【摘要】 以猪圆环病毒2型（PCV2）Cap蛋白VHH_cap基因为模板,通过PCR分别克隆下游片段VHH_down和带（G₄S）₃-linker接头序列的上游片段VHH_up,通过两步酶切连接反应,将VHH_down片段插入到pCold-SUMO载体中构建VHH_down-pCold-SUMO重组质粒,再将VHH_up片段插入到构建成功的VHH_down-pCold-SUMO重组质粒中,构建双价单特异性纳米抗体原核重组表达质粒biVHH_cap-pCold-SUMO,转化宿主菌E.coli BL21（DE3）后用IPTG进行诱导表达,表达产物经Ni²⁺亲和层析柱纯化并进行SDS-PAGE鉴定;通过间接ELISA方法测定纯化后的双价纳米抗体重组蛋白biVHH_cap的效价水平,并与相同浓度的单价纳米抗体蛋白VHH_cap的ELISA效价进行比较分析。结果表明,重组表达质粒biVHH_cap-pCold-SUMO构建正确,Cap蛋白双价单特异性纳米抗体重组蛋白在原核表达系统中以可溶性形式表达,分子质量约为45 ku;该双价纳米抗体与其单价组分相比,ELISA抗体效价提高约20倍,Cap蛋白双价纳米抗体的制备为PCV2的抗原检测和PCV2相关疾病的防控提供了新型候选抗体工具。更多还原

【Abstract】 To prepare mono-specific anti-Cap bivalent nanobody against porcine circovirus type 2（ PCV2）,VHH_up fragment and VHH_down fragment with a（ G₄S）₃-linker was amplified by using VHH_cap that was obtained by bio-panning method in the previous study as template.VHH_down fragment was inserted into pCold-SUMO vector to construct VHH_down-pCold-SUMO,and then bivalent recombinant plasmid biVHH_cap-pCold-SUMO was constructed by inserting VHH_up fragment into VHH_down-pCold-SUMO.This bivalent recombinant plasmids were transformed into E.coli BL21（ DE3） and then the recombinant bacteria were induced by IPTG.Expression products were purified by Ni²⁺affinity chromatography and detected by SDS-PAGE.The level of bivalent Cap-specific nanobody was tested by indirect ELISA method, and was compared with those of monovalent nanobodies at the same concentration or dilution level.Double enzyme digestion and sequencing results showed that the recombinant plasmid biVHH_cap-pCold-SUMO was successfully constructed.SDS-PAGE analysis results demonstrated that bivalent Cap-specific nanobody was expressed successfully in E.coli BL21（ DE3） and existed in soluble form with a relative molecular weight of 45 ku.The level of bivalent nanobody titer was improved about 20-fold for Cap protein compared with its monovalent counterpart.This study provided a novel candidate antibody tool for the detection and prevention of PCV2.更多还原