| Summary: | 碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 96 === Edwardsiella tarda is a fish pathogen that is commonly distributes in the fresh water of pound. It always cause the serious financial loss since the disease was burst. The farmers of aquaculture usually use antibiotics to control the related disease. But it may course the drug resistance of pathogen when using the antibiotics and the residue of antibiotics may threaten the healthy. It could be prevent the burst of disease and reduce the using of antibiotics if the effective vaccine have been development. The oral vaccine is the most convenient way to use, and it make the lowest stress for aquatic organism. The lower efficacy of orally administered vaccine is the antigen destruction by gastric acid digestion. Protection the antigen from digestion is a point of our experiment.
This experiment using the probe Ehly which can specific hybridization with E. tarda hemolysin gene to test the different strain of standard E.tarda. All of the E. tarad we test can be hybridize from Ehly. We transfer the cloning vector pET34b(+)-hlyA from HMS174 to expression systme BL21(DE3). We using the LB broth to incubate the BL21(DE3)/pET34b(+)-hlyA, then using IPTG induction for three hours. The recombinant target protien size is approximately 37.7 kDa when using the 12% SDS-PAGE and western blot to view the lysate protein. We using the His•Bind Column to purify the target protein, and check the efficiency of purification. The target protein which dissolves in PBS represented 0.7478% of the total protein. And the target protein from inclusion body represented 16.324% of the total protein. We using reagent X and acidic solution pH1.0, pH2.0, pH3.0, PBS(pH7.0) to precipitate the target protein. There is the better precipitation efficiency of Reagent X concentration less than 15 mM. The precipitation will not be digested by proteinase K during the reaction of precipitate. The protein which release from precipitation will be digested by proteinase K after the pH is reversion. We can see that protein which release from precipitation will be digested when treated by gastric acid. The remain precipitation protein which treated the gastric acid after 2 hour have still reveal the target protein. We feed the tilapia by 150 mg lysate target protein which precipitate by 10 mM reagent X (pH1.0), and using the western blot to view the exist of target protein. The target protein was observed from plasma after 1 hour for feeding. There was be highest level of target protein from plasma after 8 hour for feeding. We used the injection and feeding to administrate the hemolysin vaccine, and collect the antiserum. Using the antiserum to neutralize the hemolysin from E. tarda. In the injection group, we divide into two group. The no. 1, 2, 3 are inject 1 μg pure protein antigen per 100 g fish weight, and 4, 5, 6 are inject 21 μg lysate protein antigen per 100 g fish weight. The 1, 3, 7, 8 and 9 fish have22、22、21、23 and 24neutralization titer at the 4’ week after the hemolysin reaction for 2 hour. In the feeding group, we divide into two group. The no. 1, 2, 3, 4, 5 are feed 2 mg lysate protein antigen per 100 g fish weight, and 6, 7, 8, 9, 10 are feed 0.2 mg lysate protein antigen per 100 g fish weight. the 1, 3, 4, 5, 7and 9 fish at the 4’ week and 1, 2, 3, 5 and 8 fish at 5’ week have 21 neutralization titer after the hemolysin.
|
|---|
