猪丹毒丝菌天然SpaA和重组SpaA-N免疫保护效果的评价  

Immunity of native SpaA and recombinant SpaA-N against Erysipelothrix rhusiopathiae infection in mice

作　　者：吾鲁木汗.那孜尔别克 张磊[1] 何翠[1] 彭清忠[1] 恩特马克.布拉提白 

机构地区：[1]吉首大学生物资源与环境科学学院,吉首416000

出　　处：《微生物学报》2010年第3期367-372,共6页Acta Microbiologica Sinica

基　　金：国家自然科学基金(30972206);湖南省教育厅重点基金项目(07A055)~~

摘　　要：【目的】本试验以小鼠为动物模型,评估了猪丹毒丝菌重组表面保护性抗原A的N端保护区域(rSpaA-N)和天然SpaA的免疫保护效果。【方法】将猪丹毒丝菌C43311株SpaA-N以可溶形式表达在大肠杆菌BL21中,用GST Bind Resin纯化试剂盒纯化rSpaA-N,采用电洗脱法从猪丹毒丝菌C43311株NaOH提取抗原中纯化天然SpaA,将rSpaA-N、天然SpaA和NaOH提取抗原制成亚单位疫苗,同时设GST及生理盐水对照组,间隔2周分3次皮下免疫小鼠,第3次免疫后2周用100LD50猪丹毒丝菌C43065株进行腹腔攻毒,采用间接ELISA方法检测免疫组小鼠血清的抗体动态变化。【结果】SDS-PAGE结果显示,采用GST Bind Resin纯化试剂盒和电洗脱法纯化得到了66kDa的rSpaA-N和64kDa的天然SpaA,蛋白含量分别为1.34mg/mL和1.26mg/mL,而Western印迹结果表明rSpaA-N和纯化前后的SpaA具有良好的免疫反应性。保护试验结果表明,不同免疫剂量的rSpaA-N组、天然SpaA组和NaOH提取抗原组均能完全保护小鼠受强毒株C43065的致死性攻击,而GST组和生理盐水组小鼠攻毒后全部死亡。ELISA检测结果表明,在不同免疫剂量的rSpaA-N组、天然SpaA组和NaOH提取抗原组小鼠血清中的抗体效价之间无显著差异(P<0.05)。【结论】本研究结果表明rSpaA-N具有良好的免疫保护作用,可以作为猪丹毒亚单位疫苗。[Objective]We evaluated the effects of surface protective antigen A （SpaA） and its N-teminal protective domain （rSpaA-N） against Erysipelothrix rhusiopathiae infection in mice. [Methods] The SpaA was purified by electroelution from NaOH-extracted antigen of Erysipelothrix rhusiopathiae strain C43311. The rSpaA-N was expressed in E. coli BL21 as a soluble protein by IPTG inducing, and purified with GST affinity chromatography. Mice of each group were subcutaneously immunized three times with 50μg or 100 μg of native SpaA, rSpaA-N or NaOH-extracted antigen with in complete or incomplete Freund adjuvant at 2-week intervals. Five mice of each group were challenged with 100 LD_ 50 of Erysipelothrix rhusiopathiae virulent strain C43065 two weeks after the third immunization, and the specific antibody responses for SpaA was determined by indirect ELISA. [Results] Mice immunized with 50 μg or 100 μg of native SpaA,rSpaA-N,or NaOH-extracted antigen were protected completely against the challenge with strain C43065. There was no significant difference （P0.05） between the antibody responses observed for native SpaA,rSpaA-N and NaOH-extracted antigen at any dosages. Western blot results indicated that the native SpaA and rSpaA-N were recognized specifically by an antiserum against the native SpaA of strain C43311. [Coclusion] These results demonstrated that the rSpaA-N is a protective antigen of Erysipelothrix rhusiopathiae serotype 2 strains, and might be a useful vaccine candidate against swine erysipelas.

关 键 词：猪丹毒丝菌 rSpaA-N 免疫保护 亚单位疫苗 

分 类 号：S852.65[农业科学—基础兽医学]