猪流行性腹泻病毒coe基因的克隆表达及卵黄抗体的制备  

Clone and expression of coe gene and preparation of egg yolk antibody against COE of porcine epidemic diarrhea virus

作　　者：朱文姣 常琛 朱玉虎 周国丽[1] 尹志安 杨国庆[1] 刘慧敏[1] ZHU Wenjiao;CHANG Chen;ZHU Yuhu;ZHOU Guoli;YIN Zhian;YANG Guoqing;LIU Huimin(College of Life Sciences,Henan Agricultural University,Zhengzhou 450002,China;Animal Health Supervision Office of Mianchi County,Mianchi 472400,China)

机构地区：[1]河南农业大学生命科学学院,河南郑州450002 [2]渑池县动物卫生监督所,河南渑池472400

出　　处：《河南农业大学学报》2018年第4期560-565,共6页Journal of Henan Agricultural University

基　　金：河南省高等学校重点科研项目(30601522);河南农业大学创新基金项目(30601444)

摘　　要：通过RT-PCR技术扩增猪流行性腹泻病毒(Porcine Epidemic Diarrhea Virus,PEDV)coe基因,将其克隆至原核表达载体p ET32a中,构建重组表达质粒p ET32a-coe,鉴定正确后进行诱导表达。结果表明,重组菌可表达相对分子量约为33 k D的融合蛋白;Western-Blotting结果显示,COE蛋白能与抗PEDV的阳性血清发生特异性结合反应,表明研究的COE蛋白具有良好的反应原性。纯化后的COE蛋白与弗氏佐剂按比率混合作为免疫抗原,免疫产蛋鸡,获得特异性抗猪流性腹泻病毒COE蛋白的卵黄抗体(Ig Y)。氯仿抽提法提取卵黄抗体,通过SDSPAGE、Western-Blotting对提取的卵黄抗体进行鉴定并用间接酶联免疫吸附实验(ELISA)对其进行效价检测。结果显示,纯化后的卵黄抗体Ig Y具有高度的特异性,其抗体效价可达1∶1 000。研究结果表明,表达的COE蛋白具有良好的免疫原性,作为免疫抗原制备的卵黄抗体具有较高的抗体效价。The coe gene of porcine epidemic diarrhea(PEDV)was amplified by RT-PCR,and was cloned into prokaryotic expression vector pET 32 a for expression.The recombinant protein COE was induced expression after sequencing analysis.The results show that the molecular weight of the recombinant protein COE was about 33kD and Western-Blotting showed that COE could specifically react with PEDV positive serum,which indicated that COE protein had good antigenicity and specificity.Hens were immunized with the purified COE protein which was mixed with Freund’s adjuvant as an immunogen for preparation of specific IgY.The antibody IgY was extracted by chloroform,identified by SDS-PAGE and detected by Western-Blotting and ELISA.Western-Blotting showed that the purified specific IgY antibody could specifically react with the COE protein and ELISA results showed that the titer of IgY reached 1∶1 000.The results showed that COE protein had good immunogenicity and the yolk antibody prepared as immunogen had higher antibody titer.

关 键 词：猪流行性腹泻 COE蛋白 卵黄抗体 

分 类 号：S855[农业科学—临床兽医学]