新型鸭呼肠孤病毒σB 蛋白的原核表达及其多克隆抗体制备

摘要

为制备新型鸭呼肠孤病毒(new-type duck reovirus,NDRV)XX 株σB 蛋白的多克隆抗体,试验经 RT-PCR扩增 NDRV XX 株σB 基因编码序列,构建原核表达质粒 pET-32a(+)-σB,将其转化至大肠杆菌 BL21(DE3)感受态细胞后,经 IPTG 诱导获得 His-σB 重组蛋白。SDS-PAGE 显示成功表达出约55 ku 的融合蛋白,主要以包涵体形式存在,其表达时的最佳诱导时间、IPTG 诱导浓度分别为3 h 和0.25 mmol/L。经 Ni2+柱亲和层析纯化获得可溶性重组蛋白,将蛋白经 Western blotting 和蛋白质谱鉴定为高纯度的σB 重组蛋白,将纯化后σB 重组蛋白按合理免疫程序免疫家兔,获得多抗隆抗体经 Western blotting 分析显示出特异性的反应。本试验结果为 NDRV σB 蛋白功能的深入研究及基因工程疫苗的研发奠定了基础。%To prepare polyclonal antibodies againstσB protein of new-type duck reovirus (NDRV) XX strain,the encoding sequence ofσB gene of NDRV XX strain was amplified by RT-PCR and successfully inserted to expression plasmid pET-32a (+),and transformed in Escherichia coli BL21(DE3).The His-σB recombinant protein was achieved with IPTG induction.SDS-PAGE re-sult showed that the molecular weight of the expression on fusion protein was about 55 ku,was major insoluble fractions.IPTG induced time and concentration were 3 h and 0.25 mmol/L, respectively.The soluble σB recombinant protein was highly purified which was purified using Ni2 + affinity chromatography and verified by Western blotting and protein mass spectrometry. Then the polyclonal antibodies could be obtained from the rabbits which had immunized by the purified σB recombinant protein with the reasonable procedure.The Western blotting result showed that they had the specific reaction.The results built a foundation of the further study of the NDRV σB protein function and the research of genetic engineering vaccine.