中国水产科学Issue(1)：169-179,11.DOI:10.3724/SP.J.1118.2014.00169
罗非鱼无乳链球菌C5a肽酶（ScpB）的原核表达及其免疫原性
Prokaryotic expression and immunogenicity analysis of C5a peptidase (ScpB) of Streptococcus agalactiae isolated from tilapia
摘要
Abstract
Streptococcus agalactiae is a major bacterial pathogen that has caused severe economic losses in many spe-cies of freshwater, marine and estuarine fish worldwide. ScpB is a highly conserved surface protein among all group B streptococcus (GBS) strains and is an attractive surface-exposed antigen for inclusion among vaccine components against GBS. In this study, the scpB gene was amplified from the genomic DNA of an S. agalactiae strain isolated from diseased tilapia farmed in Guangdong province, China. The scpB gene contains a 2 799 bp open reading frame (ORF), encoding 932 amino acids. The molecular mass of the deduced protein was 103 kD. Blast analysis showed that it shares high similarity with scpB sequences of human GBSs registered in GenBank. Prokaryotic expression vector pET32a (+) was used to construct a recombinant expression vector pET32a (+)-scpB, which was transformed into Escherichia coli BL21 (DE3). Colonies containing the recombinant expression vector pET32a (+)-scpB were selected and then induced to express using 0.5 mmol/L IPTG for 8 h at 37℃. The expressed recombinant protein was purified by nickel chelate affinity chromatography and ultrafiltration tube enrichment. SDS-PAGE analysis and western blotting showed a spe-cific protein band of about 121 kD. The recombinant strain could produce large amounts of ScpB protein, mainly in the form of an inclusion body. To analyze the immunogenicity of the recombinant protein, the purified fusion protein and Freund’s adjuvant were mixed according to a certain proportions to produce vaccines. Three immune dosages, 1μg/g(F1), 3μg/g(F3) and 5μg/g(F5) were used. Four weeks after immunization, tilapia were challenged by artificial infection with the GBS ZP-N strain, which was previously isolated and confirmed to be a tilapia pathogen by our labo-ratory. The recorded relative percent survivals (RPS) of the vaccinated groups ranged from 69.99%to 89%, of which group F5 has the highest RPS. The lysozyme activities, superoxide dismutase (SOD) activities and antibody levels (OD450nm) were tested weekly until the end of the experiment. The results showed that after immunization, there were significantly higher lysozyme activities, superoxide dismutase (SOD) activities and antibody activities(OD450) in the vaccinated fish compared with the control group (P<0.01). The results of this study showed that recombinant protein ScpB has a strong immunogenicity and protective effect, laying the foundation for further study of polypeptide vaccines for S. agalactiae.关键词
罗非鱼/无乳链球菌/C5a肽酶/原核表达/免疫原性Key words
Tilapia/Streptococcus agalactiae/ScpB/prokaryotic expression/vaccine/immunogenicity分类
农业科技引用本文复制引用
李庆勇,可小丽,卢迈新,朱华平,高风英,刘志刚..罗非鱼无乳链球菌C5a肽酶（ScpB）的原核表达及其免疫原性[J].中国水产科学,2014,(1):169-179,11.基金项目
国家现代农业产业技术体系(CARS-49) （CARS-49）
广州市科技计划项目(2013J4100078 （）
201300000064) （）
农业部淡水渔业和种质资源利用重点实验室开放课题(KF201309) （KF201309）
