马链球菌兽疫亚种纤连蛋白结合蛋白的原核表达及其免疫原性的检测

Prokaryotic expression and immunogenicity detection of fibronectinbinding protein of Streptococcus equi ssp.zooepidemicus

根据已发表的纤连蛋白结合蛋白(fibronectin-binding protein,FNZ)基因(fnz)序列,设计并合成1对引物,以马链球菌兽疫亚种(SEZ)ATCC35246菌株基因组DNA为模板,PCR扩增fnz基因,并定向将其克隆至表达载体pET-32a(+)中。重组质粒pET-32a(+)-fnz经酶切及测序鉴定后,在大肠杆菌BL21中进行诱导表达,表达产物经Western-blot检测后进行纯化,得到了74ku的纯化蛋白。以纯化的FNZ与ISA206佐剂以1∶1的体积比混合后免疫ICR小鼠,每只小鼠背部皮下多点注射120μL(含蛋白22.5μg),14d后以同样方法进行加强免疫。间接ELISA检测结果表明,FNZ免疫组小鼠的血清FNZ抗体效价显著高于其他3个对照组。二免后第15天小鼠腹腔注射ATCC35246菌液5.0×105 CFU(10LD50),观察注射后10d内小鼠的临床表现及存活状况;结果显示,FNZ免疫组小鼠的存活率为62.5%,显著高于阴性对照组(0)和空白对照组(0),但低于SEZ灭活疫苗免疫组(87.5%)。结果表明,FNZ可使免疫小鼠对SEZ的感染产生一定的抵抗作用。

To develop a vaccine against Streptococcus equi ssp.zooepidemicus(SEZ)infection,the fibronectin-binding protein(FNZ)gene(fnz)was amplified from genomic DNA of SEZ ATCC35246 strain by PCR,and then it was cloned into the expression vector pET-32a(+)to construct a recombinant plasmid pET-32a(+)-fnz.The expression product from pET-32a(+)-fnz was analyzed by Western-blot.To evaluate the immunogenicity of the recombinant FNZ protein,32 ICR mice were immunized with the purified recombinant FNZ protein through hypodermic inoculation.The results of an indirect ELISA showed that the serum FNZ-specific antibody levels of the recombinant FNZ-immunized group were significantly higher than those of the other 3control groups.On day 15 after the booster immunization,all mice were injected intraperitoneally with the virulent strain ATCC35246.In result,the survival rate of the FNZ-immunized group was 62.5%.The result showed that the recombinant FNZ showed immune protective potency in mice against SEZ infection.