Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p...Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H2O2 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H2O2, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP. hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.展开更多
Glycidyl methacrylate (GMA)is a recently recognized mutagen. In order to explore the mutagenicity and mechanism of GMA, plasmid PBR322 was used for in vitro binding, mutant screening, restriction enzyme mapping,and DN...Glycidyl methacrylate (GMA)is a recently recognized mutagen. In order to explore the mutagenicity and mechanism of GMA, plasmid PBR322 was used for in vitro binding, mutant screening, restriction enzyme mapping,and DNA sequencing. To explore the mechamism by which an initial premutational event is converted into a stable heritable mutation, pBR322 and GMA-bound pBR322 were transformed into E. coli HB101 , and the following results were obtained : 1) GMA-bound PBR322 induced phenotype changes in competent cells. Two stable and heritable mutants were isolated (Ap￣RTc￣S and Ap￣STc￣R). 2) When restriction enzyme mapping was used to analyze the mutant Ap￣RTc￣S , four of seven maps showed changes, but no large DNA insertion or deletion were observed.3) The frequency of deletion and insertion forms counted about 10%. Sequence specificity and hot spot regions were evident in the sequence analysis of mutated plasmid. The above results indicate that the premutagenic lesions of plasmid induced by GMA can be converted into point mutations in vivo.展开更多
Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe ge...Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichiapastorisunder the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualiza-tion. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoriswith an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immuno-genicity on surface-displayed HEnAg. Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.展开更多
Plutella xylostella,a major pest of cruciferous vegetables worldwide,has developed resistance to diamide insecticides.Thiotraniliprole,a novel synthetic diamide insecticide,exhibits excellent activity against P.xylost...Plutella xylostella,a major pest of cruciferous vegetables worldwide,has developed resistance to diamide insecticides.Thiotraniliprole,a novel synthetic diamide insecticide,exhibits excellent activity against P.xylostella.In the present study,we aimed to confirm the resistance risk,cross-resistance,and mechanisms of resistance to thiotraniliprole in P.xylostella.After 40 consecutive generations of thiotraniliprole selection,we obtained a thiotraniliprole-resistance P.xylostella strain with a 5141.58-fold resistance ratio(RR)to thiotraniliprole.The overall realized heritability(h^(2))value of resistance was estimated as 0.9 using threshold trait analysis,indicating that the risk of developing resistance to thiotraniliprole is high in P.xylostella.The thiotraniliprole-resistant(TR)strain showed noticeable cross-resistance to chlorantraniliprole(RR=44670.05),cyantraniliprole(RR=7038.58),and tetrachlorantraniliprole(RR=1506.01),but no cross-resistance to tolfenpyrad,indoxacarb,diafenthiuron,or abamectin compared with the susceptible(S)strain.The enzyme assay data showed that the activities of glutathione-S transferase(GST),carboxylesterase(CarE),and the content of cytochrome P450 monooxygenase(P450s)were significantly higher in the TR strain than in the S strain.Sequencing of the full-length PxRyR cDNA revealed the gene site I4790K in the TR strain with a 100%frequency.This mutation in PxRyR likely underlies the high-level cross-resistance between thiotraniliprole and three other diamide insecticides.These findings provide valuable information for optimizing resistance management strategies to delay thiotraniliprole resistance development and ensure sustainable control of P.xylostella.展开更多
文摘Objective: To establish a sensitive and specific system for genotoxicity detection in vivo. Methods: Endogenous p53 tracer vector p53RE was constructed by using green fluorescent protein (GFP) as a reporter to trace p53 transcriptional activity under the control of base SV40 early promoter. The tracer cells 3T3-REG were established by transfecting NIH3T3 cells with tracer vector and treated with ultraviolet, H2O2 and adriamycin to induce DNA damage and the subsequent endogenous p53 expression. The GFP expression and its green fluorescence in the tracer cells were observed and measured with fluorescent microscope and FACS. Results: The GFP expression increased rapidly after various treatment and reached the maximum 1 h later, and decreased gradually afterwards. FACS analysis showed that GFP expression in 3T3-REG tracer cells was consistent with the concentration of H2O2, while that in 3T3-SVG cells, which were transfected with control vector pSV-GFP. hardly increased in response to the treatment. Conclusion: GFP tracing p53 transcriptional activity is a sensitive and specific method for genotoxicity detection.
文摘Glycidyl methacrylate (GMA)is a recently recognized mutagen. In order to explore the mutagenicity and mechanism of GMA, plasmid PBR322 was used for in vitro binding, mutant screening, restriction enzyme mapping,and DNA sequencing. To explore the mechamism by which an initial premutational event is converted into a stable heritable mutation, pBR322 and GMA-bound pBR322 were transformed into E. coli HB101 , and the following results were obtained : 1) GMA-bound PBR322 induced phenotype changes in competent cells. Two stable and heritable mutants were isolated (Ap￣RTc￣S and Ap￣STc￣R). 2) When restriction enzyme mapping was used to analyze the mutant Ap￣RTc￣S , four of seven maps showed changes, but no large DNA insertion or deletion were observed.3) The frequency of deletion and insertion forms counted about 10%. Sequence specificity and hot spot regions were evident in the sequence analysis of mutated plasmid. The above results indicate that the premutagenic lesions of plasmid induced by GMA can be converted into point mutations in vivo.
文摘Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg). MethodsThe gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichiapastorisunder the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualiza-tion. Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoriswith an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immuno-genicity on surface-displayed HEnAg. Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.
基金Supported by the Zhejiang Provincial Public Welfare Technology Application Research Program(No:LGN21C140001).
文摘Plutella xylostella,a major pest of cruciferous vegetables worldwide,has developed resistance to diamide insecticides.Thiotraniliprole,a novel synthetic diamide insecticide,exhibits excellent activity against P.xylostella.In the present study,we aimed to confirm the resistance risk,cross-resistance,and mechanisms of resistance to thiotraniliprole in P.xylostella.After 40 consecutive generations of thiotraniliprole selection,we obtained a thiotraniliprole-resistance P.xylostella strain with a 5141.58-fold resistance ratio(RR)to thiotraniliprole.The overall realized heritability(h^(2))value of resistance was estimated as 0.9 using threshold trait analysis,indicating that the risk of developing resistance to thiotraniliprole is high in P.xylostella.The thiotraniliprole-resistant(TR)strain showed noticeable cross-resistance to chlorantraniliprole(RR=44670.05),cyantraniliprole(RR=7038.58),and tetrachlorantraniliprole(RR=1506.01),but no cross-resistance to tolfenpyrad,indoxacarb,diafenthiuron,or abamectin compared with the susceptible(S)strain.The enzyme assay data showed that the activities of glutathione-S transferase(GST),carboxylesterase(CarE),and the content of cytochrome P450 monooxygenase(P450s)were significantly higher in the TR strain than in the S strain.Sequencing of the full-length PxRyR cDNA revealed the gene site I4790K in the TR strain with a 100%frequency.This mutation in PxRyR likely underlies the high-level cross-resistance between thiotraniliprole and three other diamide insecticides.These findings provide valuable information for optimizing resistance management strategies to delay thiotraniliprole resistance development and ensure sustainable control of P.xylostella.
