Sequence analysis and prokaryotic expression of zebrafish g-type lysozyme gene

【Objective】Zebrafish g-type lysozyme was expressed in Escherichia coli to obtain a fusion protein with high purity and bacteriolytic activity，which laid a foundation for the further study of antibacterial function and application of zebrafish g-type lysozyme.【Method】The biological information of g-type lysozyme gene of zebrafish was analyzed by online softwares such as ClustalW，ExPASy，SignalP-5.0 Server and PSIPRED. After coding codon optimization，g-type lysozyme gene of zebrafish was synthesized，respectively，and cloned into expression vector pET-28a（+）. The constructed plasmids were transformed into competent cells of E. coli BL21（DE3）. Then induced with IPTG to express the g-type lysozyme，whichwas used to determine its concentration and bacteriolytic activity by non-interference protein concentration determination kit and lysozyme determination kit（turbidimetry）.【Result】Zebrafish lysozyme genes g1（NM_001002706.1）and g2（XM_002664371.5）retrieved from GenBank were named Zeb-lys-g1 and Zeb-lys-g2，respectively. The open reading frame（ORF）of Zeb-lys-g1 gene was 591 bp in length and encoded 196 amino acid residues， with predicted molecular weight of 21.6 kD. The ORF of Zeb-lys-g2 was 576 bp in length and encoded 191 amino acid residues，with predicted molecular weight of 21.1 kD. The two zebrafish g-type lysozyme genes both had two cysteine residues and three conserved catalytic residues（Glu，Asp and Asp）. Zeb-lys-g1 contained 17 amino acid signal peptide at Nterminal，while Zeb-lys-g2 had no signal peptide，but its tertiary structure had multiple α-Spiral structure. In the phylogenetic tree constructed based on the similarity of lysozyme sequence，Zeb-lys-g1sequence was the closest to the g-type lysozyme sequence of goldfish，while Zeb-lys-g2 sequence was closer to the g-type lysozyme sequence of perch and flounder. Zebrafish g-type lysozyme genes（Zeb-lys-g1and Zeb-lys-g2）were subcloned intoexpression vector pET-28a（+）and transferred into competent cells of BL21（DE3）. The fusion proteins Zeb-lys-g1and Zeb-lys-g2 were obtained after induction by IPTG（final concentration 0.5 mmol/L）at 20 ℃ for 16 h. The purified concentrations were 1.01 and 1.66 mg/mL respectively，and the corresponding lysozyme activities were 689.68 and 44.39 U/mg，respectively.【Conclusion】The gene variation of fish g-lysozyme is relatively conservative. Zebrafish g-lysozyme genes zeb-lys-g1 and zeb-lys-g2 are synthesized by codon optimization and whole gene synthesis ，which cab obtain fusion proteins with high purity and high bacteriolytic activity through prokaryotic expression，which provides technical support for exploring the antibacterial mechanism of zebrafish lysozyme.