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Expression, Purification and Sublocalization of SARS-CoV Nucleocapsid Protein in insect cells

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Acta Biochimica et Biophysica Sinica 2004年第11期36卷 754-758页

作者： Ai-Xia REN, You-Hua XIE, Yu-Ying KONG, Guan-Zhen YANG, Yao-Zhou ZHANG,Yuan WANG, and Xiang-Fu WUInstitute of Biochemistry and Biotechnology, College of Life Science, Zhejiang University, Hangzhou 310029, China State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and cell Biology, Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, Shanghai 200031, China Institute of Biochemistry, Zhejiang University of Sciences, Hangzhou 310018, China Institute of Biochemistry and Biotechnology College of Life Science Zhejiang University Hangzhou 310029 China.

The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus

关键词： severe acute respiratory syndrome (SARS) SARS coronavirus nucleocapsid protein baculovirus insect cells

INFLUENCE OF MIXING DEVICE ON SERUM-FREE CULTIVATION OF insect cells IN SPINNER FLASKS

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化工学报 2000年第S1期51卷 101-104页

作者： Zhao Jiao, Dai Hu and Tan Wensong (State Key Lab of Bioreactor Engineering, East China University of Science and Technology Shanghai 200237,China)

A culture system was developed and successfully employed for the serum-free cultivation of Tn-5B1-4 (Tn5 ) insect cells. With our adaptation procedure, it was possible to obtain cells fully adapted to serum-free media in stationary T-flasks and then enable these adapted cells to grow well in spinner flasks immediately. The spinner "ask with special stirring design proved tO provide favorable culture environment that made it desirable for use in the serum-free cultivation of Tn5 cells even at low seeding density.

关键词： serum-free insect cells adaptation spinner flasks culture environment

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Expression　of　Green　Fluorescent　Protein　Gene　with　Baculovirus　Vectorin　insect　cells

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Wuhan University Journal of Natural Sciences 1997年第1期2卷 117-121页

作者： Hu Jianhong Zhu Fanxiu Qi Yipeng Huang Yongxiu Institute of Virology Wuhan University Wuhan China

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.

关键词： green fluorescent protein(GFP) baculovirus transfer vector insect cells polyhedrin gene neomycin resistance gene

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In vitro and in vivo Transient Expression in insect cells Mediated by the Cationic Liposome DDAB/DOPE

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In vitro and in vivo Transient Expression in Insect Cells Me...

Development of a New High-throughput Screening Model for Human High Density Lipoprotein Receptor (CLA-1) Agonists

中国蚕学会第三届青年学术研讨会暨浙江省第二届青年学术论坛—蚕桑分论坛

作者： Qing Li Xiao, Ya Jing Zhou, Zhi Fang Zhang, Jia Lu HeKey Laboratory of Silkworm Biotechnology, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu, 212018, P. R. China

Cationlc liposomes complexed with DNA have been extensively utilized for the delivery of reporter ortherapeutic genes both in culture and Jn *** investigated and determined the optimum conditions of a cationic liposome,composed of dimethyldioctadecylammonium bromide(DDAB)and deoleoyl phosphatidylethanolamine(DOPE),mediated areporter plasmld expressing luciferase into insect cell lines(Sf-21 and Bm-N)and silkworm *** the datademonstrated that Bombyx mori nuclear polyhedrosis virus(BmNPV)genomic DNA(128 kb)was successfully transfectedinto Bm-5 cells using this *** results suggest that DDAB/DOPE liposome will be useful as delivery agents forgene transfer to insecet cells both in vitro and in vivo.

关键词： Cationic liposome transient expression insect cells silkworm larvae

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Biomedical and Environmental Sciences 2005年第4期18卷 265-272页

作者： DE-FENG TIAN BIN HONG SHU-YI SI Institute of Medicinal Biotechnology Chinese Academy of Medical Sciences and Peking Union Medical College Beijing 100050 China

To develop a new high-throughput screening model for human high-density lipoprotein （HDL） receptor （CD36 and LIMPⅡ analogous-1, CLA-1） agonists using CLA-1-expressing insect cells. Methods With the total RNA of human hepatoma cells BEL-7402 as template, the complementary DNA （cDNA） of CLA-1 was amplified by reverse transcription-polymerase chain reaction （RT-PCR）. Bac-to-Bac baculovirus expression system was used to express CLA-1 in insect cells. CLA-1 cDNA was cloned downstream of polyhedrin promoter of Autographa californica nuclear polyhedrosis virus （AcNPV） into donor vector pFastBacl and recombinant pFastBacl-CLA-1 was transformed into E. coli DH10Bac to transpose CLA-1 cDNA to bacrnid DNA. Recombinant bacrnid-CLA-1 was transfected into Spodopterafrugiperda Sf9 insect cells to produce recombinant baculovirus particles. Recombinant CLA- 1 was expressed on the membrane of Sf9 cells infected with the recombinant baculoviruses. A series of parameters of DiI-lipoprotein binding assays of CLA-1-expressing Sf9 cells in 96-well plates were optimized. Results Western blot analysis and DiI-lipoprotein binding assays confirmed that CLA-1 expressed in insect cells had similar immunoreactivity and ligand binding activity as its native counterpart. A reliable and sensitive in vitro cell-based assay was established to assess the activity of CLA-1 and used to screen agonists from different sample libraries. Conclusion Human HDL receptor CLA-1 was successfully expressed in Sf9 insect cells and a novel high-throughput screening model for CLA-1 agonists was developed. Utilization of this model allows us to identify potent and selective CLA-1 agonists which might possibly be used as therapeutics for atherosclerosis.

关键词： HDL SR-BI CLA-1 insect cells High-throughput screening model Agonist

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High Expression of Insulin-like Growth Factor Ⅱ (IGF-Ⅱ) Using Bac-to-Bac Expression System

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The Journal of Biomedical Research 2000年第2期 53-58页

作者：赵红,汪承亚,段宇,陈家伟,P.H.Steenbergh,J.S.Sussenbach

Objective In order to obtain mature insulin like growth factor Ⅱ(IGF Ⅱ), we used Bac to Bac baculovirus expression system. Methods Firstly the IGF Ⅱ cDNA was cloned into a donor plasmid pFastBac1 and the recombinant pFastBac1 was then introduced into competent cells DH10Bac. Recombinant bacmids were constructed by transposing a mini Tn7 element from a donor plasmid pFastBac1 to the mini attTn7 attachment site on the bacmid where the Tn7 transposition functions were provided in trans by a helper plasmid, and then used to transfect Sf9 insect cells to get recombinant baculovirus. The recombinant baculovirus was used to infect insect cells. Results Agarose gel analysis showed that recombinant donor plasmid pFastBac1 was constructed successfully; Agarose gel analysis of PCR products confirmed recombinant bacmid; SDS PAGE and Western Blotting showed that a 7KD protein band appeared. Conclusion The mature IGF Ⅱ with immunogenecity has been expressed and produced by using Bac to Bac expression system.

关键词： insulin like growth factor Ⅱ Bac to Bac expression system insect cells

A Cationic Detergent Enhancing the Replication of Baculovirus and the Activity of BmNPV ie-1 Promoter In vivo and In vitro

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A Cationic Detergent Enhancing the Replication of Baculoviru...