| Enterotoxigenic E.coli(ETEC) is one of the main pathogens causing diarrhea of infant and new-born piglet. The pathogenicity of ETEC is decided by its ability to settle on the intestinal epithelial cells which mediate by the specific fimbriae on the surface of the cell, and to produce enterotoxins. K88 is one of the most important fimbriae, which can be divided into three serotype variants K88ab, K88ac and K88ad. Cell surface display is a new technology that was established in vaccine development in recent years . The hybrid fimbriae containing foreign epitope will appear on the surface of cell if the fimbriae structural genes express in the cell. This is a new way to develop live vaccine, which is so-called cell surface display. In this way, fimbriae appear as complete fimbriae structure and fimbriae antigen protein appear as polymers. So they may will increase the immunogenicity of the fimbriae antigen.In this study, the structural genes faeC-faeH of K88 fimbriae operon was amplified by Long and Accurate PCR, and the PCR product was cloned in pBR322 plasmid vector. Then the gene encoding K88 fimbriae antigen protein was reconstructed by introducing two enzyme digestion positions Apa I and Nco I in order to fusing of small exogenous DNA molecules. The ST epitope coding sequence and the epitope coding sequence of structural protein VP1 of foot and mouth disease virus (FMDV) wrere synthesized and fused in the supper variable region of K88 fimbriae antigen gene respectively. In order to assembly fimbriae on the cell efficiently, the negative regulatory gene faeA and faeB was cloned by PCR. In order to obtain ST protein that will be used for preparation of antibody for detection of STII epitope displayed on the cell surface in the next experiment, ST gene and LTB gene of ETEC were fused and expression vector pET28a-STI-STII-LTB was constructed in this study.The structural genes faeC-faeH of K88 fimbriae operon was amplified and the PCR product was cloned in pBR322 plasmid vector. Accuracy of PCR product and reconbinant plasmid pBR-fae1 was demonstrated by enzyme digestion and sequence analysis. Then enzyme digestion positions Apa I and Nco I were introduced into the major structural gene of K88 fimbriae, so plasmid pBR-fae2 was constructed in the study. The epitope coding sequence of structural protein VP1 of foot and mouth disease virus (FMDV) wrere fused in Apa I and Nco I of plasmid pBR-fae2. Consequently, the plasmids pBR-fae-STII and pBR-fae-VP1 respectively containing K88pili-ST epitope and K88 pili-VP1 epitope fusion gene were constructed in this study. The result of sequence analysis of faeA of K88ab, K88ac showed that there is no difference between the two serotypes. Compared with the negative regulatory protein sequence of other fimbriae produced by E.coli, there is a conservative motif containing seven amino acids. This domain may be the binding site to regulatory sequences. The regulatory gene faeB and its promoter region were cloned in this study. The result of sequence analysis indicated that there contains the characteristic sequence -35 and -10 of promoter and there are three GATC-related sites that related fimbriae expression and assembly. ST and LTB fused gene and expression vector pET28a-STI-STII-LTB were demonstrated by enzyme digestion and sequence analysis. Recombinant plasmid pET28a-STI-STII-LTB were transformed into E.coli BL21(DE3). Added 0.8mM IPTG and induced 4h, SDS-PAGE demonstrated that STI-STII-LTB reached the highest expression level.Construction of plasmid pBR-fae-STII and pBR-fae-VP1and cloning of two regulatory gene faeA and faeB will lay the foundation for assembly of K88 fimbriae .Expression of STI-STII-LTB protein provided a preparation for preparation of ST antibody for testing the displayed STII epitope in the following experiments . |
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