Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin p...Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 element from the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells were infected with the recombinant virus containing p24 to express the target protein. The target protein expressed was analyzed on a 15% polyacrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positive result shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISA and other reliable diagnostic methods of HIV-1 infection.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
A 1500 bp DNA fragment of LsNPV genome was sequenced by silver sequencing system.A whole coding region of ORF1 and an incomplete ORF2 were found.ORF1 was 345 bp long and was predicted to encode a protein with 115 amin...A 1500 bp DNA fragment of LsNPV genome was sequenced by silver sequencing system.A whole coding region of ORF1 and an incomplete ORF2 were found.ORF1 was 345 bp long and was predicted to encode a protein with 115 amino acid residues,the 5'regulatory region of ORF1 contained early transcriptional consensus sequence such as AT/ACGTGT,CGTGC and a stem loop structure.On the 3'terminus of ORF1,a poly(A)tail signal was found.Incomplete ORF2 was 354 bp long and predicted to encode 118 amino acid residues,the 5'regulatory region of ORF2 contained two conserved late transcriptional motif ATAAG.Compared with all the genes from AcNPV,incomplete ORF2 showed 56.5%nucleotide sequence homology and 44.9%amino acid sequence homology with AcNPV PDV E66 gene.Two conserved ATAAG motif on 5'regulatory region of ORF2 was similar to those in AcNPV PDV E66 gene.ORF1 showed no high identity with AcNPV and other baculoviruses.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
Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study,we clone...Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study,we cloned,expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then,the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly,all of the 89 clones from monoclonal ELISA were positive (S/N>2.1) and the result was further confirmed experimentally once again. Six N protein-binding pep-tides,designated separately as SNA1,SNA2,SNA4,SNA5,SNA9 and SNG11,were selected for se-quencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4,SNA2,SNA9 and SNA1. In addition,the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also,the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program,and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur sub-units (UCRI or UQCR),otherwise known as the Rieske iron-sulfur proteins (RISP). Notablely,in the 2Fe-2S redox centre of UCRI,there were 6 residues GGW(Y)F(Y)CP compatible to the residues (po-sition 2→7,GGWFCP7) of the NH2-terminal of the 15-mer peptide,which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here,the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.展开更多
文摘Recombinant transposing vector pFHIV24 was constructed by cloning the HIV-1 p24 gene into the Multiple cloning site (MCS) of the transposing vector pFastBac1 in the correct orientation with respect to the polyhedrin promoter. Recombinant bacmid bHIV24 was obtained by transposing a mini-att Tn7 element from the recombinant pFHIV24 to the mini-att Tn7 attachment site on the bacmid by Tn7 transposition functions provided by the helper plasmid. Minipreparation of recombinant bacmid DNA was transfected intoSpodoptera frugiperda (Sf9) cells to get the recombinant virus. Fresh insect Sf9 cells were infected with the recombinant virus containing p24 to express the target protein. The target protein expressed was analyzed on a 15% polyacrylamide gels and then used as antigen to check HIV-1 positive serum by ELISA. Our positive result shows that the expressed p24 protein could be used as standard antigen for HIV-1 diagnosis by ELISA and other reliable diagnostic methods of HIV-1 infection.
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A 1500 bp DNA fragment of LsNPV genome was sequenced by silver sequencing system.A whole coding region of ORF1 and an incomplete ORF2 were found.ORF1 was 345 bp long and was predicted to encode a protein with 115 amino acid residues,the 5'regulatory region of ORF1 contained early transcriptional consensus sequence such as AT/ACGTGT,CGTGC and a stem loop structure.On the 3'terminus of ORF1,a poly(A)tail signal was found.Incomplete ORF2 was 354 bp long and predicted to encode 118 amino acid residues,the 5'regulatory region of ORF2 contained two conserved late transcriptional motif ATAAG.Compared with all the genes from AcNPV,incomplete ORF2 showed 56.5%nucleotide sequence homology and 44.9%amino acid sequence homology with AcNPV PDV E66 gene.Two conserved ATAAG motif on 5'regulatory region of ORF2 was similar to those in AcNPV PDV E66 gene.ORF1 showed no high identity with AcNPV and other baculoviruses.
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.
基金the Science and Technology Project of Chongqing Municipal Educa- tion Commission, China (Grant No. KJ071109)the Key Project of the Ministry of Science and Technology of China (Grant No. 2003CB514120)
文摘Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study,we cloned,expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then,the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly,all of the 89 clones from monoclonal ELISA were positive (S/N>2.1) and the result was further confirmed experimentally once again. Six N protein-binding pep-tides,designated separately as SNA1,SNA2,SNA4,SNA5,SNA9 and SNG11,were selected for se-quencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4,SNA2,SNA9 and SNA1. In addition,the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also,the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program,and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur sub-units (UCRI or UQCR),otherwise known as the Rieske iron-sulfur proteins (RISP). Notablely,in the 2Fe-2S redox centre of UCRI,there were 6 residues GGW(Y)F(Y)CP compatible to the residues (po-sition 2→7,GGWFCP7) of the NH2-terminal of the 15-mer peptide,which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here,the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.
