猪流行性腹泻病毒3种抗原卵黄抗体的制备

Preparation of yolk antibodies against three different antigens of porcine epidemic diarrhe virus

【目的】探讨猪流行性腹泻病毒（PEDV）3种不同抗原制备的卵黄抗体对PEDV的中和能力,为抗猪流行性腹泻卵黄抗体的制备提供新的方法和理论基础。【方法】根据PEDV S糖蛋白基因设计引物扩增PEDVS534基因（636~789位氨基酸）和PEDV COE基因（499~638位氨基酸）,并构建原核表达质粒pET32a-PEDV S534和pET32a-COE,分别转化表达菌株BL21（DE3）进行诱导表达。以原核表达的PEDV S534蛋白、COE蛋白以及PEDV灭活病毒为抗原,分别免疫产蛋鸡并制备卵黄抗体,通过病毒中和试验评价各卵黄抗体的中和效力。【结果】成功表达了PEDV S534蛋白和COE蛋白,Western-Blotting分析显示,2种蛋白均具有很好的免疫原性;制备的3种特异性卵黄抗体均具有一定的病毒中和能力,PEDV S534蛋白特异性卵黄抗体的中和效价为1∶12,COE蛋白特异性卵黄抗体的中和效价为1∶25,而PEDV灭活病毒的卵黄抗体中和效价达到1∶120。【结论】PEDV灭活病毒的中和能力最强,其次为COE蛋白特异性卵黄抗体,PEDV S534卵黄抗体的中和能力较弱。

【Objective】This study investigated the ability of yolk antibodies to neutralize three different antigens of PEDV to provide new method and theoretical basis for preparing swine PED yolk antibody.【Method】Two pairs of primers were designed to clone PEDV S534 gene（636-789aa）and COE gene（499-638aa）before they were inserted into pET32 ato acquare recombinant plasmids,pET32a-PEDV S534 and pET32a-COE.After the recombinant plasmids were transformed into BL21（DE3）,IgY against PEDV was obtained from chicken egg yolk immunized with three different antigens,PEDV S534 protein,COE protein and PEDV inactivated virus.Then,their abilities to neutralize PEDV were tested by neutralization test.【Result】The PEDV S534 protein and COE protein were expressed successfully,and Western-Blotting indicated that the immunogenicity of the proteins were high.The virus neutralization test indicated that allthree egg yolk antibodies could neutralize PEDV.The anti-PEDV S534 IgY neutralization titer was 1∶12,the anti-COE IgY neutralization titer was 1∶25,and the anti-PEDV inactivated virus IgY neutralization titer was 1∶120.【Conclusion】The anti-PEDV inactivated virus IgY had the highest neutralization titer,followed by the anti-COE IgY and the anti-PEDV S534 IgY.