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
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
家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是一种具有较强传染性的病毒,在感染的极晚期超高水平表达一种约29 kD的碱溶性多角体蛋白。但该蛋白为病毒复制的非必需蛋白,因此,利用该基因可以被删除或被外源基因替换而...家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是一种具有较强传染性的病毒,在感染的极晚期超高水平表达一种约29 kD的碱溶性多角体蛋白。但该蛋白为病毒复制的非必需蛋白,因此,利用该基因可以被删除或被外源基因替换而不影响病毒的复制的原理构建重组病毒,可以实现外源基因的高水平表达。然而,目前虽已经有较多文献研究多角体蛋白高水平表达的分子机制,但迄今对于多角体蛋白基因编码区的结合蛋白尚不明确。本研究通过DNA pull-down联合质谱技术对结合在多角体蛋白编码区的蛋白质进行鉴定,共鉴定到78个宿主来源的蛋白和49个病毒自身编码的蛋白。对这些蛋白进行GO注释,结果表明这些蛋白除了具有共同的核酸结合功能外,还有其他不同的生物学功能,共同在多角体蛋白的超高水平表达过程中发挥作用。进一步从上述蛋白中筛选出了最值得深入研究的9个宿主蛋白和10个病毒蛋白。对这些蛋白进行深入研究,将为深入揭示多角体蛋白超高水平表达的分子机制提供新的线索。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’
文摘家蚕核型多角体病毒(Bombyx mori nucleopolyhedrovirus,BmNPV)是一种具有较强传染性的病毒,在感染的极晚期超高水平表达一种约29 kD的碱溶性多角体蛋白。但该蛋白为病毒复制的非必需蛋白,因此,利用该基因可以被删除或被外源基因替换而不影响病毒的复制的原理构建重组病毒,可以实现外源基因的高水平表达。然而,目前虽已经有较多文献研究多角体蛋白高水平表达的分子机制,但迄今对于多角体蛋白基因编码区的结合蛋白尚不明确。本研究通过DNA pull-down联合质谱技术对结合在多角体蛋白编码区的蛋白质进行鉴定,共鉴定到78个宿主来源的蛋白和49个病毒自身编码的蛋白。对这些蛋白进行GO注释,结果表明这些蛋白除了具有共同的核酸结合功能外,还有其他不同的生物学功能,共同在多角体蛋白的超高水平表达过程中发挥作用。进一步从上述蛋白中筛选出了最值得深入研究的9个宿主蛋白和10个病毒蛋白。对这些蛋白进行深入研究,将为深入揭示多角体蛋白超高水平表达的分子机制提供新的线索。
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.
