摘要
以尼罗罗非鱼（Oreochromisniloticus）为鱼体样本，克隆了罗非鱼源无乳链球菌（Streptococcusagalactiae）scpB基因，构建了原核表达质粒pET32a（＋）-scpB，转入大肠杆菌BL2I（DE3）后，IPTG诱导表达，表达的重组蛋白经镍离子金属螯合柱纯化及超滤管浓缩后，进行SDS-PAGE和Westernblot分析鉴定。纯化的重组蛋白以不同剂量（1μg/g、3μg/g、5μg／g）免疫尼罗罗非鱼后，每周检测各实验组鱼体血清非特异性免疫指标[溶菌酶活性、超氧化物歧化酶（SOD）活力]及抗体水平变化情况，并于免疫4周后以4倍LD50的剂量对其进行人工攻毒。结果显示，该重组蛋白在大肠杆菌中以包涵体的形式存在；对尼罗罗非鱼的相对免疫保护率为69．66％-89．00％，其中5pg儋组的相对保护率最高；受免鱼体血清中溶菌酶活性、SOD活力和抗体水平较对照组有极显著提高（P〈0．01），结果表明，重组蛋白ScpB具有较强的免疫原性和保护作用。本研究旨在为GBS多肽疫苗的研制奠定基础。
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°C. 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, 1 μg/g（F1）, 3 μg/g（F3） and 5 μg/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 act
出处
《中国水产科学》
CAS
CSCD
北大核心
2014年第1期169-179,共11页
Journal of Fishery Sciences of China
基金
国家现代农业产业技术体系(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)
关键词
罗非鱼
无乳链球菌
C5a肽酶
原核表达
免疫原性
Tilapia
Streptococcus agalactiae
ScpB
prokaryotic expression
vaccine
immunogenicity
