To construct the bi-valent genetic engineering vaccine against pseudorabies virus（PRV）and porcine reproductive and respiratory syndrome virus（PRRSV）,the modified PRRSV ORF5 gene（ORF5M） and the VP22 gene of bovine herpesvirus 1（BHV-1）,which encodes VP22 protein and has been demonstrated to exhibit the unusual protein transduction property,were inserted into a PRV universal transfer vector pIECMV by turns.A recombinant virus transfer vector pIECMV-VP22ORF5M possessing VP22-ORF5M fusion gene was generated.The recombinant virus transfer vector pIECMV-VP22ORF5M co-transfected the IBRS-2 cells with PRV TK-/gE-/LacZ+ genomic DNA digested by EcoRⅠusing liposome method.Based on homologous recombination,the recombinant virus was generated and then purified by the plaque assay and PCR amplification.After three rounds of plaque purification,the recombinant virus was further confirmed by PCR,Southern blot and Western blot.A recombinant PRV（rPRV）TK-/gE-/VP22GP5+ expressing VP22-GP5 fusion protein was constructed.The results of TCID50 tests showed that the insertion of the foreign genes had no influence on the propagation of rPRV in IBRS-2 or PK-15 cells.The construction of rPRV TK-/gE-/VP22GP5+ provides a basis for further study of bi-valent genetic engineering vaccines against PRRSV and PRV,and that this strategy may also be useful to develop more efficient genetic engineering vaccines against other pathogens.
拟验证天然断裂内含肽Npu Dna E、Ssp Dna E的反式剪接反应对嵌合蛋白形成的影响,探讨依据此方法表达抗菌肽的可行性,规避抗菌生物表达中对宿主的毒害作用.将花蝉属抗菌肽ADP-1(Amblyomma defensin peptide 1)拆分为N端和C端两部分,其N端与断裂型内含肽Npu Dna E的N端融合,C端与Ssp Dna E的C端融合,并分别构建到原核表达载体p ET-23a(Amp+)和p ET-30a(Kan+)上,获得重组表达质粒p ET-23a-ADP-1-N-Npu和p ET-30a-Ssp-ADP-1-C,然后单独或共转化大肠杆菌感受态细胞BL21.实验结果表明,两个重组质粒分别单独或组合转化至Bl21(DE3),经IPTG诱导后,SDS-PAGE检测转化的菌株中都表达了目的蛋白,共转化的菌株诱导表达破菌后其上清液有抑菌活性.该实验验证以两种天然断裂型内含肽Ssp Dna E和Npu Dna E及搭载的ADP-1 N端和C端可在大肠杆菌中获得表达,且共转化的两段内含肽可在离体上清中发生反式剪接,促进融合在内含肽上的ADP-1两部分形成完整具有抑菌活性产物.
A 0.5kb antisense RNA fragment was designed targeting the 5’ non-coding region (NCR), translation initiation site and potential transcriptional active region of the sole immediate early gene(IE180) of Pseudorabies virus(PRV). The antisense RNA fragment was amplified by PCR and inserted into the downstream of hCMV IE promoter/enhancer of the eukaryotic expression vector pCI-neo. The resulting antisense RNA expression plasmid(pCI-0.5) was transfected into PK-15 cells and the transfected cells were selected by G418.The expression of antisense RNA was confirmed by RT-PCR. In order to assess the antiviral potency of the cell lines expressing the antisense RNA, the antisense RNA cell lines and the control cell lines were infected with 0.1 pfu of PRV strain Ea per cell. The infected supernatant were collected at different time points post-infection(p.i.) and the TCID\-(50) were further determined. The data showed that the cell lines expressing the antisense RNA could markedly inhibit the replication of PRV. The viral titers in cells expressing the antisense RNA was 156-fold less than control cells at 40 hp i.
