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Recombinant baculovirus

Ju-Chun Chang, Se Jin Lee, Jae Su Kim, Chung-Hsiung Wang, Yu-Shin Nai
The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV)...
February 22, 2018: Journal of Visualized Experiments: JoVE
Daniel M P Ardisson-Araújo, Ana Maria Rodrigues da Silva, Fernando L Melo, Ethiane Rozo Dos Santos, Daniel R Sosa-Gómez, Bergmann M Ribeiro
In this report, we described the genome of a novel baculovirus isolated from the monocot insect pest Mocis latipes , the striped grass looper. The genome has 134,272 bp in length with a G + C content of 38.3%. Based on the concatenated sequence of the 38 baculovirus core genes, we found that the virus is a betabaculovirus closely related to the noctuid-infecting betabaculoviruses including Pseudaletia unipuncta granulovirus (PsunGV), Trichoplusia ni granulovirus (TnGV), Helicoverpa armigera granulovirus (HearGV), and Xestia c-nigrum granulovirus (XecnGV)...
March 16, 2018: Viruses
Peggy Stolt-Bergner, Christian Benda, Tim Bergbrede, Hüseyin Besir, Patrick H N Celie, Cindy Chang, David Drechsel, Ariane Fischer, Arie Geerlof, Barbara Giabbai, Joop van den Heuvel, Georg Huber, Wolfgang Knecht, Anita Lehner, Regis Lemaitre, Kristina Nordén, Gwynn Pardee, Ines Racke, Kim Remans, Astrid Sander, Judith Scholz, Magda Stadnik, Paola Storici, Daniel Weinbruch, Isabel Zaror, Linda H L Lua, Sabine Suppmann
Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E...
March 12, 2018: Journal of Structural Biology
Sarah L Irons, Adam C Chambers, Olga Lissina, Linda A King, Robert D Possee
Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed 'BacMam', with considerable potential in biomedical applications. This chapter explains the process of making a recombinant baculovirus, encompassing production of a recombinant virus by homologous recombination in insect cells, followed by amplification and titration of the virus-all steps needed before commencing gene expression and protein production...
February 21, 2018: Current Protocols in Protein Science
Adam C Chambers, Mine Aksular, Leo P Graves, Sarah L Irons, Robert D Possee, Linda A King
This unit provides information on the replication cycle of insect baculovirus to provide an understanding of how this virus has been adapted for use as an expression vector for recombinant proteins in insect cells. We provide an overview of the virus structure and its unique bi-phasic replication cycle, which has been exploited in developing the virus as an expression vector. We also review the development of the baculovirus expression vector system (BEVS), from the mid-1980s to the present day in which the BEVS is now an established tool for the production of a range of recombinant proteins and multi-protein complexes including virus-like particles...
February 21, 2018: Current Protocols in Protein Science
Masatoshi Suganuma, Tsuyoshi Nomura, Yukiko Higa, Yukiko Kataoka, Shunsuke Funaguma, Hironobu Okazaki, Takeo Suzuki, Kazuhito Fujiyama, Hideki Sezutsu, Ken-Ichiro Tatematsu, Toshiki Tamura
A silkworm-baculovirus system is particularly effective for producing recombinant proteins, including glycoproteins. However, N-glycan structures in silkworm differ from those in mammals. Glycoproteins in silkworm are secreted as pauci-mannose type N-glycans without sialic acid or galactose residues. Sialic acid on N-glycans plays important roles in protein functions. Therefore, we developed pathways for galactosylation and sialylation in silkworm. Sialylated N-glycans on proteins were successfully produced in silkworm by co-expressing galactosyltransferase and sialyltransferase and providing an external supply of a sialylation-related substrate...
February 9, 2018: Journal of Bioscience and Bioengineering
Krisztina Koczka, Philipp Peters, Wolfgang Ernst, Heinz Himmelbauer, Lisa Nika, Reingard Grabherr
The baculovirus insect cell expression system has become a firmly established production platform in biotechnology. Various complex proteins, multi-subunit particles including veterinary and human vaccines are manufactured with this system on a commercial scale. Apart from baculovirus infected Spodoptera frugiperda (Sf9) cells, the Trichoplusia ni (HighFive) cell line is alternatively used as host organism. In this study, we explored the protein production capabilities of Tnms42 insect cells, a new derivative of HighFive, which is free of latent nodavirus infection...
February 9, 2018: Journal of Biotechnology
Anping Wang, Lingling Gu, Shuang Wu, Shanyuan Zhu
Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity...
February 2018: Veterinary Microbiology
Yuriko Egashira, Satoru Nagatoishi, Masato Kiyoshi, Akiko Ishii-Watabe, Kouhei Tsumoto
Silkworm-baculovirus expression systems are efficient means for production of recombinant proteins that provide high expression levels and post-translational modifications. Here, we characterized the stability, glycosylation pattern, and antibody-dependent cell-mediated cytotoxicity activity of anti-HER2 monoclonal antibodies containing native or glycoengineered mammalian-like N-glycans that were produced by using a silkworm-baculovirus expression system. Compared with a monoclonal antibody produced by using a Chinese hamster ovary cell expression system, the glycoengineered monoclonal antibody had comparable thermal stability and a higher antibody-dependent cell-mediated cytotoxicity activity...
February 5, 2018: Journal of Biochemistry
Pedro Bullón, Beatriz Castejón-Vega, Lourdes Román-Malo, María Paz Jimenez-Guerrero, David Cotán, Tamara Y Forbes-Hernandez, Alfonso Varela-López, Antonio Pérez-Pulido, Francesca Giampieri, José L Quiles, Maurizio Battino, José A Sánchez-Alcázar, Mario D Cordero
BACKGROUND: Cathepsin C (CatC) is a lysosomal enzyme involved in the activation of serine proteases from immune and inflammatory cells. Several mutations with loss of function in the Cathepsin C (CatC) gene have been shown to be the genetic mark of Papillon-Lefèvre syndrome (PLS), a rare autosomal recessive disease characterized by severe early-onset periodontitis, palmoplantar hyperkeratosis and increased susceptibility to infections. Deficiencies or dysfunction in other cathepsin family proteins such as B or D have been associated with autophagic and lysosomal disorders...
February 1, 2018: Journal of Allergy and Clinical Immunology
Cuitlahuac Chavez-Pena, Amine A Kamen
The baculovirus expression vector system (BEVS) is a popular manufacturing platform for the production of recombinant proteins, antiviral vaccines, gene therapy vectors, and biopesticides. Besides its successful applications in the industrial sector, the system has also played a significant role within the academic community given its extensive use in the production of hard-to-express eukaryotic multiprotein complexes for structural characterization for example. However, as other expression platforms, BEVS has to be continually improved to overcome its limitation and adapt to the constant demand for manufacturing processes that provide recombinant products with improved quality at higher yields and lower production cost...
January 29, 2018: Biotechnology Advances
Wei-Feng Ding, Xin Zhang, Xian Li, Zhi-Gang Liu, Shi-Cong Xie, Ying Feng
This paper used recombinant baculoviruses that carried three reporter genes, green fluorescent protein (GFP), β-galactosidase, and secreted alkaline phosphatase (SEAP), to infect four new cell lines from Papilio demoleus Linnaeus larvae (named RIRI-PaDe-1, RIRI-PaDe-2, RIRI-PaDe-3, and RIRI-PaDe-4). The expression levels of the three recombinant proteins were detected at 24, 48, 72, 96, 120, and 144 h after infection and compared with Sf9 and High Five cells to evaluate the characteristics of these four cell lines as host cells...
January 24, 2018: In Vitro Cellular & Developmental Biology. Animal
María Florencia Gottardo, Matías L Pidre, Camila Zuccato, Antonela S Asad, Mercedes Imsen, Gabriela Jaita, Marianela Candolfi, Víctor Romanowski, Adriana Seilicovich
Pituitary tumors are the most common primary intracranial neoplasms. Humanin (HN) and Rattin (HNr), a rat homolog of HN, are short peptides with a cytoprotective action. In the present study, we aimed to evaluate whether endogenous HNr plays an antiapoptotic role in pituitary tumor cells. Thus, we used RNA interference based on short-hairpin RNA (shRNA) targeted to HNr (shHNr). A plasmid including the coding sequences for shHNr and dTomato fluorescent reporter gene was developed (pUC-shHNr). Transfection of somatolactotrope GH3 cells with pUC-shHNr increased apoptosis, suggesting that endogenous HNr plays a cytoprotective role in pituitary tumor cells...
January 19, 2018: Apoptosis: An International Journal on Programmed Cell Death
Mallory E Heimerman, Maria V Murgia, Ping Wu, Andre D Lowe, Wei Jia, Raymond R Rowland
Protein p72 is the major capsid protein of African swine fever virus (ASFV) and is an important target for test and vaccine development. Monoclonal antibodies (mAbs) were prepared against a recombinant antigenic fragment, from amino acid (aa) 20-303, expressed in baculovirus. A total of 29 mAbs were recovered and tested by immunofluorescent antibody (IFA) staining on ASFV Lisbon-infected Vero cells. Six antibodies were IFA-positive and selected for further characterization. Epitope mapping was performed against overlapping polypeptides expressed in E...
January 1, 2018: Journal of Veterinary Diagnostic Investigation
Chikako Ono, Junki Hirano, Toru Okamoto, Yoshiharu Matsuura
Insect expression systems based on baculovirus are widely used for the generation of recombinant proteins. Here, we evaluated the infectivity of baculoviruses under the physiological stresses of 'freeze- thaw' and sonication, and the baculoviral contamination of recombinant proteins after protein purification. Our findings suggest that Nonidet™ P-40 (NP-40) treatment of baculoviruses completely abolishes their infectivity and that recombinant proteins purified with affinity beads do not include infectious baculoviruses...
January 11, 2018: Microbiology and Immunology
Shuai Zhang, Yanhong Huang, Jingling Zhu, Liya Shan, Jianfeng Gao, Yunhua Zhang, Na Yu, Lei Yang, Jin Huang
Neuritin plays an important role in the development and regeneration of the nervous system, and shows good prospects in the treatment and protection of the nervous system. To characterize neuritin function, we constructed a baculovirus expression system of neuritin, and identified the biological activity of the neuritin protein. The results and showed that the expression product could promote the neurite growth of dorsal root ganglion in chicken embryos. The neuritin open reading frame was amplified and cloned into the plasmid pFastBac™HTA...
January 7, 2018: Gene
Lorena C S Chaves, Bergmann M Ribeiro, Gary W Blissard
The retroviral Gag protein is frequently used to generate 'virus-like particles' (VLPs) for a variety of applications. Retroviral Gag proteins self-assemble and bud at the plasma membrane to form enveloped VLPs that resemble natural retrovirus virions, but contain no viral genome. The baculovirus expression vector system has been used to express high levels of the retroviral Gag protein to produce VLPs. However, VLP preparations produced from baculovirus-infected insect cells typically contain relatively large concentrations of baculovirus budded virus (BV) particles, which are similar in size and density to VLPs, and thus may be difficult to separate when purifying VLPs...
January 4, 2018: Journal of General Virology
Wenlong Dai, Chao Zhang, Xueyang Zhang, Pei Xiong, Qingwei Liu, Sitang Gong, Lanlan Geng, Dongming Zhou, Zhong Huang
Enterovirus D68 (EV-D68) is increasingly associated with severe acute respiratory infection and acute flaccid myelitis (AFM) in children around the world. However, neither vaccines nor therapeutic drugs are available for EV-D68. Here we report the development of a virus-like particle (VLP) based experimental EV-D68 vaccine. We found that EV-D68 VLPs could be successfully generated in insect cells infected with a recombinant baculovirus co-expressing the P1 precursor and 3CD protease of EV-D68. Biochemical and electron microscopic analyses revealed that EV-D68 VLPs were composed of VP0, VP1, and VP3 capsid proteins derived from precursor P1 and were visualized as spherical particles of ∼30 nm in diameter...
December 30, 2017: Vaccine
Nirit Mor-Vaknin, Miguel Rivas, Maureen Legendre, Smriti Mohan, Ye Yuanfan, Theresa Mau, Anne Johnson, Bin Huang, Lili Zhao, Yukiko Kimura, Steven J Spalding, Paula W Morris, Beth S Gottlieb, Karen Onel, Judyann C Olson, Barbara S Edelheit, Michael Shishov, Lawrence K Jung, Elaine A Cassidy, Sampath Prahalad, Murray H Passo, Timothy Beukelman, Jay Mehta, Edward H Giannini, Barbara S Adams, Daniel J Lovell, David M Markovitz
OBJECTIVE: The nuclear oncoprotein DEK is an autoantigen associated with juvenile idiopathic arthritis (JIA), especially the oligoarticular subtype. DEK is a secreted chemoatactic factor. Abundant DEK and DEK autoantibodies are found in the inflamed JIA synovium. In screening sera samples from two different JIA patient cohorts for DEK autoantibodies, we now further characterize the nature of DEK autoantibodies. METHODS: DEK autoantibody levels were analyzed in sera from 33 JIA patients, 13 patients with other inflammatory conditions, and 11 healthy controls and 89 sera samples from JIA patients undergoing anti-TNF therapy...
December 29, 2017: Arthritis & Rheumatology
Xingjian Liu, Yinü Li, Xiaoyuan Hu, Yongzhu Yi, Zhifang Zhang
The baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) has been investigated as a possible tool for gene therapy, but its inhibition by complement proteins in human serum limits its applicability. Here, we used the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV) to construct a gene delivery vector in which a reporter gene is driven by a cytomegalovirus IE promoter. Enhanced green fluorescent protein (EGFP) and luciferase reporter genes were used to test the efficiency of gene delivery...
December 1, 2017: Oncotarget
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