Hyphantria cuea nucleopolyhedrovirus(HcNPV) and several other nucleopolyhedrosis was extracted by differential centrifugation,and purified with sucrose gradient centrifugation method.Polyhedrin was gotten and analysed...Hyphantria cuea nucleopolyhedrovirus(HcNPV) and several other nucleopolyhedrosis was extracted by differential centrifugation,and purified with sucrose gradient centrifugation method.Polyhedrin was gotten and analysed by SDS-PAGE.It indicated that most of those proteins had 32 KD protein band,and with 64 KD protein,and might be the main protein dimer with the structure and found Hyphantria cunea nuclear polyhedrosis virus protein in 18 KD department had a specific band.展开更多
Baculoviruses have been widely used as biological agents because of their specificpathogenicity for target insect and harmlessness to mammals, birds and plants as well asadvantages with persistence and epidemics as pe...Baculoviruses have been widely used as biological agents because of their specificpathogenicity for target insect and harmlessness to mammals, birds and plants as well asadvantages with persistence and epidemics as pestcides. A major drawback for morewide-spread use of these viruses is their low virulenee and slow speed of action, which re-stricted their use. No enhancement in pathogenicity of recombinant viruses was observedwith insertion of the Bacillus thuringiensis full-length endotoxin cryIA(c) and cryIA(b) geneinto the AcNOV (Autographa californica nuclear polyhedrosis virus) genome under the con-trol of polyhedrin gene promoter. The reason may be that protoxin, full-length genesproduct of recombinant virus in infected insect cells, which was short of insect gut alkalienvironment, cannot be degraded into active toxic polypeptide. Expression level of 3’展开更多
The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells...The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.展开更多
The polyhedrin (polh) gene is often used to analyse evolution of baculovirus. In this report, the polh of Antheraea pernyi nucleopolyhedro-virus (AnpeNPV) was cloned and sequenced. The Open reading frame (ORF) of the ...The polyhedrin (polh) gene is often used to analyse evolution of baculovirus. In this report, the polh of Antheraea pernyi nucleopolyhedro-virus (AnpeNPV) was cloned and sequenced. The Open reading frame (ORF) of the AnpeNPV consists of 738 nucleotides encoding 245 amino acids with molecular masses of 29 kDa. The deduced amino acids were significant homol-ogy with other baculoviruses, such as Attacus ricini NPV (ArNPV) and Autographa californica NPV (AcNPV). A strongly hydrophilic region was predicted at positions from 30 to 50 of the An-peNPV Polh protein by bioinformatics analysis. Expression of the polh gene of AnpeNPV in E. coli was examined by SDS–PAGE, Western blot and Mass-spectrum analysis. The result showed that the bacterium expression system was suitable for the virus gene expression. It indi-cated that the products of the polh gene ex-pressed in this system can be easier to use for raising antibodies.展开更多
The intact 741 hp polyhedrin gene of LsNPV was sequenced by Silver Sequencing System, and shares 90.6% and 97.0% nucleotide identity, 97.2% and 97. 6% amino acid identity with PfNPV and MdNPV polh genes respectively....The intact 741 hp polyhedrin gene of LsNPV was sequenced by Silver Sequencing System, and shares 90.6% and 97.0% nucleotide identity, 97.2% and 97. 6% amino acid identity with PfNPV and MdNPV polh genes respectively. The 14 hp conservative sequence with the core element GTAAG,is located in the 5'untranslated region of the gene. The polh gene was predicted to encodes a 246 amino sold residures with molecular weight of 29.0 kd, in which the number of acidic amino acids and alkaline amino acids was roughly equal resulting in almost no charges in polyhedrin protein molecule and hence occlusion body. It gives a valuable implication that ionic bonds as well as hydrophobic bonds and hydrogen bond may Play an important role in the crystallization or polyhedrin, by comparing amino acid variation of twenty-one polyhedrin. The comparison of promoter regions of polyhedrin gene and class Ⅲ gene shown that they are very similar, but also have differences in GC content.This could explain that both categories of gene are highly expressed, and polyhedrin genes are expressed more higher than class Ⅲ gene.展开更多
LdMNPV-NEFU isolate collected from the forestry farm of Northeast Forestry University was purified and the genomic DNA of LdMNPV was extracted. The LdMNPV polyhedrin gene was cloned by PCR. The results showed that the...LdMNPV-NEFU isolate collected from the forestry farm of Northeast Forestry University was purified and the genomic DNA of LdMNPV was extracted. The LdMNPV polyhedrin gene was cloned by PCR. The results showed that the sequence was an open reading frame (ORF) of 735bp capable of encoding 245 amino acids. The polyhedrin gene sequences of the LdMNPV-NEFU isolate and a Canada strain, LdMNPV-G differed in 5 bases. The polyhedrin gene of the LdMNPV-NEFU isolate contained C, G, T, C and G at 54, 109,379, 508 and 701 sites from the start codon, but the LdMNPV-G isolate contained G, C, C, T and T at the corresponding sites respectively. The same amino acids were encoded by the two ORF sequences, with the exception that Asp and His are encoded by GAC on the polyhedrin gene sequence of the LdMNPV-NEFU isolate and by CAC in the LdMNPV-G isolate. The LdMNPV polyhedrin gene was expressed in E.coli BL21 (DE3) by the pT7-7 plasmid vector.展开更多
DNA of Syngrapha falcifera nuclear polyhedrosis virus D-clone (Sfa-D clone) was extracted and digested by three kinds of restriction endonuclease. We calculated its molecular weight and measure its melting temperature...DNA of Syngrapha falcifera nuclear polyhedrosis virus D-clone (Sfa-D clone) was extracted and digested by three kinds of restriction endonuclease. We calculated its molecular weight and measure its melting temperature, G+C%. virus particle and polyhedrin were purified. The structural polypeptides and polyhedrin are analysed by SDS-PAGE.展开更多
文摘Hyphantria cuea nucleopolyhedrovirus(HcNPV) and several other nucleopolyhedrosis was extracted by differential centrifugation,and purified with sucrose gradient centrifugation method.Polyhedrin was gotten and analysed by SDS-PAGE.It indicated that most of those proteins had 32 KD protein band,and with 64 KD protein,and might be the main protein dimer with the structure and found Hyphantria cunea nuclear polyhedrosis virus protein in 18 KD department had a specific band.
文摘Baculoviruses have been widely used as biological agents because of their specificpathogenicity for target insect and harmlessness to mammals, birds and plants as well asadvantages with persistence and epidemics as pestcides. A major drawback for morewide-spread use of these viruses is their low virulenee and slow speed of action, which re-stricted their use. No enhancement in pathogenicity of recombinant viruses was observedwith insertion of the Bacillus thuringiensis full-length endotoxin cryIA(c) and cryIA(b) geneinto the AcNOV (Autographa californica nuclear polyhedrosis virus) genome under the con-trol of polyhedrin gene promoter. The reason may be that protoxin, full-length genesproduct of recombinant virus in infected insect cells, which was short of insect gut alkalienvironment, cannot be degraded into active toxic polypeptide. Expression level of 3’
文摘The green fluorescence of bioluminescent jellyfish Aequorea victoria is due to the presence of the green fluorescent protein (GFP). To examine whether the GFP gene can be applied as a reporter gene in insect cells, a baculovirus transfer vector containing the neomycin resistance gene (neo) was established. The GFP gene was subcloned into the vector downstream of the polyhedrin gene (ocu) promoter. In the presence of G418, the recombinant virus can be purified. Expression of the GFP gene in the recombinant virus should give rise to synthesis of the GFP with a molecular weight of 30×10 3 dalton, and is observable by the strong green light irradiated by ultraviolet or blue light in viable intact insect cells. The GFP produced in insect cells has typical fluorescent spectra indistinguishable from those of the purified native GFP. The GFP gene as a good reporter gene can be applied to the baculovirus insect cell expression system.
文摘The polyhedrin (polh) gene is often used to analyse evolution of baculovirus. In this report, the polh of Antheraea pernyi nucleopolyhedro-virus (AnpeNPV) was cloned and sequenced. The Open reading frame (ORF) of the AnpeNPV consists of 738 nucleotides encoding 245 amino acids with molecular masses of 29 kDa. The deduced amino acids were significant homol-ogy with other baculoviruses, such as Attacus ricini NPV (ArNPV) and Autographa californica NPV (AcNPV). A strongly hydrophilic region was predicted at positions from 30 to 50 of the An-peNPV Polh protein by bioinformatics analysis. Expression of the polh gene of AnpeNPV in E. coli was examined by SDS–PAGE, Western blot and Mass-spectrum analysis. The result showed that the bacterium expression system was suitable for the virus gene expression. It indi-cated that the products of the polh gene ex-pressed in this system can be easier to use for raising antibodies.
文摘The intact 741 hp polyhedrin gene of LsNPV was sequenced by Silver Sequencing System, and shares 90.6% and 97.0% nucleotide identity, 97.2% and 97. 6% amino acid identity with PfNPV and MdNPV polh genes respectively. The 14 hp conservative sequence with the core element GTAAG,is located in the 5'untranslated region of the gene. The polh gene was predicted to encodes a 246 amino sold residures with molecular weight of 29.0 kd, in which the number of acidic amino acids and alkaline amino acids was roughly equal resulting in almost no charges in polyhedrin protein molecule and hence occlusion body. It gives a valuable implication that ionic bonds as well as hydrophobic bonds and hydrogen bond may Play an important role in the crystallization or polyhedrin, by comparing amino acid variation of twenty-one polyhedrin. The comparison of promoter regions of polyhedrin gene and class Ⅲ gene shown that they are very similar, but also have differences in GC content.This could explain that both categories of gene are highly expressed, and polyhedrin genes are expressed more higher than class Ⅲ gene.
文摘LdMNPV-NEFU isolate collected from the forestry farm of Northeast Forestry University was purified and the genomic DNA of LdMNPV was extracted. The LdMNPV polyhedrin gene was cloned by PCR. The results showed that the sequence was an open reading frame (ORF) of 735bp capable of encoding 245 amino acids. The polyhedrin gene sequences of the LdMNPV-NEFU isolate and a Canada strain, LdMNPV-G differed in 5 bases. The polyhedrin gene of the LdMNPV-NEFU isolate contained C, G, T, C and G at 54, 109,379, 508 and 701 sites from the start codon, but the LdMNPV-G isolate contained G, C, C, T and T at the corresponding sites respectively. The same amino acids were encoded by the two ORF sequences, with the exception that Asp and His are encoded by GAC on the polyhedrin gene sequence of the LdMNPV-NEFU isolate and by CAC in the LdMNPV-G isolate. The LdMNPV polyhedrin gene was expressed in E.coli BL21 (DE3) by the pT7-7 plasmid vector.
文摘DNA of Syngrapha falcifera nuclear polyhedrosis virus D-clone (Sfa-D clone) was extracted and digested by three kinds of restriction endonuclease. We calculated its molecular weight and measure its melting temperature, G+C%. virus particle and polyhedrin were purified. The structural polypeptides and polyhedrin are analysed by SDS-PAGE.
