枯草芽孢杆菌表达柱状黄杆菌lip基因口服疫苗对草鱼免疫保护效果的研究

Oral vaccination for Ctenopharyngodon idella against Flavobacterium columnare using Bacillus subtilis expressing lip gene

关键词:  柱状黄杆菌  枯草芽孢杆菌  草鱼  口服疫苗

Key words:  Flavobacterium colunmare  Bacillus subtilis  Grass carp  Oral vaccine

摘要:

枯草芽孢杆菌(Bacillus subtilis)是一种水产益生菌，最近也被用作口服疫苗的投递载体。本研究利用前期筛选到的柱状黄杆菌(Flavobacterium columnare)保护性抗原基因lip，与pBE2R载体连接后，以质粒的方式转入枯草芽孢杆菌WB800中，构建了重组菌株WB800 (pBE2R-lip)。聚丙酰胺电泳和免疫印迹实验表明，枯草芽孢杆菌WB800 (pBE2R-lip)能表达lip蛋白，其分子量约为32 kDa。利用枯草芽孢杆菌WB800 (pBE2R-lip)口服免疫草鱼(Ctenopharyngodon idellus)后，通过酶联免疫吸附实验，在受免草鱼血清中检测到特异性抗体效价的上升，在人工感染实验中，受免草鱼的免疫保护率为52.4%。本研究表明，利用枯草芽孢杆菌能有效表达柱状黄杆菌的lip基因，以其作为口服疫苗能引起草鱼的免疫应答并提供免疫保护效果。

Abstract:

Bacillus subtilis is used as a probiotic bacterium in aquaculture and has been explored as a live carrier for the expression and oral delivery of antigen proteins. Flavobacterium columnare, a gram-negative rod-shaped bacterium, is the causative agent of columnaris disease in fish. It is ubiquitous in aquatic environments and causes disease in a variety of freshwater fish worldwide, which leads to severe economic loss. In a previous study, we discovered a protective antigen gene, lip, against F. columnare. In this study, the lip gene was inserted into a shuttle expression vector pBE2R, and then expressed as a recombinant fusion protein in B. subtilis WB800. The expressed protein of 32-kDa, which was estimated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western-blotting, was consistent with the molecular weight deduced from amino acid sequence. Grass carp (Ctenopharyngodon idella) was immunized through oral administration of B. subtilis WB800 (pBE2R-lip). The effects of protection and immune responses were evaluated. Enzyme-linked immunosorbent assay results showed that lip-specific antibodies could be detected in the sera of immunized fish, and the antibody levels were highest in the fourth week, with an average of 1:117. The outcome of immunization with B. subtilis WB800 (pBE2R-lip) resulted in a relative percent survival of 52.4% against F. columnare. After being immunized via oral administration, the quantity of B. subtilis in the hindgut of grass carp was measured using spread plate count method. Results showed that B. subtilis could not colonize the gut of grass carp. Our study demonstrated that oral administration of B. subtilis expressing lip gene could produce an effective immune response and offer good resistance against F. columnare.