Study On The Surface Display Of WSSV Protein Vp19 And Vp28 Using CotX As A Molecular Vector On Bacillus Subtilis Spores

White spot syndrome virus (WSSV) causes white spot syndrome, a major disease in crustaceans, particularly shrimp.The previous studies suggest that vaccination strategy will have potential utility against WSSV infection in aquaculture. However, intramuscular injection of protein vaccines is costly and laborious, not being practically feasible for application in shrimp farming. Therefore, more effective and practical method to deliver DNA vaccine such as oral administration would be highly desirable. The gram-positive bacterium B. subtilis has been extensively studied as a model prokaryotic system. This organism is not regarded as a pathogen and is classified as a novel food that is currently being used as a probiotic for both human and animal consumption. The single, distinguishing feature of this microorganism is that it produces an endospore as part of its developmental life cycle when starved of nutrients. The mature spore can survive in a metabolically dormant form for hundreds or thousands of years. The spore offers unique resistance properties and can survive extremes of temperature, desiccation, and exposure to solvents and other noxious chemicals. These unique characheristics would make the spore an attractive vehicle for delivery of heterologous antigens or, indeed, any bioactive molecule to extreme environments such as the gastrointestinal tract. The goal of this approach was toward the construction of recombinant Bacillus subtilis spores to deliver Vp19/Vp28 vaccine against WSSV to crayfish.The researches and results are in four parts:1.To investigate whether CotX could carry target protein onto the spore surface, we constructed a recombinant integrative plasmid, designated as pJS749, which carries a recombinant cotX-gfp gene under the control of the cotX promoter. We transformed pJS749 into Bacillus subtilis 168(trp-), anα-amylase inactivated mutant DRJS749 was selected and confirmed to be a double crossover integrant, where cotX-gjp fragment was integrated into the chromosome. After induction of spore formation, significant green fluorescence was observed on spore surface of strain DRJS749 under fluorescent microcopy.2.Spore coat gene cotX of Bacillus subtilis were recombined with envelop gene vpl9 and vp28 of WSSV, The integrative plasmid pJS747and pJS748 were constructed for fusion expression of CotX-Vpl9 and CotX-Vp28.3.The integrative plasmids were transformed into Bacillus subtilis 168, the resulting recombinant bacteria named DRJS747 and DRJS748 were induced to produce recombinant spore displaying Vp19 or Vp28 which were identified by immunofruorescence.4.The recombinant spores were used to orally immunize crayfish with coated feed. Challenge experiments with WSSV demonstrated the protection in immunized crayfish by recombinant spores presenting Vp28, but not Vp19.The main conclusions of this study are as follows:1.CotX is associated with the outer part of the coat. CotX can therefore be used as a molecular vehicle for spore surface display of exogenous proteins.2.Bacillus subtilis spores displaying Vpl9 or Vp28 using CotX as molecular vectors can be used as oral vaccine to protect the crayfish against WSSV, and provides a new way to design more practical strategies for the control of WSSV and other invertebrate pathogens.