表达口疮病毒B2L基因自杀性DNA疫苗的构建及鉴定

Construction and identification of a suicidal DNA vaccine expressing B2L gene of orf virus

根据口疮病毒AH-GY13株B2L基因序列设计特异性引物,两端分别添加Bam HⅠ和XmaⅠ酶切位点。提取该毒株的DNA并以其为模板,扩增出B2L基因,再将该基因克隆至自杀性DNA疫苗载体pSCA1中,构建重组真核表达质粒pSCA-B2L。将该重组质粒转染BHK-21细胞,并通过间接免疫荧光试验(IFA)和Western-blot验证B2L在体外的表达情况。IFA和Western-blot结果表明,B2L蛋白在体外能够正常表达,且具有良好的反应原性。试验中成功构建的真核表达质粒pSCA-B2L,为进一步研制口疮病毒自杀性DNA疫苗奠定了基础。

A suicidal DNA vaccine expressing the B2L gene of ORFV was constructed and the protein expressed was identified in vitro.A pair of specific primers was designed based on the B2L gene of Orf virus AH-GY13 strain.Bam H I and Xma I were added to the primers.Then,B2L gene was amplified using the DNA of clinical isolated Orf virus as templates.The B2L gene was cloned into pSCA1 plasmid,a suicidal DNA vaccine vector.Moreover,the recombinant plasmid pSCA-B2L was transfected into BHK-21 cells.IFA and Western-blot were used for identification of the B2L protein expression in vitro.The results demonstrated that a suicidal DNA vaccine expressing the B2L gene of Orf virus was successfully constructed.The results of IFA and Western-blot showed that the recombinant plasmid pSCA-B2L could express the B2L protein in vitro.And the expressed protein has good reactogenicity.The present study successfully constructed a eukaryotic expression plasmid pSCA-B2L,which would lay the foundation for the further study on nucleic acid vaccine against Orf virus.