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

【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 prokaryotic 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 c DNA 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 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. hongkongensis. 【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 expression 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.