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目的制备2型猪链球菌(S.suis 2)表面抗原一(Sao)重组蛋白的多克隆抗体,鉴定其免疫学特性。方法通过PCR从2型猪链球菌中国强毒株05ZYH33基因组中扩增基因Sao,构建重组表达质粒pET28a-Sao,转化E.coli BL21,异丙基硫代半乳糖苷诱导表达目的蛋白,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳鉴定表达产物,His亲和层析柱纯化重组蛋白,利用纯化后Sao蛋白制备多克隆抗体,间接酶联免疫吸附试验检测抗体效价,Western blot鉴定抗体特异性,评价Sao多克隆抗体的促全血杀菌作用。结果构建的重组质粒可以在宿主菌中高效表达,纯化后获得的重组蛋白纯度可达90%;间接酶联免疫吸附试验测定Sao多克隆抗体效价为1∶102400;Western blot结果显示,Sao多克隆抗体有较好的特异性;全血杀菌实验显示,05ZYH33在含有Sao多抗血清的全血中相对存活率下降85%。结论本研究制备的2型猪链球菌重组Sao蛋白多克隆抗体效价高,特异性好,可明显增强全血对细菌的杀伤作用。
Objective To prepare a polyclonal antibody against the S. suis 2 serotype 2 protein and identify its immunological properties. Methods Gene Sao was amplified by PCR from the genome of Streptococcus suis type 2 Streptococcus suis serotype 05ZYH33. The recombinant plasmid pET28a-Sao was constructed and transformed into E. coli BL21. The induced protein was expressed by isopropyl thiogalactoside. Twelve The expressed product was identified by sodium alkyl sulfonate - polyacrylamide gel electrophoresis, His recombinant protein was purified with His affinity chromatography, polyclonal antibody was prepared by purified Sao protein, indirect ELISA was used to detect antibody titer, and Western blot was used to identify Antibody specificity to evaluate the Sao whole blood anti-Sao polyclonal antibody. Results The constructed recombinant plasmids could be efficiently expressed in host bacteria and the purity of purified recombinant protein was up to 90% after purification. The titer of Sao polyclonal antibody was 1:102400 by indirect enzyme-linked immunosorbent assay. Western blot results showed that Sao Clonal antibodies have better specificity; whole blood bactericidal experiments showed that, 05ZYH33 Sao plasma antiserum containing the relative survival rate decreased 85%. Conclusions The recombinant polyclonal Sao protein of Streptococcus suis type 2 is highly potent and specific, which can significantly enhance the killing effect of whole blood on bacteria.