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 bovin...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.展开更多
To elucidate the characteristics of molecular epidemiology of PRRSV in China, the nucleotide sequence of open reading frame 5 (ORF5) coding regions from two wild PRRSV strains, J1 and S1, isolated in northern and mid-...To elucidate the characteristics of molecular epidemiology of PRRSV in China, the nucleotide sequence of open reading frame 5 (ORF5) coding regions from two wild PRRSV strains, J1 and S1, isolated in northern and mid-eastern regions of China was determined by RT-PCR. Comparison showed that only 4 different bases between J1 and S1 strains resulted in 3 amino acid (aa. )changes in their deduced proteins. There were 3 and 2 different aa. between the deduced aa. sequences of J1, S1 strains and the American prototype ATCC virus VR2332 respectively, while there existed 16 and 15 aa. changes between the 2 isolates and the modified live vaccine strains derived from VR2332. The putative protein molecular weight, isoelectric point, signal region, glycosylation site and possible transmembrane helices of ORF5 of each strains were similar to that of VR2332 strain. These results showed that the two isolates belong to the same genotype and they may come from USA.展开更多
文摘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.
文摘To elucidate the characteristics of molecular epidemiology of PRRSV in China, the nucleotide sequence of open reading frame 5 (ORF5) coding regions from two wild PRRSV strains, J1 and S1, isolated in northern and mid-eastern regions of China was determined by RT-PCR. Comparison showed that only 4 different bases between J1 and S1 strains resulted in 3 amino acid (aa. )changes in their deduced proteins. There were 3 and 2 different aa. between the deduced aa. sequences of J1, S1 strains and the American prototype ATCC virus VR2332 respectively, while there existed 16 and 15 aa. changes between the 2 isolates and the modified live vaccine strains derived from VR2332. The putative protein molecular weight, isoelectric point, signal region, glycosylation site and possible transmembrane helices of ORF5 of each strains were similar to that of VR2332 strain. These results showed that the two isolates belong to the same genotype and they may come from USA.
