Bovine Coronavirus N Protein As A Model Antigen In The Slow-release Effect Of The Chitosan Microspheres

With the development of the immunology and the genetic engineering, new genetic engineering vaccines have been developed while these vaccines always have the disadvantages for small molecules, poor immunogenicity and difficult to induce effective immune response. So some substances are needed to compensate for this deficient, then adjuvants have been created. Traditionary adjuvants have many shortcomings, for example, they can cause local phlogosis reacts, carcinogenesis and hard to degradate in the body. So new types of adjuvants need to be created to overcome the disadvanges above. Chitosan, as a natural positively charged polymer which is non-toxic, biodegradable, non-immunogenic, is a partially deacetylated polymer obtained from the alkaline deacetylation of chitin. Therefore the chitosan have the potential value to develop a new adjuvant to prolong the antigen release for enhancing the immune response. Model antigen influences apparently to evaluate the adjuvants. It′s important to search for a better model antigen which is easy to be obtained with high immunogenicity and purity. Coronavirus nucleocapsid protein is not only conservative and better- immunogenic but also not influenced by modification of the protein. It also meets the basic requirement for model antigen. In conclusion, the bovine coronavirus N protein, which was cloned and expressed in E.Coli. as a model antigen, was used to evaluate the immunity response for the slow-release effect of the chitosan microspheres.Blank chitosan microspheres were prepared by cross-linking method using glutaraldehyde as crosslinkers and followed by emulsification. The microspheres were in good shape, smoth surface, good dispersibility with average size at 5.0μm, electric potential distribute in 36mV. NO. 487～1287 bases at 3′side of N protein gene from bovine coronavirus DB2 strain were artificially synthesized and highly expressed at 50.5% in E. Coli. The expressed N protein was separated by SDS-PAGE and prepared with high purity and average concentration of 14.77 mg/mL by electronic elution. BCV N-loaded chitosan microspheres were developed with embedding ratio of 16.49% and drug loading of 65.62% by absorptive method. These microspheres were used to immunize BALB/c mice by injection, and 206 adjuvant-N protein, only N protein and blank chitosan microspheres were used as controls. The dynamic antibody level in serum was monitored by indirect ELISA to evaluate the slow-release and adjuvanted effect of the BCV N-loaded chitosan microspheres.Results showed that BCV N-loaded chitosan microspheres induced better antibody response than the only N protein group in the inoculated mice, indicating that the BCV N-loaded chitosan microspheres had slow-release effect as adjuvant. However, chitosan microspheres were not so good in comparison with the 206 adjuvant with limit of the diameter distribution or the specific surface area, which is under research.