摘要:
Our experiment was to express recombi-nant antibody fused with EGFP fluorescent in E.coli for de-tecting porcine circovirus type 2( PCV2) in direct immu-nofluorescence assay ( DIF). The plasmid pET28a- V22-EGFP vector was transformed into Escherichia coli BL21 (DE3) and V22-EGFP protein was induced by IPTG. Ex-pression and solubility of recombinant V22-EGFP protein was analyzed by SDS-PAGE and Western blot and purified by Ni-NTA His?Bind Resin. The purified V22-EGFP protein was used to establish PCV2 direct immunofluorescence assay. SDS-PAGE and Western blot results revealed that recombi-nant V22-EGFP protein was successfully expressed with 60% soluble protein. With a obviously green appearances the puri-fied V22-EGFP protein showed great and stable fluorescence activity, even stored at -20 °C for 6 months. The DIF assay based on V22-EGFP showed good specifity to PCV2-infected PK-15 cells only. Compared to the conventional PCV2 IFA method, the PCV2 DIF based on recombinant V22-EGFP was more stable, sensitive and specific, so it enjoys a great poten-tial application in PCV2 research.%本研究旨在利用原核大肠杆菌系统表达1株猪圆环2型病毒(Porcine circovirus type 2,PCV2)小分子荧光抗体,以及该荧光抗体用于PCV2病毒直接免疫荧光(Direct immunofluorescence assay,DIF)检测.选取1株筛选到的PCV2特异性纳米抗体基因V22,按照E.coli原核表达系统进行密码子优化重新合成,与荧光蛋白基因融合后插入pET28(a)中,20°C条件下诱导表达16 h,利用SDS-PAGE、Western-blot技术鉴定蛋白是否表达及其可溶性,并对可溶荧光抗体蛋白进行镍柱亲和层析纯化,纯化蛋白用于建立PCV2特异性DIF检测方法,同时对其工作条件、工作浓度、特异性以及稳定性进行验证,并与PCV2经典间接免疫荧光检测方法进行比较,验证其灵敏度.结果表明,PCV2荧光抗体基因在E.coli原核表达系统中表达,可溶蛋白占目的蛋白的60%左右;纯化后荧光抗体蛋白呈现明显绿色,具有良好的荧光活性,不同批次表达及纯化的荧光抗体效果一致,-20°C保存6个月后抗体效价不变;PCV2特异性DIF试验结果表明该荧光抗体只对PCV2病毒感染细胞具有良好的反应原性,利用该抗体建立的直接免疫荧光法能够用于PCV2病毒滴度的测定,滴度测定结果与间接免疫荧光法检测结果一致.说明,在原核系统中成功表达PCV2的小分子荧光抗体,该荧光抗体蛋白具有良好的特异性、灵敏度及稳定性,用该抗体建立的PCV2特异性DIF操作简单,且具有较好的特异性和敏感性,对PCV2的检测有潜在应用价值.