| Colibacillus diarrhea of newborn piglet is a kind of the most common infectious disease.which was caused by enterotoxigenic E.coli (ETEC) and characterized by '"yellow scour" of the 1 to 7-day-old newborn piglet and high morbidity and mortality. With the development and application of vaccine. colibacillus diarrhea of newborn piglet has been controlled to a certain extent. but the innocuity and the immunogenicity of the vaccine are still unsatisfied so that it is exigent to develop a new multivalence gene engineering inactivated vaccine which has high innocuity and immunogenicity.In this study. the genes of ST1 K88ac and LTB were linked and the trivalent gene engineering vaccine K88ac-ST1-LTB was constructed successfully with molecular bio-technology. The purpose of this study was to improve the innocuity and the immunity efficacy of the vaccine and promote prevention level against the colibacilius diarrhea of newborn piglet.(1)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 C DE3 )(pXK88acST3LT5) expressing fusion protein K88ac-ST1-LTB was constructed . 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 BL21 (DE3 )( pXK88acST3LT5) was inoculated into LB medium at the ratio of 1%, and was cultured for 2 hours at 37 . then was induced bv 1mmol L-1 1PTG for 4 h, finally the fusionprotein 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 method. With the supernatant and lysate of the recombinant strain BL21 (DE3) (pXK88acST3LT5) respectively, the test of 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 STi toxin. Therefore, the recombinant strain could be used as a candidate strain of gene engineering inactivated vaccine against colibacillus diarrhea of newborn piglet.(2)The recombinant strain BL21 (DE3) (pXK88acST3LT5) was tested for the properties of bacterial staining, culturing, plasmid stability, preservation condition and so on in laboratory. The properties of bacterial staining, culturing, biochemistry of the bacterium seeds from the first to the tenth generation were all in accord with E.coli; Under the unselected pressure (Kan") , the recombinant strain BL21 (DE3) (pXK88acST3LT5) can be cultured for twenty generations continually and the reserved rate of recombinant plasmid was 100%.The result indicated that the recombinant plasmid pXK88acST3LT5 can be transmitted stably in BL21 (DE3) .The induced culture of the recombinant strain BL21 (DE3) (pXK88acST3LT5) was analyzed with SDS-PAGE and thin-layer gel scan, the result showed that the relative content of fusion protein was above 73% of total protein; The mice immunized respectively with the culture of the first and the tenth basic seeds were challenged with 1 MLD (5 108 CFU) virulent bacterium liquid, and the protected rate was 90%, which indicated all of the seeds from the first to the tenth generation can be used to produce vaccine. So they can be chosen as basic seeds .By use of kept term detecting test, the term of seed kept in glycerin at -20 癈 was two years,while it... |
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