【摘要】 根据GenBank报道的停乳链球菌免疫分泌蛋白基因(immunogenic secreted protein,ISP)序列设计引物,PCR扩增得到1 550bp的isp片段,其基因序列与GenBank提交序列的同源性为98.13%.将isp基因与表达载体pET-25b(+)连接,成功构建重组质粒pET-25b(+)-isp.转化大肠杆菌BL21(DE3),成功得到阳性表达重组菌株BL21(pET-25b(+)-isp).经IPTG诱导后,SDS-PAGE与Western blot检测得到约55ku的目的蛋白.经优化确定,isp蛋白的最优表达条件:1mmol/L IPTG,37℃诱导18h,且此时表达的蛋白具有良好的抗原性.该研究制备了奶牛乳房炎停乳链球菌的基因工程疫苗,并对其免疫原性进行了探究,为奶牛乳房炎的免疫防治奠定了基础.更多还原

【Abstract】 To express the immunogenic secreted protein(ISP)of Streptococcus dysgalactiae and provide target protein for further immunological study,ispgene of S.dysgalactiae was amplified by PCR from its genomic DNA with specific primers designed according to the coding sequence of ISP retrieved from GenBank,and was inserted into pET-25b(+)vector.The length of the ispgene was about 1 550 bp,with98.13%identity with the sequence reported in GenBank.The recombinant plasmid pET-25b(+)-isp was then transformed into Escherichia coli BL21(DE3)for expression of ISP fusion protein.After induction with IPTG,expression of the target protein was identified by SDS-PAGE and western blot,and the molecular weight of the target protein was determined to be 55 ku.After optimization,the optimal expression condition of ISP was established as induction at 37℃ for 18 hwith the addition of 1mmol/L IPTG,and the protein thus obtained had good immunogenicity.In summary,the genetically engineered vaccine of S.dysgalactiae was prepared and its immunogenicity was studied in this study.It has laid a foundation for the prevention and cure of bovine mastitis.更多还原