Expression And Immunogenic Study Of E.coli F18 Fimbriae FedF Protein And LTB-FedF Fusion Protein

1. Cloning and Sequence Analysis of LT Gene of EnterotoxigenicE.coliOne pair of primers was designed according to LT sequences published in Genbankto amplify LT gene by PCR from a clinic isolated Escherichia coli strain containingthe LT gene. The PCR product was cloned into pMD18-T vector, the recombinantplasmid was named pMDTLT. The cloned LT gene was sequenced. Results ofhomology comparison showed that LT gene cloned in this study share 97.6 % to 98.8% identity with the LT gene published on GenBank in nucleotide sequence level.2. Expression and immunogenic study of E.coli F18 fimbriae FedFproteinAccording to the fedF gene sequence of F18 accessed on GenBank, a pair of primerswas designed. Then fedF gene products were amplified from field strain isolated frompiglet suffered from diarrhea. After purification, ligation and transformation, fedFgene was successfully cloned into pMD18-T, the cloned gene was confirmed to have97.8%～99.2% identity in gene sequence level with other fedF gene in GenBank.Then the cloned fedF gene was subsequently sub-cloned into expressing vectorpBV220 by using another set of primers via biological engineering'methods and theresulting recombinant plasmid was then transformed into BL21(DE3) LysS. bacteria.It was confirmed by SDS-PAGE analysis and Western-blotting that the recombinantBL21 bacteria induced by IPTG were able to express the FedF protein with moleculeweight of 30kDa. The induced FedF protein account 11.17% of total bacterial protein.Mice were immunized with purified FedF protein, and the immunized mice allprotected from lethal E.coli strain challenge.3. Expression and immunogenic study of E.coli LTB-FedF fusionproteinThe gene coding for the LTB without signal peptide cloned into plasmid vectorpET28a(+) named pETLTB by genetic engineering methods. The 5' terminus of thegene that code for F18 fedF was genetically fused to the 3' terminus of the genecoding for the LTB, resulted in a recombinant plasmid pETLTBFedF which was wastransformed into E.coli BL21(DE3) LysS. The recombinant bacteria containingpETLTBFedF was able to express large quantities of fusion protein (LTB-FedF) underinduction of IPTG, the amount of the induced fusion protein whose expected moleculeweight was about 40kDa account for23.17% of total bacterial protein. The LTB-FedFprotein was further confirmed by SDS-PAGE analysis and Western-blotting. Theinduced LTB-FedF protein was purified and immunized into 6 week mice, and theimmunized mice showed partial protection to lethal challenge of E.coli strain, whichsuggest the LTB exhibited suppression on immune system, further study of LTB isnecessary to explain this phenomena.