The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was...The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low- copy vector pOK12, producing pOKShimen-RzTФ. Direct transfection of pOKShimen-RzTqb into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.展开更多
Determination of isocarbophos by cathodically sweeping oscilliopolarography is described. In a 1.0×10?5 mol/L sodium dodecylbenzene sulfonate (SDS)+0.1 mol/L HAC-NaAC (pH=4.0) buffer medium, isocarbophos and its ...Determination of isocarbophos by cathodically sweeping oscilliopolarography is described. In a 1.0×10?5 mol/L sodium dodecylbenzene sulfonate (SDS)+0.1 mol/L HAC-NaAC (pH=4.0) buffer medium, isocarbophos and its alkaline hydrolysate exhibited sensitive second derivative wave at ?0.50 V and ?0.48 V respectively. The peak current was linearly proportional to the concentration of isocarbophos in the range of 5.40×10?6?1.05×10?4 mol/L by detecting isocarbophos directly. The detection limit was 3.60×10?6 mol/L with the relative standard derivation (RSD) of 7.3%. By employing an alkaline hydrolysis, the peak current was linearly proportional to the concentration of isocarbophos in the range from 7.70×10?7 mol/L to 1.24×10?4 mol/L, and the detection limit was 5.80×10?7 mol/L with RSD of 3.1%. The hydrolysis procedure and the electrode reaction were studied by voltammetry.展开更多
基金supported by the National Basic Research Program of China (2005CB523202)
文摘The reverse genetics for classical swine fever virus （CSFV） is currently based on the transfection of in vitro transcribed RNA from a viral genomic cDNA clone, which is inefficient and time-consuming. This study was aimed to develop an improved method for rapid recovery of CSFV directly from cloned cDNA. Full-length genomic cDNA from the CSFV Shimen strain, which was flanked by a T7 promoter, the hepatitis delta virus ribozyme and T7 terminator sequences, was cloned into the low- copy vector pOK12, producing pOKShimen-RzTФ. Direct transfection of pOKShimen-RzTqb into PK/T7 cells, a PK-15- derived cell line stably expressing bacteriophage T7 RNA polymerase, allowed CSFV to be rescued rapidly and efficiently, i.e., at least 12 h faster and 31.6-fold greater viral titer when compared with the in vitro transcription-based rescue system. Furthermore, the progeny virus rescued from PK/T7 cells was indistinguishable, both in vitro and in vivo, from its parent virus and the virus rescued from classical reverse genetics. The reverse genetics based on intracellular transcription is efficient, convenient and cost-effective. The PK/T7 cell line can be used to rescue CSFV directly from cloned cDNA and it can also be used as an intracellular transcription and expression system for studying the structure and function of viral genes.
文摘Determination of isocarbophos by cathodically sweeping oscilliopolarography is described. In a 1.0×10?5 mol/L sodium dodecylbenzene sulfonate (SDS)+0.1 mol/L HAC-NaAC (pH=4.0) buffer medium, isocarbophos and its alkaline hydrolysate exhibited sensitive second derivative wave at ?0.50 V and ?0.48 V respectively. The peak current was linearly proportional to the concentration of isocarbophos in the range of 5.40×10?6?1.05×10?4 mol/L by detecting isocarbophos directly. The detection limit was 3.60×10?6 mol/L with the relative standard derivation (RSD) of 7.3%. By employing an alkaline hydrolysis, the peak current was linearly proportional to the concentration of isocarbophos in the range from 7.70×10?7 mol/L to 1.24×10?4 mol/L, and the detection limit was 5.80×10?7 mol/L with RSD of 3.1%. The hydrolysis procedure and the electrode reaction were studied by voltammetry.
