罗非鱼无乳链球菌gap基因的原核表达及免疫原性研究

Prokaryotic expression and immunogenicity of gap gene from Streptococcus agalactiae in Tilapia

试验旨在探究罗非鱼无乳链球菌(GBS)膜蛋白gap的免疫原性。试验提取GBS的DNA,利用PCR扩增出gap基因,并与表达载体pET-28a-SUMO构建重组质粒,转化至大肠杆菌BL21;经IPTG诱导、SDS-PAGE分析和Western blot检测确定其表达效果。采用纯化的gap蛋白与弗氏佐剂乳化腹腔注射免疫罗非鱼,以间接ELISA法检测血清效价并使用GBS进行攻毒试验。结果显示,PCR扩增出1011 bp大小的单条带gap基因,与预期一致;重组载体pET-28a-gap在23℃、0.4 mmol/L IPTG、12 h条件下成功表达49.8 ku的可溶性蛋白;ELISA检测显示,重组蛋白gap能够产生较高的血清抗体效价水平,对GBS攻毒后的罗非鱼相对保护率为64.1%。研究表明,重组蛋白gap具有较好的免疫原性,为鱼类的链球菌病的预防提供新的理论依据。

The aim of the study was to investigate the immunogenicity of GBS membrane protein gap in tilapia.GBS DNA was extracted,gap gene was amplified by PCR,and the recombinant plasmid was constructed with the expression vector pET-28a-SUMO and transformed into E.coli BL21.The expression was determined by IPTG induction,SDSPAGE analysis and Western blot analysis.Tilapia was immunized with purified gap protein and Fredrick adjuvant by intraperitoneal injection.Serum titer was detected by indirect ELISA and GBS was used for challenge test.The results showed that a single gap gene with a size of 1011 bp was amplified by PCR,which was consistent with the expectation.Recombinant vector pET-28a-gap successfully expressed 49.8 ku of soluble protein at 23℃,0.4 mmol/L IPTG and 12 h.ELISA showed that recombinant protein gap could produce higher titer level of serum antibody,and the relative protection rate against GBS of Tilapia was 64.1%.The study indicates that the recombinant protein gap has good immunogenicity,which provides a new theoretical basis for the prevention of streptococcal disease in fish.