电泳和免疫印迹分析副溶血弧菌和溶藻弧菌主要外膜蛋白和多糖抗原

目的　考察副溶血弧菌和溶藻弧菌的主要外膜蛋白 (MOMP)及其多糖抗原 (PSA)在不同菌株间的结构特征及其免疫学特性 ,从而进一步揭示其共同的保护性抗原。方法　一步超速离心法和蛋白酶K消化法分别制备 10株副溶血弧菌和7株溶藻弧菌的MOMP和PSA ,SDS -PAGE和免疫印迹分析其结构特点。结果　两种细菌的MOMP和PSA的结构和形态菌株间差别明显 ,但也有MOMP一致的菌株 ,免疫印迹显示 ,全部的 10株副溶血弧菌和 5株溶藻弧菌都具有分子量约为36kD的主要外膜蛋白 ,他们能够被副溶血弧菌全菌抗血清所识别 ,是两种细菌共同的具有强免疫原性的主要抗原。PSA分析未见经典LPS的O侧链结构。多糖成份在菌株间的交叉反应有限 ,可能受弧菌自身表面抗原复杂性的影响 ,凝集反应结果与免疫印迹试验结果不完全一致。此外 ,副溶血弧菌与溶藻弧菌的PSA也会产生免疫学上的交叉反应。结论　 36kD的MOMP是广泛存在于副溶血弧菌和溶藻弧菌的高丰度蛋白 ;与主要外膜蛋白相比 ,多糖抗原在菌株间具有更大的异质性 ;副溶血弧菌和溶藻弧菌的MOMP可能比PSA更具研制疫苗潜力。

Electrophoretic and immunobot analysis of the major outer membrane protein (MOMP) and polysaccharides antigen(PSA) of Vibrio paraheamolyticus and Vibrio alginolticus

To investigate the structural profiles of the major membrane protein(MOMP) and polysaccharides antigen (PSA) of Vibrio parahaemolyticus and Vibrio alginolticus among different strains,and then to reveal the common protective antigen.Ten Vibrio parahaemolyticus and 7 Vibrio alginolticus MOMPs and PSAs were prepared through one-step ultracentrifuging method and protase K treatment respectively.The structural polymorphology of MOMPs and PSAs were analyzed by SDS-PAGE and Western blot analysis.The results showed that few strains were observed with the completely same MOMP and PSA patterns.But all 10 Vibrio parahaemolyticus had the common strong protein band with molecular weight about 36-kilodalton observed on NC membrane by Western blot analysis.Interestingly,5 Vibrio alginolticus MOMP could also be recognized near 36-kilodalton area by rabbit anti-serum R-WC-ND-8 against the whole cell of Vibrio parahaemolyticus ND-8.PSA analysis indicated that no typical O-specific side chains were observed in both Vibrio parahaemolyticus and Vibrio alginolticus.Affected by surface antigen complexity of Vibrio itself,agglutination reaction were not all identical with Western-blot reaction.Moreover,R-WC-ND-8 could recognize one Vibrio alginolticus PSA.In conclusion,the 36-kilodalton MOMP is the common main protein among Vibrio parahaemolyticus and Vibrio alginolticus.Contrast to MOMP,PSA has more extensive heterogeneity among different Vibrio strains.Correspondingly,MOMP may be the superior candidate to PSA for vaccine preparation.