Preliminary Construction Of The Type 1 Fimbriae Vector Of E.coli From Chickens Expressing Foreign Antigenic Gene

Bacterial surface display, in which heterologous peptides and proteins are fused withthe bacterial surface proteins and expressed on the surface of recombinant bacteria hasprovided a new method to construct live recombinant vaccines.Compared with other kinds of bacterial surface proteins used as exposure carriers forthe foreign epitopes, fimbriae are advantageous of constructing engineering vaccines.Fimbriae are adhesive bacterial surface structures and the amount of fimbriae is largewhich are surface polymer having up to about 500 copies per cell, every copy is consistsof about 1000 subunits, fimbriae normally are very good immunogens and there arespecific receptors on the intestinal epithelial cells, so if the gene coding for foreignantigenic epitopes can be inserted into the subunit gene, they can easily be recognized andcaptured by the immune system and the amount of epitopes presented on the cell surfaceare much larger which may be favourable for the induction of stronger immune response,so it is good expression vector to display foreign epitopes and construct engineeringvaccines.To select wild-type Eschorichia coli with type 1 fimbriae, structural gene limA of type1 fimbriae was amplified by PCR from 101 strain wild-type Eschorichia coli isolates' schromosomal samples. Finally we obtained 63 strain wild-type Eschorichia coli with type1 fimbriae.The secondary structure antigen epitopes, hydrophilicity and flexibility of fimAsubunit were predicted with the DNAstar software. Based on the prediction, the sitebetween amino acid residues 68 and 69 for inserting heterologous epitopes was chosen inwhich inserting can not influence the expression and assembly of type 1 fimbriae. Toconstruct homologous recombination suicide vector, the recombination directingsequence which lie on the upstream and downstream region of the inserting site F wasfirstly selected, then the site-directed mutation was performed with the overlappingextention PCR and the KpnI restriction site was introduced into fimA to insert the foregnepitopes, the gene fragment coding for the HNS for Newcastle disease virus (NDV) wasamplified by PCR and inserted into fimA, finally we obtained the inserting mutantfragment for fimALR-HNS gene. FimALR-HNS gene was amplified by PCR and clonedinto a suicide vector pDM4, which containing the Chloramphenicol resistance gene andsacB gene. The homologous recombination suicide vector plasmid with Chloramphenicolresistance gene as single crossover recombinants negative selective marker and sacB gene as double crossover recombinants positive selective marker include homologous sequenceand inserted gene HNS.The results indicate that the homologous recombination suicide vector plasmid wassuccessfully constructed and we try to transform it into wild-type Eschorichia coli fromchickens by conjugal transfer and electrotransformation method. It may lay thefoundations for further study of the inserting mutation Eschorichia colias live vectorvaccine.