Cloning and expression of FimA-c3d recombinant protein

Abstract

Abstract 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- mC3d 2 and FimA- mC3d 3 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- mC3d 2 and pCold-TF-fimA- mC3d 3 were 70, 100, 130 and 160 kDa, respectively. The fusion protein was recognized by rabbit anti-fimbriae polyclonal antibodies, and then visualized by goat anti-rabbit polyclonal antibodies with a chrome appearance by enzyme-subtract interaction. The recombinant proteins were separately purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography (IMAC). Finally we conclude that antigen conjugated with mC3d can be functionally expressed in prokaryotic vectors.