciavalle R M, Kelly J W, Dillin A. Opposing activities protect against age-onset proteotoxicity. Science, 2006, 313(5793): 1604-1610.

[15]Pinkston J M, Garigan D, Hansen M, Kenyon C. Mutations that increase the life span of C. elegans inhibit tumor growth. Science, 2006, 313(5789): 971-975.

[16]Chen Y, Cui T, Knosel T, Yang L, Zoller K, Petersen I. IGFBP7 is a p53 target gene inactivated in human lung cancer by DNA hypermethylation. Lung Cancer, 2011, 73: 38-44.

[17]Chen Y, Pacyna-Gengelbach M, Ye F, Knosel T, Lund P, Deutschmann N, Schluns K, Kotb W F, Sers C, Yasumoto H, Usui T, Petersen I. Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) has potential tumour-suppressive activity in human lung cancer. The Journal of Pathology, 2007, 211(4): 431-438.

[18]Ruan W, Xu E, Xu F, Ma Y, Deng H, Huang Q, Lü B, Hu H, Lin J, Cui J, Di M, Dong J, Lai M. IGFBP7 plays a potential tumor suppressor role in colorectal carcinogenesis. Cancer Biology and Therapy, 2007, 6(3): 354-359.

[19]Ruan W J, Lin J, Xu E P, Xu F Y, Ma Y, Deng H, Huang Q, Lü B J, Hu H, Cui J, Di M J, Dong J K, Lai M D. IGFBP7 plays a potential tumor suppressor role against colorectal carcinogenesis with its expression associated with DNA hypomethylation of exon 1. Journal of Zhejiang University Science B, 2006, 7(11): 929-932.

[20]Wajapeyee N, Serra R W, Zhu X, Mahalingam M, Green M R. Oncogenic BRAF induces senescence and apoptosis through pathways mediated by the secreted protein IGFBP7. Cell, 2008, 132: 363-374.

[21]Akaogi K, Sato J, Okabe Y, Sakamoto Y, Yasumitsu H, Miyazaki K. Synergistic growth stimulation of mouse fibroblasts by tumor-derived adhesion factor with insulin-like growth factors and insulin. Cell Growth and Differentiation, 1996, 7: 1671-1677.

[22]Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 72: 248-254.

[23]马良. 家蚕BmIBP基因的原核表达及其亚细胞定位研究[D]. 杭州: 浙江理工大学, 2010.

Ma L. Expression analysis and subcellular localizaton of BmBP from silkworm Bombyx mori[D]. Hangzhou: Zhejiang Sci-Tech University, 2010. (in Chinese)

[24]Miyajima S, Kawase S. Changes in virus-infectivity titer in the hemolymph and midgut during the course of a cytoplasmic polyhedrosis in the silkworm. Journal of Invertebrates Pathology, 1968, 12: 329-334.

[25]张建红, 高玮, 张立人. 家蚕质型多角体病毒在蚕体内的形态发生及其细胞病理变化. 蚕业科学, 1990, 16(3): 140-144.

Zhang J H, Gao W, Zhang L R. Morphogenesis of cytoplasmic polyhedrsis virus of Bombyx mori in vivo and cytopathologic changes. Acta Sericologica Sinica, 1990, 16(3): 140-144. (in Chinese)

[26]Garbe J C, Yang E, Fristrom J W. IMP-L2: an essential secreted immunoglobulin family member implicated in neural and ectodermal development in Drosophila. Development, 1993, 119: 1237-1250.

[27]Gao K, Deng X, Qian H, Wu P, Qin G, Guo X. Cloning, characterization, and expression analysis of a novel BmGDAP1 gene from silkworm, Bombyx mori, involved in cytoplasmic polyhedrosis virus infection. Gene, 2012, 497: 208-213.

[28]Wu P, Wang X, Qin G X, Liu T, Jiang Y F, Li M W, Guo X J. Microarray analysis of the gene expression profile in the midgut of silkworm infected with cytoplasmic polyhedrosis virus. Molecular Biology Reports, 2011, 38: 333-341.

[29]吴萍, 刘挺, 覃光星, 郭锡杰. 家蚕中肠组织感染质型多角体病毒的应答基因分析. 中国农业科学, 2011, 44(13): 2814-2822.

Wu P, Liu T, Qin G X, Guo X J. Analysis of responsive genes in the midgut of silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus. Scientia Agricultura Sinica, 2011, 44(13): 2814-2822. (in Chinese)

[30]Li N, Zhang Z P, Zhang L L, Wang S H, Zou Z H, Wang G D, Wan Y L. Insulin-like growth factor binding protein 7, a member of insulin-like growth factor signal pathway, involved in immune response of small abalone Haliotis diversicolor. Fish and Shellfish Immunology, 2012, 33: 229-242.