猪圆环病毒2型Cap蛋白特异性纳米抗体的原核表达研究

Prokaryotic Expression of Specific Nanobody against Porcine Circovirus Type 2 Cap Protein

关键词:  猪圆环病毒2型  纳米抗体  原核表达  ELSIA效价

Key words:  Porcine circovirus type 2  Nanobody  Prokaryotic expression  ELISA titer

摘要:

为利用大肠埃希菌表达猪圆环病毒2型(PCV2)Cap蛋白特异性纳米抗体，并对其抗原结合活性进行鉴定，以Cap蛋白特异性纳米抗体基因(vhhcap)序列为模板，利用Primer 5.0软件设计一对通用vhhcap特异性扩增引物，通过PCR方法扩增vhhcap基因片段，经双酶切处理后分别克隆到pCold-SUMO和pET-32a(+)载体上，转化宿主菌E.coli BL21(DE3)，使用IPTG进行诱导表达，表达产物通过Ni2+亲和层析柱进行纯化，经SDS-PAGE分析和Western blotting鉴定，同时使用ELISA方法对两种载体表达的纳米抗体ELISA效价进行检测和对比分析。SDS-PAGE结果显示pCold-SUMO和pET-32a(+)载体均能实现VHHcap纳米抗体重组蛋白的原核可溶性表达，Western blotting表明纳米抗体重组蛋白可通过6×His-Tag进行检测；ELISA结果显示pCold-SUMO载体表达的纳米抗体重组蛋白效价为1∶500，ELISA效价明显高于pET-32a(+)载体表达产物。PCV2 Cap特异性纳米抗体成功表达为PCV2检测及治疗用新型的抗体开发奠定了基础。

Abstract:

One strain of nanobody sequence acquired from previous studies was used to express specific nanobody against porcine circovirus type 2 Cap protein by prokaryotic expression system of E.coli and its antigen binding activity was analyzed.A pair of specific universal primers was designed according to the nanobody sequence by using software Primer 5.0 and the target sequence fragment was amplified by PCR method.The amplified target DNA was digested by restriction enzymes and then was cloned into vector pCold-SUMO and pET32a(+) to generate vhhcap-pCold-SUMO and vhhcap-pET32a(+),respectively.The recombinant plasmid was transformed into E.coli BL21(DE3) separately.Recombinant bacteria was induced by IPTG,respectively.Expression products were purified by Ni2+affinity chromatography column.These specimens were analyzed by SDS-PAGE.The purified recombinant nanobody protein was further identified by Western blotting and its antigen binding activity was analyzed by ELISA method.SDS-PAGE results showed that VHHcap recombinant protein was expressed in soluble form by vector pCold-SUMO and pET32a(+).Western blotting result indicated that purified VHHcap recombinant protein reacted well with 6×His-Tag.And ELISA result demonstrated that the antigen binding activity titer of VHHcap-pCold-SUMO was 1∶500 and it was better than that of VHHcap-pET32a(+).This study provided a basis for the development of novel antibodies for PCV2 detection and treatment.