CAJ | 학술논문

以尼罗罗非鱼(Oreochromis niloticus)为鱼体样本,克隆了罗非鱼源无乳链球菌(Streptococcus agalactiae) scpB基因,构建了原核表达质粒pET32a(+)-scpB,转入大肠杆菌BL21(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Western blot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5μg/g 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。
이니라라비어(Oreochromis niloticus)위어체양본,극륭료라비어원무유련구균(Streptococcus agalactiae) scpB기인,구건료원핵표체질립pET32a(+)-scpB,전입대장간균BL21(DE3)후, IPTG유도표체,표체적중조단백경얼리자금속오합주순화급초려관농축후,진행SDS-PAGE화Western blot분석감정。순화적중조단백이불동제량(1μg/g、3μg/g、5μg/g)면역니라라비어후,매주검측각실험조어체혈청비특이성면역지표[용균매활성、초양화물기화매(SOD)활력]급항체수평변화정황,병우면역4주후이4배LD50적제량대기진행인공공독。결과현시,해중조단백재대장간균중이포함체적형식존재；대니라라비어적상대면역보호솔위69.66%~89.00%,기중5μg/g 조적상대보호솔최고；수면어체혈청중용균매활성、SOD활력화항체수평교대조조유겁현저제고(P<0.01),결과표명,중조단백ScpB구유교강적면역원성화보호작용。본연구지재위GBS다태역묘적연제전정기출。
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.