Study On Construction Of Novel CpG Immunostimulatory Molecule And Its Immunological Effects

In order to develop safe and effective immune adjuvant of animals, a novel CpG oligodeoxynucleotides (ODN) contains 11 CpG motifs was designed and synthesized, a pair of primers were applied to introduce the endonucleases digestion sites of PstI to the two ends of CpG ODN. After amplification by PCR, the bioactivity of this purified CpG ODN was detected by MTT assay to stimulate the proliferation of lymphocytes from bloods of porcine, cattle and yak .The results were founded that the CpG ODN could provoked remarkable proliferation of lymphocytes in vitro in comparison with that of control group(P<0.05).The Chitosan nanoparticle was prepared by ion—crossing method, and this CpG ODN was entrapped with chitosan nanoparticles (CNP) and utilized to immunize Kunming mice which were co-inoculated with paratyphoid vaccine at the age of 21 days. The peripheral blood was weekly collected from the tail vein of inoculated mice to detect the effects of CpG ODN Chitosan-nanoparticles on immune responses of mice. The results showed that the CpG ODN and its Chitosan-nanoparticles could remarkably enhance the immune responses of mice co-administrated with paratyphoid vaccine. Then the CpG ODN was digested by PstI endonuclease and ligated to VR1012 plasmid which was also digested with the PstI, to construct a novel immunostimulatory molecule CpG-VR1012. The recombinant plasmid was digested with PstⅠ and HindⅢ to identify whether the CpG ODN was inserted into the plasmid. Then the CpG-VR1012 packed with CNP of was prepared to immunize experimental mice solely or with vaccines against Swine fever, the Pasteurellosis and streptococcus. The results were founded that, compared with those of the control, the content of immunoglobulins, dominantly IgG, including IgA and IgM, and specific antibodies to the vaccines against Swine fever, the Pasteurellosis and streptococcus mounted significantly in the sera from the VRC1-CNP vaccinatedmice, the amount of IL2, IL4 and IL6 increased remarkably in the sera of immunized mice, so were the number of white blood cells, lymphocytes and monocytes, granuloytes of the mice immunized with VRC1-CNP. After the challenge by virulent Pasteurellosis and streptococcus, these immune values were respectively elevated to different extent in the mice vaccinated with VRC1-CNP.. The most of immunized mice survived, while the control mice fell ill with the evident lesions with diffuse hemorrhage in stomach, small intestines and peritoneum. Then VRC1-CNP was used to immunize pig along with Swine fever vaccine and Porcine Reproductive and Respiratory Syndrome (PRRS). The effects of VRC1-CNP were investigated on cellular and humoral immune levels of pig and antigen-specific antibody responses. The results showed that, in comparison with the control, the amounts of immunglobulins, specific antibody, contents of interleukins and numbers of immunocytes were all elevated remarkably. These indicated that VRC1-CNP could obviously boost the immune responses of pig and increase protective immunity and resistance against infection by virulent pathogen. All these results suggested that VRC1-CNP could remarkably magnify immunity and resistance of animals against infectious pathogens, and be new promising effective DNA molecular immunoadjuvant for future vaccination and control against infectious diseases of porcine.