羊口疮病毒119蛋白表达、纯化和多克隆抗体的制备

发布时间：2018-11-22 18:15

【摘要】：[目的]克隆表达羊口疮病毒ORFV119蛋白,以纯化重组蛋白为免疫原制备鼠多克隆抗体,并鉴定抗体的特性。[方法]PCR扩增ORFV119基因,克隆入原核表达载体p ET-28a(+)中构建重组质粒p ET28a-119。经双酶切和测序正确后,转化感受态大肠杆菌BL21,IPTG诱导表达,SDS-PAGE鉴定融合蛋白表达情况。表达产物进行超声破碎和Ni柱纯化,之后目的蛋白免疫BALB/c小鼠,制备ORFV119多克隆抗体并对其通过中和实验进行鉴定。[结果]成功构建重组质粒p ET28a-119,在大肠杆菌BL21中ORFV119融合蛋白以部分可溶形式表达。可溶性目的蛋白纯化后作为免疫原制备鼠多克隆抗体,抗体效价达1∶12 800,中和实验显示该多抗具有保护作用,可减轻宿主细胞在病毒感染时的病变效应(中和效价66 ND50/m L)。[结论]成功诱导表达、纯化ORFV119蛋白并制备其多克隆抗体,为深入研究ORFV感染、发病机理及羊口疮疾病的临床诊断奠定基础。
[Abstract]:[objective] to clone and express the ORFV119 protein of sheep mouth sore virus and to prepare mouse polyclonal antibody using purified recombinant protein as immunogen, and to identify the characteristics of the antibody. [methods] ORFV119 gene was amplified by PCR and cloned into prokaryotic expression vector p ET-28a () to construct recombinant plasmid p ET28a-119.. After double enzyme digestion and sequencing correctly, the expression of the fusion protein was induced by BL21,IPTG and identified by SDS-PAGE. The expressed product was purified by ultrasonic fragmentation and Ni column. Then BALB/c mice were immunized with the target protein. The polyclonal antibody of ORFV119 was prepared and identified by neutralization experiment. [results] Recombinant plasmid p ET28a-119, was successfully constructed to express ORFV119 fusion protein in Escherichia coli BL21 in partially soluble form. The purified soluble target protein was used as immunogen to prepare mouse polyclonal antibody. The titer of antibody reached 1:12 800. The neutralization experiment showed that the polyclonal antibody had protective effect. It can alleviate the pathological effect of virus infection in host cells (neutralizing titer 66 ND50/m L). [conclusion] the expression, purification and polyclonal antibody of ORFV119 protein were successfully induced and purified, which laid a foundation for the further study of ORFV infection, pathogenesis and clinical diagnosis of sheep aphthous ulcer.
【作者单位】：南方医科大学检验与生物技术学院抗体工程研究所;佛山科学技术学院口腔医学院医学检验学系;
【基金】：国家自然科学基金项目(“羊痘病毒ORFV119蛋白与宿主细胞相互作用的分子机制”,No.31170147;“羊口疮病毒ORFV118蛋白调控宿主细胞凋亡的分子机制研究”,No.31672536)
【分类号】：S852.654

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