猪丹毒丝菌天然SpaA和重组SpaA-N免疫保护效果的评价

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

摘要:

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66 kDa的rSpaA-N和64 kDa的天然SpaA，蛋白含量分别为1.34 mg/mL 和1.26 mg/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。

Abstract:

Abstract: [Objective] The purpose of this study was to evaluate the protective 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 LD50 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 (P<0.05) 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.