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
口蹄疫的有效控制关键在于早期检测 ,然而有很多疾病症状与口蹄疫相似 ,仅靠临床症状难以确诊 ,因此必须进行实验室诊断。实验室诊断包括病毒学诊断和血清学诊断。病毒学诊断方法有病毒分离、补体结合试验、酶联免疫吸附试验 ( EL ISA)...口蹄疫的有效控制关键在于早期检测 ,然而有很多疾病症状与口蹄疫相似 ,仅靠临床症状难以确诊 ,因此必须进行实验室诊断。实验室诊断包括病毒学诊断和血清学诊断。病毒学诊断方法有病毒分离、补体结合试验、酶联免疫吸附试验 ( EL ISA)、RT-PCR以及乳胶凝集试验 ( L AT)。 RT-PCR有待进一步完善 ,而用于野外检测的现场诊断方法已取得可喜进展。血清学诊断包括中和试验和 EL ISA,中和试验已经被 EL ISA方法取代 ,并且通过检测非结构蛋白的抗体可以区分感染动物和免疫动物。更加快速、敏感、可靠以及用于检测潜伏感染的诊断技术将是今后研究的热点。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.
文摘口蹄疫的有效控制关键在于早期检测 ,然而有很多疾病症状与口蹄疫相似 ,仅靠临床症状难以确诊 ,因此必须进行实验室诊断。实验室诊断包括病毒学诊断和血清学诊断。病毒学诊断方法有病毒分离、补体结合试验、酶联免疫吸附试验 ( EL ISA)、RT-PCR以及乳胶凝集试验 ( L AT)。 RT-PCR有待进一步完善 ,而用于野外检测的现场诊断方法已取得可喜进展。血清学诊断包括中和试验和 EL ISA,中和试验已经被 EL ISA方法取代 ,并且通过检测非结构蛋白的抗体可以区分感染动物和免疫动物。更加快速、敏感、可靠以及用于检测潜伏感染的诊断技术将是今后研究的热点。
