抗PEDV Fab抗体库筛选、乳酸菌表达及中和活性分析

Porcine epidemic diarrhea virus(PEDV) is a member of the Coronaviridae and belongs to group I coronavirus. PEDV is a causative agent of severe diarrhea which leads to death in piglets. Because of the high mortality which is up to 100% in suckling piglets, PED is an important porcine disease. Unfortunately, difficulties of in vitro propagation of PEDV have limited viral research of PEDV and hampered vaccine development.Lactic acidbacteria(LAB),which are generally recognized as safe by the U.S. Food and Drug Administration, are microorganisms that are present innumerous food fermentations and are also normal constituents of the intestinal habitat. In addition,some strains of LAB exhibit probiotic properties. These characteristic been exploited for the use of LAB as live vectors for the expression of different peptides and the delivery of peptides to mucosal surfaces in animal models..In the present study, we aim for the development and evaluation of a novel antibody expressed on lactococcus lactis to protect piglets against PEDV. To achieve the goal, PEDV specific Fab antibody can be isolated by the E. coli display technique after panning of E. coli libraries on immobilized S antigen. We can construct lactococcus lactis strains expressing extracellular or cell-wall-attached anti-PEDV neutralizing antibodies, which might be useful to deliver passive immunity against PEDV.

猪流行性腹泻病毒（porcine epidemic diarrhea，PEDV)是I型冠状病毒，仔猪感染后可引起严重的腹泻，甚至死亡，目前还没有疫苗能够完全保护仔猪免于感染。乳酸菌是食品级微生物，该表达系统可将异源蛋白直接在口腔、胃肠道表达，与粘膜系统或靶蛋白直接作用。本研究根据PEDV猪小肠绒毛上皮细胞内复制这一特点，利用乳酸菌表达异源蛋白可直接口服治疗胃肠道疾病这一优势，表达了三段S基因的中和表位，采用流式细胞术从高容量大肠杆菌展示系统筛选抗PEDV高亲和力Fab抗体，并将其分泌表达或表面表达于乳酸乳球菌中，重点研究乳酸乳球菌表达的Fab或scFv对PEDV的中和活性。本研究旨在实现乳酸菌表达重组抗体对仔猪腹泻产生保护这一目标，为乳酸菌表达重组抗体发展成治疗腹泻疾病抗体药物奠定一定理论基础。

猪流行性腹泻病毒（porcine epidemic diarrhea，PEDV)是引起仔猪腹泻的重要病毒，致死率高，是世界上重要的猪病病毒，目前还没有很好的防治措施。乳酸菌是食品级微生物，该表达系统可将异源蛋白直接在口腔、胃肠道表达，与粘膜系统或靶蛋白直接作用。在本实验中，我们选取了抗原抗体共表达的方式，将抗原基因S1和anti-PEDV scFv基因共同表达在大肠杆菌周质腔内，利用流式细胞术进行三轮淘选，获得具有高亲和力抗体的阳性菌。用大肠杆菌表达阳性菌scFv基因后纯化，用ELISA方法检测抗PEDV ScFv抗体特异性，实验结果表明表达的抗体具有中和活性。之后我们构建了表达抗PEDV scFv抗体的乳酸菌表达载体，分别为L. lactis NZ9000/pNZ8148-scFv和L.lactis NZ9000/pMG36e-scFv,通过乳酸菌表达产物的SDS－PAGE分析可以确定抗PEDV scFv均有表达。ELISA检测实验结果显示乳酸菌表达的scFv抗体具有中和活性。本研究初步表明乳酸菌表达anti-PEDV scFvs可成为预防和治疗仔猪感染PEDV的一项重要措施。