羊口疮病毒青海QH02株ORF011基因的克隆、原核表达及鉴定

Cloning, prokaryotic expression and identification of Orf virus ORF011 gene from QH02 strain

目的克隆羊口疮病毒QH02株ORF011基因并进行序列分析及原核表达、鉴定。方法提取羊口疮病毒QH02株的DNA,根据GenBank中(GU320351.1)的序列设计并合成引物,PCR扩增ORF011基因。将测序正确的序列用生物信息学软件对其基因及蛋白的结构进行预测,并将基因构建至pET-32a(+)原核表达载体,转化至大肠埃希菌BL21(DE3),挑取阳性克隆测序正确后用异丙基-β-D-硫代半乳糖苷(isopropyl-β-d-thiogalactoside, IPTG)诱导表达,经十二烷基硫酸钠-十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)、Western-blot验证重组蛋白反应原性。结果 PCR扩增得到1 137 bp的ORF011基因片段,重组蛋白在大肠埃希菌中以包涵体的形式存在,重组蛋白相对分子质量为60×10~3,且纯度较高,Western-blot检测结果表明目的蛋白具有较好的反应原性。结论构建的原核表达系统可以成功表达B2L蛋白,经Western-blot验证具有良好的反应原性,可作为羊口疮防控产品研发的候选分子。

Objective To clone and express the Orf virus ORF011 gene from the QH02 strain.Methods The genomic DNA was extracted from the QH02 strain of Orf virus and was used as the template to amplify the fragment of the ORF011 gene.The primers for PCR amplification were designed and synthesized according to the sequence of GU320351.1 in GenBank.The structure of the Orf virus ORF011 gene was predicted using bioinformatics software;then the gene was constructed into pET-32 a(+)vector and transformed into Escherichia coli BL21(DE3).The recombinant protein was induced by IPTG after the positive clones were selected and sequenced,and the expression of recombinant protein was verified by SDS-PAGE and Western-blot.Results The gene fragment of 1 137 bp was amplified by PCR,and the recombinant protein was expressed as inclusion body in E.coli.The target protein was 60×10~3 in weight with high purity.The result of Western-blot showed that the recombinant protein had good reactivity.Conclusions B2 L protein of Orf virus can be expressed in the prokaryotic system with high quality and good reactivity.