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王晶, 李敏, 刘文静, 杜骁杰, 王玲, 李先富, 王长军, 潘秀珍, 高基民. Sao蛋白多克隆抗体制备鉴定及促全血杀菌作用[J]. 中国公共卫生, 2014, 30(3): 364-367. DOI: 10.11847/zgggws2014-30-03-35
引用本文: 王晶, 李敏, 刘文静, 杜骁杰, 王玲, 李先富, 王长军, 潘秀珍, 高基民. Sao蛋白多克隆抗体制备鉴定及促全血杀菌作用[J]. 中国公共卫生, 2014, 30(3): 364-367. DOI: 10.11847/zgggws2014-30-03-35
WANG Jing, LI Min, LIU Wen-jing.et al, . Preparation and identification of polyclonal antibodies against protein Sao of Streptococcus suis serotype 2[J]. Chinese Journal of Public Health, 2014, 30(3): 364-367. DOI: 10.11847/zgggws2014-30-03-35
Citation: WANG Jing, LI Min, LIU Wen-jing.et al, . Preparation and identification of polyclonal antibodies against protein Sao of Streptococcus suis serotype 2[J]. Chinese Journal of Public Health, 2014, 30(3): 364-367. DOI: 10.11847/zgggws2014-30-03-35

Sao蛋白多克隆抗体制备鉴定及促全血杀菌作用

Preparation and identification of polyclonal antibodies against protein Sao of Streptococcus suis serotype 2

  • 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 express the gene encoding surface antigen one(Sao)in highly virulent strains of Streptococcus suis serotype 2(S.suis 2)and to prepare the polyclonal antibodies against Sao to assess the immune characteristics of the anti-Sao polyclonal antibodies.Methods The target gene sao was amplified by PCR using the genomic template of 05ZYH33.Sao gene was subsequently inserted into pEASY-T1-simple vector and subcloned into expression vector pET28a.The prokaryotic expression plasmid pET28a-sao was transformed into E.coli BL21 after the identification with double digestion of BamH I and Sal I and DNA sequencing.With the induced expression by isopropyl β-D-1-thiogalactopyrannoside(IPTG),the recombinant protein Sao was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophresis(SDS-PAGE).The Sao protein was purified with His-Binding-resin.The titer and specificity of the polyclonal antibodies were determined by enzyme-linked immunosorbent assay(ELISA)and Western blot.Bactericidal assay was used to identify the bactericidal activity of rabbit polyclonal antibodies against Sao.Results The expression vector pET28a-sao was successfully constructed.The fused gene was highly-expressed in E.coli BL21.ELISA detection results indicated that the titer of polyclonal antibodies to Sao was about 1:102400.Western blot results showed that the anti-Sao polyclonal antibodies could bind to recombinant Sao protein specifically.The polyclonal antibodies against the recombinant Sao protein could promote bactericidal ability of human whole-blood.Conclusion The anti-Sao polyclonal antibodies are of good specificity and high titer and could promote bactericidal ability of human whole-blood.

     

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