Immunogenicity Of ETEC K99 Protein And Fusion Protein K88ac-STII

Enterotoxin Escherichia coli is main pathogen causing piglet diarrhea,which has result in tremendous economic loss, especially in our stockbreeding manufacture.Many researches have proved that K88ac,K99 pilus and ST enterotoxin are major protective protein against Enterotoxin Escherichia coli. So all of them play a crucial role in vaccinal research.Specific primers was designed to amphfy the nucleotide sequence of K99 gene(M35282)of Enterotoxin Escherichia coli(ETEC) reported by GenBank.With the specific primers by PCR , the was interesting gene amplified of target fragment was obtained .The target frament was doned into the multiply plasmid vector pBS-T to generate.The recombinant plasmids were proved through analysis, PCR,enzyme cut and sequencing,it was named pBS-K99.The recombinant plasmids and pET-28a were degisted by XhoI/NcoI, then the fragment was identified and cloned into prokaryotic expression vector pET-28a by T4 DNA ligatse .The recombinant plasmid pET-28a-K99 was transformed into E.coli BL21(DE3) and induced to express with IPTG, and analyzed the product of expression through SDS-PAGE and Western-blotting. Base on recombinant strains BL21(DE3)(pET28a-K88ac-STâ…¡)and BL21(DE3)(pET28a-K99),we also carried out the following experiment:Recombinant strains were beared and the expression was induced by IPTG.As a result, each of them can express differential protein, which have the same molecular weight as prospective protein. The prospective protein was purified with dialytic membrane and the primary of concentration was adjusted to 3mg/ml, then Freund's incomplete adjuvant was added into the purified fusion protein to become experimental vaccine. Piglets of each groups were vaccinated respectively with recombinant protein vaccine,commodity vaccine and PBS as contrast. The serum IgG anti-protein of each groups were detected by indirect ELISA.The recombinant plasmid BL21(DE3)(pET28a-K99)was induced to express with IPTG,the result of SDS-PAGE showed that the product of protein express molecular weight was about 18KDa and the product of express was abundance , the product of protein express through Western blotting showed that it can combine with special serum produced in rabbit of immuned by ETEC(C83912). Piglets of each groups were vaccinated respectively with recombinant protein vaccine, commodity vaccine and PBS,the result clearly demonstrated that the IgG content of recombinant protein vaccine and commodity vaccine group was significantly higher than that of PBS piglets (P < 0.01), and the remarkable difference did not appear between recombinant protein vaccine and commodity vaccine group (P > 0.05).