Construction And Expression Of Fusion Gene Of Heat-Labile Enterotoxin B Subunit And K99 Fimbrial Of Escherichia Coli

Primers were designed according to the nucleotide sequence of LTB gene and K99 gene of enterotoxin Escherichia coli(ETEC) reported respectively. With the specific primers, target fragments were amplified from ETEC. The target framents were inserted into the linearized plasmid vector PBS-T respectively. By enzyme analysis, PCR and sequencing, the recombinant plasmids were proved to be true. The recombinant plasmids LTB and k99 were used to construct the fusion gene.In the middle of the two genes, a linker, as a bridge, were fused them. The fuse gene(LTB-K99) and pET-28a were degisted with BamHI/XhoI, then the fragment was identifeied and cloned into prokaryotic expression vector PET-28a, the combinant plasmids were transformed into E.coliBL21(DE3) and induced to express with IPTG, and analyzed the products of expression through SDS-PAGE and Weste-blotting .Result: the structural gene LTB and K99 were were amplified from ETEC. After analysis of the sequence, the gene of LTB and K99 has high homology. The LTB-K99 fusion gene is 966bp.By analysis of SDS-PAGE and Western-blot the fragments were coloed into prokaryotic expression vector pET-28a with the right ORF, and can express the products expected successfully in E.coli.The result of study discover: the products of fusion gene are abundance and can combine with serum produced in rabbit. The result of study accord with that of oversea.The fusion gene possibily become candidate genes of ETEC engineering vaccine.