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
将 I型牛疱疹病毒 (BHV- 1 ) LA株DNA H ind III A片段中的 Sal I- Sal I亚片段(含 TK基因 )克隆到载体质粒 p BluescriptSK中 ,再用 Bgl II和 Sac I切去 347bp,获得含 TK基因部分缺失的重组质粒 p Sd TK,然后用 H ind III和 Xba I切去...将 I型牛疱疹病毒 (BHV- 1 ) LA株DNA H ind III A片段中的 Sal I- Sal I亚片段(含 TK基因 )克隆到载体质粒 p BluescriptSK中 ,再用 Bgl II和 Sac I切去 347bp,获得含 TK基因部分缺失的重组质粒 p Sd TK,然后用 H ind III和 Xba I切去其中的多克隆位点 ;将来源于 p CR3- Uni的 CMV启动子、多克隆位点和 BGH poly A信号插入 p Sd TK的Xho I位点上 ,构建了 BHV- 1通用转移载体p Sd TK- CMB,此载体可用来表达牛其它病毒的抗原基因 。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.
文摘将 I型牛疱疹病毒 (BHV- 1 ) LA株DNA H ind III A片段中的 Sal I- Sal I亚片段(含 TK基因 )克隆到载体质粒 p BluescriptSK中 ,再用 Bgl II和 Sac I切去 347bp,获得含 TK基因部分缺失的重组质粒 p Sd TK,然后用 H ind III和 Xba I切去其中的多克隆位点 ;将来源于 p CR3- Uni的 CMV启动子、多克隆位点和 BGH poly A信号插入 p Sd TK的Xho I位点上 ,构建了 BHV- 1通用转移载体p Sd TK- CMB,此载体可用来表达牛其它病毒的抗原基因 。
