猪附红细胞体粘附蛋白及其红细胞受体的筛选与鉴定

Eperythrozoonosis is a globally widespread zoonosis caused by Eperythrozoon, which is not only harmful to human health, but also causes animal morbidity and mortality. Especially the pigs infected with Eperythrozoon, can result in meat yield reduction, growth retardation, productivity decrease and death. And the recessive infected pigs or resistant pigs can be long-term, life-long spreading Eperythrozoon, therefore it is caused enormous loss and harm to the development of animal husbandry and public health security. To date, no method has been found to control Eperythrozoonosis, so it is important to prevent and control the disease by elucidating the pathogenic mechanism of E.suis. In this study, we use pig Eperythrozoon (E. suis) as a model to construct E.suis signature-tagged mutagenesis library, then screen the library in vivo and in vitro to obtain E.suis adhesion protein genes. After that, further identification of the adhesion protein genes were performed by phage display technology, red cell adherence inhibition assay, electron microscopy etc. In the end, the screening and identification of erythrocyte receptors were performed by Far-Western blot, Pull-Down etc. We hope this study can elucidate the pathogenic mechanism of E.suis, and lay foundation for Eperythrozoonosis prevention and control.

附红细胞体病是由附红细胞体引起的一种呈全球性分布的人兽共患病，该病不仅危害人类健康，而且还能引起动物的发病和死亡。尤其是猪感染附红细胞体后，可致生长发育受阻，生产能力下降，严重时引起死亡，并且隐性感染猪和耐过猪还可长期带毒、终生散毒，因此给我国的畜牧业发展和公共卫生安全带来了巨大的损失和危害。但时至今日，尚未有效控制附红细胞体病的方法，阐明附红细胞体的致病机制对附红细胞体病的防控具有积极意义。因此本研究以猪附红细胞体为模型，首先构建猪附红细胞体信号标签突变文库，然后对其进行体内筛选和体外筛选，获得猪附红细胞体粘附蛋白基因。然后通过噬菌体展示技术、红细胞粘附抑制实验、电镜技术等对所获得的粘附蛋白基因做进一步鉴定。最后利用Far-Western blot、Pull-Down等技术筛选和鉴定附红细胞体粘附蛋白的红细胞受体。从而为阐明猪附红细胞体致病机制，进而为猪附红细胞体病的防控奠定基础。

猪附红细胞体病是一种呈全球性分布的人兽共患病，该病不仅危害人类健康，而且还能引起动物的发病和死亡。尤其是猪感染附红细胞体后，可致生长发育受阻，生产能力下降，严重时引起死亡，给我国的畜牧业发展和公共卫生安全带来了巨大的损失和危害。没有高效、廉价的疫苗是造成该病尚未被控制的主要原因，而阻碍疫苗研制的主要原因是目前附红细胞体的致病机制尚未明确。本研究首先构建了T7噬菌体展示猪附红细胞体基因组文库，然后利用猪红细胞对其进行了4轮筛选，获得了16个猪附红细胞体粘附蛋白基因，其中VP蛋白结合红细胞的能力和识别该病阳性血清的能力最强。然后通过ELISA、结合实验、Pull down和Far-western blot技术，我们发现VP蛋白的受体为PLG。接着我们又利用重组VP蛋白对噬菌体展示单域抗体库进行了5轮筛选，发现单域抗体DAB5的结合效率最高，然后通过Far-western blot、ELISA和结合实验进一步证明了DAB5确实能够与VP蛋白结合，并且anti-VP抗血清和DAB5蛋白都能够抑制M.suis粘附和侵入红细胞。最后我们通过Far-western blot和结合抑制实验证明了band3和spectrin为VP蛋白的红细胞受体。本研究为阐明猪附红细胞体致病机制和研发猪附红细胞体疫苗、治疗药物及免疫学诊断方法奠定了基础。