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High-Level Production of a Functional Recombinant Hepatitis B Virus Polymerase in Insect Cells with a Baculovirus Expression System 被引量:1
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作者 王晓燕 高琳琳 +3 位作者 邓菲 张艳芳 李岩 林菊生 《Journal of Huazhong University of Science and Technology(Medical Sciences)》 SCIE CAS 2007年第3期269-273,共5页
HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express fu... HBV polymerase has intrinsic RNA-dependent reverse transcriptase, DNA-dependent DNA polymerase as well as RNaseH activity. Analysis of HBV polymerase has been hampered for many years due to the inability to express functional enzyme in a recombinant system. To obtain ac- tive polymerase at a high level, we have taken advantage of baculovirus expression system. The gene of HBV polymerase was amplified by PCR and cloned into pFastBac Dual to construct the recombi- nant plasmid pFastbac Dual-pol. The recombinant donor plasmid, pFastbac Dual-pol, was constructed by inserting HBV polymerase gene into EcoRI and PstI sites controlled by polyhedrin promoter. The recombinant donor plasmid was transformed into DH10Bac competent cells for transposition. Re- combinant bacmid was constructed by inserting of the mini-Tn7 element from the donor plasmid into the mini-attTn7 attachment site on the bacmid. The recombinant bacmid DNA was isolated and transfected into the Sf9 cells to produce the recombinant virus, and healthy insect Sf9 cells were in- fected with the recombinant virus containing HBV polymerse gene to express the target protein. HBV polymerse expressed in insect cells was analyzed by SDS-PAGE. PCR results showed recombinant donor plasmid, pFastbac Dual-pol, was constructed successfully. The recombinant hepatitis B virus polymerase was expressed in insect cells at high level. The recombinant hepatitis B virus polymerase should facilitate the analysis of HBV polymerase biological characteristics, allow the investigation for new anti-HBV drugs specifically blocking HBV polymerase. 展开更多
关键词 hepatitis B virus POLYMERASE baculovirus insect cell
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Expression of Green Fluorescent Protein Gene with Baculovirus Vectorin Insect Cells
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作者 Hu Jianhong Zhu Fanxiu +1 位作者 Qi Yipeng Huang Yongxiu 《Wuhan University Journal of Natural Sciences》 CAS 1997年第1期117-121,共5页
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
关键词 green fluorescent protein(GFP) baculovirus transfer vector insect cells polyhedrin gene neomycin resistance gene
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Expression of p30, the major surface antigen of Toxoplasma gondii, in baculovirus-insect cell system and the evaluation of immune response induced by p30
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作者 陈晓光 陈兆明 +3 位作者 马鑫 彭红娟 沈树满 刘国章 《Journal of Medical Colleges of PLA(China)》 CAS 2000年第3期157-160,共4页
Objective: To produce the major surface antigen (p30) of Toxoplasma gondii from the Baculovirus Expression System. Methods: The p30 coding sequence was cloned into a transfer vector, then the recombinant baculovirus c... Objective: To produce the major surface antigen (p30) of Toxoplasma gondii from the Baculovirus Expression System. Methods: The p30 coding sequence was cloned into a transfer vector, then the recombinant baculovirus containing p30 gene was cloned and purified by the co-transfection and plaque assay. The expression and immunoactivity of the recombinant p30 were analyzed by SDS-PAGE and Western blot. The immune responses in mice for being immunized with recombinant p30 were tested. Results: About 750μg of purified (95% purity) p30 was obtained from a culture of 108 in- sect Sf21 cells. Mice in injected with the recombinant protein produced specific humoral and cellular immune responses. Immunization with p30 also prolonged the period of mice survival infected by Toxoplasma gondii. Conclusion: It is indicated that the recombinant p30 from baculovirus expression system can stimulate mice to produce effective protection from Toxo- plasma gondii infection. 展开更多
关键词 TOXOPLASMA GONDII MAJOR surface antigen baculovirus expression SYSTEM immune
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Recombinant expression and purification of functional vascular endothelial growth factor-121 in the baculovirus expression system 被引量:3
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作者 Nastaran Mohseni Ali Jahanian Najafabadi +4 位作者 Fateme Kazemi-Lomedasht Roghaye Arezomand Mahdi Habibi-Anbouhi Delavar Shahbazzadeh Mahdi Behdani 《Asian Pacific Journal of Tropical Medicine》 SCIE CAS 2016年第12期1170-1174,共5页
Objective: To express human Vascular endothelial growth factor121(VEGF121) in insect cells. Methods: A gene construct containing VEGF was cloned in the p Fast Bac-HTA vector, followed by transformation in DH10 BAC. Th... Objective: To express human Vascular endothelial growth factor121(VEGF121) in insect cells. Methods: A gene construct containing VEGF was cloned in the p Fast Bac-HTA vector, followed by transformation in DH10 BAC. The recombinant bacmid was then extracted, and transfected into Sf9 insect cells. The transfected cells were harvested, and then VEGF expression was confirmed by Western blotting using specific antibodies. The tube formation assay was used for functional assessment of VEGF. Results: Our results showed that VEGF could be successfully expressed in the baculovirus system. Purified VEGF was able to stimulate in vitro tube formation of human endothelial cells. Conclusions: Results from this study demonstrated that the recombinantly-produced VEGF can be considered as a promising candidate for therapeutic purposes. 展开更多
关键词 Vascular endothelial growth factor baculovirus expression system Recombinant bacmid
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Efficient expression of histidine-tagged large hepatitis delta antigen in baculovirus-transduced baby hamster kidney cells 被引量:4
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作者 Ying-Wei Chiang Jaw-Chin Wu +3 位作者 Kuei-Chun Wang Chia-Wei Lai Yao-Chi Chung Yu-Chen Hu 《World Journal of Gastroenterology》 SCIE CAS CSCD 2006年第10期1551-1557,共7页
AIM: To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen (L-HDAg). METHODS: A recombinant baculovirus expressing histidine-tagged L-HDAg (L-HDAgH) ... AIM: To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen (L-HDAg). METHODS: A recombinant baculovirus expressing histidine-tagged L-HDAg (L-HDAgH) was constructed to transduce baby hamster kidney (BHK) cells by a simplified transduction protocol. RESULTS: The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly, the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy. Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles. CONCLUSION: The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive posttranslational modifications. 展开更多
关键词 baculovirus Hepatitis delta virus L-HDAg Mammalian cell Protein expression TRANSDUCTION
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Genetic Modification of Baculovirus Expression Vectors 被引量:4
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作者 Shu-fen Li Hua-lin Wang +1 位作者 Zhi-hong Hu Fei Deng 《Virologica Sinica》 CAS CSCD 2012年第2期71-82,共12页
As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expre... As a protein expression vector, the baculovirus demonstrates many advantages over other vectors. With the development of biotechnology, baculoviral vectors have been genetically modified to facilitate high level expression of heterologous proteins in both insect and mammalian cells. These modifications include utilization of different promoters and signal peptides, deletion or replacement of viral genes for increasing protein secretion, integration of polycistronic expression cassette for producing protein complexes, and baculovirus pseudotyping, promoter accommodation or surface display for enhancing mammalian cell targeting gene delivery. This review summarizes the development and the current state of art of the baculovirus expression system. Further development of baculovirus expression systems will make them even more feasible and accessible for advanced applications. 展开更多
关键词 baculovirus Protein expression Promoters Signal peptides Gene delivery
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Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system
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作者 Huiyuan Lu Jun Ma Nian Liu Shoulin Wang 《The Journal of Biomedical Research》 CAS 2010年第3期242-249,共8页
Objective:CYP1A2 and NADPH-CYP450 oxidoreductase(POR)were expressed in the baculovirus/ Spodoptera frugiperda(sf9)system.The aim of this study was to investigate the effects of heme precursors on the expression o... Objective:CYP1A2 and NADPH-CYP450 oxidoreductase(POR)were expressed in the baculovirus/ Spodoptera frugiperda(sf9)system.The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR.Methods:The heme precursors[δ-Aminolaevulinic Acid(5-ALA),Fe^3+and hemin]were introduced into the system to evaluate their effects on the expression of CYP1A2,POR and their co-expression.All the proteins were identified using immunoblotting,CO-difference spectroscopy,or cytochrome c assay.Results:In the present study,functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system,and both of them showed high activities.Co-addition of 5-ALA and Fe^3+significantly improved expression of CYP1A2 by about 50%compared with the addition of 5-ALA,Fe^3+or hemin alone.Either co-addition of 5-ALA and Fe^3+or addition of 5-ALA or Fe^3+alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control(no addition).However,unlike CYP1A2,there was no difference between the co-addition and addition of these heme precursors alone.Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR,with a 3:1 ratio of BvCYP1A2/BvPOR significantly increasing their co-expression.Surprisingly,the addition of 0.1 mM 5-ALA or Fe^3+alone,but not their co- addition,could significantly improve the CYP1A2 and POR co-expression(P〈0.05).Conclusion:5-ALA and Fe^3+increased the expression of CYP1A2 and POR in a baculovirus/sf9 system,but the pattern of their expression was different between their expression alone and co-expression. 展开更多
关键词 CYP1A2 POR baculovirus/Spodopterafrugiperda system CO-expression heme precursors
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Baculovirus-mediated Expression of p35 Confers Resistance to Apoptosis in Human Embryo Kidney 293 cells
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作者 Jian-hua SONG Chang-yong LIANG Xin-wen CHEN 《中国病毒学》 CSCD 2007年第5期389-396,共8页
Baculovirus has many advantages as vectors for gene transfer.We demonstrated that recombinant baculovirus vectors expressing p35(Ac-CMV-p35) and eGFP(Ac-CMV-GFP) could be transduced into human kidney 293 cells efficie... Baculovirus has many advantages as vectors for gene transfer.We demonstrated that recombinant baculovirus vectors expressing p35(Ac-CMV-p35) and eGFP(Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently.The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter.MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells.Cell growth curve showed the Ac-CMV-p35 and Ac-CMV-GFP transduced and non-transduced cells had similar proliferation rate,so baculovirus-mediated p35 expression had no adverse effect on cell proliferation.In addition,baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D,UV or serum-free media.These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy. 展开更多
关键词 细胞凋亡 基因治疗 P35基因 基因表达 胚胎肾细胞 杆状病毒
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Expression of the Human Growth Hormone Genein Insect Cells
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作者 耿朝晖 俞新大 +4 位作者 周卫东 张宝珠 李建民 余慕贞 王英 《Developmental and Reproductive Biology》 1998年第1期32-39,共8页
The insect cellvirus (AcNPV) system was used to express heterologous protein. The recombinant transfer vector pVL1393/hGH was reconstructed in which the human Growth Hormone (hGH) gene was inserted under the control ... The insect cellvirus (AcNPV) system was used to express heterologous protein. The recombinant transfer vector pVL1393/hGH was reconstructed in which the human Growth Hormone (hGH) gene was inserted under the control of the polyhedron gene promoter. The Spodoptera frugiperda (Sf9) cells was cotransfected with the plasmid DNA containing hGH gene and wildtype AcNPV DNA. The hGH gene was transferred to the AcNPV genome DNA through homologous recombination, and the recombinant virus rAcVhGH was obtained by multiple plaque purification. The high level of production of hGH (40 μg/mL) in supernatant of the infected monolayer culture was determined by immunochemiluminescent assay. 展开更多
关键词 HGH GENE ACNPV transfer VECTOR insect cells GENE expression
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Baculovirus-expressed FAdV-4 penton base protein protects chicken against hepatitis-hydropericardium syndrome 被引量:2
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作者 ZHANG Jun-qin WEI Yan-ming +3 位作者 HUANG Kun SUN Xiao-mei ZOU Zhong JIN Mei-lin 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2019年第11期2598-2604,共7页
Hepatitis-hydropericardium syndrome(HHS)is an infectious disease caused by fowl adenovirus serotype 4(FAdV-4).Several structural and non-structural proteins of FAdV-4 have been expressed in Escherichia coli and baculo... Hepatitis-hydropericardium syndrome(HHS)is an infectious disease caused by fowl adenovirus serotype 4(FAdV-4).Several structural and non-structural proteins of FAdV-4 have been expressed in Escherichia coli and baculovirus expression system to develop candidate subunit vaccines.However,the protective efficiency of baculovirus-expressed penton base protein has not been assessed.In this study,two recombinant capsid proteins,penton base and fiber-2,were constructed.And then,penton base and fiber-2 were administrated alone or together to specific pathogen-free(SPF)chickens at 14 days of life and boosted at 28 days of life.At 42 days of life,the immunized groups and the control group were challenged with FAdV-4 virulent strain.Results show that inoculating penton base or penton base+fiber-2 provided 100%protection to the chickens.All groups vaccinated with the recombinant protein produced detectable antibodies and showed no apparent lesions.Thus,baculovirus-expressed penton base protein is a promising candidate subunit vaccine. 展开更多
关键词 FOWL adenovirus SEROTYPE 4 hepatitis-hydropericardium SYNDROME PENTON base subunit vaccine baculovirus expression system
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Porcine Interleukin-2 Expression in Insect Cells and Its Enhancement of Pig Immunity to Swine Influenza Virus Inactivated Vaccine 被引量:3
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作者 CHEN Hong-ying ZHANG Hong-ying HUANG Yan-quan CUI Bao-an WANG Zhen-ya WANG Yan-bin LIU Jin-peng CHAO An-jun 《Agricultural Sciences in China》 CAS CSCD 2010年第8期1211-1220,共10页
Mature porcine interleukin-2 (pIL-2) gene was amplified by PCR from the plasmid pGEM-T-pIL2 and cloned into the baculovirus pFastBacTM Dual vector of the Bac-to-Bac baculovirus expression system under the control of... Mature porcine interleukin-2 (pIL-2) gene was amplified by PCR from the plasmid pGEM-T-pIL2 and cloned into the baculovirus pFastBacTM Dual vector of the Bac-to-Bac baculovirus expression system under the control of the PH promoter. Recombinant plL-2 (rpIL-2) expressed in Sf9 insect cells was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunofluorescence assay. Western blot analysis confirmed that the rpIL-2 protein had a molecular mass of 20 kDa, which was larger than the molecular mass of the mature protein predicted based on its peptide sequence. The rpIL-2 protein induced in vitro proliferation of ConA-stimulated porcine splenocytes and enhanced in vivo protective immune responses induced by vaccinating the pigs with inactivated oil emulsion vaccine against swine influenza virus. The results showed that the rpIL-2 expressed in Sf9 insect cells has immunoenhancement effects; the finding lays the foundation for the preparation of a specific recombinant IL-2 protein and the development of a novel immune adjuvant of vaccines against various infectious porcine pathogens to increase the immunoprotective efficacy of vaccines. 展开更多
关键词 porcine interleukin-2 Sf9 insect cells expression inactivated vaccine swine influenza virus
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Secretive Expression of Insect Antifungal Peptide-Encoded Genes in Pichia pastoris and Activity Assay of the Products 被引量:2
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作者 SANG Yan-xia DENG Xiao-juan +5 位作者 YANG Wan-ying WANG Wen-xian WEN Shuo-yang LIU Wen-quan HUANG Ya-dong CAO Yang 《Agricultural Sciences in China》 CAS CSCD 2007年第10期1209-1216,共8页
The antifungal peptides, drosomycin (Drs) and its isoform drosomycin-like C (Drs-lC) from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamento... The antifungal peptides, drosomycin (Drs) and its isoform drosomycin-like C (Drs-lC) from Drosophila melanogaster and Thanatin from Podisus maculiventris, have potent activity with broad spectrum against filamentous fungi. Secretive expression of these genes in yeasts makes it possible to utilize the supernatants of yeast culture as protective reagents on fruit, vegetable, food and other agricultural products. So the study of effective secretion by yeast expression system is of great importance. Three genes, Drs, Drs-lC, and Thanatin, were cloned into pPICZαA and the recombinant vectors, pPICZαA-Drs, pPICZαA-Drs-lC, and pPICZαA-Thanatin were transformed into Pichia pastoris by the electric transfer method. The recombinant P. pastoris, which was screened by phenotype selection and PCR amplification, was induced to express antifungal peptide by methanol. The expressive products of the three recombinants showed antifungal activity against 5 out of 6 test fungi strains, and the products of Thanatin also had strong activity against the tested bacteria. The three antifungal peptide genes, Drs, Drs-lC, and Thanatin, were constructed into yeast P. pastoris. The expressed peptides were successfully secreted into the culture medium and exhibited potent activities against the test strains. 展开更多
关键词 insect antimicrobial peptides Pichia pastoris secretive expression activity assay
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Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells 被引量:13
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作者 Qin FANG Eng Khuan Seng +1 位作者 Wen DAI Lan-lan ZHANG 《中国病毒学》 CSCD 2007年第5期397-404,共8页
Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inne... Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro. 展开更多
关键词 草鱼呼肠孤病毒 VP6蛋白 增强绿色荧光蛋白 杆状病毒表达系统 共表达
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Cloning and Expression of HIV-1 p24 Gene in Insect Cells by Using BAC-TO-BAC System
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作者 Mallam Nock Joshua Qi Yipeng +1 位作者 Huang Yongxiu Liu Ziye 《Wuhan University Journal of Natural Sciences》 EI CAS 1998年第1期113-118,共6页
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
关键词 CLONING expression baculovirus ANTIGEN ANTIBODY ELISA
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Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells
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作者 Guo-cheng FAN Fang-luan GAO +5 位作者 Tai-yun WEI Mei-ying HUANG Li-yan XIE Zu-jian WU Qi-ying LIN Lian-hui XIE 《Virologica Sinica》 SCIE CAS CSCD 2010年第6期401-408,共8页
To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8... To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity,its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system.The S8 gene was subcloned into the pFastBacTM1 vector,to produce the recombinant baculovirus transfer vector pFB-S8.After transformation,pFB-S8 was introduced into the competent cells (E.coli DH10Bac) containing a shuttle vector,Bacmid,generating the recombinant bacmid rbpFB-S8.After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection,Sf9 cells were collected at different times and analyzed by SDS-PAGE,Western blotting and immunofluorescence microscopy.The expression level of the P8 protein was highest between 48-72 h after transfection of Sf9 cells.Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells. 展开更多
关键词 Rice gall dwarf virus (RGDV) Outer coat protein baculovirus expression system Spodoptera frugiperda (Sf9) insect cells
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Expression,purification and characterization of enterovirus-71 virus-like particles 被引量:43
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作者 Yao-Chi Chung Jen-Huang Huang +4 位作者 Chia-Wei Lai Heng-Chun Sheng Shin-Ru Shih Mei-Shang Ho Yu-Chen Hu 《World Journal of Gastroenterology》 SCIE CAS CSCD 2006年第6期921-927,共7页
AIM: Enterovirus 71 (EV71) has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. ... AIM: Enterovirus 71 (EV71) has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region. METHODS: The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses: Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD. RESULTS: Both single infection by Bac-P1-3CD and coinfection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins selfassembled into clusters of virus-like particles (VLP) resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling. CONCLUSION: Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics. 展开更多
关键词 Enterovirus 71 Virus-like particle VLP VACCINE baculovirus insect cell
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Expression of Aminopeptidase N1(APN1),the Main Receptor Protein for Bacillus thuringiensis CrylA Toxin from Helicoverpa armigera Larval Midgut in Trichoplusia ni cells 被引量:1
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作者 CHANG Hong-lei LIANG Ge-mei WANG Gui-rong YU Hong-kun GUO Yu-yuan WU Kong-ming 《Agricultural Sciences in China》 CAS CSCD 2008年第3期329-335,共7页
The aim of this article is to successfully express the Bt (Bacillus thuringiensis) toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm (Helicoverpa armigera Hübner) wit... The aim of this article is to successfully express the Bt (Bacillus thuringiensis) toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm (Helicoverpa armigera Hübner) within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance. The fragments of aminopeptidase N1 (APN1) gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method, and were separately cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac. It was cultured in LB medium, which contained Te, Kan, Ge, X-gal, and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationshio of resistance with Bt. 展开更多
关键词 APN1 baculovirus expression vector Trichoplusia ni expression
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The Parasitoid Factor in the Virulence and Spread of Lepidopteran Baculoviruses 被引量:1
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作者 J. E. Cossentine 《Virologica Sinica》 SCIE CAS CSCD 2009年第4期305-314,共10页
Insect parasitoids and baculoviruses play important roles in the natural and strategic biological control of insects. The two parasites are frequent competitors within common hosts and much research has focused on the... Insect parasitoids and baculoviruses play important roles in the natural and strategic biological control of insects. The two parasites are frequent competitors within common hosts and much research has focused on the negative impact that baculoviral host infections have on parasitoids. This review summarizes the impacts that parasitoids may have on the virulence and spread of lepidopteran baculoviruses. By changing host behavior and development, parasitoids have been shown to decrease baculovirus virulence and productivity within parasitized baculovirus-susceptible hosts; however, studies of the tools used by hymenopteran parasitoids to overcome their hosts'immune systems, suggest that parasitoids may, in some cases, facilitate baculoviral infections in less susceptible hosts. Laboratory and field research have demonstrated that parasitoids can mechanically transmit baculoviruses between insects, and in this way, increase the efficacy of the viruses. Instances of new, more virulent isolates of baculoviruses have been recorded from specifically parasitoid-targeted hosts suggesting other possible benefits from the transmission or activation of baculoviruses by parasitoids. 展开更多
关键词 insect parasitoid baculovirus Interactions
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Characterization of T-complex polypeptide 1 (TCP-1) from the Chilo suppressalis HSP60 family and its expression in response to temperature stress 被引量:3
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作者 YU Tong-ying LU Ming-xing CUI Ya-dong 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2018年第5期1032-1039,共8页
Many proteins require assistance from molecular chaperones at various stages to attain correctly folded states and functional conformations during protein synthesis. In this study, the gene encoding T-complex polypept... Many proteins require assistance from molecular chaperones at various stages to attain correctly folded states and functional conformations during protein synthesis. In this study, the gene encoding T-complex polypeptide 1(TCP-1), which belongs to the heat shock protein 60(HSP60) family, was isolated and characterized from the rice stem borer, Chilo suppressalis, by RACE and q PCR, respectively. The full-length c DNA of Tcp-1 was 2 144 bp and encoded a 1 635-bp ORF; the deduced translational product contained 545 amino acids with 5′-and 3′-UTRs and an isoelectric point of 5.29. Cluster analysis confirmed that the deduced amino acid sequence shared high identity(60–99%) with TCP-1 from other insects. To investigate Tcp-1 expression in response to abiotic stress, q PCR was used to analyze expression levels of Tcp-1 m RNA in C. suppressalis larvae exposed to temperatures ranging from –11 to 43°C. With respect to heat shock, Tcp-1 expression was higher than the control after a 2-h exposure to 30 and 36°C and declined at 39 and 43°C. Difference in Tcp-1 expression was observed at temperatures ranging from –11 to 27°C. q PCR analyses revealed that Tcp-1 expression was the highest in hindgut tissue as compared to heads, epidermis, fat body, foregut, midgut, and malpighian tubules. Our results indicated that Tcp-1 expression was differentially expressed in C. suppressalis tissues, and was impacted by temperature stress. 展开更多
关键词 TCP-1 Chilo suppressalis expression analysis temperature stress insect tissues
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Susceptibility to AcMNPV and Expression of Recombinant Proteins by a Novel Cell Clone Derived from a Trichoplusia ni QAU-BTI-Tn9-4s Cell Line 被引量:1
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作者 Ming Shan Shi-ying Zhang +2 位作者 Lei Jiang Ming Ma Guo-xun Li 《Virologica Sinica》 SCIE CAS CSCD 2011年第5期297-305,共9页
It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But ... It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But the characteristics of the cell line do not always remain stable and may change upon continuous passage.Recently an alphanodavirus,named Tn5 Cell Line Virus (or TNCL Virus),was identified in High Five cells in particular.Therefore,we established a new cell line,QB-Tn9-4s,from Trichoplusia ni,which was determined to be free of TNCL virus by RT-PCR analysis.In this paper,we describe the development of a novel cell clone,QB-CL-B,from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV,and the level of recombinant protein production.This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate;although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies,there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells. 展开更多
关键词 Cell line insect virus Recombinant protein expression RT-PCR TNCL virus
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