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
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
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
家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)对蚕丝业的危害极大。该病毒的多角体蛋白编码基因polyhedrin(polh)能够在病毒感染极晚期超高水平表达,并在细胞核内形成包涵体,多角体蛋白转运到宿主细胞核中的详细机制...家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus, BmNPV)对蚕丝业的危害极大。该病毒的多角体蛋白编码基因polyhedrin(polh)能够在病毒感染极晚期超高水平表达,并在细胞核内形成包涵体,多角体蛋白转运到宿主细胞核中的详细机制目前未知。为阐明病毒蛋白的入核机制,本研究通过分子克隆、免疫共沉淀(co-immunoprecipitation, CO-IP)、免疫荧光等方法,详细分析了多角体蛋白(polyhedrin, Polh)的表达规律,并进一步揭示了宿主蛋白importin α通过蛋白互作的方式参与多角体蛋白的入核转运,为进一步深入阐明包括Polh在内的诸多杆状病毒入核蛋白的入核机制提供了参考。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
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
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.
