抗猪圆环病毒2b/2d亚型衣壳蛋白纳米抗体的制备与鉴定  

Preparation and characterization of the nanobody against both PCV2b and PCV2d capsid protein

作　　者：苏文静 毕明芳 莫小兵 SU Wenjing;BI Mingfang;MO Xiaobing(College of Veterinary Medicine,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学动物医学学院,吉林长春130062

出　　处：《中国兽医学报》2023年第11期2197-2202,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32071476)。

摘　　要：采用原核表达系统表达了PCV2b/2d-Cap的重组纳米抗体,并对其与PCV2b和PCV2d亚型Cap蛋白的结合能力进行了初步探索。以羊驼筛选到的纳米抗体基因为模板,通过PCR将纳米抗体基因片段进行扩增。经双酶切及连接反应后,将纳米抗体基因片段(VHH)插入到经改造的pET28a-SUMO载体中,构建了pET28a-SUMO-VHH重组质粒。将构建好的质粒转化到大肠杆菌BL21(DE3)感受态细胞中诱导表达。表达产物通过亲和层析、除去SUMO标签以及分子筛凝胶过滤层析进行纯化。并通过Bio-dot Western blot和间接ELISA方法检测了该纳米抗体与PCV2b/2d-Cap蛋白的结合能力。结果显示,构建的重组质粒测序正确;经纯化得到的纳米抗体蛋白约为14 kDa;间接ELISA证实该纳米抗体对PCV2b/2d-Cap蛋白检测下限均为0.68 mg/L。结果表明,该纳米抗体与PCV2b和PCV2d亚型Cap蛋白均具有交叉反应,为PCV2b/2d亚型的临床检测建立新型诊断方法奠定了基础。The recombinant nanobody of PCV2b/2d Cap was expressed using the prokaryotic expression system,and its binding ability with Cap proteins of PCV2b/2d subtype was preliminarily explored.The selected VHH gene was amplified and cloned into a pET28a-SUMO vector modified by adding a ULP1 enzyme cleavage site to express soluble protein with an N-terminal SUMO-tag.The transformed E.coli BL21(DE3)with recombinant plasmid(pET28a-SUMO-VHH)were cultured in LB medium,and IPTG was added to induce the recombinant protein expression.The nanobody was then purified by Ni-NTA affinity chromatography,followed by SUMO-tag removal and size-exclusion chromatography.As expected,the molecular weight of purified nanobody protein wasabout 14 kDa.Moreover,the ability of nanoantibody to bind with PCV2b/2d-capsid proteins was tested by Bio-dot Western blot assay and indirect ELISA assay,the results showed that the overall detection sensitivity of our indirect ELISA method for both PCV2b/2d-Cap was 0.68 mg/L.Therefore,the cross reactivity of this nanobody enables it to detect both PCV2b and PCV2d clinical samples,which can provide clear clue for next generation PCV2 diagnostic kits development.

关 键 词：猪圆环病毒2型 CAP蛋白 纳米抗体 交叉反应 

分 类 号：S852.4[农业科学—基础兽医学] S852.65[农业科学—兽医学]