Effect Of 6 Kinds Of Targeting Molecules FaeG-mSTa-LTB-STB Fusion Protein Inclusion Bodies Oral Immunization

Weaned piglets diarrhea is an important disease affecting the development of the pig industry, with high disease incidence rate and serious economic loss,has become a problem in the swine industry worldwide.Enterotoxigenic Escherichia coli(ETEC) is an important factor in causing diarrhea in weaned piglets.According to recent epidemiological survey,F4 fimbriae genes occupy a dominant position in the diarrhea.About enterotoxin genes,STa,STb and LT are the most important virulence factors.Currently dealing with piglet diarrhea mainly relies on antibiotics treatment,not only prone to drug resistance,also has a great influence on piglet,so an economical and effective vaccine to prevent diarrhea of weaned piglets is extremely necessary developed.In this study,we use the method of SOE PCR to get the fusion of Fae G-m STa-LTb-STb tetravalent antigen gene,and connected the fusion gene into an expression vector p Pro EXHTa, transformed into E. coli BL21.After induced,SDS-PAGE and Weastern blot proved the fusion gene was highly expressed in E. coli in inclusion bodies,and has a reactionogenicity.To study the immunogenicity of the fusion protein,the inclusion bodies were extracted and purified and immunied BALB/c mice by oral directly.Then in serum specific Ig G,Ig M and fecal s Ig A specific antibody levels after immunization at different times and spleen lymphocyte proliferation assay were measured.Then the duration of bacteria existence in feces after challenge were determined.In three months after immunization,mucosal immune memory response was measured.The results showed that immunized mice were produced specific antibodies and cellular immune responses, duration of bacteria existence significantly reduced after challenge.Immune memory response tests showed that immunized mice at least within three months kept the memory of the immune response to specific antigens.In order to enhance the effect of tetravalent antigen genes,C5 a L,Cpe17,Cpe30,Yad A3,sfim H targeting molecules were added by PCR at the end of the fusion gene to enhance the transmission efficiency of antigen.Similarly,DCpep targeting molecule was added at the front of the single antigen target groups,for targeting dendritic cells,to enhance the efficiency of M- presenting cells and strengthen immunity further.Each group of recombinant gene with single and double target molecular was detemined by SDS-PAGE and Western blot,showed that all of recombinant genes can be highly expressed in E. coli,existed in the form of inclusion bodies,with reactogenicity.In order to compare the immune effect between variety target molecular,to choose the target molecular with best enhance effect.BALB/c mice was oral immunized with the inclusion bodies of each group.At different times serum specific Ig G,Ig M and fecal s Ig A specific antibody levels and spleen lymphocytes proliferation assay after immunization,and the duration of bacteria existence after challenge were measured.The results showed that the antibody levels of the groups of single and double target molecules were significantly higher than that of no target,except for the sfim H target molecule groups.The levels of specific antibodies increased in all groups,and the duration of bacteria existence was significantly shorter than control group.The proliferation of spleen lymphocyte showed that the stimulation index of each experiment group were significantly higher than control group.This shows that the target molecules of Cpe17,C5 a L,Cpe30 and Yad A3 have the effect of immunity enhancement.However,these targeted molecules are not all coordinated with the DCpep target molecules,and only the Yad A3 target molecules have a synergistic effect with the DCpep targeting molecules,thus improving the immune effect.After choosing the best target molecules,we conducted a piglet immunity experiment, respectively,immuned by oral immunization,nasal cavity immunization and Injection immunization. 7 days after twice immunization and challenge experiments,respectively,each immunization pigs were bled from the anterior vena cava and the levels of Ig G antibodies in serum was measured by indirect ELISA.The incidence of piglets,feces excretion and daily gain were observed before and after chanllenge.The results showed that the levels of the specific antibodies in the piglets of the experimental group were significantly higher than control group in 7 days after twice immunization and challenge experiments(**p<0.01).The levels of antibody in oral Yad A3-DT group were significantly higher than those in no target group,and the difference was significantly(**p<0.01). After chanllenge,the control group had a quick onset,diarrhea was obvious,and the immune group had no obvious diarrhea,and the duration of bacteria existence was significantly shorter than control group,and the daily gain was more. This shows that the tetravalent fusion antigen in three different routes of immunization can be induced higher levels of specific immune responses in piglets,in a certain extent can protect piglets are not infected by ETEC,and the effect of double target molecular enhance immunication was demonstrated.To sum up,recombined Fae G-m STa-LTb-STb tetravalent antigen has good immunogenicity.It confirms the oral inclusion body immunity is a feasible route of immunization,and various target molecules have a certain immunity enhancement,suggesting that recombinant tetravalent protein as an oral vaccine antigens has potential application value,which lays a foundation for the next step for the development of a new type of ETEC oral vaccine.