绿盲蝽表皮蛋白基因AlCP17的克隆、多克隆抗体制备及表达谱分析  

Cloning,polyclonal antibody preparation and expression profiling of cuticular protein gene AlCP17 from Apolygus lucorum(Hemiptera:Miridae)

作　　者：谭永安[1] 张杰钰 姜义平 赵静[1] 肖留斌[1] 戈林泉[2] TAN Yong-An;ZHANG Jie-Yu;JIANG Yi-Ping;ZHAO Jing;XIAO Liu-Bin;GE Lin-Quan(Institute of Plant Protection,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;School of Horticulture and Plant Protection,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]江苏省农业科学院植物保护研究所,南京210014 [2]扬州大学园艺与植物保护学院,扬州225009

出　　处：《昆虫学报》2023年第1期37-44,共8页Acta Entomologica Sinica

基　　金：江苏省自主创新资金[cx(21)3088,cx(22)2038];国家自然科学基金项目(31301668);江苏省重点研发计划(现代农业)面上项目(BE2021303)。

摘　　要：【目的】本研究旨在通过克隆绿盲蝽Apolygus lucorum表皮蛋白(cuticular protein,CP)基因AlCP17、制备其多克隆抗体和分析其时空表达特性,为AlCP17蛋白功能的研究奠定基础,也为进一步解析绿盲蝽表皮发育过程中的信号通路图谱提供依据。【方法】基于前期转录组数据克隆绿盲蝽AlCP17的cDNA全长序列并进行生物信息学分析;构建AlCP17原核表达载体pCZN1-AlCP17并转化大肠杆菌Escherichia coli进行体外表达,利用纯化后的重组蛋白制备AlCP17蛋白多克隆抗体;qRT-PCR检测AlCP17在绿盲蝽不同发育阶段(1-5龄若虫和成虫)和3龄初期若虫不同组织(头、胸、足、中肠、脂肪体和表皮)中的表达谱。【结果】克隆获得绿盲蝽AlCP17全长cDNA序列(GenBank登录号:OM302231),其开放阅读框长594 bp,编码197个氨基酸,预测蛋白分子量为21.69 kD,理论等电点为6.11。编码的蛋白质具有典型的表皮蛋白结构特征,含有节肢动物表皮蛋白几丁质保守结合域(non-cysCBD),即Chitin_bind_4结构域;氨基酸序列比结果对显示,AlCP17与半翅目(Hemiptera)臭虫科(Cimicidae)温带臭虫Cimex lectularius的CPA2B-like的氨基酸序列一致性最高(76.29%);系统发育进化树显示,CP是一种高度保守的蛋白,与温带臭虫的CPA2B-like和茶翅蝽Halyomorpha halys的CP7-like相似性高。IPTG诱导获得原核表达重组蛋白AlCP17,经纯化后制备的AlCP17多克隆抗体纯化效果较好,可以用于后续实验。AlCP17在绿盲蝽不同发育阶段和3龄初期若虫不同组织中均有表达,在初孵1龄若虫中表达量达到峰值,且在若虫各龄期的末期表达量显著上升;在3龄初期若虫中肠中表达量最高。【结论】克隆了绿盲蝽AlCP17基因cDNA全长序列,AlCP17具有昆虫表皮蛋白的典型特征,AlCP17在绿盲蝽体内的表达具有发育阶段特异性和组织特异性。这些结果为今后研究这一蛋白在绿盲蝽表皮发育过程中的生理功能奠定了基础。【Aim】This study aims to lay the foundation for the functional study of cuticular protein(CP)17 of Apolygus lucorum(AlCP17)and to provide a preliminary basis for further analyzing the signal pathway map of cuticular development of A.lucorum by cloning its gene AlCP17,preparing its polyclonal antibody and analyzing its expression profiles.【Methods】Based on the previous transcriptome data,the full-length cDNA sequence of AlCP17 of A.lucorum was cloned and subjected to bioinformatics analysis.The prokaryotic expression vector pCZN1-AlCP17 was constructed and transformed to Escherichia coli to conduct in vitro expression.The purified recombinant protein was used to prepare polyclonal antibody of AlCP17.qRT-PCR was used to analyze the expression profiles of AlCP17 in different developmental stages(1st-5th instar nymphs and adult)and different tissues(head,thorax,leg,midgut,fat body and cuticle)of the early 3rd instar nymphs of A.lucorum.【Results】The full-length cDNA sequence of AlCP17(GenBank accession no.:OM302231)was cloned from A.lucorum with an open reading frame of 594 bp in length,encoding a putative protein of 197 amino acids with the predicted molecular weight of 21.69 kD and the theoretical isoelectric point of 6.11.AlCP17 has the typical structure characteristics of cuticular protein,containing the conserved chitin binding domain(non-cysCBD)of arthropod cuticular protein,namely Chitin_bind_4 domain.Amino acid sequence alignment result showed that AlCP17 shows the highest amino acid sequence identity(76.29%)with the CPA2B-like of Cimex lectularius of Cimicidae of Hemiptera.Phylogenetic tree showed that CP is a highly conserved protein with high similarity to CPA2B-like of C.lectularius and CP7-like of Halyomorpha halys.The prokaryotically expressed recombinant AlCP17 was obtained by IPTG induction.After purification,the polyclonal antibody of AlCP17 was obtained and had good purity for subsequent experiments.AlCP17 was expressed in different developmental stages and different tissues of the early 3

关 键 词：绿盲蝽 表皮蛋白 RACE 原核表达 多克隆抗体 

分 类 号：Q966[生物学—昆虫学]