猪流行性腹泻病毒S1基因的截段克隆表达及其在卵黄抗体制备上的应用

Prokaryotic expression of porcine epidemic diarrhea virus truncated S1 gene and the application in hyperimmunized yolk antibody production

通过RT-PCR技术扩增了猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)S1截段基因序列,将扩增的S1基因截段克隆到原核表达载体pET-32α(+)中,构建了重组表达质粒pET-32α-S1,经测序鉴定正确后,转化至感受态细胞BL21(DE3)中,并进行IPTG诱导表达。结果显示:重组菌可表达相对分子质量为45 000的融合蛋白,蛋白表达量较高;Western blotting结果显示,表达的S1蛋白能与PEDV阳性血清发生特异性的免疫反应,表明原核表达的S1蛋白具有良好的反应原性;将纯化后的S1蛋白与蜂胶佐剂按比例混合,作为免疫抗原,免疫产蛋鸡,每隔2周免疫1次,共免疫3次,定期利用ELISA方法检测抗体效价水平,琼脂扩散试验抗体效价最高达到1∶64,收集并纯化卵黄抗体(IgY),SDS-PAGE结果分析纯化效果较好;在人工感染仔猪试验中,卵黄抗体试验组的治愈率为100%,说明卵黄抗体对猪流行性腹泻病具有较好的治疗效果。结果表明:表达的重组S1蛋白具有良好的抗原性,作为免疫抗原免疫产生的卵黄抗体具有较高的抗体效价,为进一步研制猪流性腹泻卵黄抗体生物制剂奠定了基础。

In the experiment, PEDV truncated S1 samples. Furthermore,the truncated S1 gene was BL21 （ DE3）,the recombinant protein S1 was IPTG. The recombinant protein S1 was about 45 gene was amplified and sequenced from positive cloned into pET-32α（＋） vector for expression in expressed in soluble form after induction with 090 and could specifically reacted to PEDV positive serum. Western-blotting showed that the purified recombinant protein S1 retains better antigenicity and specificity. The hens were immunized with the PEDV S1 protein propolis vaccine. Af- ter the antibodies titer reached to 1 ： 64 tested by double diffusion, eggs of hyperimmunity were collected. The hyperimmunized yolk antibodies were purified by chloroform extraction and salt precipitation with ammonium sulfate. SDS-PAGE showed that the purification effect of truncated S1 protein is better. In artificially infected piglets trials, the cure rate of yolk antibody achieves 100% . The results proved the protein S1 had good immunogenicity and could provide a basis of hyperimmunized yolk antibody production