羊口疮病毒F1L和B2L基因真核表达质粒构建及其在MDBK细胞中的表达

目的 为构建羊口疮病毒F1L和B2L基因真核表达质粒,同时验证其在MDBK细胞中的表达。方法 本试验以pVAX1为真核表达载体,在前期构建的克隆质粒pMD18-T-B2L和pMD18-T-F1L基础上,构建真核表达质粒pVAX1-F1L和pVAX1-B2L,对其分别进行双酶切和测序鉴定,将鉴定正确的真核表达质粒pVAX1-F1L和pVAX1-B2L转染至MDBK细胞,采用RT-PCR和间接免疫荧光试验(IFA)检测目的基因表达。结果 双酶切鉴定和序列测定表明真核表达质粒pVAX1-F1L和pVAX1-B2L构建成功,RT-PCR和IFA同时验证目的基因在MDBK细胞浆中能瞬时表达。结论 本试验构建成功的真核表达质粒为下一步羊口疮病毒核酸疫苗研究奠定基础。

Construction and expression of recombinant plasmids of OrfV F1L and B2L genes in MDBK cells

In this study, we constructed eukaryotic recombinant expression plasmids of F1L and B2L genes of Orf Virus, and identified their expression in MDBK cells. The recombinant plasmids pVAX1-B2L and pVAX1-F1L were constructed based on the pMD18-T-F1L and pMD18-T-B2L which were constructed before. Then, they were identified by digestion with restriction enzymes and sequencing. The MDBK cells were transfected with pVAX1-B2L and pVAX1-F1L plasmids coated by liposome, and the RT-PCR and indirect immunofluoresence assay (IFA) were adopted to detect the expressions of pVAX1-B2L and pVAX1-F1L. Results indicated that the recombinant plasmids pVAX1-B2L and pVAX1-F1L were constructed and confirmed successfully by sequencing and digestion, and their instantaneous expressions were verified in the cytoplasm of MDBK cells by RT-PCR and IFA. The eukaryotic recombinant expression plasmids constructed in this study would lay the foundation for the further study on nucleic acid vaccine of OrfV.