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Order Blubervirales: Surface Protein
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Next, an optimization of the HBs DNA vaccine plasmid vector was tried by modification of the content of immunostimulatory CpG motifs (Payette et al. 2006). However, two doses of DNA vaccine did not generate any detectable anti-HBs antibody response in either of two chimpanzees in this study.
Adenoviral Vectors for Gene Therapy of Inherited and Acquired Disorders of the Lung
Published in Kenneth L. Brigham, Gene Therapy for Diseases of the Lung, 2020
David T. Curiel, Robert I. Garver
The details of plasmid design and common methods of use have been described in recent reviews (18,19) and therefore will not be reiterated in detail here (Fig. 1). In brief, the most widely used method involves three major steps. First, the new coding sequence with appropriate transcriptional start and stop regulatory sequences is added to a multiple cloning site within the deleted El region of a plasmid containing a portion of the left-hand (5') end of the adenovirus genome. Second, this plasmid vector containing the new coding sequence is cotransfected into 293 cells with a second plasmid that contains the entire adenovirus genome with an El deletion modified to contain a “stuffer fragment” of plasmid DNA. The stuffer fragment not only contains the plasmid origin of replication and antibiotic resistance gene for bacterial propagation, but it is sufficiently large to prevent that adenoviral DNA from being packaged into a stable viral particle. Homologous recombination occurs be tween the two plasmids so that the El region containing the coding sequence of interest replaces the plasmid stuffer within the otherwise intact genome, and the E1 proteins made by the 293 cells activate the recombinant genome replication with the result that recombinant virus is made. The third step is a series of plaque purifications with screening assays at each step to eliminate undesired wild type virus that is generated by homologous recombination between the viral sequences within 293 cells and the adenoviral plasmid with the stuffer fragment.
Vaccinia Virus as a Carrier of Vaccine Antigens
Published in F. Y. Liew, Vaccination Strategies of Tropical Diseases, 2017
The large size of the vaccinia virus genome and its noninfectious nature, when stripped of transcriptional enzymes, make construction of recombinant genomes in vitro impracticable. Moreover, to obtain expression of foreign genes it is necessary to engineer the gene downstream of a vaccinia promoter since the virus-coded RNA polymerase does not recognize heterologous promoters. Recombinant viruses are therefore constructed in two steps. First, the foreign gene is inserted downstream of a vaccinia promoter in a plasmid vector. Second, this plasmid is transfected into virus-infected cells and the foreign gene is transferred to the virus genome by homologous recombination. Recombinant viruses are obtained from the progeny of this transfection and distinguished from wild-type virus by selective methods.
Cloning, characterization, and inhibition of the novel β-carbonic anhydrase from parasitic blood fluke, Schistosoma mansoni
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Susanna Haapanen, Andrea Angeli, Martti Tolvanen, Reza Zolfaghari Emameh, Claudiu T. Supuran, Seppo Parkkila
The plasmid vector was prepared according to the manufacturer’s instructions and then transformed with heat shock into BL21 StarTM (DE3) cells (Invitrogen, Carlsbad, USA), as described previously.58 The production of recombinant protein was executed manually in LB broth (Sigma-Aldrich, St. Louis, MO, USA) with 1:1000 gentamicin (Sigma-Aldrich) and 1:100 glycerol (VWR International, Radnor, PA, USA)/glucose (Sigma-Aldrich) as proposed in Kopp et al.59 at +37 degrees and shaking with 200 rpm. Both glycerol and glucose proved to be equally effective additives in the growth medium to reduce the number of impurities. The OD (Fisher Scientific Colorimeter Model 45 (WA12173), Fisherbrand, Thermo Fisher Scientific, Waltham, MA, USA) was measured and at the OD 0.4–0.5 1 M isopropyl-β-D-thiogalactopyranoside (IPGT, Thermo Fisher Scientific) was added in relation of 1:1000 to LB medium. Growth continued overnight and was terminated the next day by pelleting the cells by centrifugation at 5000 × g for 20 min resulting in a total incubation and production time of 24 h.
Establishment of Cell-Based Assay System for Evaluating Cytotoxic Activity Modulated by the Blockade of PD-1 and PD-L1 Interactions with a Therapeutic Antibody
Published in Immunological Investigations, 2023
Haruka Hirosaki, Yosuke Maeda, Masahiro Takeyoshi
To generate the lentiviral vectors carrying the PD-1 or PD-L1 gene, the full length of PD-1 (NCBI CCDS No. 33428.1) or PD-L1 (NCBI CCDS No. 6464.1) was subcloned into the plasmid vector pLVSIN containing a puromycin-resistant gene (TaKaRa Bio Inc., Shiga, Japan). To generate the lentiviral vector carrying the IL-2 gene, the full length of IL-2 (NCBI CCDS No. 3726.1) was subcloned into pLVSIN containing a hygromycin-resistant gene (TaKaRa Bio Inc., Shiga, Japan). Each plasmid vector carrying a target gene was co-transfected with Lentiviral High Titer Packaging Mix (TaKaRa Bio Inc., Shiga, Japan) into Lenti-XTM 293T cells (TaKaRa Bio Inc., Shiga, Japan) using TransIT®-293 transfection reagent (TaKaRa Bio Inc., Shiga, Japan). The culture supernatants were used as lentiviral vectors for each gene transfer.
Osthole exhibits an antitumor effect in retinoblastoma through inhibiting the PI3K/AKT/mTOR pathway via regulating the hsa_circ_0007534/miR-214-3p axis
Published in Pharmaceutical Biology, 2022
Xiufang Lv, Haojiang Yang, Hui Zhong, Li He, Li Wang
The has_circ_0007534 overexpression plasmid (oe-circ_0007534) was constructed using pCD5‐ciR vector (Geneseed Biotech Co., Ltd., Guangzhou, China). Meanwhile, a control plasmid (vector) was also constructed. The miR-214-3p mimics and negative controls (miR-NCs) were designed and obtained from GenePharma (Shanghai, China). Small interfering RNA (siRNA) against hsa_circ_0007534 (si-circ_0007534) and scrambled negative control (si-NC) were provided by RiboBio Co., Ltd. (Guangzhou, China). The aforementioned plasmids were transfected into Y-79 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. After transfection for 48 h, transfected cells were treated with different concentrations of osthole for an additional 48 h. Then, cell-viability assay, cell apoptosis assay, cell colony formation assay, qRT-PCR and western blot analysis were performed as the methods described above.