Prokaryotic expression, purification and immunogenicity of LrrG protein of Streptococcus agalactiae based on codon optimization
CHEN Zhiting, KE Xiaoli, CHEN Gang, HUANG Lifang, LIU Zhigang，LU Maixin#br#
1.Key Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation, Ministry of Agriculture and Rural Affairs, Guangdong Provincial Key Laboratory of Aquatic Animal Immune Technology, Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou 510380, China; 2.College of Fisheries, Guangdong Ocean University, Zhanjiang 524025, China; 3.College of Animal Science and Technology, Zhongkai University of Agricultural Engineering, Guangzhou 510550, China
Abstract: In order to improve the supernatant expression of the recombinant protein leucine-rich repeat sequence (LrrG), the pCzn1-LrrG recombinant expression vector was constructed and transfected into the competent cell BL21 (Plys). First, according to the codon preference of Escherichiacoli, the whole LrrG gene sequence was artificially synthesized and inserted into the expression vector through double enzyme digestion. The optimized recombinant plasmid pCzn1-LrrG was transfected into competent cells for prokaryotic expression. The supernatant recombinant protein LrrG obtained by lysis and purification had the optimized LrrG gene fragment of 2 286 bp via PCR amplification. The recombinant plasmid pCzn1-LrrG constructed by double enzyme digestion contained 2 fragments of about 4 400 bp and 2 286 bp. The sequenced recombinant vector was completely consistent with the optimized gene sequence, and the encoded amino acid sequence remained unchanged. The SDS-PAGE and western blot revealed that the relative molecular mass of the LrrG recombinant protein was about 108 000, showing GBS antigenicity. After purified by affinity chromatography, the expression quantity of LrrG recombinant supernatant protein in Escherichiacoli BL21 (Plys) was increased by 2.2-3.8 times compared to that of the original type. Moreover, the optimized LrrG recombinant protein supplied a 61.54%-69.23% RPS against GBS infection in tilapia, indicating that the codon optimization effectively improved the expression level of LrrG recombinant protein in E.coli, and the optimized LrrG recombinant protein still maintained the original immunogenicity. The findings laid a foundation for the preparation and application of the genetic engineering vaccine based on the surface protein of S.agalactiae.
陈徵婷, 可小丽, 陈刚, 黄丽芳, 刘志刚, 卢迈新. 基于密码子优化策略的无乳链球菌表面蛋白LrrG的原核表达、纯化及免疫原性[J]. 大连海洋大学学报, 2021, 36(6): 920-928.
CHEN Zhiting, KE Xiaoli, CHEN Gang, HUANG Lifang, LIU Zhigang, LU Maixin. Prokaryotic expression, purification and immunogenicity of LrrG protein of Streptococcus agalactiae based on codon optimization. Journal of Dalian Ocean University, 2021, 36(6): 920-928.
2021, Vol. 36 
