家蚕卵黄原蛋白受体BmVgR的体外表达

doi:10.3864/j.issn.0578-1752.2014.19.022

摘要/Abstract

摘要： 【目的】克隆家蚕卵黄原蛋白受体（vitellogenin receptor, VgR），分析其在昆虫细胞中的表达特征，了解正常VgR和突变VgR的表达模式，为阐明其生物学功能提供参考依据。【方法】基于家蚕VgR的基因编码序列设计特异引物，扩增的目的片段连接到载体pET28a上，构建原核表达载体，转化E. coil BL21（DE3）表达菌株，IPTG诱导获得目的蛋白；用镍柱亲和层析方法纯化诱导表达的家蚕VgR肽蛋白；将纯化后的肽蛋白制备BmVgR的兔源多克隆抗体；PCR扩增获得大造和突变型白妙卵（scanty vitellin，vit）的LBD1+EGF1结构域（C11D和C11V）片段，连接到细胞表达载体上，通过细胞转染表达目的蛋白，采用细胞免疫组化检测大造和突变型vit的VgR在细胞里的表达情况；通过Western blot检测细胞培养液中大造和突变型vit VgR的表达量。【结果】原核表达获得了一个大小约为22 kD的融合蛋白，以包涵体形式表达；镍柱亲和层析初纯化得到BmVgR的肽蛋白，进一步切胶纯化后，以此为抗原制备BmVgR的兔源多克隆抗体，其抗体效价＞1﹕512 000，纯度为95%以上。转录水平检测表明Sf9和Spli221昆虫细胞中没有BmVgR和BmVg内源基因的表达；在Sf9昆虫细胞中成功表达了家蚕野生型和突变型vit BmVgR的结构域SP+LBD1+EGF1肽段，家蚕突变型vit在BmVgR第1个表皮生长因子同源区域的第3个ClassB区域有编码50个氨基酸残基的核酸序列缺失；细胞免疫组化试验表明，突变型和正常型的C11V和C11D都能在Sf9细胞质中正常表达；制备的BmVgR兔源多克隆抗体和Myc标签抗体同时检测到细胞表达的目的蛋白，表明试验所制备的VgR抗体特异性较好；由于SP+LBD1+EGF1肽段存在信号肽，细胞表达的蛋白会分泌进入细胞培养液中，Western blot对培养液进行蛋白检测，表明突变型vit存在氨基酸缺失，但并不影响肽蛋白的正常表达，且表达量没有明显差异。【结论】EGF1结构域的缺失并不影响SP+LBD1+EGF1肽蛋白的正常表达，可能是其蛋白功能异常导致突变型vit表型产生。

关键词: 家蚕, 卵黄原蛋白受体, 原核表达, 细胞表达

Abstract: 【Objective】 The objective of this study is to clone the silkworm BmVgR, to analyze its expression characteristics in insect cells and to investigate the expression patterns of the normal and mutational VgRs, thus providing a basis for its function identification. 【Method】 Specific primer sequences were designed according to BmVgR coding sequence, the amplified fragment was connected to the pET28a, and then transformed into E. coil BL21 (DE3), the fusion protein was obtained by IPTG induction. Ni-NTA affinity chromatography was used to purify the BmVgR protein peptide. Then the anti-BmVgR rabbit polyclonal antibody was obtained by BmVgR protein peptide as an antigen. The LBD1+EGF1 (C11D and C11V) domain of the normal and mutational VgRs was connected to the cell expression vector, the target protein was expressed with cell transfection. Immunohistochemistry was used to analyze the expression characteristics of the normal and mutational VgRs in insect cells. Western blot was used to detect the expression levels of the normal and mutational VgRs in the cell culture medium.【Result】 Prokaryotic expression gained a size of about 22 kD fusion protein, which was expressed as inclusion bodies. BmVgR protein peptide was expressed and purified, the anti-BmVgR rabbit polyclonal antibody was obtained, its titer was higher than 1﹕512 000. Purity was more than 95%. The endogenous genes of BmVgR and BmVg did not express in Sf9 and Spli221. The normal and mutational BmVgR-SP+LBD1+EGF1 was successfully expressed in the Sf9 cell (the BmVgR of the vit mutant, which was mutated in the 3rd Class B region of the EGF1 domain, coding 50 amino acid residuces). Immunohistochemistry showed that vit mutant and normal BmVgR could express normally in Sf9 cytoplasm. BmVgR rabbit polyclonal antibody and Myc-tagged antibody simultaneously detected the target protein, which indicated that the VgR antibody was better. As the SP + LBD1 + EGF1 peptide existed signal peptide, the expressed protein was secreted into the cell culture medium. Western blot indicated that the presence of amino acid deletion of the mutant vit did not affect the expression of the normal protein peptide, and the expression levels of the normal and mutational VgRs were not significantly different.【Conclusion】 The deletion of EGF1 domain didn’t affect the normal expression of SP+LBD1+EGF1 peptide protein, which may lead to the phenotype of the vit.

Key words: Bombyx mori, vitellogenin receptor, prokaryotic expression,

')">cell expression

罗娟，陈恩祥，刘红玲，彭芷昕，杨从文，沈关望，张海燕，邢润苗，林英，夏庆友. 家蚕卵黄原蛋白受体BmVgR的体外表达[J]. 中国农业科学, 2014, 47(19): 3922-3928.

LUO Juan, CHEN En-xiang, LIU Hong-ling, PENG Zhi-xin, YANG Cong-wen, SHEN Guan-wang, ZHANG Hai-yan, XING Run-miao, LIN Ying, XIA Qing-you. Expression of Silkworm Vitellogenin Receptor in Vitro[J]. Scientia Agricultura Sinica, 2014, 47(19): 3922-3928.

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参考文献

[1] Tufail M, Takeda M. Molecular characteristics of insect vitellogenins. Journal of Insect Physiology, 2008, 54: 1447-1458.
[2] Sappington T W, Raikhel A S. Molecular characteristics of insect vitellogenins and vitellogenin receptors. Insect Biochemistry and Molecular Biology, 1998, 28: 277-300.
[3] Schonbaum C P, Lee S, Mahowald A P. The Drosophila yolkless gene encodes a vitellogenin receptor belonging to the low density lipoprotein receptor superfamily. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92: 1485-1489.
[4] DiMario P J, Mahowald A P. Female sterile (1) yolkless: a recessive female sterile mutation in Drosophila melanogaster with depressed numbers of coated pits and coated vesicles within the developing oocytes. The Journal of Cell Biology, 1987, 105: 199-206.
[5] Bujo H, Yamamoto T, Hayashi K, Hermann M, Nimpf J, Schneider W J. Mutant oocytic low density lipoprotein receptor gene family member causes atherosclerosis and female sterility. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92: 9905-9909.
[6] Ciudad L, Piulachs M D, Belles X. Systemic RNAi of the cockroach vitellogenin receptor results in a phenotype similar to that of the Drosophila yolkless mutant. The FEBS Journal, 2006, 273: 325-335.
[7] Shu Y H, Wang J W, Lu K, Zhou J L, Zhou Q, Zhang G R. The first vitellogenin receptor from a Lepidopteran insect: molecular characterization, expression patterns and RNA interference analysis. Insect Molecular Biology, 2011, 20: 61-73.
[8] Tufail M, Takeda M. Molecular cloning, characterization and regulation of the cockroach vitellogenin receptor during oogenesis. Insect Molecular Biology, 2005, 14(4): 389-401.
[9] Tufail M, Takeda M. Molecular cloning and developmental expression pattern of the vitellogenin receptor from the cockroach, Leucophaea maderae. Insect Biochemistry and Molecular Biology, 2007, 37: 235-245.
[10] Ferenz H J. Yolk protein accumulation in Locusta migratoria (R. & F.) (Orthoptera: Acrididae) oocytes. International Journal of Insect Morphology and Embryology, 1993, 22(2/4): 295-314.
[11] Tufail M, Takeda M. Insect vitellogenin/lipophorin receptors: molecular structures, role in oogenesis, and regulatory mechanisms. Journal of Insect Physiology, 2009, 55: 88-104.
[12] Hiesberger T, Hermann M, Jacobsen L, Novak S, Hodits R A, Bujo H, Meilinger M, Hüttinger M, Schneider W J, Nimpf J. The chicken oocyte receptor for yolk precursors as a model for studying the action of receptor-associated protein and lactoferrin. The Journal of Biological Chemistry, 1995, 270(31): 18219-18226.
[13] Kounnas M Z, Church F C, Argraves W S, Strickland D K. Cellular internalization and degradation of antithrombin III-thrombin, heparin cofactor II-thrombin, and α1-antitrypsin-trypsin complexes is mediated by the low density lipoprotein receptor-related protein. The Journal of Biological Chemistry, 1996, 271(11): 6523-6529.
[14] Stifani S, Barber D L, Nimpf J, Schneider W J. A single chicken oocyte plasma membrane protein mediates uptake of very low density lipoprotein and vitellogenin. Proceedings of the National Academy of Sciences of the United States of America, 1990, 87: 1955-1959.
[15] Strickland D K, Kounnas M Z, Argraves W S. LDL receptor-related protein: a multiligand receptor for lipoprotein and proteinase catabolism. FASEB Journal, 1995, 9: 890-898.
[16] Rudenko G, Henry L, Henderson K, Ichtchenko K, Brown M S, Goldstein J L, Deisenhofer J. Structure of the LDL receptor extracellular domain at endosomal pH. Science, 2002, 298: 2353-2358.
[17] Raikhel A S, Dhadialla T S. Accumulation of yolk proteins in insect oocytes. Annual Review of Entomology, 1992, 37: 217-251.
[18] Fujikawa K, Kawaguchi Y, Kusakabe T, Koga K. Histological studies on the yolk granule formation in the egg character mutant, vit, of Bombyx mori. Journal of Insect Biotechnology and Sericology, 2003, 72: 111-115.
[19] Kawaguchi Y, Tatsuke T, Oike Y, Taniguchi A, Kusakabe T, Lee J M, Koga K. Fertility and hatching of the vit mutant eggs in Bombyx mori. Journal of Insect Biotechnology and Sericology, 2008, 77: 121-124.
[20] 林英, 赵萍, 侯勇, 李娟, 龚达平, 孙全, 夏庆友, 向仲怀. 家蚕卵黄原蛋白及其受体基因. 动物学报, 2005, 51(1): 117-125.
Lin Y, Zhao P, Hou Y, Li J, Gong D P, Sun Q, Xia Q Y, Xiang Z H. Vitellogenin and vitellogenin receptor gene of the silkworm Bombyx mori. Acta Zoologica Sinica, 2005, 51(1): 117-125. (in Chinese)
[21] 王艳霞. 家蚕卵黄原蛋白受体 (BmVgR) 的全长克隆及功能分析[D]. 重庆: 西南大学, 2010.
Wang Y X. The cloning and functional analysis of vitellogenin receptor in silkworm, Bombyx mori[D]. Chongqing: Southwest University, 2010. (in Chinese)
[22] Jeon H, Blacklow S C. An intramolecular spin of the LDL receptor β propeller. Structure, 2003, 11: 133-136.
[23] Van Hoof D, Rodenburg K W, Van der Horst D J. Intracellular fate of LDL receptor family members depends on the cooperation between their ligand-binding and EGF domains. Journal of Cell Science, 2005, 118: 1309-1320.