Reverse transcription polymerase chain reaction (RT-PCR) was used for the detection of classical swine fever virus (CSFV) in blood and tissue samples of field cases and experimentally inoculated pigs. The distribution...Reverse transcription polymerase chain reaction (RT-PCR) was used for the detection of classical swine fever virus (CSFV) in blood and tissue samples of field cases and experimentally inoculated pigs. The distribution of CSFV in different organ samples showed some discrepancies in infected pigs. Four weaner pigs were inoculated with C-strain vaccine virus, then samples of spleen, tonsil, lung, mesenteric lymph node, kidney and brain were collected after slaughter and tested for E2 and NS5B genes using one-step RT-PCR and nested RT-PCR. Using the same method, 12 field cases were simultaneously studied. A discrepancy of CSFV in different samples was found upon detecting the target gene. The most reliable diagnostic organs were spleen and tonsil, and the nested RT-PCR assay provided a highly sensitive and specific method with comparable performance to the one-step RT-PCR assay.展开更多
In order to develop swine hepatitis E （HE） genetically engineering vaccines, specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplieons generated by PC...In order to develop swine hepatitis E （HE） genetically engineering vaccines, specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplieons generated by PCR assays were directly cloned into T-A plasmid and expressed using pEASY-M1 expression vector. Three recombinant eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3 were constructed. The eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2, and pEASY-LB3 were transfected into 293T cells, and three target genes were detected by real-time fluorescent quantitative RT-PCR. The results confirmed that three eukaryotic expression plasmids were transfected into 293Teells and target protein was expressed. Analysis by SDS-PAGE electrophoresis and Western-blot indicated that three target proteins were expressed in 293T cells transfected with eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3. Antigenicity studies indicated good HEV responses. Therefore, three recombinant DNAs of HEV ORF2 nucleic acid vaccine candidates were ob- tained, which might lay the foundation for further studies in the future.展开更多
为了研究猪瘟病毒E 2糖蛋白的立体结构及生物学特性,将增强型荧光蛋白基因(EGFP)和猪瘟兔化弱毒疫苗株(HCLV)E 2基因经PCR扩增后克隆至pB lueB acH is2A质粒,与杆状病毒DNA共转染后经PCR鉴定获得了含有EGFP和HCLV E 2融合基因(GFPTE 2)...为了研究猪瘟病毒E 2糖蛋白的立体结构及生物学特性,将增强型荧光蛋白基因(EGFP)和猪瘟兔化弱毒疫苗株(HCLV)E 2基因经PCR扩增后克隆至pB lueB acH is2A质粒,与杆状病毒DNA共转染后经PCR鉴定获得了含有EGFP和HCLV E 2融合基因(GFPTE 2)的重组杆状病毒rBACTE 2-339,并将其感染sf9细胞后在荧光显微镜下观察到了亮绿色荧光,说明融合基因已初步表达。展开更多
In order to understand the incidence reasons in a scaled pig farm of Guizhou Province, the infected pigs were diagnosed with epidemiological investigation, clinical observation, pathological diagnosis, enzyme-linked i...In order to understand the incidence reasons in a scaled pig farm of Guizhou Province, the infected pigs were diagnosed with epidemiological investigation, clinical observation, pathological diagnosis, enzyme-linked immunosorbent assay （ELISA）, PCR/RT-PCR and bacterial isolation and identification. The results showed that the pigs were infected mixedly with classical swine fever virus （CSFV）, pseudorabies virus （PRV） and E. coli. Therefore, some measures should be adopted to control the incidence of CSF, PR and colibacillosis in the pig farm.展开更多
基金supported by the grants from the National"863" Programme (2006AA10A2041)Development Programme of Science and Technology,Chengguan District of Gansu province (08-5-4)
文摘Reverse transcription polymerase chain reaction (RT-PCR) was used for the detection of classical swine fever virus (CSFV) in blood and tissue samples of field cases and experimentally inoculated pigs. The distribution of CSFV in different organ samples showed some discrepancies in infected pigs. Four weaner pigs were inoculated with C-strain vaccine virus, then samples of spleen, tonsil, lung, mesenteric lymph node, kidney and brain were collected after slaughter and tested for E2 and NS5B genes using one-step RT-PCR and nested RT-PCR. Using the same method, 12 field cases were simultaneously studied. A discrepancy of CSFV in different samples was found upon detecting the target gene. The most reliable diagnostic organs were spleen and tonsil, and the nested RT-PCR assay provided a highly sensitive and specific method with comparable performance to the one-step RT-PCR assay.
基金Supported by the Basal Research Fund of Guangxi(10-111-1)+2 种基金the Guangxi Science and Technology Project(10100014-4)the Scientific Research Project of Guangxi Bureau of Livestock,Fisheries and Veterinary Services(12049031)the Systemic Research Project of Guangxi Key Laboratory of Animal Vaccines and New Technology(12-071-28-A-5)
文摘In order to develop swine hepatitis E （HE） genetically engineering vaccines, specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplieons generated by PCR assays were directly cloned into T-A plasmid and expressed using pEASY-M1 expression vector. Three recombinant eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3 were constructed. The eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2, and pEASY-LB3 were transfected into 293T cells, and three target genes were detected by real-time fluorescent quantitative RT-PCR. The results confirmed that three eukaryotic expression plasmids were transfected into 293Teells and target protein was expressed. Analysis by SDS-PAGE electrophoresis and Western-blot indicated that three target proteins were expressed in 293T cells transfected with eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3. Antigenicity studies indicated good HEV responses. Therefore, three recombinant DNAs of HEV ORF2 nucleic acid vaccine candidates were ob- tained, which might lay the foundation for further studies in the future.
文摘为了研究猪瘟病毒E 2糖蛋白的立体结构及生物学特性,将增强型荧光蛋白基因(EGFP)和猪瘟兔化弱毒疫苗株(HCLV)E 2基因经PCR扩增后克隆至pB lueB acH is2A质粒,与杆状病毒DNA共转染后经PCR鉴定获得了含有EGFP和HCLV E 2融合基因(GFPTE 2)的重组杆状病毒rBACTE 2-339,并将其感染sf9细胞后在荧光显微镜下观察到了亮绿色荧光,说明融合基因已初步表达。
文摘In order to understand the incidence reasons in a scaled pig farm of Guizhou Province, the infected pigs were diagnosed with epidemiological investigation, clinical observation, pathological diagnosis, enzyme-linked immunosorbent assay （ELISA）, PCR/RT-PCR and bacterial isolation and identification. The results showed that the pigs were infected mixedly with classical swine fever virus （CSFV）, pseudorabies virus （PRV） and E. coli. Therefore, some measures should be adopted to control the incidence of CSF, PR and colibacillosis in the pig farm.
