【摘要】 以江苏分离株HA9801基因组DNA为模板,扩增猪链球菌2型(SS2)胞外因子ef基因的1423～2203bp片段,并将其亚克隆至表达载体pGEX-4T-1上,构建了重组质粒pGEX-4T-1-ef。将鉴定阳性的质粒转化大肠杆菌BL21(DE3),0.5mmol/LIPTG诱导8h,表达的融合蛋白rGST-sEF大小为56ku,以包涵体形式存在。Western-blotting显示,rGST-sEF能与全菌兔抗血清发生特异性反应。用自制rGST-sEF亚单位疫苗免疫新西兰兔,以最小致死量HA9801攻击,保护率为60%。结果表明,表达的融合蛋白rGST-sEF具有良好的免疫原性和免疫反应性。更多还原

【Abstract】 A fragment(sequence positions 1423-2203)of extracellular factor(EF)gene was amplified by PCR from the genomic DNA of Streptococcus suis type 2(SS2)strain HA9801.The recombinant plasmid pGEX-4T-1-ef was constructed by inserting this fragment into plasmid pGEX-4T-1.After identification by PCR,restriction endonuclease digestion and sequencing,the recombinant plasmid pGEX-4T-1-ef was transformed into Escherichia coli BL21(DE3).The fragment was expressed under induction with 0.5 mmol/L IPTG for 8 h.It was confirmed that the fusion protein of 56 ku named as rGST-sEF was expressed in the form of inclusion bodies by SDS-PAGE,and the rGST-sEF could specifically react with antiserum against whole bacterial cells of SS2.The rabbits were immunized with rGST-sEF protein emulsified with Freund’s complete adjuvant,and then challenged with the strain HA9801 at 1 MLD.60% of the immunized rabbits survived.The results demonstrated that the expressed rGST-sEF had good antigenicity and reactogenicity in rabbits.更多还原