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Expression of Hantaan virus 26 kD fragment of nucleocapsid protein in insect cells and prelimimary study on its immunogenicity

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Journal of Medical Colleges of PLA(China) 2003年第5期18卷 267-272页

作者：罗雯张芳琳阎岩吴兴安刘勇白文涛王海涛徐志凯 Department of Microbiology Fourth Military Medical UniversityXi'an 710033China Department of Microbiology Fourth Military Medical UniversityXi'an 710033Chinabjective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major an-tigenic epitopes in insect cells and make a preliminary analysis of its immunological characteristics. Methods: The re-combinant baculovirus bac-S0. 7 with the 700 bp fragment of S gene 5'terminal of Hantaan virus was constructed and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0. 7 infecting insect cells specific antibody with the highest titer of 1:1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0. 7 gene fragment in insect cells is immunogenic.

Objective: To express the 26 kD fragment of Hantaan virus nucleocapsid protein that contains the major antigenic epitopes in insect cells, and make a preliminary analysis of its immunological characteristics. Methods: The recombinant baculovirus bac-S0.7 with the 700 bp fragment of S gene 5' terminal of Hantaan virus was constructed, and the antigenicity of the expression product was tested. Mice were injected with Sf9 cells infected by the recombinant baculovirus. The humoral and cellular immunological effects were identified by indirect immunofluorescence assay, micro-cell culture neutralization test and T lymphocytes stimulation test. Results: Immunized by bac-S0.7 infecting insect cells, specific antibody with the highest titer of 1∶1 600 was observed. The stimulation indexes of splenocytes of immunized mice to nucleocapsid protein of Hantaan virus was higher than the negative control. Conclusion: The expression product of S0.7 gene fragment in insect cells is immunogenic.

关键词： Hantaan virus nucleocapsid protein insect cell immunogenicity

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High-Level Production of a Functional Recombinant Hepatitis B Virus Polymerase in insect cells with a Baculovirus Expression System

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2007年第3期27卷 269-273页

作者：王晓燕高琳琳邓菲张艳芳李岩林菊生 Institute of Liver Diseases Tongji Hospital Tongii Medical College Huazhong University of Science and Technology Wuhan Institute of Virology Chinese Academy of Sciences

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.

关键词： hepatitis B virus polymerase baculovirus insect cell

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Baculovirus as a highly efficient expression vector in insect and mammalian cells

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Acta Pharmacologica Sinica 2005年第4期26卷 405-416页

作者： Yu-chen HU~1 Department of Chemical Engineering,National Tsing Hua University,Hsinchu,Taiwan,China Department of Chemical Engineering National Tsing Hua University Hsinchu Taiwan China. ychu@mx.nthu.edu.tw

Baculovirus has been widely used for the production of recombinant proteins ininsect *** the finding that baculovirus can efficiently transduce mamma-lian cells,the applications of baculovirus have been greatly *** pros-pects and drawbacks of baculovirus-mediated gene expression,either in insect orin mammalian cells,are *** progresses in expanding the applicationsto studies of gene regulation,viral vector preparation,in vivo and ex vivo genetherapy studies,generation of vaccine vectors,etc are discussed and the effortsdirected towards overcoming the existing bottlenecks are particularly emphasized.

关键词： baculovirus insect cell mammalian cell gene therapy vaccine recombinant protein

Differences in nutrient uptake between the fat body and embryonic primary cultures of silkworm （Bombyx mori）

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insect Science 2006年第1期13卷 19-24页

作者： LEILA MATINDOOST JALAL J. SENDI HOORIEH SOLEIMAN JAHI KAYVAN ETEBARI Department of Plant Protection Faculty of Agriculture University of Guilan Rasht Department of Virology School of Medical Science Tarbiat Modarres Tehran Department of Sericulture Faculty of Natural Resources University of Guilan Somehesara Iran

Nutrition utilization and by-product formation in cultured insect cells has been investigated in several insect cells and has been of great interest to cell culturists and physiologists. In this research the biochemical changes in embryonic and fat body primary cultures of silkworm, Bombyx mori, have been compared. TC-100 medium supplemented with 10% and 20% FBS was used in embryonic and fat body primary cultures, respectively. Medium was renewed every week and the amount of glucose, uric acid, urea, total protein and alkaline phosphatase were measured in the samples from medium of primary cultures using spectrophotometeric methods. All biochemical macromolecules except uric acid showed significant changes. Glucose decreased in embryonic tissues, while in fat body culture its amount increased. Urea accumulation in embryonic culture was higher than in the fat body cultures. Since urea is a by-product, this accumulation could be due to higher utilization of amino acids. Total protein showed considerable changes and was consumed by embryonic culture more than the fat body＇ s. Alkaline phosphatase showed stronger activity in embryonic cells.

关键词： biochemical Bombyx mori embryo fat body insect cell primary culture,silkworm

Simulation,construction and characterization of a multi-functional thrombolytic agent with anti-thrombosis activity

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Frontiers in Biology 2008年第3期3卷 265-268页

作者： Weiran YU Jian JING Academy of Military Medicine Beijing Institute of BioengineeringBeijing 100071China Department of Biochemistry and Molecular Biology Beijing Key LaboratoryBeijing Normal UniversityBeijing 100875China

Prourokinase(scu-PA),a thrombolytic agent,was inserted between Gly118 and Ile119 with foreign anti-thrombosis functional motif(Lys-Gly-Asp-Trp-motif)to construct a multi-functional chimeric *** molecular model of a chimera was simulated and *** recombinant chimeric protein was expressed by the baculovirus-insect cell expression system and puri-fied by affinity *** physico-chemical characteristics of the chimeric molecule were *** thrombolytic activity was determined to be 90000 IU/mg of fibrinolytic special activity by the fibrin-plate *** anti-thrombosis activities were also assayed with IC50 of 9.6 μM by an inhibition test of ADP-induced platelet aggregation.

关键词： anti-thrombosis thrombolytic agent insect cell baculovirus prourokinase(scu-PA) foreign functional motif

Expression,purification and characterization of enterovirus-71 virus-like particles

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World Journal of Gastroenterology 2006年第6期12卷 921-927页

作者： Yao-Chi Chung Jen-Huang Huang Chia-Wei Lai Heng-Chun Sheng Shin-Ru Shih Mei-Shang Ho Yu-Chen Hu Department of Chemical Engineering National Tsing Hua University Hsinchu 300 Taiwan China School of Medical Technology Chang GungUniversity Taoyuan 333 Taiwan China Institute of Biomedical Sciences AcademiaSinica Taipei 11529 Taiwan China

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.

关键词： Enterovirus 71 Virus-like particle VLP Vaccine Baculovirus insect cell