Molecular Mechamism Of Action Research About Differences Of WSSV Infection Of Two Strains Of Vibrio Alginolyticus

Probiotics in aquaculture industry has been widely used and accepted, and ithas became one of the main means of shrimps disease controlling. At present,probiotic species are very abundant, but the mechanism of action of probiotics is notvery clear. Dissolved in Vibrio alginolyticus as aquaculture probiotics has beenrecognized and applied. Based on laboratory experiment, feeding Vibrio alginolyticusto shrimp,that can enhance the infection resistance of shrimp with WSSV. On thebasis of the research results are differently splend idus strain vary widely, bycomparing differences between two bacterial strains, using molecular biologytechniques, this study find the strain between the extracellular and intracellular protein,polysaccharide, the difference between further determine the serine protease of theextracellular protein and extracellular polysaccharide substances. For the screening ofprobiotic bacteria played a certain instruction function, and the metabolites ofprobiotic bacteria and probiotic fermented using provides a certain reference value.This experiment will be divided into several parts: using SDS-PAGE, massspectrometry technology for separation of bacteria protein, artificial inductedreorganization protein protein purification, and through infected immuneexperiment, verify the function of the protein. RNAi in combination withexperimental verification ferritin to TRX early immune system function.Divided into the following several aspects:content and method:1Through16sRNA gene sequence analysis, molecular means. Combining with16s RDNA will dissolve two strains of Vibrio alginolyticus identification tosubspecies. Two strains isolated from the intestinal tract of shrimps, two strains ofbacteria to different strains of Vibrio alginolyticus, the differences between16-23S DNA and there are many techniques for bacterial identification.2Using16s rDNA molecular techniques and RFLP technique, that will dissolvemany strains Vibrio alginolyticus. Separation will dissolve algae strains of vibrioappraisal still has a lot of technology, using RFLP technique to16-23srDNA canseparate strains which cannot be separated, the molecular techniques will be a goodcombination subspecies further separation, for dissolving strain of Vibrioalginolyticus identification has a certain role.3By SDS-PAGE and mass spectrometry analysis of soluble protein separationVibrio alginolyticus,find out the differences between proteins by mass spectrometryanalysis, and identified protein bands differences of serine protease, serine proteaseis an important substance in the immune, it have natural antiviral effect, we furtherspeculated that dissolve in Vibrio alginolyticus serine protease of the extracellularproducts have important effect in antiviral effects.4By SDS-PAGE, mass spectrometry analysis of soluble protein separation, Vibrioalginolyticus identify the proteins by mass spectrometry analysis differencesidentified protein bar is iron carrier protein.By the technique of artificial recombinantprotein iron grain, FER gene clone and expression to the carrier pBAD/gIIIA,constitute a restructuring pBAD/gIIIA-FER. Carrier gene will restructure into e. coliprokaryotic expression in the top10, FER protein was purified to human recombinantprotein, under the induction of arabinose. through, according to the results of Westernblot experiments-get the expected target protein, confirmed that the recombinantprotein expression experiments successfully.5By using synthetic recombinant bacteria, under the induction of arabinose, ferritinfor function verification, the results show that ferritin was itself has certain improveimmune function, delay the median lethal time of prawn, the immune response toinfection results all death, further illustrate bacterial iron carrier itself mainly throughthe competition of iron ion inhibition of other bacteria breeding, also suggests thatprobiotics is through the overall role to achieve the effect of live.6Using bacterial extracellular polysaccharide extraction technology, the xtracellularpolysaccharide from the two strains exsit larger differences, although only for lactobacillus extracellular polysaccharide immune action research, this experimentfurther confirmed that the soluble polysaccharide of algal metabolites of vibrio alsohas certain immune function, To a certain extent to stimulate prawn nonspecific immunemechanism,for the development of probiotic products have a certain potential value.From what has been discussed above, we can draw the following conclusionprobiotics itself by integrity work, a certain ingredients from probiotics are not ableto completely replace the probiotics resisting for WSSV infection, this experimentwill help further promote the application of probiotics in aquaculture.