香港牡蛎cyclin B基因组织表达特征及其多克隆抗体制备  

Tissue distribution characteristics of cyclin B gene in Hong Kong oyster(Crassostrea hongkongensis)and preparation of its polyclonal antibody

作　　者：邱衡通 许贵林 李宛怡 周齐家 刘小宁 朱俊霖 QIU Heng-tong;XU Gui-lin;LI Wan-yi;ZHOU Qi-jia;LIU Xiao-ning;ZHU Jun-lin(Key Laboratory of Environment Change and Resources Use in Beibu Gulf,Ministry of Education/Institute of Geography and Oceanography,Nanning Normal University,Nanning,Guangxi 530100,China;Guangxi Academy of Marine Sciences(Guangxi Mangrove Research Center),Guangxi Academy of Sciences/Research Center for Carbon Sink and Low-carbon Engineering in the Beibu Gulf of Guangxi,Nanning,Guangxi 530007,China)

机构地区：[1]北部湾环境演变与资源利用教育部重点实验室/南宁师范大学地理与海洋研究院,广西南宁530100 [2]广西科学院广西海洋科学院(广西红树林研究中心)/广西北部湾碳汇与低碳工程研究中心,广西南宁530007

出　　处：《南方农业学报》2024年第4期909-919,共11页Journal of Southern Agriculture

基　　金：国家重点研发计划“海洋农业与淡水渔业科技创新”重点专项(2022YFD2401200);广西科技基地和人才专项(桂科AD21075012);北部湾环境演变与资源利用教育部重点实验室系统基金项目(KLOP-X1910);广西大学生创新创业计划项目(S202310603072)。

摘　　要：【目的】明确香港牡蛎(Crassostrea hongkongensis)B型细胞周期蛋白基因(cyclin B)组织表达特征,并通过原核表达融合蛋白制备多克隆抗体,为探究香港牡蛎cyclin B基因在环境胁迫下的表达特征及揭示cyclin B调控贝类细胞周期的作用机制提供理论依据。【方法】克隆香港牡蛎cyclinB基因c DNA序列,通过SignalP-5.0、ProtParam、TMHMM2.0、NetSurfP2.0及Bioedit7.0等软件进行生物信息学分析,采用实时荧光定量PCR检测cyclinB基因在香港牡蛎不同组织中的表达情况;构建重组质粒pET32a-cyclin B并运用原核表达系统诱导表达融合蛋白,经Ni-NTA镍离子亲和层析柱纯化后免疫BALB/c小鼠制备鼠源多克隆抗体,然后以间接ELISA检测多克隆抗体效价、Western blotting检测多克隆抗体特异性。【结果】香港牡蛎cyclinB基因cDNA序列全长2353bp,包括1299bp的开放阅读框(ORF)、103 bp的5’非编码区(5’-UTR)和951 bp的3’非编码区(3’-UTR),共编码432个氨基酸残基。香港牡蛎cyclin B蛋白相对分子量为49.13 kD,理论等电点为7.16,不含信号肽和跨膜结构域。香港牡蛎cyclin B氨基酸序列包含2个保守的周期蛋白框,与来自同属的长牡蛎cyclin B氨基酸序列相似性最高(98.8%),基于cyclin B氨基酸序列相似性构建的系统发育进化树也显示香港牡蛎与长牡蛎的亲缘关系最近。cyclin B基因在香港牡蛎的闭壳肌、外套膜、性腺、鳃、唇瓣、消化腺等组织中均有表达,以性腺中的相对表达量最高,显著高于其他组织中的相对表达量(P<0.05)。通过原核表达获得的融合蛋白cyclin B具备良好免疫原性,且主要以包涵体形式存在;以其免疫BALB/c小鼠制备出的鼠源多克隆抗体效价大于1∶64000,能特异识别香港牡蛎性腺中的cyclin B蛋白。【结论】香港牡蛎cyclin B基因具有较高的保守性,其氨基酸序列包含2个保守的周期蛋白框;香港牡蛎cyclin B基因表达具有广谱性和组织差异【Objective】The objective of this study was to clarify the tissue expression characteristics of cyclin B protein gene(cyclin B)in Hong Kong oyster(Crassostrea hongkongensis),and to prepare polyclonal antibody by prokaryo-tic expression fusion protein,so as to provide a theoretical basis for exploring the expression characteristics of cyclin B in C.hongkongensis under environmental stress and revealing the mechanism of cyclin B in regulating the cell cycle of shellfish.【Method】The cDNA sequence of cyclin B gene was cloned from C.hongkongensis,and the bioinformatics analysis was performed by softwares such as SignalP-5.0,ProtParam,TMHMM 2.0,NetSurfP 2.0 and Bioedit 7.0,and the expression of cyclin B gene in different tissues was detected by real-time fluorescence quantitative PCR.The recombinant plasmid pET32a-cyclin B was constructed,the fusion protein was induced by the prokaryotic expression.Polyclonal antibodies were prepared by immunizing BALB/c mice after purification by Ni-NTA nickel ion affinity chromatography column,and then the polyclonal antibody titer was detected by indirect ELISA and the specificity of polyclonal antibody was detected by Western blotting.【Result】The cDNA sequence of cyclin B gene in C.hongkongensis was 2353 bp in length,including 1299 bp open reading frame(ORF),103 bp 5'non-coding region(5'-UTR)and 951 bp 3'non-coding region(3'-UTR),encoding a total of 432 amino acid residues.The calculated molecular weight of cyclin B protein in C.hongkongensis was 49.13 kD,the theoretical isoelectric point was 7.16,and it did not contain signal peptide and transmembrane domain.The amino acid sequence of cyclin B contained two conserved cyclin boxes,and the amino acid sequence of cyclin B was the most similar to that of Pacific oyster(98.8%),the phylogenetic tree based on amino acid sequence also showed that cyclin B of C.hongkongensis was the most closely related to that of Pacific oyster.The cyclin B gene was expressed in the adductor muscle,mantle,gonad,gill,labial palp and digestive gl

关 键 词：香港牡蛎 cyclin B基因 表达特征 多克隆抗体 减数分裂 

分 类 号：S968.31[农业科学—水产养殖]