Study About Influence On Immunogenicity Of Molecular Adjuvant C3d To Major Subunit FimA Of Type Ⅰ Fimbriae In Salmonella Enteritidis

Salmonella enteritidis is an important foodborne enteric pathogen,which cause substantial economic loss both directly through infected farm animals, and indirectly from animal carriage leading to cases of human Salmonella infections.It is generally accepted that fimbriae are an important factor in bacterial survival and persistence in the host. They are involved in the adhesion of bacteria to different cells/surfaces that is often the initial step in the colonization of host tissue and an essential stage in pathogenesis of salmonellosis.The main fimbriae of the Salmonella enteritidis are typeⅠ, typeⅡ, typeⅢ, typeⅣ, SEF14,SEF18.Most Salmonella enteritidis express type I fimbriae,which are built from FimA with the coding gene fimA.The final degradation product of the third (C3)component of complement, C3d, can bind the complement receptor 2 (CR2)on antigen presenting cells directly, reducing the activation threshold of B lymphocytes, thereby enhancing antigen immunogenicity.Thus,C3d is considered as a novel molecular adjuvant.Some reports support its adjuvant effect,but these reports are mainly based on C3d conjugated eukaryotic protein vaccine and some DNA vaccine immunization,there is no reserch report about its effect of immunogenicity to protein expressed in prokaryotic expression vector.To explore the influence on immunogenicity of C3d to protein expressed in prokaryotic expression vector, we choose FimA as target protein from major subunit of typeⅠfimbriae,to express FimA and fusion protein of FimA with tadem of mC3d of one,two and three copies respectively. The target gene fimA was used to construct recombinant plasmids as following:the fimA gene was amplified by PCR using the pair of primers and the DNA template from Salmonella enteritidis standard strain SD-2 genomic DNA.The PCR product of fimA gene were digested and then cloned into the expression vector pCold-TF to construct recombinant plasmid pCold-fimA, which was confirmed by the means of combination with restriction endonuclease analysis and sequencing.The different copy of genes of mC3d, mC3d.2 and mC3ds, which obtained through the digestion of recombinant plasmids of pUC-mC3d,pUC-mC3d2 and pUC-mC3d3,were cloned into the recombinant plasmid pCold-fimA to construct the recombinant plasmids,i.e. pCold-fimA-mC3d,pCold-fimA-mC3d2 and pCold-fimA-mC3d3.On the basis of the above described constructs,the FimA and the FimA-mC3d fusion protein which carried tadem of mC3d with one, two and three copies respectively, were optimally induced and expressed by IPTG at 15℃for 24 hours.SDS-PAGE showed that the recombinant proteins were 70 kDa,100 kDa,130 kDa and 160 kDa respectively. The antigenicity of the recombinant proteins expressing FimA were demonstrated by Western-blot with the rabbit polyclonal antibody of type I fimbriae.The recombinant proteins were separatedly purified by Ni-TED (tris-carboxymethyl ethylene diamine) immobilized metal iron affinity chromatography(IMAC).For further exploring the influence on antigenicity of C3d to FimA,the purified proteins of FimA and FimA-mC3d which carried tadem of mC3d with one,two and three copies respectively, were subcutaneously immunized to BABL/c mice.In the first immunization, we use soluble proteins of FimA, FimA-mC3d,FimA-mC3d2 and FimA-mC3d3 respectively. In the second immunization, the groups were all used for FimA soluble protein.In two immunizations,the control group was immunized with sterile saline.One week after second immunizaion, all groups were challenged with 10 cfu Salmonella enteritidis standard strain SD-2,then observed all mice tested every day.The results showed that,when the immunization dose of 10μg,antibody levels in mice with FimA-mC3d2 and FimA-mC3d3 fusion protein groups were raised and increasd by two times and four times compared with the FimA protein groups.In addition, in this immunization dose,antibody level in FimA-mC3d3 groups was increasd by four times and two times compared with the FimA-mC3d and FimA-mC3d2 groups.The challenge test results showed that immune protection ratio in mice with FimA, FimA-mC3d2 and FimA-mC3d3 groups were 50%,75% and 100% respectively.Therefore,we confirm that C3d can enhance the immune effects of the conjugated protein which are expressed in prokaryotic vector. It may be very promising for the futher study of C3d as the novel molecular adjuvant for prokaryotic protein vaccine,and also for the application in the prevention of Salmonellosis in the future.