The Establish Of A New Delivery System Of Aquatic Vaccine

Objective:We expect to establish a new aquatic vaccine delivery system to improve the aquatic vaccine inoculation effect by using the transmembrane transduction ability of the protein transduction domain of the TAT (TAT-PTD).Methods:We used the method of the genetic engineering to construct and express the recombinant protein GFP-TAT, and compare their transmember efficiency and immune results respectively to explore the possibility of TAT applications in aquatic vaccine. After then, we continued to construct the recombinant vectors pET28a(+)-OmpK and pET28a(+)-OmpK-TAT, and induced the highly express of these two recombinant proteins. By using these two proteins to immunize European eel, the valence of antibody in the serum and the immune protection efficiency were evaluated when the Vibrio parahaemolyticus attacked European eel.Results:(1) We have cloned the full sequence of GFP and linked TAT sequences at the C-terminal of GFP gene sequences through PCR, The recombinant plasmid of pET28a(+)-GFP-TAT was constructed and the recombinant protein GFP-TAT was expressed, and the recombinant protein GFP-TAT was purified by the Nickel column;(2) Cells were incubated with GFP and GFP-TAT respectively for 1 hour, and we could find that GFP-TAT can transduce into cells faster than GFP;(3) Brocade carps were immunized with GFP or GFP-TAT respectively, by three different ways (injection application, oral application and immersion application) for three weeks. Then we have detected the valence of antibody in the serum. We can find that the titer of the injection group was the highest, the immerse group was second highest and the oral group was minimum, and we can also find that the titer of the GFP-TAT immersion application was higher than the group of GFP injection application. TAT in immerse application can improve the immune effect of the GFP;(4) We have constructed the recombinant plasmid pET28a(+)-OmpK and pET28a(+)-OmpK-TAT by methods of genetic engineering, and induced the expression of these two recombinant protein respectively. Then we have purified these two proteins through nickel column;(5) We used OmpK and OmpK-TAT as vaccine to immunize European eel by immersion application respectively for two times. The results showed that the titer of the OmpK-TAT immersion group is 10 fold higher than the titer of the OmpK immersion group;(6) We used the Vibrio parahaemolyticus to attack those immunized fishes. The results showed that the survival rate of OmpK-TAT group was significantly higher than the OmpK group and the control group. The results indicated that TAT can mediate the OmpK into the fish body more effectively, and at the same time it can also enhance the immune protection during the immersion application.Our results provided a solid foundation for the further study in establishing a new type of aquatic vaccine delivery system, and the basis for the research and development of the immersion application vaccine.