Construction And Immunogenicity Studies Of GE-3 Gene Engineering Strain Against Diarrhea In Neonatal Swine Caused By ETEC

Colibacillus diarrhea of newborn piglet is a kind of the most common infectious disease,which is caused by enterotoxigenic E.coli (ETEC) and characterized by"yellow scour"of the 1 to 7-day-old newborn piglet with high morbidity and mortality. With the development and application of vaccines, colibacillus diarrhea of newborn piglet has been controlled to a certain extent, but the safe and immunogenicity of the vaccines are still unsatisfied so that it is exigent to develop a new gene engineering multivalent inactivated vaccine which has high safe and immunogenicity.In this study, the genes of ST1,K88ac and LTB were linked and the gene engineering trivalent vaccine K88ac-ST1-LTB was constructed successfully with molecular biology technology. The purpose of this study was to improve the safe and immunogenicity of the vaccine against the colibacillus diarrhea of newborn piglet.According to the published DNA sequence of K88ac,ST and LTB, one pair of K88ac primer, three pairs of ST1 primers and one pair of LTB primer had been designed and synthesized .With the technology of PCR, K88ac gene, ST1 mutant gene and LTB gene were amplified respectively. After isolating , purifying, restriction endonuclease-digesting and linking with T4 DNA Ligase ,the recombinant plasmid pXK88acST3LT5 was obtained and transformed into BL21(DE3),thus the recombinant strain BL21(DE3)( pXK88acST3LT5 ) expressing fusion protein K88ac-ST1-LTB wasconstructed. It was proved that the recombinant plasmid pXK88acST3LT5 contained the fusion gene K88ac-ST1-LTB which had correct sequence and ORF by identification of endonuclease-digesting and sequence analysis. The recombinant strain GE-3 was inoculated into LB medium at the ratio of 1%, and was cultured for 2 hours at 37℃, then was induced by 1 mmol·L-1 IPTG for 4 h, finally the fusion protein K88ac-ST1-LTB was expressed at high level. The expressed protein can be recognized by MAb of ST1, antibodies of LTB and K88ac with ELISA. With the supernatant and the lysate of the recombinant strain GE-3 respectively, the pouring stomach of sucking mice was done and the result was negative(G/C≤0.083),which indicated that the fusion protein lost the toxicity of ST1.The mice had been protected which were immunized with inclusion body or the inactivated vaccine and then challenged with C83902 virulent strain. The result showed that the fusion protein K88ac-ST1-LTB can stimulate the mice to produce antibody which has the function of neutralizing the natural ST1 toxin. Therefore, the recombinant strain could be used as a candidate strain of gene engineering inactivated vaccine against colibacillus diarrhea of newborn piglet.On the basis of lab techniques, the industry producing process was studied. According to the induced test, the 100 mmol·L-1 lactose was the most ideal inducement in the industry condition. After adding 100 mmol·L-1 lactose inducement, GE-3 strain could express fusion protein at the second hour. With the prolongation of the induced time, the total amount of bacterium and target protein increased too, and were stable from the fifth hour to the sixth hour. Thereby, we confirmed that the concentration of lactose inducement was 100 mmol·L-1, and the induced time was at least six hours. Three batches of the safe and efficiency vaccine produced in the industrialized condition indicated that the industrialized producing process was scientific and feasible.The study on the gene engineering trivalent vaccine K88ac-ST1-LTB against colibacillus diarrhea of newborn piglet has some advantages as following.Firstly, the gene engineering vaccine has been deleted the toxicity of ST1. By PCR mutant method, Cys was changed into Ser, which made one of the three pairs of double-sulfide bond vanished and the protein's space conformation changed. Secondly, the problem of low immunogenicity of ST1 was solved. By gene engineering technology three ST1 genes were linked together and two antigenic structures were added in the expression, which improved the immunogenicity. Thirdly, the fusion protein K88ac-ST1-LTB expressed by the recombinant strain GE-3 constructed in the study has more immune range,and the recombinant strain GE-3 could be used as a candidate strain of vaccine.