Immune Effection Of NcGRA7-IL-18 Recombinant Protein Against Neospora Caninum Infection In Balb/c Mice

Neosporosis is a global disease,which seriously affects the animal husbandry development in the world.Neosporosis mainly causes abortion of pregnant animals,stillbirth and congenital motor nerve damage of newborn animals.However,there is still no vaccine available for neosporosis.So,the research try to find a vaccine to control neosporosis.Since interferon a(IFN-a)was approved by FDA in 1986,many cytokines have been used for disease prevention and treatment.Combination of cytokines and antigens could improve the level of cellular immunity and improve the effect of immune protection.It has been reported that interleukin-18(IL-18)could induce the immune response to Th1 type and production of interferon-gamma(IFN-?),which is lethal to intracellular pathogens,so it has been researched as an important vaccine adjuvant.Neospora caninum dense granule 7(Nc GRA7)is one of the main effective antigens for the neosporosis vaccine research.Therefore,the research try to explore Nc GRA7 and IL-18 recombinant expression protein(Nc GRA7-IL-18)against neosporosis infection ability in the host.In this research,Nc GRA7 and IL-18 genes were amplified by PCR method,the Nc GRA7-IL-18 gene was amplified by in-Fusion Cloning technology,p MD18-T-Nc GRA7,p MD18-T-IL-18 and p MD18-T-Nc GRA7-IL-18 cloning vector need to be constructed and p ET-32a-Nc GRA7,p ET-32a-IL-18,p ET-32a-Nc GRA7-IL-18 expression vectors also need to be constructed.Then these expression vector were transfected into BL21(DE3)competent cells to induce protein expression.The target proteins were identified by SDS-PAGE and western blot.The immune effect was evaluated by immune type(Th1/Th2),survival percent of mice after infected with neospora caninum and real time PCR identify the neospora caninum in mice organs(heart,liver,spleen,lung,kidney,brain).The results were show as follows,(1)Gene amplification and vector construction results.Nc GRA7,IL-18,Nc GRA7-IL-18 genes were successfully amplified,p MD18-T-Nc GRA7,p MD18-T-IL-18,p MD18-T-Nc GRA7-IL-18 cloning vectors were successfully constructed,p ET-32a-Nc GRA7,p ET-32a-IL-18,p ET-32a-Nc GRA7-IL-18 expression vectors were also successfully constructed.(2)Protein expression results.Nc GRA7,IL-18 and Nc GRA7-IL-18 proteins were successfully expressed in BL21(DE3)competent cells and purified by NI-NTA,and the protein expression was optimized.(3)Antibody test results.Balb/c mice were immunized three times,and the specific antibody titer of Nc GRA7-IL-18 immunization group was above 1:409600.7 days after second immunization,Ig G1 of Nc GRA7-IL-18 immunization group was significantly higher than PBS group(P < 0.01).However,there was no existence difference(P > 0.05)between two groups in Ig G2 a antibody titer.(4)Detection results of cytokines.The results of cytokine detection showed that IFN-? was not detected in the serum of mice after immunization.There was no difference about spleen lymphocyte in the amount of IFN-? produced by Nc GRA7-IL-18 immunized group compared with PBS group stimulated by lysate antigen of neospora caninum tachyzoites(P > 0.05).The amount of IL-4 produced show Nc GRA7-IL-18 immunized group was higher than PBS group(P < 0.01).The purified Nc GRA7-IL-18 protein and Nc GRA7 protein were used to stimulate splenic lymphocytes of mice.The amount of IFN-? produced in Nc GRA7-IL-18 protein was higher than Nc GRA7 protein(P<0.05).(5)The results of neospora caninum infection.Four mice in each group were infected 2×107 neospora caninum tachyzoites,one died on the seventeenth day in the Nc GRA7-IL-18 group,and the rest of three mice did not die after infection during 40 days.The protection percent could reach 75%.However,all mice died in 8 days in PBS group.Real-time PCR was used to detect neospora caninum DNA in heart,liver,spleen,lung,kidney and brain of balb/c mice.Neospora caninum was detected from the liver,spleen and lungs in the died mouse of Nc GRA7-IL-18 group,and was not found in the one of the rest surviving mice.However,neospora caninum DNA was found in PBS group mice.Conclusions: The Balb/c mice were immunized by recombinant Nc GRA7-IL-18 protein,it tends to be Th2 type immunization response.The protein own immune protective effection against neospora caninum in Balb/c mice.