摘要
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.
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 EcoR Ⅰ using 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.
出处
《病毒学报》
CAS
CSCD
北大核心
2006年第1期62-65,共4页
Chinese Journal of Virology
基金
国家自然科学基金(3030025730400322)
国家"973"项目(2005CB523200)资助
关键词
牛疱疹病毒1型VP22
猪繁殖与呼吸综合征病毒
ORF5M基因
重组伪狂犬病毒
bovine herpesvirus 1 VP22 （BHV-1 VP22）
porcine reproductive and respriatory syndrome virus （PRRSV）
modified ORF5 gene （ORF5M）
recombinant pseudorabies virus（rPRV）
