Protective Effect Of Recombinant Protein Of NcMIC2-like1 On Mice Infected With Neospora Caninum

Neospora caninum is an intracellular protozoan, which has a wide range of hosts and mainly infects cattle and dogs etc. Neosporosis can cause abortion, stillbirth and mummification. Neospora caninum infection is extremely harmful for cattle which has been considered as the main reason causes fetal abortion and causes a huge economic lost to livestock [1-2]. However, there still do not have effect therapeutic medicine to eradicate Neospora caninum. Therefore, immunization has become a more and more significant way to control it. In this study, we used the Neospora NcMIC2-like1 gene as a vaccine candidate, and fused it with the IL-2 gene. We expressed the recombinant proteins by using the prokaryotic expression vector pGEX-4T-1, and identified by SDS-PAGE and Western blot, indicated that the proteins were expressed successfully and can be specifically identified by mouse anti-Neospora serum. After vaccinated with NcMIC2-like1 and NcMIC2-like1-IL-2recombinant protein respectively, mice were infected with Neospora. By detecting the expression levels of IgG, IgG1, IgG2 a, IL-4 and IFN-γ before challenge, additionally survival rate, the growth rate and the number of Neospora caninum in brain of mice after challenge to evaluate its immune protective effects of the recombinant protein to Neospora caninum.Construction of recombinant plasmids pGEX-NcMIC2-like1 and pGEXNcMIC2-like1-IL-2 and expression of recombinant proteins IL-2 gene and NcNcMIC2-like1 gene were amplified, cloned and analyzed. Results showed that, the ORF of murine IL-2 gene and NcNcMIC2-like1 gene was 204 bp and 1134 bp respectively, and both homologies were 100% when compared with interleukin 2（Il2）mRNA（NM₀08366.3）and Liverpool hypothetical protein（NCLIV₀33690） mRNA in GenBank. After that, recombinant plasmids pGEX-NcMIC2-like1, pGEX-IL-2 and pGEX-NcMIC2-like1-IL-2 were transformed into BL21（DE3） competent cells. After analysis by SDS-PAGE electrophoresis, molecular weights of these three proteinswere 70 KDa,33KDa and 74 KDa, respectively. Western blot results indicated, the recombinant proteins pGEX-NcMIC2-like1 and pGEX-NcMIC2-like1-IL-2 can be specifically identified by murine anti-Neospora serum with certain reactogenicity.The immune protective effect of recombinant protein NcMIC2-like1-IL-2and NcMIC2-like1 We purified the recombinant proteins pGEX-NcMIC2-like1-IL-2,pGEX-NcMIC2-like1 and pGEX-IL-2. The results showed that the levels of IgG、IgG1 and IgG2 a improved in varying degrees after immunizing the mice with recombinant proteins pGEX-NcMIC2-like1-IL-2 and pGEX-NcMIC2-like1, the expression levels of cytokines also increased and were both significantly different（P≤0.05） compared with the adjuvantcontrol blank group and the control group, which means that both kinds of recombinant proteins can stimulate the body to produce Th1/ Th2 immune responses. After infected with Neospora caninum for 40 days, the survival rate and weight gain rate of pGEX-NcMIC2-like1-IL-2 group mices were90% and 80.8% respectively, and were significantly different（P ≤ 0.05） with the control group;the survival rate and weight gain rate of pGEX-NcMIC2-like1 group were 70% and 62.5% respectively, significantly different（P≤0.05） with the control group. Extracted the brain genomes of mice in each group were performed fluorescence quantitative PCR, the Neospora reduction rates of pGEX-NcMIC2-like1-IL-2 group and pGEX-NcMIC2-like1 group were 64.7% and 61.4%respectively. Both of two groups are significantly different（P≤0.05） compared with the control group. The results indicated that PGEX-NcMIC2-like1-IL-2 and pGEX-NcMIC2-like1 recombinant proteins both had certain protective effects to resist against Neosporosis. Protective effect of pGEX-NcMIC2-like1-IL-2recombinant protein was better than that of pGEX-NcMIC2-like1. Therefore, IL-2 can enhance the protective effect of recombinant protein of NcMIC2-like1 in mice infected with Neospora caninum.