新型鸭细小病毒样颗粒的制备及鉴定

Preparation and identification of novel duck parvovirus virus-like particles

【背景】鸭短喙侏儒综合征(beak atrophy and dwarfism syndrome,BADS)是由新型鸭细小病毒(novel duck Parvovirus,NDPV)感染导致雏鸭生长发育迟缓、上下喙萎缩的疾病。BADS的暴发给我国养鸭业造成了巨大的经济损失。【目的】利用大肠杆菌表达系统制备NDPV病毒样颗粒(virus-like particles,VLPs),为研制NDPV相关疫苗奠定基础。【方法】对NDPV VP2序列全长进行密码子优化、合成,连接至pColdTF表达载体,获得pColdTF-NDPV-VP2重组质粒,酶切、测序鉴定正确后将重组质粒转化至大肠杆菌BL21(DE3)中进行诱导表达,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulphate-polyacrylamide gel electrophoresis,SDS-PAGE)对蛋白表达进行可溶性分析;使用凝血酶(thrombin)切除trigger factor(TF)标签,利用镍柱(Ni-NTA)亲和层析方法纯化重组蛋白;利用Western blotting对纯化后的VP2蛋白进行反应原性分析;利用透射电镜、动态光散射观察重组蛋白形态以及能否形成VLPs。【结果】构建了pColdTF-NDPV-VP2重组质粒,在大肠杆菌中主要以可溶性形式表达,融合蛋白TF-VP2大小约为115 kDa,去除TF标签经镍柱纯化后得到67 kDa的VP2蛋白;Western blotting试验表明VP2蛋白能与NDPV鸭阳性血清发生特异性结合;通过透射电镜可以观察到形状规则、直径约为20−25 nm的病毒样颗粒。【结论】利用大肠杆菌表达系统制备了NDPV VLPs,为下一步研发BADS相关亚单位疫苗及生物相关制品提供了基础。

[Background]Beak atrophy and dwarfism syndrome(BADS)caused by novel duck parvovirus(NDPV)infection leads to stunted growth and atrophy of the upper and lower beaks of ducklings.The outbreak of BADS has caused huge economic losses to the duck industry in China.[Objective]To construct NDPV virus-like particles(VLPs)in Escherichia coli,laying the foundation for the development of NDPV-related vaccines.[Methods]The full-length NDPV VP2 sequence was codon-optimized,synthesized,and then ligated into the pColdTF vector to obtain the pColdTF-NDPV-VP2 recombinant plasmid.After being identified by enzyme digestion and sequencing,the recombinant plasmid was transformed into E.coli BL21(DE3)for expression.SDS-PAGE was employed to analyze the solubility of the recombinant protein.The TF tag was removed by thrombin protease,and then recombinant protein was purified by Ni-NTA affinity chromatography.Western blotting was employed to examine the reactogenicity of purified VP2 protein and dynamic light scattering and transmission electron microscopy to observe the morphology of the recombinant protein and the formation of VLPs.[Results]The recombinant plasmid pColdTF-NDPV-VP2 was constructed successfully and expressed in E.coli mainly in the soluble form.The TF-VP2 fusion protein had a molecular weight of 115 kDa,and the molecular weight was 67 kDa after removal of the TF tag.Western blotting showed that the VP2 protein specifically bound to NDPV-positive duck serum.The VP2 VLPs with diameters of 20 nm to 25 nm could be observed by transmission electron microscopy.[Conclusion]We used the E.coli expression system to design NDPV VLPs,which can provide a basis for the development of subunit vaccines and biological-related products against BADS.