Prokaryotic expression and acetylation identification of phosphomethylpyrimidine synthase (ThiC) in Vibrio alginolyticus
WANG Xudong,FAN Chenlong,DING Yu*
College of Fisheries，Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemilogy for Aquatic Economic Animals，Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes,Guangdong Ocean University,Zhanjiang 524088,China
Abstract: To understand whether there is acetylation of phosphomethylpyrimidine synthase (ThiC) in pathogenic Vibrioalginolyticus,the target gene ThiC was amplified via PCR basing on the genome of V.alginolyticus HY 9901.The prokaryotic expression vector pET-28a-ThiC were constructed,which was successfully expressed in Escherichiacoli BL21 by induction,and its expression conditions were optimized.Then,the protein expression and acetylation level were analyzed by SDS-PAGE and Western blot.The results showed that the ThiC gene had length of 1 941 bp,and that the recombinant Escherichiacoli BL21 was expressed as the recombinant protein(72 500).The optimal expression of recombinant protein was under conditions of 0.1 mmol/L IPTG for 3 hours at 37 ℃.Western blot results revealed that ThiC was acetylated protein,and there was no deacetylation in vitro.In conclusion,the recombinant expression strain for V.alginolyticus ThiC was successfully constructed,ThiC was acetylated protein,and there was no deacetylation in vitro.The findings lay the foundation for studying the post-translational regulation mechanism of vitamin nutrition of V.alginolyticus.
2022, Vol. 37 
