腺病毒介导的新孢子虫抗原NcSRS2在实验小鼠中的免疫功能

Neospora caninum (N. caninum) belongs to apicomplexican parasite and causes diseases in many animals including abortion and stillbirth of cattle, which brings great economic loss in the world. However,so far, no effective vaccine has been used to prevent and control the neosporosis caused by N.caninum. Thus,in the present study, recombinant adenovirus designated as rAdv-NcSRS2 expressing the NcSRS2 antigen of N.caninum was constructed.Subsequently,mice were immunized with rAdv-NcSRS2 and challenged by N.caninum. Immune response was evaluated by determining IgG2a/IgG1 antibody level and NO concentration in the peripheral blood as well as cytokine (IL-2,IL-12,IL-4 and IFN-γ)level and transformation rate of spleen lymphocytes.Besides,protection function was also evaluated by analyzing pathogenic change,immunohistochemical change and real-time PCR (based on specific gene Nc-5 of N.caninum) for different tissues including heart,liver,spleen,lung,kidney and brain of mice challenged with N.caninum.Immune function of NcSRS2 antigen of N.caninum mediated by adenovirus in mice can provide theoretical basis and applification foundation to develop an effective novel vaccine for prevention and control of neosporosis.

新孢子虫属于顶复门寄生原虫，可导致多种动物的疾病，在临床上主要导致奶牛和肉牛的流产和死胎，在世界范围内造成极大的经济损失。然而，到目前为止，尚无有效疫苗预防与控制该病。为此，本项目拟构建表达新孢子虫（Neospora caninum）抗原NcSRS2的重组腺病毒（命名为rAdv-NcSRS2），然后对小鼠进行重组腺病毒免疫与寄生虫的攻毒，测定外周血IgG2a/IgG1抗体水平、一氧化氮浓度以及脾脏淋巴细胞的细胞因子(IL-2,IL-12,IL-4,IFN-γ)水平和转化率，借以评估该重组腺病毒激发的免疫应答；对攻毒后小鼠的不同组织（心、肝、脾、肺、肾与脑）进行病理学、免疫组织化学以及寄生虫特异基因的荧光定量PCR分析，借以评估该重组腺病毒对攻毒的保护效果。本研究预期获得腺病毒介导的新孢子虫抗原NcSRS2在实验小鼠中的免疫功能，为进一步开发防控新孢子虫病的新型疫苗提供理论依据与物质基础。

针对目前临床上缺乏新孢子虫病有效疫苗，该项目在血清5型腺病毒中成功地整合与表达了新孢子虫有效表面抗原NcSRS2，并在实验动物模型小鼠中对重组腺病毒的免疫功能进行了评估。结果表明，新孢子虫抗原基因NcSRS2高效地整合进了血清5型腺病毒载体pAV.Des1d中，重组腺病毒 rAdv-NcSRS2能够在HEK293A细胞系中进行高效地组装与表达，通过对重组腺病毒的扩增和浓缩获得了滴度为4×109 TCID50的高浓度腺病毒。该重组腺病毒免疫实验小鼠后，利用重组蛋白NcSRS2作为抗原，通过间接ELISA方法测定免疫组比对照组具有较高的IgG水平，说明重组腺病毒可激发较强的体液免疫应答；外周血中NO浓度比对照组具有明显地提高，证实重组腺病毒具有增强吞噬细胞的免疫学功能；采集腺病毒免疫后小鼠脾脏，通过淋巴细胞转化实验得知，实验组与对照组的淋巴细胞转化率具有统计学上的显著差别，此外，免疫组小鼠脾脏中细胞因子IL-2、IFN-γ等较对照组也具有明显提高，说明重组腺病毒可激发有效的细胞免疫应答反应。本研究的科学意义在于，获得的重组腺病毒能够在实验小鼠模型中激发高效的体液与细胞免疫应答反应，为以后该疫苗应用于大型动物如奶牛中以防控新孢子虫病带来的经济损失提供重要的参考。