伪狂犬活/灭活病毒接种小鼠诱导免疫效力及差异因子比较

Comparison of immunity and cytokine difference in mice induced by live or inactivated Pseudorabies virus

[目的]本研究旨在比较活/灭活病毒进入体内后对免疫系统活化的差异,为免疫增强剂的筛选和评价建立高效动物模型。[方法]本试验利用伪狂犬病毒(PRV)Bartha-K61株建立小鼠伪狂犬人工感染模型,筛选活/灭活病毒接种小鼠后免疫相关差异因子。将PRV Bartha-K61活毒以不同剂量、不同方式感染小鼠,通过计算小鼠存活率确定最佳感染剂量与感染方式;将筛选的最佳剂量的伪狂犬病毒活毒与相同剂量下灭活病毒按照前述筛选的最佳感染方式接种小鼠,利用ELISA试剂盒检测免疫7 d后小鼠血清中TNF-α、IL-1β、IFN-β和IL-6水平;利用PRV gB ELISA检测免疫7 d后小鼠血清PRV特异性gB抗体水平;利用灭活PRV作为特异性抗原进行淋巴细胞增殖试验并计算各组淋巴细胞刺激指数(SI)。[结果]在保证小鼠存活的前提下,6~8周龄小鼠的PRV的安全感染剂量为5×104TCID50;同等剂量下,相比于肌肉和腹腔注射,皮下注射为最安全的感染方式;同等剂量的活毒和灭活病毒接种小鼠后,与灭活病毒组相比,活病毒感染7 d后小鼠血清中TNF-α、IL-1β和IFN-β水平显著升高(P<0.001,P<0.01),与灭活病毒组相比,活病毒组小鼠血清PRV gB特异性抗体IgG水平和SI也显著升高(P<0.05,P<0.001);不同灭活方式间各细胞因子和SI差异不显著(P>0.05)。[结论]成功建立小鼠伪狂犬人工感染模型,明确活/灭活伪狂犬病毒接种小鼠后,机体早期存在与免疫相关差异因子,为后续免疫增强剂的评价筛选提供依据。

[Objectives]In order to clarify the difference in activation of the immune system by live or inactivated viruses after entering the body, a high-efficiency animal model was established for the evaluation of the efficacy of immune enhancers to better improve the immune efficacy of inactivated vaccines. In this experiment, the Pseudorabies virus(PRV)Bartha-K61 strain was used to establish a PRV artificial infection mouse model, and to screen the immune-related difference factor after inoculation with live or inactivated antigens. [Methods]PRV Bartha-K61 was inoculated to experimental mice in different doses and methods, and the survival rate was calculated to screen the safe dose. Mice were vaccinated with live virus or inactivated virus at a safe dose, and ELISA kits were used to detect the level of serum PRV-specific gB antibodies and the level of serum TNF-α,IL-1β,IFN-β and IL-6 of mice 7 days after immunization;inactivated PRV was used as a specific antigen to perform lymphocyte proliferation experiments and calculate the lymphocyte proliferation index(stimulation index, SI)of each group to screen the best vaccination route. [Results]5×10~4 TCID50was a safe challenge dose for 6-8 weeks mouse;subcutaneous injection was the best way of infection. Compared with the live virus and the inactivated virus group after 7 days of infection, the serum levels of TNF-α,IL-1β and IFN-β significantly increased(P<0.001,P<0.01),the levels of PRV gB IgG antibody and the lymphocyte SI in the mouse serum also significantly increased(P<0.05,P<0.001);There is no significant difference in cytokines and SI between different inactivation methods(P>0.05). [Conclusions]The experiment successfully established an artificial pseudorabies virus infection model in mice, and clarified that after live or inactivated pseudorabies virus immunized mice, there were differential immune-related factors in the early stage of the body, providing a research platform for the evaluation and screening of subsequent immunopotentiator.