Construction And Expression Of Stx2eB-LTB-ST Fusion Gene And Analysis Immunogenicity Of The Fusion Protein

Colibacillosis is a kind of important zoonosis. It is susceptivity especially in the newborn domestic animals. It can cause growth retardation, low productivity and even death. So it is a tremendous threaten to the animal husbandry. As a kind of pathogenic bacteria, according to its pathogenesis, Escherichia coli usually may be divided into ETEC, EPEC, EIEC, EHEC and UPEC. Above all, as a pathogeny, ETEC is very important to porcine, it induces porcine edema disease and the diarrhea. Approximately 40% cases of porcine diarrhea were caused by ETEC, and about 35% in China. The major virulence factors of edema disease and the diarrhea are Stx2e, LT and ST produced by E.coli. It is already clear that the subunit B of Stx2e can specifically bind to Gb4 acceptor in the Vero cell membrane and display nontoxic. So we can prevent Stx2e out of the target cells by neutralizing subunit B with its antibodies. The research about ST shows that its mature protein peptide chain is too short to display strong immunogenicity, we can enhance its immunogenicity by linking it to a big carrier protein molecule. This research used the principle of recombination PCR technology. We employed the strain E. coli O138 (Stx2e+), the plasmid EWD299 carrying LT gene, and O141 (ST+) as the templates to amplify the fragments of stx2eB, LTB and ST. With base-overlapping in these gene fragments, we constructed the stx2eB-LTB-ST fusion gene and the stx2eB-LTB-ST(L) fusion gene by PCR. After cloned into pMD18-T and DNA sequenced, the two fusion genes were subcloned into pET28a in frame. The recombinant plasmids pET28a::stx2eB-LTB-ST and pET28a::stx2eB-LTB-ST(L) were constructed and then transformed into host strain BL21(DE3). Analysis of SDS-PAGE and thin layer scanning showed the amount of the two...