基于兔球虫载体的兔瘟病毒VP60特异性免疫应答模式的研究

Rabbit Hemorrhagic Disease (RHD) already has caused serious harm to rabbit industry.And the traditionally used inactivated tissue vaccine has some disadvantages. Live oral carrier vaccine is one focus in development of RHD novel vaccine. Coccidia is one ideal live carrier. However, there are some differences in patterns of immune response between the coccidia carrier and natural infection of RHDV. So, these differences and their influence to vaccine efficacy need to be investigated. A low-virulence Eimeria intestinalis will be used as a model coccidia in the study. And YFP or DHFR-TS (Dihydrofolate reductase-thymidylate synthase) as screenable markers, a double-cassette expression vector carrying RHDV VP60 gene will be constructed. And then stable transfection of Eimeria will be used to gain VP60 transgenic E. intestinalis strain. Furtherly, genomic PCR, reverse-transcription PCR and Western-blot will be used to determine the expression level of RHDV VP60 protein in trangenic E. intestinalis. And animal oral inoculation, enzyme-linked immunosorbent assay ( ELISA), Flow cytometric analysis, T lymphocyte proliferation assay and cytokine assay will be used to determine the level of specific immune response pattern induced by VP60 protein of trangenic E. intestinalis. Finally, animal immunization-challenge testing will be used to determine the influence to vaccine efficacy from different patterns of immune response. The study will provide foundation for successful development of a novel oral RHD vaccine with coccidia as live carrier.

兔瘟对家兔养殖业危害较大，传统灭活苗存在诸多不足，口服活载体疫苗是兔瘟新型疫苗的研究重点之一，球虫是理想的活载体，然而，球虫载体在免疫应答模式与兔瘟病毒感染存在差异，这种差异对球虫载体疫苗的保护性效力影响值得探讨。本研究拟以兔肠艾美耳球虫弱毒株为模型，以YFP或DHFR-TS为筛选标记，构建携带兔瘟病毒VP60基因的双框表达载体，然后通过球虫稳定电转染技术获得兔瘟病毒VP60转基因兔肠艾美耳球虫。在此基础上，利用基因组PCR、RT-PCR和Western-blot技术对转基因虫株进行分子鉴定，以明确VP60在兔球虫中的表达情况；并通过动物口服接种、ELISA、流式细胞分析、淋巴细胞增殖、细胞因子试验检测各项免疫指标，以明确口服VP60转基因虫株的病毒特异性免疫应答模式；最后通过动物接种和攻毒试验来评价免疫应答模式差异对疫苗保护效力的影响。为最终球虫载体兔瘟新型疫苗的研制奠定基础。

兔瘟对家兔养殖业危害较大，兔瘟传统灭活苗存在诸多不足，口服活载体疫苗是兔瘟新型疫苗的研究重点之一，而兔球虫能够激发宿主较强的黏膜免疫应答，是理想的肠道黏膜免疫活载体。本研究以兔肠艾美耳球虫弱毒株为基础，通过转基因技术构建能够表达兔瘟病毒抗原蛋白的转基因兔肠艾美耳虫株，并对该虫株的兔瘟免疫保护效果和免疫应答模式做一研究。目前，我们在构建携带兔瘟病毒VP60基因和筛选标记（YFP和DHFR-TS）质粒载体的基础上，通过限制性内切酶介导的子孢子电转染、十二指肠接种、体内乙胺嘧啶筛选和体外流失细胞仪分选等方法步骤，获得了VP60转基因兔肠艾美耳球虫虫株，第六代卵囊发光率可达92%，genome-walking、Western-blot和IFA方法对转基因虫株分子鉴定结果显示，VP60基因插入了兔肠艾美耳球虫基因组的多个位点，VP60蛋白主要分布于虫体细胞质部位，表达量高，动物免疫和攻毒试验结果表明，VP60转基因兔肠艾美耳球虫能够激发兔瘟病毒VP60特异性免疫应答，且能够针对兔瘟病毒的致死性攻击产生一定的免疫保护效果，本研究为最终基于球虫载体的兔瘟新型疫苗的最终研制奠定基础。