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Immunogenicity and Immunostimulating Complexs(ISCOMs) Induced Protective Immunity to Eels(Anguillia Anguillia) of Outer Membrane Proteins (OMP) of Vibrio Vulnificus

Author: TianDing
Tutor: LinTianLong
School: Fujian Normal University
Course: Developmental Biology
Keywords: Vibrio vulnificus Outer membrane protein Immunogenicity Immune stimulating complexes European eel
CLC: R392
Type: Master's thesis
Year: 2009
Downloads: 48
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Abstract


FJ03-X2 strain-based Laboratory trauma vibrios (Vibrio Vulnificus) separation, identification, conservation, type I strains belonging to Vibrio vulnificus, a pathogen of the European eel (Anguilla anguilla). The median lethal dose (LD 50 ) the strains with hemolytic activity of, casein protease activity, European eel elvers (about 30 g / tail) of about 7.1 × 10 3 The CFU / tail, and has been using 16S rDNA sequence and comparative analysis of Vibrio vulnificus hemolysin specific Nested-PCR identified joint. First, this study used a different method to compare the effect of various methods of extraction separation Vibrio vulnificus outer membrane protein the (outer membraneproteins OMP), screening the best of Vibrio vulnificus outer membrane protein extraction methods. Rabbit anti-Vibrio vulnificus FJ03-X2 whole cell serum prepared and applied to SDS-PAGE and Western-blotting analysis compared two methods for extraction of the outer membrane protein composition and antigenicity. SDS-PAGE results showed that Sarkosyl separation of the major outer membrane protein of relative molecular weight is concentrated in the 66 ~ 14kDa; relative molecular weight of 38kDa outer membrane protein of 36 kDa protein of high abundance. The results showed that Western-blotting with rabbit anti-bacteria hyperimmune serum antiserum can recognize most of the outer membrane protein fraction, indicating that the strain of the FJ03-X2 outer membrane protein antigen. The relative molecular weight were 66 kDa, 47kDa, 44 kDa was 38kDa (superscript TM) Resin. The antiserum against, 34kDa outer membrane of the egg Bai Chengjiang positive reaction is a major immunogen component. Vibrio vulnificus outer membrane protein production under different culture conditions, a comparative analysis of the results indicate that there is a direct correlation with the temperature and time of Vibrio vulnificus outer membrane protein production. Followed in this study were prepared immunostimulating complexes (ISCOMs) subunit vaccine, intraperitoneal injection of immune European eel Vibrio vulnificus outer membrane protein (OMP), indirect ELISA measured immune 7d, 14d, 21d, 28d, 35d, 42d collected serum antibody titers were 1:100,1:3200,1:6400,1:12800,1:3200,1:1600 European eel serum antibody response level upward trend first 28d reached after immunization peak and then begin to decline; corresponding antibodies could be detected in 42d. Vibrio vulnificus in 28d challenge of OMP-ISCOMs immunohistochemistry protective immunity can reach 100%; OMPs immunohistochemistry immune protection force for 70%; control group all died; description of OMP-ISCOMs has a strong immunogenic, can induce the European eel generated higher immune protection. This is also consistent with the highest the 28d European eel serum antibody level. The western blot results showed that the band and the immune response, its relative molecular weight, respectively, for the 47kDa, 38kDa, 36kDa, 34kDa OMP of four major protein bar can be detected in a position corresponding to blots. The test results prompted trauma paralyzed  the cake さ saddle-owned invasion plaque  Mou; ば key behaved? Prepared ISCOMs subunit vaccine, a small dose of a single immunization can play a significant immune protection. Third order to further investigate whether different Vibrio outer membrane proteins have common characteristics, the Sarkosyl method Vibrio vulnificus separation (Vibrio vulnificus), Vibrio alginolyticus (Vibrio alginolyticus), Vibrio parahaemolyticus (Vibrio parahaemolyticus) , Vibrio anguillarum (Vibrio anguillarum) outer membrane protein (OMP) conducted a preliminary comparative analysis. SDS-PAGE and Western blotting spectrum of the four kinds of Vibrio outer membrane protein similarity there are differences. SDS-PAGE showed four Vibrio except Vibrio parahaemolyticus can be isolated from the outer membrane protein of 47kDa, 38kDa; Vibrio vulnificus and Vibrio alginolyticus there are four kinds of common outer membrane protein, relative molecular weight of 47kDa 40kDa, 38kDa, 36kDa; Vibrio alginolyticus and Vibrio anguillarum 75kDa, 47kDa, 43kDa, 38kDa four kinds of common outer membrane protein. Preparation of rabbit anti-Vibrio vulnificus outer membrane proteins of hyperimmune serum indirect ELISA serum titer of 1:51200. Immunoblotting with the antiserum, rabbit anti-Vibrio vulnificus outer membrane protein serum with Vibrio vulnificus outer membrane proteins, outer membrane proteins of Vibrio alginolyticus anguillarum outer membrane proteins of the immune response, which dissolved algae Vibrio outer membrane protein reactive relatively strong, there are three clear reaction to hair ribbon, relative molecular weight of 38kDa, 30kDa, 29kDa; antiserum hydrophila Aeromonas outer membrane protein reaction with; while 38kDa Vibrio vulnificus and Vibrio alginolyticus common immune reaction with; outer membrane protein is both Vibrio may have a common protective antigen. In this study, the culture conditions and outer membrane protein extraction method Vibrio vulnificus, filter out the optimal incubation temperature and time as well as extraction method; experimental analysis of Vibrio vulnificus outer membrane protein antigen and immunogenicity ; and further confirmed Vibrio vulnificus outer membrane protein the immunostimulatory complex vaccine capable of inducing the European eel generated good immune protection, and laid the foundation for the development of trauma Vibrio subunit vaccine; while also looking for different Vibrio common outside membrane protein antigen useful attempt.

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