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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。
The scpB gene of Streptococcus agalactiae was cloned from the fish of Oreochromis niloticus. The prokaryotic expression plasmid pET32a (+) - scpB was constructed and transformed into Escherichia coli BL21 ( DE3). After IPTG induction, the expressed recombinant protein was purified by Ni2 + metal chelate column and concentrated by ultrafiltration. The recombinant protein was identified by SDS-PAGE and Western blot. After being immunized with purified recombinant proteins at different doses (1μg / g, 3μg / g, 5μg / g), the nonspecific immune indexes of serums in each experimental group were detected weekly [lysozyme activity, superoxide dismutase Enzyme (SOD) activity] and antibody level changes, and four weeks after immunization with four times the LD50 dose artificial challenge. The results showed that the recombinant protein existed as a inclusion body in E. coli. The relative immunoprotection against Nile tilapia was 69.66% ~ 89.00%, of which 5μg / g had the highest relative protection rate. The lysozyme activity, SOD activity and antibody level in serum were significantly higher than those in the control group (P <0.01). The results showed that the recombinant protein ScpB had strong immunogenicity and protective effect. This study aims to lay the foundation for the development of GBS polypeptide vaccine.