羊口疮病毒B2L基因的原核表达、纯化及鉴定

Prokaryotic Expression,Purification and Identification of ORFV B2L Gene

[目的]获取羊口疮病毒B2L基因编码蛋白。[方法]根据GenBank上羊口疮病毒ORFV/ShanXi/2011/China株B2L基因序列,设计并合成1对特异性引物,利用PCR方法扩增B2L全基因序列,然后将扩增的B2L基因克隆到PMD18-T载体上;对构建的重组克隆质粒(PMD18-T-B2L)经过测序鉴定后,将目的基因亚克隆到PET-32a(+)原核表达载体上,获得重组表达质粒PET-32a-B2L,然后通过双酶切和测序进行鉴定;将重组菌于37℃、终浓度含1 mM IPTG的LB培养基中,诱导表达3h后进行SDS-PAGE分析;表达的目的蛋白经过层析纯化后,进行Western Blot鉴定。[结果]原核表达重组质粒构建成功;SDS-PAGE鉴定发现目的条带的分子量与预期大小相符;纯化的目的蛋白经Western Blot鉴定正确,表明蛋白表达成功。[结论]本研究成功构建了羊口疮病毒B2L基因原核表达重组质粒,并成功表达和纯化了B2L蛋白,为后续羊口疮病毒检测方法的建立打下了基础。

[Objective]To obtain the B2L protein of Orf virus(ORFV).[Methods]Based on ORFV/ShanXi/2011/China strain B2L gene sequence in GenBank,a pair of specific primers were designed and synthesized.The primers were used to amplify the B2L ORF fragment of ORFV by PCR.The PCR product was cloned into PMD18-T vector to form recombinant PMD18-T-B2L,which was identified by enzymatic digestion and sequencing.The B2L fragment of interest was digested and subcloned into Prokaryotic Expressing Vector PET-32a(+)and was verified by sequencing.The recombinant plasmid(PMD18-T-B2L)was cloned and sequenced,and the target gene was subcloned into PET-32a(+)prokaryotic expression vector.Recombinant expression plasmid PET-32a-B2L was obtained and identified by double restriction digestion and sequencing.The recombinant strain was induced at37℃and the final concentration of1mM IPTG in LB medium.After3h expression,SDS-PAGE analysis was carried out.The expressed protein was purified by chromatography and identified by Western and Blot.[Results]The B2L protein was expressed with the expected size successfully.The SDS-PAGE identification results showed that the molecular weight of the target band was in line with the expected size.The purified protein was identified by Western Blot,indicating the successful expression of the protein.[Conclusion]The recombinant plasmid of ORFV B2L gene was constructed successfully in this study,and the B2L protein were expressed and purified successfully,which laid a foundation for the establishment of sheep ORFV detection method.