南方农业学报2024，Vol.55Issue(4)：909-919,11.DOI:10.3969/j.issn.2095-1191.2024.04.001
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
摘要
Abstract
[Objective]The objective of this study was to clarify the tissue expression characteristics of cyclin B pro-tein 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 recombi-nant 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 trans-membrane domain.The amino acid sequence of cyclin B contained two conserved cyclin boxes,and the amino acid se-quence of cyclin B was the most similar to that of Pacific oyster(98.8% ),the phylogenetic tree based on amino acid se-quence 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 gland of C.hongkongensis,and the relative expression level in the gonad was the highest,which was significantly higher than that in other tissues(P<0.05).The fusion protein cyclin B obtained by prokaryotic expression system exhibited good immunogenicity and mainly existed in the form of an inclusion body.The titers of the polyclonal antibody detected via immunizing BALB/c mice were above 1∶64000,and the polyclonal antibody could specifically recognize cyclin B protein in the gonad of C.hongkongen-sis.[Conclusion]The cyclin B gene of C.hongkongensis is highly conserved,and its amino acid sequence contains two conserved cyclin boxes.The expression of cyclin B gene in C.hongkongensis is broad-spectrum and tissue specific,and plays a role in regulating the process of meiosis of germ cells.The murine polyclonal antibody prepared by prokaryotic ex-pression and immunization of BALB/c mice exhibits high titers,it can specifically recognize the cyclin B protein in the gonad of C.hongkongensis,and provides technical support for further exploring the biological function and expression regulation mechanism of cyclin B in C.hongkongensis.关键词
香港牡蛎/cyclin B基因/表达特征/多克隆抗体/减数分裂Key words
Crassostrea hongkongensis/cyclin B gene/expression characteristics/polyclonal antibody/meiosis分类
农业科技引用本文复制引用
邱衡通,许贵林,李宛怡,周齐家,刘小宁,朱俊霖..香港牡蛎cyclin B基因组织表达特征及其多克隆抗体制备[J].南方农业学报,2024,55(4):909-919,11.基金项目
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) （2022YFD2401200）
广西科技基地和人才专项(桂科AD21075012) （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) （KLOP-X1910）
广西大学生创新创业计划项目(S202310603072) "Marine Agriculture and Freshwater Fishery Science and Technology Innovation"Project of Na-tional Key Research and Development Program of China(2022YFD2401200) （S202310603072）
Guangxi Science and Technology Base and Talent Special Project(Guike AD21075012) （Guike AD21075012）
Project of Key Laboratory of Environment Change and Resources Use in Beibu Gulf,Ministry of Education(KLOP-X1910) （KLOP-X1910）
Guangxi College Student Innovation and Entrepreneurship Training Project(S202310603072) （S202310603072）
