Academy of Scientific Research & Technology and National Research Center, Egypt

Recombinant protein consisting of an antigen fused to C3d may elicit a more robust immune response than the antigen alone. The objective of the present study was to clone FimA- c3d recombinant DNA into pCold-TF vector and successfully express in prokaryotic expression vector. FimA subunit of type I fimbriae from Salmonella enterica serovar Enteritidis was conjugated to mouse complement fragment molecule C3d. FimA and FimA-mC3d, FimA-mC3d2 and FimA- mC3d3 were cloned into the expression vector pCold-TF. Constructions of the recombinants pCold-TF-fimA with different copies of C3d were confirmed by digestion with restriction enzymes and sequencing. Soluble fusion proteins of FimA with different copies of C3d were induced by IPTG and were expressed in Escherichia coli BL21 (DE3) under optimal conditions. The results showed that the proteins induced from recombinants pCold-TF-fimA, pCold-TF-fimA-mC3d, pCold-TF-fimA-mC3d2 and pCold-TF-fimA-mC3d3 were 70, 100, 130 and 160 kDa, respectively. The fusion protein was recognized by rabbit anti-fimbriae polyclonal antibodies, and then visual- ized by goat anti-rabbit polyclonal antibodies with a chrome appearance by enzyme-subtract inter- action. The recombinant proteins were separately purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC). Finally we conclude that anti- gen conjugated with mC3d can be functionally expressed in prokaryotic vectors. a 2014 Production and hosting by Elsevier B.V. on behalf of Academy of Scientific Research & Technology.