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 p ET32 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 33 k D 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 Ig Y. The antibody Ig Y was extracted by chloroform,identified by SDS-PAGE and detected by Western-Blotting and ELISA. Western-Blotting showed that the purified specific Ig Y antibody could specifically react with the COE protein and ELISA results showed that the titer of Ig Y reached 1 ∶ 1 000. The results showed that COE protein had good immunogenicity and the yolk antibody prepared as immunogen had higher antibody titer.
引文
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