Study On Outer Membrane Proteins And Lipopolysaccrides Of Vibrio Harveyi

In the paper, outer membrane proteins (OMP) and Lipopolysaccrides (LPS) were extracted from V. harveyi using immunological technology, microbiological technology and proteinic technology, their texture and immune feature were analyzed. The influences of culture condition and extracting method on expression of OMP by were tested. Simultaneously, the influences of protective antigens on immunological function of Pseudosciaena crocea were studied. The results were summarized as follow.1. LPS and OMP were extracted from 3 strains of V. harveyi by means of water-phenol method and Sarkosyl method. Their components and immunogenicities were analyzed by means of SDS-PAGE and Western-blot in this study. The result showed that the major OMP of V. harveyi were composed by proteins with molecular weight (MW) of 31-43kD and LPS varied from 20kD to 120kD. Though significantly difference was found from LPS and OMP SDS-PAGE characteristics of the three strains, common bands were present. Western-blot test with rabbit antiserum (using V. harveyi formalin killed whole cell as antigen) detected two or one band of OMP from the three strains respectively; the common one was the band with MW of about 64kD. Western-blot of LPS from the strains showed seven, two and four reaction bands, respectively, and the 31kD band was the common one.2. LPS was extracted from the bacterium cultured with different time duration. The LPS production increased gradually in 48h. SDS-PAGE showed the differences of the samples from various culture times, more bands were present at 30h than other time. Western-blot analysis detected two common bands from all samples. Nevertheless, no significant change was found from OMP extracted from the bacteria cultured with various time durations. Western-blot test detected one common band with MW of 64kD. The results indicated that the expression and immunogenicity of OMP was not significantly affected by culture time, while the production, expression and immnogenicity of LPS were markedly influenced.3. The OMP was extracted from V. harveyi 100608 in different culturing and extracting conditions. The profiles of OMP analyzed with SDS-PAGE showed there were no obvious differences among groups with different culturing conditions and extracting method. The major bands were 74kD, 42kD, 40kD, 37kD and 34kD. Simultaneously, which showed slight difference in different temperatures. Four methods were used to extracting OMP. The profiles of OMP extracted with PMSF method had more bands; SDS method had fewest band and lack definition. There was no obvious difference between Sarkosyl method and Triton X-100 method, but the bands in high molecule quantitydistrict of OMP extracted with Triton X-100 were more than with Sarkosyl. The result hinted that the most suitable bacteria cultured condition and extracted method was: pH 6.5-7.6, shake culturing, the culture temperature ranging from 25 °C to 34 °C, culture time was about 30 hours, Sarkosyl method.4. P. Crocea was immunized by injecting with LPS, OMP and formalin killed Vibrio karveyi. Three antigens all came from V. harveyi of three different serotypes. Different time after the injection, influence of three antigens on immunological function of P. Crocea was determined by testing the agglutinating antibody titers of serum, lysozyme activity, phagocytic activity and the relative percentage survival. The result showed that three antigens had had higher immunogenicity and antigenicity. The agglutinating antibody titers of immune group were highest after 28 days. The lysozyme activity and phagocytic activity reached top value after 21 days, and then the index reduced gradually. The immunological indexes of all immune groups were higher than the control group significantly. Formalin killed V. harveyi could get the highest agglutinating antibody titers. The agglutinating antibody titers of LPS group and OMP group were lower than FKC group. But, the relative percentage survival indicated that the relative percentage survival of LPS group and OMP group were higher than of FKC. The relative percentage survival was in the sequence of group LPS > group OMP > group FKC > > control group. LPS and OMP groups got better effect, indicating that LPS and OMP were important protective antigens of V. harveyi.