Cloning and Prokaryotic Expression Analysis of glyA Gene of Vibrio harveyi

【Objective】Vibrio harveyi is a normal flora present in seawater, however, it has been one of the reasons for the disease and even death of seawater fish in recent years. The main purpose of this study was to clone and analyze the prokaryotic expression of glyA gene in V. harveyi ZJ0603 strain in order to lay a foundation for the development of subunit vaccine.【Method】The PCR technique was used to clone the glyA gene of V. harveyi strain ZJ0603, and the analysis on the physicochemical properties, signal peptides, subcellular localization and advanced structural of its encoded serine hydroxylmethyltransferase（SHMT）were performed. The obtained glyA gene was connected to the prokaryotic expression vector pET-28a to construct the recombinant plasmid pET-28a-glyA, and then the recombinant strain BL21-pET-28aglyA was constructed. After sequencing, the proper strains were selected and induced with IPTG and Western blot analysis and identification of the recombinant protein were conducted. And the expression conditions of the recombinant strain BL21-pET-28a-glyA were optimized to obtain a large number of proteins.【Results】Bioinformatics analysis showed that the glyA gene had a total length of 1 296 bp, a total of 431 amino acids, a theoretical isoelectric point of 6.18, an instability coefficient of 28.66, and a total average hydrophilicity of -0.200. Generally, the glyA gene was hydrophilic and distributed in the cytoplasm, and the expected molecular weight of the protein was 46.60 ku. The recombinant plasmid pET-28a-glyA was successfully constructed and transferred into the expression strain. After induction by IPTG, Western blot results showed that the SHMT recombinant protein was successfully obtained.【Conclusion】The optimal induction time, concentration and temperature of IPTG were 4 h, 0.4 mmol/L and 37 ℃ after optimizing the expression conditions by the variable-controlling method.