Preparation And Preliminary Application Of Classical Swine Fever Virus E2 Protein Using Carbon Nanotubes As Carrier

As a member of the graphene family, carbon nanotubes(CNTs) have the ability to cross cell membranes, CNTs with choicely designed which are able to enter cells as carriers of a variety of organic or inorganic particles, such as proteins, nucleic acids and chemical drugs. CNTs were shown to activate cells from the innate immune system, such as monocytes, macrophages and dendritic cells. The researches of carbon nanotubes always focused on tumor diagnosis and treatment. With the in-depth study of carbon nanotubes, more and more scholars begin to pay attention to the application of carbon nanotubes as vaccination scaffolds. The classical swine fever(CSF) caused by the classical swine fever(CSFV) infection in pigs is highly virulent and contact disease. It brings serious hazard in the pig industry. The glycoprotein E2 of CSFV is recognized as a kind of major protective antigen at present, and can induce neutralization antibody against CSFV. To explore the impact of carbon nanotubes on animal immunization, we used chemical approaches to covalently append a recombinant protein E2 onto carbon nanotubes as a antigen to immune mice. After two immunizations, Enzyme-Linked Immunosorbent Assay(ELISA) for antibody detection was carried out, to assess the impact of carbon nanotubes on immunity.The experiment consists of three parts: 1)We used E. coli to express the CSFV-E2.SDS-PAGE was used to analysis of the expression product. And His tag Ni-NTA affinity chromatography was used to purify the expression product, Western Blot was used to analysis the specific of antigen. 2) EDC / NHS as a coupling agent were used to covalently append a recombinant protein E2 onto multi-walled carbon nanotubes with carboxyl reaction. The protein content of E2 protein concentration of antibody conjugated to CNTs was determined using Coomassie blue protein assay. 3) Carbon nanotubes-E2 protein as an immunogen was used to immunize mice, at the same time protein E2 group, Freund’s adjuvant with E2 protein as the control groups. After two treatments, with two weeks apart. The protein-specific serum IgG responses were evaluated by ELISA assay. Result:Coomassie blue assay measured the coupling E2 protein is 480μg. Among the treatment groups,the antibody titers of E2 group is1:3200, the antibody titers of carbon nanotube-E2 is 1:12800, the antibody titers of Freund’s adjuvant-E2 is 1: 51200.Conclusion: We preparated a carbon nanotube-E2 as antigens. And the result of ELISA, proved that carbon nanotubes can enhance the immune response, induce specific antibodies response.