Based on the nucleotide sequence of porcine reproductive and respiratory syndrome virus (PRRSV) CH 1a strain, a pair of primers was designed. The GP3 gene of PRRSV HB 1 strain was cloned by RT PCR. The sequences analy...Based on the nucleotide sequence of porcine reproductive and respiratory syndrome virus (PRRSV) CH 1a strain, a pair of primers was designed. The GP3 gene of PRRSV HB 1 strain was cloned by RT PCR. The sequences analysis showed that the GP3 genes of HB 1 and CH 1a strain had 91% and 89% homology, at the levels of the nucleotide and amino acid, respectively. The GP3 gene was digested by Bam HⅠ and Eco RⅠ, and the fragment was inserted into the same sites of the universal vector pPgG uni. A recombinant virus transfer vector pPgG GP3 expressing PRRSV GP3 gene was constructed. The transfer vector pPgG GP3 digested by Kpn Ⅰ was co transfected PK 15 cells with the PRV TK -/gG -/LacZ + genomic DNA digested by Eco RⅠ using liposome method. The recombinant virus was purified by the phage test and PCR amplification. The expression of GP3 gene was indicated by Western blot analysis using anti sera. A recombinant PRV virus expressing PRRSV GP3 gene was obtained. The recombinant virus is very useful in the research of the genetic engineering vaccine against pseudorabies virus and PRRS virus.展开更多
Based on the nucleotide sequence of pseudorabies virus (PrV) Ka strain, a pair of primers was designed. PrV Ea strain PK gene was cloned and inserted into retroviral expression vector pLXSN (neo r). The recombinant pl...Based on the nucleotide sequence of pseudorabies virus (PrV) Ka strain, a pair of primers was designed. PrV Ea strain PK gene was cloned and inserted into retroviral expression vector pLXSN (neo r). The recombinant plasmid pLXSNPK was co transfected with packaging cell PA317 using lipofectin method. The transfectants were selected by G418 (300 mg/L) for 2 weeks. Viral supernatants gaining stable cell clones were collected, the total RNA was extracted and the PK gene was amplified by RT PCR. It was shown that the PK gene had been inserted into the retroviral genome. The positive viral supernatant was collected to infect the interesting target cell MDBK. The infected MDBK cells were selected by G418 (800 mg/L) and analyzed by indirect immunofluorescence and SDS PAGE and Western blotting. It was confirmed that a MDBKPK cells expressing PK protein was obtained. It is useful to study the biological function of PrV PK gene in the process of PrV inducing culture cells apoptosis.展开更多
文摘Based on the nucleotide sequence of porcine reproductive and respiratory syndrome virus (PRRSV) CH 1a strain, a pair of primers was designed. The GP3 gene of PRRSV HB 1 strain was cloned by RT PCR. The sequences analysis showed that the GP3 genes of HB 1 and CH 1a strain had 91% and 89% homology, at the levels of the nucleotide and amino acid, respectively. The GP3 gene was digested by Bam HⅠ and Eco RⅠ, and the fragment was inserted into the same sites of the universal vector pPgG uni. A recombinant virus transfer vector pPgG GP3 expressing PRRSV GP3 gene was constructed. The transfer vector pPgG GP3 digested by Kpn Ⅰ was co transfected PK 15 cells with the PRV TK -/gG -/LacZ + genomic DNA digested by Eco RⅠ using liposome method. The recombinant virus was purified by the phage test and PCR amplification. The expression of GP3 gene was indicated by Western blot analysis using anti sera. A recombinant PRV virus expressing PRRSV GP3 gene was obtained. The recombinant virus is very useful in the research of the genetic engineering vaccine against pseudorabies virus and PRRS virus.
文摘Based on the nucleotide sequence of pseudorabies virus (PrV) Ka strain, a pair of primers was designed. PrV Ea strain PK gene was cloned and inserted into retroviral expression vector pLXSN (neo r). The recombinant plasmid pLXSNPK was co transfected with packaging cell PA317 using lipofectin method. The transfectants were selected by G418 (300 mg/L) for 2 weeks. Viral supernatants gaining stable cell clones were collected, the total RNA was extracted and the PK gene was amplified by RT PCR. It was shown that the PK gene had been inserted into the retroviral genome. The positive viral supernatant was collected to infect the interesting target cell MDBK. The infected MDBK cells were selected by G418 (800 mg/L) and analyzed by indirect immunofluorescence and SDS PAGE and Western blotting. It was confirmed that a MDBKPK cells expressing PK protein was obtained. It is useful to study the biological function of PrV PK gene in the process of PrV inducing culture cells apoptosis.
