家蚕表皮蛋白BmCPAP3-G的表达特征及其与几丁质的结合特性  

Expression Pattern and Chitin-Binding Mode Analyses of Cuticle Protein BmCPAP3-G in the Silkworm(Bombyx mori)

作　　者：张薇薇[1] 董照明[1] 张艳[1] 张晓璐[1] 张守亚[1] 赵萍[1] 

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室,重庆400716

出　　处：《中国农业科学》2017年第9期1723-1733,共11页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31530071;31472154);西南大学博士基金项目(SWU116076)

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 k D,等电点为4.82,位于第15号染色体上,由3个相同的Cht BD2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[ 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 westem 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_l-2, domain_2-3 were successf

关 键 词：家蚕 表皮蛋白 CPAP 表达谱 几丁质结合 

分 类 号：S881.2[农业科学—特种经济动物饲养]