绿盲蝽AlEcR-A的单克隆抗体制备及在外源20E诱导下的应答

doi:10.3864/j.issn.0578-1752.2017.01.008

摘要/Abstract

摘要： 【目的】获得绿盲蝽（Apolygus lucorum）蜕皮激素受体（AlEcR-A）原核表达的重组蛋白，制备单克隆抗体，分析绿盲蝽AlEcR-A在外源蜕皮激素(20E)诱导下mRNA及蛋白表达量的变化趋势，为进一步研究EcR的功能奠定前期基础，同时也为构建20E信号传导的网络图谱提供理论依据。【方法】在前期获得绿盲蝽AlEcR-A的基础上，将含有其基因的T载体经Nde I和XbaI双酶切，构建AlEcR-A原核表达载体（pCzn1-AlEcR-A），将该表达载体经IPTG诱导表达和蛋白Ni-IDA亲和纯化，以获得AlEcR-A基因功能区的纯化蛋白。进一步用其进行免疫反应，取小鼠的脾细胞与SP2/0细胞进行细胞融合，采用间接ELISA及Western blot筛选验证是否为目的抗体，通过抗体纯化，Western blot检测该抗体能否特异性结合AlEcR-A重组蛋白及绿盲蝽总蛋白，从而制备得到AlEcR-A蛋白单克隆抗体。最后利用RT-PCR及Western blot方法，进行20E微注射绿盲蝽2龄若虫，分析8 d内AlEcR-A的mRNA及蛋白含量的变化，明晰20E诱导下AlEcR-A的应答反应。【结果】经Nde I和XbaI双酶切后原核表达载体pCzn1-AlEcR-A在大肠杆菌Arctic express中能高效表达一个约为55 kD的蛋白，且该重组蛋白经IPTG诱导后主要以包涵体的形式存在；经Ni-IDA亲和层析后，pCzn1-AlEcR-A重组蛋白的包涵体纯度仅在55 kD附近有一条明显的特异性条带，说明靶蛋白已得到纯化。进一步通过小鼠免疫、细胞融合及腹水制备，获得了1株能稳定分泌抗AlEcR-A蛋白单克隆抗体的细胞株，命名为8H7；Western blot分析表明，该细胞株不仅能与绿盲蝽总蛋白结合，还可特异性与AlEcR-A重组蛋白反应，且条带大小一致，说明制备的AlEcR-A单克隆抗体准确、有效；RT-PCR及Western blot结果表明，与注射蒸馏水相比，20E微注射处理后的绿盲蝽AlEcR-A mRNA表达量及蛋白表达量均显著升高，且随着处理时间的延长，其增值幅度也逐渐增高。【结论】获得了一株高特异性的能稳定分泌AlEcR-A单克隆抗体的细胞株，20E有诱导AlEcR-A mRNA及蛋白表达的作用。

关键词: 绿盲蝽, 蜕皮激素受体, 20-羟基蜕皮酮, 单克隆抗体, 表达量

Abstract: 【Objective】The objectives of this study are to prepare the monoclonal antibody against ecdysone receptors protein and clarity the response of AlEcR-A under exogenous ecdysterone hormone (20E) in Apolygus lucorum.【Method】The vector containing AlEcR-A was double enzyme restricted by Nde Iand Xba I, then the cDNA identified by sequencing was constructed to pCzn1 vector and transformed into Arctic express of E. coli. The target recombinant protein has over expressed and has been purified by using Ni-NTA agarose. BALB/c mice were immunized with the purified recombinant fusion protein. The spleen cells of mouse were fused with SP2/0 cells. The specificity of hybridoma cell line was characterized by ELISA and Western blot analysis. Finally, the mRNA relative expression and protein content of AlEcR-A treated with exogenous 20E by RT-PCR and Western blot were analyzed, respectively.【Result】The recombinant plasmid pCzn1-AlEcR-A was high-efficient expression in Arctic express when induced by 37℃ and 5.0 mmol·L^-1IPTG. The 55 kD target protein from the strain containing AlEcR-A was obtained by the Ni-NTA agarose. One hybridoma cell line, named 8H7, was obtained and characterized by ELISA and Western blot analysis, which could specifically combine with the total protein of A. lucorum and recombinant protein of AlEcR-A. By using RT-PCR and Western blot analysis, both the mRNA relative expression and protein content of AlEcR-A were remarkably increased after treating with 20E which compared with control.【Conclusion】the high specificity monoclonal antibody of AlEcR-A protein was obtained, and 20E could induce the mRNA and protein expressions of AlEcR-A.

Key words: Apolygus lucorum, ecdysone receptor, 20E, monoclonal antibodies, expression level

谭永安，肖留斌，郝德君，赵静，孙洋，柏立新. 绿盲蝽AlEcR-A的单克隆抗体制备及在外源20E诱导下的应答[J]. 中国农业科学, 2017, 50(1): 86-93.

TAN YongAn, XIAO LiuBin, HAO DeJun, ZHAO Jing, SUN Yang, BAI LiXin. Preparation of Monoclonal Antibody Against AlEcR-A Protein and Its Response Induced by Exogenous 20-Hydroxyecdysone in Apolygus lucorum[J]. Scientia Agricultura Sinica, 2017, 50(1): 86-93.

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参考文献

[1] Lu Y H, Wu K M, Jiang Y Y, Xia B, Li P, Feng H Q, Wyckhuys K A G, Guo Y Y. Mirid bug outbreaks in multiple crops correlated with wide-scale adoption of Bt cotton in China. Science, 2010, 328(5982): 1151-1154.

[2] Lu Y H, Wu K M, Wyckhuys K A G, Guo Y Y. Overwintering hosts of Apolygus lucorum (Hemiptera: Miridae)in northern China. Crop Protection, 2010, 29(9): 1026-1033.

[3] Wu K M, Li W, Feng H Q, Guo Y Y. Seasonal abundance of the mirids, Lygus luconun and Adelphocoris spp. (Hemiptera: Miridae) on Bt cotton in northern China. Crop Protection, 2002, 21(10): 997-1002.

[4] Lu Y H, Wu K M, Jiang Y Y, Guo Y Y, Desneux N. Widespread adoption of Bt cotton and insecticide decrease promotes biocontrol services. Nature, 2012, 487(7407): 362-365.

[5] Nedjoua Z, Noureddine S. Environmental risks of two chitin synthesis inhibitors on Gambsia affinis: chronic effects on growth and recovery of biological responses. Biological Control, 2011, 59: 106-113.

[6] Li Y Q, Qin Y G, Yang N, Sun Y F, Yang X L, Sun R F, Wang Q M, Ling Y. Studies on insecticidal activities and action mechanism of novel benzoylphenylurea candidate NK-17. PLoS ONE, 2013, 8(6): e66251.

[7] Levine R B, Fahrback S E, Weeks J C. Steroid hormones and the reorganization of the nervous system during metamorphosis. Seminars in Neuroscience, 1991, 3(6): 437-447.

[8] Billas I M L, Browning C, Lawrence M C, Graham L D, Moras D, Hill R. The structure and function of ecdysone receptor//Smagghe G. Ecdysone: structures and functions. Springer, Berlin, 2009: 335-360.

[9] Riddiford L M, Cherbas P, Truman J W. Ecdysone receptors and their biological actions. Vitamins & Hormones, 2000, 60: 1-73.

[10] Reinking J, Lam M M S, Pardee K, Sampson H M, Liu S, Yang P, Williams S, White W, Lajoie G, Edwards A, Krause H M. The Drosophila nuclear receptor E75 contains heme and is gas responsive. Cell, 2005, 122(2): 195-207.

[11] Riddiford L M, Hiruma K, Zhou X F, Nelson C A. Insights into the molecular basis of the hormonal control of molting and metamorphosis from Manduca sexta and Drosophila melanogaster. Insect Biochemistry and Molecular Biology, 2003, 33(12): 1327-1338.

[12] 郭恩恩, 李胜, 曹阳. 昆虫蜕皮的激素网络调控. 蚕业科学, 2008, 34(2): 370-374.

Guo E E, Li S, Cao Y. The hormonal regulation network of insect molting. Science of Sericulture, 2008, 34(2): 370-374. (in Chinese)

[13] Spindler K D, Hönl C, Tremmel C, Braun S, Ruff H, Spindler-Barth M. Ecdysteroid hormone action. Cellular and Molecular Life Sciences, 2009, 66(24): 3837-3850.

[14] 武强. 双水相萃取分离免疫球蛋白和单克隆抗体研究[D]. 杭州: 浙江大学, 2014.

WU Q. Separation of immunoglobulin and monoclonal antibody by aqueous two-phase extraction[D]. Hangzhou: Zhejiang University, 2014. (in Chinese)

[15] Song N N, Ding W H, Chu S Y, Zhao J, Dong X, Di B B, Tang C S. Urotensin II stimulates vascular endothelial growth actor secretion from adventitial fibroblasts in synergy with angiotensin II. Circulation Journal, 2012, 76(5): 1267-1273.

[16] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-△△CT method. Methods, 2001, 25(4): 402-408.

[17] Schluepmann H, Pellny T, van Dijken A, Semmkens S, Paul M. Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100 (11): 6849-6854.

[18] 谭永安, 肖留斌, 孙洋, 柏立新. 绿盲蝽水溶性海藻糖酶ALTre-1基因原核表达、纯化与酶学特性. 中国农业科学, 2013, 46(17): 3587-3593.

Tan Y A, Xiao L B, Sun Y, Bai L X. Prokaryotic expression, purification and functional activity assay in vitro of soluble trehalse from Apolygus lucorum. Scientia Agricultura Sinica, 2013, 46(17): 3587-3593. (in Chinese)

[19] 谭永安, 肖留斌, 柏立新, 孙洋. 绿盲蝽膜结合型海藻糖酶ALTre-2基因表达、纯化及酶学特性. 棉花学报, 2013, 25(5): 396-402.

Tan Y A, Xiao L B, Bai L X, Sun Y. Expression, purification and enzymatic activity of a membrane-bound trehalase gene from Apolygus lucorum. Cotton Science, 2013, 25(5): 396-402. (in Chinese)

[20] Hoskins R A, Carlson J W, Kennedy C, Acevedo D, Evans-Holm M, Frise E, Wan K H, Park S, Mendez-Lago M, Rossi F, Villasante A, Dimitri P, Karpen G H, Celniker S E. Sequence finishing and mapping of Drosophila melanogaster heterochromatin. Science, 2007, 316(5831): 1625-1628.

[21] Tan Y A, Xiao L B, Zhao J, Sun Y, Bai L X. Molecular and functional characterization of the ecdysone receptor isoform-A from the cotton mirid bug, Apolygus lucorum (Meyer-Dür). Gene, 2015, 574(1): 88-94.

[22] Tan Y A, Xiao L B, Zhao J, Xiao Y F, Sun Y, Bai L X. Ecdysone receptor isoform-B mediate soluble trehalase expression to regulate growth and development in the mirid bug, Apolygus lucorum (Meyer-Dür). Insect Molecular Biology, 2015, 24(6): 611-623.

[23] Arbeitman M N, Hogness D S. Molecular chaperones activate the Drosophila ecdysone receptor, an RXR heterodimer. Cell, 2000, 101(1): 67-77.

[24] Kimura S, Sawatsubashi S, Ito S, Kouzmenko A, Suzuki E, Zhao Y, Yamagata K, Tanabe M, Ueda T, Fujiyama S, Murata T, Matsukawa H, Takeyama K, Yaegashi N, Kato S. Drosophila arginine methyltransferase 1 (DART1) is an ecdysone receptor co-repressor. Biochemical and Biophysical Research Communications, 2008, 371(4): 889-893.

[25] Sun Y N, An S H, Henrich V C, Sun X P, Song Q S. Proteomic identification of PKC-mediated expression of 20E-induced protein in Drosophila melanogaster. Journal of Proteome Research, 2007, 6(11): 4478-4488.

[26] Jing Y P, Liu W, Wang J X, Zhao X F. The steroid hormone 20-hydroxyecdysone via non-genomic pathway activates Ca²⁺/calmodulin- dependent protein Ⅱ to regulate gene expression. The Journal of Biological Chemistry, 2015, 290(13): 8469-8481.

[27] Manabu K, Shuichiro T, Makoto K, Haruhiko F. Tissue-specific and stage-specific expression of two silkworm ecdysone receptor isoforms ecdysteroid-dependent transcription in cultured anterior silk glands. European Journal of Biochemistry, 1997, 248(3): 786-793.

[28] Lu Y H, Wu K M, Wyckhuys K A G, Guo Y Y. Temperature- dependent life history of the green plant bug, Apolygus lucorum (Meyer-Dür) (Hemiptera: Miridae). Applied Entomology and Zoology, 2010, 45(3): 387-393.