猪繁殖与呼吸综合征病毒GP5蛋白特异性纳米抗体的原核表达及鉴定

Prokaryotic Expression and Identification of Specific Nanobodies against GP5 Protein of PRRSV

为获得针对猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)GP5蛋白的特异性纳米抗体,并鉴定其抗原结合活性,以本实验室前期获得的PRRSV GP5蛋白特异性纳米抗体VHHGP5基因片段为模板,使用PCR对目的片段进行扩增,将扩增的片段经双酶切处理后连接到pColdSUMO-FLAG载体上,随后转化至E.coli TOP 10感受态细胞。经菌液PCR鉴定,将构建成功的重组质粒转化至E.coli BL21(DE3)感受态细胞,用IPTG作为诱导剂对目的蛋白进行诱导表达,表达产物通过Ni-NTA亲和层析柱纯化。通过SDA-PAGE凝胶电泳分析表达及纯化后的蛋白,同时使用ELISA方法检测纯化后的纳米抗体效价,并使用Western blot鉴定纯化后的蛋白。SDS-PAGE结果显示,成功获得了大小约36 kDa的重组蛋白,利用原核表达系统制备的PRRSV GP5特异性纳米抗体重组蛋白以可溶性形式表达;ELISA结果显示,该纳米抗体结合抗原的效价为1:50000;Western blot结果显示,该纳米抗体具有较好的反应性和特异性。本试验为PRRSV检测用及治疗用新型抗体开发奠定了基础。

In order to prepare specific nanobodies against GP5 protein of porcine reproductive and respiratory syndrome virus(PRRSV),and to identify its antigen-binding activity,VHH_(GP5)gene fragment encoding the nanobody previously obtained by the laboratory was used as a template to amplify the target fragment using PCR,after double digestion by restrict enzymes,the amplified fragment was ligated to pCold-SUMO-FLAG vector and transformed into E.coli Top 10competent cells.After PCR identification,the recombinant plasmid successfully constructed was transformed into E.coli BL21(DE3)competent cells,IPTG was used as an inducer for inducible expression of the target protein,then the expressed protein was purified by Ni-NTA affinity chromatography column.The expressed and purified protein was analyzed by SDS-PAGE gel electrophoresis,while the titer of the purified nanobodies was determined using ELISA,and the purified protein was identified by Western blot.SDS-PAGE results showed that the recombinant protein of nanobodies against GP5 protein of PRRSV,prepared in a prokaryotic expression system,was expressed in a soluble form;ELISA results showed that the titer of the nanobody-binding-antigen was 1:50000;and Western blot results showed that the prepared nanobodies had good reactivity and specificity.In conclusion,a foundation was laid for the detection of PRRSV and the development of novel antibodies against the virus.