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Polyhedrin promoter

María Martínez-Solís, Silvia Gómez-Sebastián, José M Escribano, Agata K Jakubowska, Salvador Herrero
The baculovirus expression vector system (BEVS) has been widely used to produce a large number of recombinant proteins, and is becoming one of the most powerful, robust, and cost-effective systems for the production of eukaryotic proteins. Nevertheless, as in any other protein expression system, it is important to improve the production capabilities of this vector. The orf46 viral gene was identified among the most highly abundant sequences in the transcriptome of Spodoptera exigua larvae infected with its native baculovirus, the S...
2016: PeerJ
Yingli Shi, Jianhai Xiang, Guangzhou Zhou, Tetsuzan Benny Ron, Hsin-I Tong, Wen Kang, Si Sun, Yuanan Lu
A newly isolated Pacific white shrimp (Litopenaeus vannamei) beta-actin promoter SbaP and its derivative compact construct SbaP (ENX) have recently been demonstrated to promote ectopic gene expression in vitro and in vivo. To further explore the potential transduction application, this newly isolated shrimp promoter SbaP was comparatively tested with cytomegalovirus (CMV), simian virus 40 (SV40), polyhedrin (Polh), and white spot syndrome virus immediate early gene 1 (WSSV ie1) four constitutive promoters and a beta-actin promoter (TbaP) from tilapia fish to characterize its promoting function in eight different cell lines...
June 2016: Marine Biotechnology
Bo Liu, Yongchang Gong, Zhen Li, Xiaolong Hu, Guangli Cao, Renyu Xue, Chengliang Gong
Grass carp hemorrhagic disease is a common fish disease and often results in significant economic losses in grass carp aquaculture in China. This study was aimed to develop a novel oral vaccine against grass carp reovirus (GCRV). GCRV vp6 and vp7 genes with β-actin promoter of Megalobrama amblycephala and polyhedrin promoter (Ph10) of baculovirus, respectively, were cloned into plasmid pFast™-Dual to construct a vector pFast-PHVP7-AVP6, which was used to generate a recombinant baculovirus BacFish-vp6/vp7 via Bac-to-Bac system...
June 2016: Molecular Biology Reports
Ya-Bin Chen, Wei Xiao, Ming Li, Yan Zhang, Yang Yang, Jian-Sheng Hu, Kai-Jun Luo
The hemichannel and gap junction channel are major portals for the release of factors responsible for the effects of apoptotic cells on the spread of apoptosis to neighboring cells and apoptotic corpse clearance, typically by phagocytes. The N-terminal cytoplasmic domain in the connexins, gap junction proteins in vertebrate, has been implicated in regulating channel closure. However, little is known about how the hemichannel close responds to apoptotic signaling transduction leading to the reduction of neighboring cellular apoptosis in an invertebrate...
May 2016: Archives of Insect Biochemistry and Physiology
Linda A King, Richard Hitchman, Robert D Possee
Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3 ), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac(®) or BaculoDirect™, Invitrogen) or virus genome and transfer vector. In the latter case, this allows the natural process of homologous recombination to transfer the foreign gene, under control of the polyhedrin or other baculovirus gene promoter, from the transfer vector to the virus genome to create the recombinant virus...
2016: Methods in Molecular Biology
Barbara J Kelly, Linda A King, Robert D Possee
The development of baculovirus expression vector systems has accompanied a rapid expansion of our knowledge about the genes, their function and regulation in insect cells. Classification of these viruses has also been refined as we learn more about differences in gene content between isolates, how this affects virus structure and their replication in insect larvae. Baculovirus gene expression occurs in an ordered cascade, regulated by early, late and very late gene promoters. There is now a detailed knowledge of these promoter elements and how they interact first with host cell-encoded RNA polymerases and later with virus-encoded enzymes...
2016: Methods in Molecular Biology
Tamer Z Salem, Craig P Seaborn, Colin M Turney, Jianli Xue, Hui Shang, Xiao-Wen Cheng
The simian virus 40 polyadenylation signal (SV40 polyA) has been routinely inserted downstream of the polyhedrin promoter in many baculovirus expression vector systems (BEVS). In the baculovirus prototype Autographa californica multiple nucleopolyhedrovirus (AcMNPV), the polyhedrin promoter (very late promoter) transcribes its gene by a viral RNA polymerase therefore there is no supporting evidence that SV40 polyA is required for the proper gene expression under the polyhedrin promoter. Moreover, the effect of the SV40 polyA sequence on the polyhedrin promoter activity has not been tested either at its natural polyhedrin locus or in other loci in the viral genome...
2015: PloS One
Zhihong Huang, Ao Li, Mengjia Pan, Wenbi Wu, Meijin Yuan, Kai Yang
In the baculovirus shuttle vector (bacmid) system, a helper plasmid and a donor plasmid are employed to insert heterologous genes into a cloned baculovirus genome via Tn7 transposition in Escherichia coli. The helper and donor plasmids are usually cotransfected with constructed bacmids into insect cells, which will lead to integration of these plasmids into the viral genome, and hence to the production of defective virions. In this study, to facilitate the preparation of plasmid-free recombinant bacmids, we modified a set of helper and donor plasmids by replacing their replication origins with that of a temperature-sensitive (ts) plasmid, pSIM6...
October 2015: Virologica Sinica
Javier López-Vidal, Silvia Gómez-Sebastián, Juan Bárcena, Maria del Carmen Nuñez, Diego Martínez-Alonso, Benoit Dudognon, Eva Guijarro, José M Escribano
Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells...
2015: PloS One
Guohui Li, Mangmang Li, Qian Zhou, Zhaoyang Hu, Qi Tang, Qin Yao
To improve the expression of heterologous genes using baculovirus expression system, we constructed a novel shuttle vector based on the Bm-Bacmid. In the Bm-Bacmid, partial sequences of Chitinase and Cystein Protease were replaced with a tandem cassette of Cm and egfp through homologous recombination. Bombyx mori bidensovirus (BmBDV) ns1 under the control of polyhedrin promoter was inserted into the modified Bm-bacmid by transposition. For comparison, BmBDV ns1 under the control of polyhedrin promoter was also cloned in the wild type Bm-bacmid...
April 2015: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
Huan Yu, Jiao Meng, Jian Xu, Tong-Xian Liu, Dun Wang
A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus). RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected HzAM1 cells. In order to investigate the influence of foreign gene insertion in HearNPV, including the ar1b gene, chloramphenicol resistance gene, lacZ, kanamycin resistance gene, and the gentamicin resistance gene, two virus strains (HZ8-HearNPV and wt-HearNPV) were used as controls in the cell transfection analysis...
2015: PloS One
Sujit M Kolangath, S H Basagoudanavar, M Hosamani, P Saravanan, R P Tamil Selvan
The recombinant baculoviruses were constructed to investigate the necessity of VSV-G pseudotyping for mammalian cell transduction. The viruses were designed to express green fluorescent protein (GFP) gene under the control of cytomegalovirus promoter, with or without pseudotyping with VSV-G. VSV-G was placed under the control of polyhedrin promoter that is recognized by insect cells, allowing the formation of pseudotyped baculovirus. The study findings demonstrate that the pseudotyping of baculovirus significantly enhanced transduction efficiency compared to non-pseudotyped baculovirus, resulting in consequent distinction in the expression of GFP in mammalian cells...
December 2014: Virusdisease
Guohui Li, Mangmang Li, Peng Wang, Zhaoyang Hu, Qin Yao, Qi Tang, Keping Chen
ns1 gene of Bombyx mori bidensovirus (BmBDV) consisted of 951 nucleotides encoding a deduced 316-amino aicd protein. In this study, the gene was cloned and fused in frame with a N-terminal 6×His tag under control of the polyhedrin promoter, which was transposed into the mini-attTn7 locus of a modified baculovirus vector. Transfection of Sf-9 cells with the resulting recombinant DNA was performed to prepare recombinant virus and the resultant supernatant of transfection with fluorescent signal was harvested...
2014: Acta Biochimica Polonica
Huiqing Chen, Mei Li, Weijun Mai, Qi Tang, Guohui Li, Keping Chen, Yajing Zhou
In our previous study, Orf101 (Bm101) of Bombyx mori nucleopolyhedrovirus (BmNPV) was identified as a component of the budded virions important for viral late gene expression. In this study we demonstrate that Bm101 is actually a previously unrecognized core gene and that it is essential for mediating budded virus production. To determine the role of Bm101 in the baculovirus life cycle, a Bm101 knockout bacmid containing the BmNPV genome was generated through homologous recombination in Escherichia coli. Furthermore, a Bm101 repair bacmid was constructed by transposing the Bm101 open reading frame with its native promoter region into the polyhedrin locus of the Bm101 knockout bacmid...
December 2014: Cytotechnology
Monique M van Oers, Gorben P Pijlman, Just M Vlak
In December 1983, a seminal paper appeared on the overexpression of human IFN-β in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produced massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes were dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years, major improvements were achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells and the scale-up of the cell culture process...
January 2015: Journal of General Virology
Masashi Iwanaga, Keita Tsukui, Koudai Uchiyama, Susumu Katsuma, Shigeo Imanishi, Hideki Kawasaki
We previously established the first Bombyx mori macula-like virus (BmMLV)-free cell line (BmVF cells) from a B. mori embryo. In this study, we evaluated the expression of recombinant proteins in BmVF cells using a B. mori nucleopolyhedrovirus (BmNPV)-derived expression vector. Our results showed that BmVF cells are susceptible to BmNPV, and both the promoter activity of the polyhedrin gene and the post-translated modifications of a recombinant protein are equivalent between BmMLV-negative BmVF and -positive BmN4 cells...
November 2014: Journal of Invertebrate Pathology
Yueh-Lung Wu, Carol-P Wu, Yu-Hui Huang, Sheng-Ping Huang, Huei-Ru Lo, Hao-Shuo Chang, Pi-Hsiu Lin, Ming-Cheng Wu, Chia-Jung Chang, Yu-Chan Chao
UNLABELLED: The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells...
November 2014: Journal of Virology
V Ruiz, A C Mignaqui, M C Nuñez, E Reytor, J M Escribano, A Wigdorovitz
Recombinant FMDV empty capsids have been produced in insect cells and larvae using the baculovirus expression system, although protein yield and efficiency of capsid assembly have been highly variable. In this work, two strategies were compared for the expression of FMDV A/Arg/01 empty capsids: infection with a dual-promoter baculovirus vector coding for the capsid precursor (P12A) and the protease 3C under the control of the polyhedrin and p10 promoters, respectively (BacP12A-3C), or a single-promoter vector coding the P12A3C cassette (BacP12A3C)...
November 2014: Molecular Biotechnology
Masashi Iwanaga, Yuka Adachi, Koudai Uchiyama, Keita Tsukui, Susumu Katsuma, Hideki Kawasaki
Bombyx mori ovary-derived BmN4 cells have been successfully adapted to a commercial serum-free medium (SFM; SF900-II) by gradually reducing the serum-containing TC-100 medium content from 100 to 0% (v/v). The BmN4 cells adapted to the SFM (BmN-SFM) adhered strongly to the culture flask and showed altered cell morphology. The BmN-SFM was subcultured 200 times, and the population doubling time was 4.70 d. Infection studies showed that BmN-SFM cells were easily susceptible to B. mori nucleopolyhedrovirus (BmNPV), and both the multiplication of budded virus and the promoter activity of the polyhedrin gene in BmN-SFM cells were almost the same as those in BmN4 cells before adaptation...
October 2014: In Vitro Cellular & Developmental Biology. Animal
Silvia Gómez-Sebastián, Javier López-Vidal, José M Escribano
Here we describe the development of a baculovirus vector expression cassette containing rearranged baculovirus-derived genetic regulatory elements. This newly designed expression cassette conferred significant production improvements to the baculovirus expression vector system (BEVS), including prolonged cell integrity after infection, improved protein integrity, and around 4-fold increase in recombinant protein production yields in insect cells with respect to a standard baculovirus vector. The expression cassette consisted of a cDNA encoding for the baculovirus transactivation factors IE1 and IE0, expressed under the control of the polyhedrin promoter, and a homologous repeated transcription enhancer sequence operatively cis-linked to the p10 promoter or to chimeric promoters containing p10...
2014: PloS One
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