The Fusion Of Novel CpG Molecule And Procine Interleukin-6 Gene And Its Regulation Of Immune Response

To develop a safe and novel fusion immunoadjuvant to enhance the immunity and resistance of animal, an 88 bases immunostimulatory oligodeoxynuleotides containing eleven CpG motifs (CpG ODN) was synthesized and amplified by PCR. The chitosan nanoparticles (CNP) was prepared by ion linking method to entrap the CpG ODN that significantly promoted the proliferation of lymphocytes of pig in vitro. Then the CpG- CNP was used to inoculate muscularly 21-day old Kunming mice, which were orally challenged with virulent K88 K99 E.Coli at 35 days post inoculation. The blood was collected from the tail vein of mice on 0, 7, 14, 21, 28 ,35, 42 and 49 days after inoculation to detect the changes of and content of IgG, IgA, IgM, IL-2, IL-4 and IL-6 by ELISA, and immune cells were sorted out by routine method. The results showed the CpG provoked remarkable proliferation of lymphocytes of pig in vitro in comparison with that of control group (P<0.05). The inoculation with CpG-CNP significantly raised the content of IgG, IgM and IgA in the sera of immunized mice( P<0.05); the level of IL—2, IL—4 and IL — 6 of the mice significantly increased in comparison with those of controls(P<0.05); So were the number of white blood cells and lymphocytes in immunized mice. The challenge results were found that, compared with those of control, the humoral and cellularimmunity were significantly enhanced in immunized mice, which resisted the infection of E.coli and survived, while the control mice manifested evident symptoms and lesions of infection. The CpG-CNP could significantly promote the cellular and humoral immunity and resistance of mice against E.coli infection.Then we used Bglllto digest the CpG ODN, and linked it with VPIL6 which was a eukaryotic VR1020 expression plasmid containing porcine IL6 gene. The recombinant plasmid (VPIL-6C) was digested by Stu I to identify whether the CpG ODN were correctly inserted into the plasmid. After prepared the chitosan nanoparticles containing VPIL-6C(VPIL-6C-CNP), the VPIL-6C-CNP was used to inoculate muscularly 21-day old Kunming mice singly or with special vaccines, which were challenged with Type II haemolytic streptococcus and Pasteurella bacilli C44-8 at 28 days post inoculation. The blood was collected from the tail vein of mice on 0, 7, 14, 21, 28, 35 and 42 days after inoculation to detect the changes of immunoglobulins, cytokines and immune cells. The results showed the inoculation with VPIL-6C-CNP significantly raised the content of IgG, IgM and IgA in the sera of immunized mice( PO.05); the level of IL —2, IL~4 and IL—6 of the mice significantly increased in comparison with those of controls(P<0.05); So were the number of white blood cells and lymphocytes in immunized mice. The challenge results were found that, in comparison with those of control, the humoral and cellular immunity were significantly enhanced in immunized mice, which resisted the infection of streptococcus and Pasteurella bacilli, while the control mice manifested evident symptoms and lesions and finaly died of infection ?In order to evaluate the effect of VPIL-6C on immune response of swine, VPIL-6C-CNP was used to inoculate the swines with Swine Fever Vaccine and Procine Reproductive and Respiratory Syndrome (PRRS) Vaccine. The results indicated that the humoral and cellular immune responses of immunized pigs were significantly increased, including the content of immunoglobulins, specific antibodies aginst Swine Fever and PRRS virus, interleukins and immune cells; the fusion immunoregulating molecule could significantly enhance the specific immunity of swine to vaccination, suggesting that the novel DNA molecule is apotentially promising immunoenhancing adjuvant for the prevention and control of porcine infectious diseases.