牛乳腺炎无乳链球菌PI-2a菌毛岛屿AP1-AP2-BP基因亚单位抗体的制备

目的 构建牛乳腺炎无乳链球菌菌毛岛屿PI-2a亚单位重组抗原AP₁-AP₂-BP,并制备其多亚单位抗体,为后期研制新型免疫疫苗和检测试剂提供实验基础。方法 利用延伸PCR技术构建了AP₁-AP₂-BP三联基因,并将该串联基因进行转化,诱导,表达,纯化,并将纯化蛋白制作为抗原,对家兔免疫,制备多亚单位抗体,并通过亲和层析的方式从免疫后的血清中获得纯化的IgG,完成多亚单位抗体的制备。结果 AP₁-AP₂-BP三基因串联重组工程菌株通过诱导、表达,对产物亲和层析等方法获得的纯化蛋白,其相对分子质量为65 kDa,其含量达到3.3 mg/mL,并具有较好的免疫原性。经间接ELISA可知,经过4周的免疫抗体的滴度已达到1∶5 600的水平。通过 Protein A280测量数据表明,纯化后的IgG的含量高达9.1 mg/mL。结论 牛乳腺炎无乳链球菌的菌毛岛屿AP₁-AP₂-BP三基因串联重组工程菌经诱导、表达后获得的纯化蛋白AP₁-AP₂-BP多亚单位抗原有较好的免疫原性,为制备相应多亚单位抗体的制备提供了良好的多价抗原,同时为将研制新型工程疫苗及检测试剂提供了科学研究基础。

Preparation of bovine mastitis Streptococcus agalactiae PI-2a pili islands AP1-AP2-BP subunit antibody

In this experiment, the subunit recombinant antigen AP₁-AP₂-BP of the pili island PI-2a of bovine mastitis Streptococcus agalactiae was constructed and its multi-subunit antibody was prepared, which provided the experimental basis for the development of a new immune vaccine and detection reagent in the later stage. The triplex gene of AP₁-AP₂-BP was constructed by extended PCR technique, and the tandem gene was transformed, induced, expressed and purified, and the purified protein was made into an antigen, the rabbits were immunized with it, and prepare multi-subunit antibodies. The purified IgG was obtained from the immunized serum by affinity chromatography, and the preparation of multi-subunit antibodies. The AP₁-AP₂-BP three Gene tandem Recombinant Engineering strains was transformed, induced, expressed. And the purified protein obtained by Affinity Chromatography. The relative molecular weight of purified protein was 65 kDa. The content of purified protein was 3.3 mg/mL. The purified polyunit antigen had good immunogenicity. The results of EILSA showed that the titer of antibody in the serum reached 1∶5600 after the fourth immunization. The measured data of Protein A280 showed that the content of purified IgG was as high as 9.1 mg/mL. The purified protein AP₁-AP₂-BP multi-subunit antigen obtained from the AP₁-AP₂-BP three Gene tandem Recombinant Engineering strains of bovine mastitis Streptococcus agalactiae has good immunogenicity, which provides a good polyvalent antigen for the preparation of the corresponding multi-subunit antibody. At the same time, it provides a scientific basis for the development of new engineering vaccines and detection reagent.