家蚕表皮蛋白BmCPAP3-G的表达特征及其与几丁质的结合特性

doi:10.3864/j.issn.0578-1752.2017.09.018

摘要/Abstract

摘要： 【目的】探讨家蚕（Bombyx mori）表皮蛋白BmCPAP3-G的表达特征及与几丁质的结合方式，为家蚕表皮蛋白的功能研究提供依据。【方法】应用生物信息学方法分析家蚕CPAP家族表皮蛋白及BmCPAP3-G的结构域的序列特征；利用原核表达的方法表达BmCPAP3-G的融合蛋白，通过镍柱亲和层析技术纯化可溶性蛋白，利用5800 MALDI-TOF/TOF质谱鉴定正确后进行多克隆抗体制备，并利用几丁质亲和层析技术验证BmCPAP3-G与几丁质的结合；利用半定量RT-PCR和Western blot方法对BmCPAP3-G的组织和时期表达情况进行检测；利用几丁质亲和层析技术探讨体外BmCPAP3-G的结构域与几丁质结合能力强弱的关系。【结果】BmCPAP3-G蛋白具有18个氨基酸组成的信号肽，分子量为27 kD，等电点为4.82，位于第15号染色体上，由3个相同的ChtBD2结构域组成，并且其结构域中的半胱氨酸和芳香族氨基酸的同源性较高；对BmCPAP3-G进行了克隆和原核表达，并利用镍柱亲和层析技术纯化到了较纯的可溶性蛋白，经5800 MALDI-TOF/TOF鉴定正确后制备了其多克隆抗体，通过几丁质亲和层析技术验证了BmCPAP3-G能够与几丁质进行结合；利用半定量RT-PCR和Western blot的方法对BmCPAP3-G的组织和时期表达情况进行检测，发现转录水平和蛋白水平的结果一致，BmCPAP3-G在头和表皮中的表达量较高，在中肠、生殖腺和丝腺中的表达量较低，从表皮和丝腺的时期表达情况分析BmCPAP3-G在4龄眠期的表达量最高，随着5龄期的进行表达量呈逐渐下降的趋势；为了探讨BmCPAP3-G结构域与几丁质结合的关系，对该蛋白的结构域进行原核表达及镍柱亲和层析纯化，成功纯化了有活性的结构域3、结构域1-2、结构域2-3，将这3个结构域进行几丁质亲和层析验证其与几丁质的结合能力，发现它们均能够与几丁质进行结合，但是结合能力有差异，结构域3相比于结构域1-2和结构域2-3的结合能力要弱，即两个结构域比单个结构域与几丁质的结合能力强。【结论】BmCPAP3-G为典型的CPAP家族的表皮蛋白，可能参与几丁质层在眠期的降解再形成过程。BmCPAP3-G单个ChtBD2结构域就能够与几丁质结合，但多个结构域的同时存在加强了其与几丁质的结合能力。

关键词: 家蚕, 表皮蛋白, CPAP, 表达谱, 几丁质结合

Abstract: 【Objective】The objective of this study is to explore the expression pattern of BmCPAP3-G and the binding mode of BmCPAP3-G with chitin, which will lay a foundation for the research of the cuticle proteins of silkworm (Bombyx mori).【Method】The sequence features of CPAP motif cuticular proteins and the conserved domains of BmCPAP3-G were analyzed by bioinformatics methods. The recombinant proteins were expressed by prokaryotic expression and purified by Ni affinity chromatography. The protein was identified by 5800 MALDI-TOF/TOF mass spectrometry, and then was used to prepare the polyclonal antibodies. The chitin-binding activity of BmCPAP3-G was verified by chitin affinity chromatography. The spatial and temporal expression patterns of BmCPAP3-G were analyzed by semi-quantitative RT-PCR and western blot. The binding mode of the domains of BmCPAP3-G with chitin was detected by using chitin affinity chromatography. 【Result】The BmCPAP3-G protein has a signal peptide consisting of 18 amino acids, the molecular weight of 27 kD and the isoelectric point of 4.82, the encoding gene located on the chromosome No.15. BmCPAP3-G protein has three ChtBD2 domains, in which cysteine and aromatic amino acid showed very high homology. the BmCPAP3-G was cloned, expressed and the active recombinant protein was purified. After being identified by mass spectrometer, the polyclonal antibody against BmCPAP3-G was prepared. The BmCPAP3-G was found to bind chitin by using chitin affinity chromatography. The spatial and temporal expression patterns of BmCPAP3-G were analyzed by semi-quantitative RT-PCR and western blot, revealing similar results at the transcriptional and protein levels. BmCPAP3-G was expressed highly in the head and cuticle, and minimally in the midgut, gonad, and silk gland. In the silk gland, BmCPAP3-G had a high expression level in the fourth molting and its expression decreased in the cuticle and silk gland as the fifth instar goes on. active recombinant proteins domain_3, domain_1-2, domain_2-3 were successfully expressed and it was found that all the domain_3, domain_1-2, and domain_2-3 could bind to chitin in vitro, but their binding abilities were different. Individual domains could bind with chitin, and two domains showed stronger chitin binding capacity than the single domain. 【Conclusion】BmCPAP3-G is a typical cuticular protein of CPAP family and may be involved in the degradation and formation process of chitin layer in the silk gland during molting stage. BmCPAP3-G could bind with chitin by a single ChtBD2 domain, and multiple domains in which makes it have stronger chitin binding capacity.

Key words: silkworm (Bombyx mori), cuticular proteins, CPAP, expression pattern, chitin-binding

张薇薇，董照明，张艳，张晓璐，张守亚，赵萍. 家蚕表皮蛋白BmCPAP3-G的表达特征及其与几丁质的结合特性[J]. 中国农业科学, 2017, 50(9): 1723-1733.

ZHANG WeiWei, DONG ZhaoMing, ZHANG Yan, ZHANG XiaoLu, ZHANG ShouYa, ZHAO Ping. Expression Pattern and Chitin-Binding Mode Analyses of Cuticle Protein BmCPAP3-G in the Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2017, 50(9): 1723-1733.

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