Sao蛋白多克隆抗体制备鉴定及促全血杀菌作用

发布时间：2018-07-12 16:51

本文选题：型猪链球菌 + 原核表达　；参考：《中国公共卫生》2014年03期

【摘要】：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。
[Abstract]:Objective to prepare the polyclonal antibody against the surface antigen 1 (Sao) recombinant protein of S.suis 2 and to identify its immunological properties. Methods the gene Sao. was amplified from the genome of strain 05ZYH33 of Streptococcus suis by PCR. The recombinant expression plasmid pET28a-Saowas constructed and transformed into E. coli BL21, and the target protein was induced by isopropylthiogalactoside. Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis was used to identify the recombinant protein by his affinity chromatography. The polyclonal antibody was prepared by using the purified Sao protein. Indirect enzyme-linked immunosorbent assay (Elisa) was used to detect the titer of antibodies. Western blot was used to identify the specificity of antibodies and to evaluate the bactericidal effect of Sao polyclonal antibodies on whole blood. Results the recombinant plasmid could be highly expressed in the host bacteria. The purity of the purified recombinant protein was 90%, and the titer of Sao polyclonal antibody was 1: 102400 by indirect enzyme-linked immunosorbent assay (Elisa). The results showed that the Sao polyclonal antibody had good specificity. Whole blood bactericidal test showed that the relative survival rate of ZYH33 in whole blood containing Sao polyantibody decreased 85%. Conclusion the recombinant Sao protein polyclonal antibody prepared in this study has high titer and good specificity, and can obviously enhance the killing effect of whole blood on bacteria.
【作者单位】：温州医科大学检验医学院生命科学学院;南京军区军事医学研究所;
【基金】：国家自然科学基金(81172794;81071317;31170124) 江苏省自然科学基金(BK2011098;BK2011097) 浙江省新苗人才计划(2013R413039) 南京军区医学科技创新课题(ZX39)
【分类号】：R392

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