Search results for "Expression vector"
showing 10 items of 31 documents
Identification, sequence and mRNA expression pattern during metamorphosis of a cDNA encoding a glycine-rich cuticular protein in Tenebrio molitor
1995
The study of insect cuticular proteins and their sequences is of interest because they are involved in protein-protein and protein-chitin interactions which confer the mechanical properties and fine architecture of the cuticle. Moreover, in the coleopteran Tenebrio molitor there is a dramatic change in cuticular architecture between pre- and postecdysial secretion. We report the isolation, by differential screening, and the sequence characterization of a cDNA clone encoding a cuticular protein of T. molitor, ACP17. After insertion in the expression vector pEX1, the recognition of the fusion protein by an anti-cuticular monoclonal antibody confirmed the cuticular nature of ACP17. Northern hy…
Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing
2019
The clustered regularly interspaced short palindromic repeats (CRISPR) system?associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout se…
The human p53 gene mutated at position 249per se is not sufficient to immortalize human liver cells
1999
A particular point mutation of the tumor suppressor gene p53, namely a G→T transversion at the third base of codon 249, is frequently detected in primary hepatocellular carcinomas from patients living in areas where the levels of dietary exposure to aflatoxin B 1 and the rates of infection with the hepatitis B virus are very high. Very recently, a nontumorigenic liver epithelial cell line (HACL-1) with a finite life-span and expressing a number of hepatocyte-specific markers was established from a human hepatocellular adenoma in our laboratory. To analyze the role of mutated p53 in the immortalization of human liver cells, we transfected HACL-1 cells with an expression vector containing a h…
Cloning and Sequencing of a cDNA Encoding a Larval-Pupal-Specific Cuticular Protein in Tenebrio Molitor (Insecta, Coleoptera). Developmental Expressi…
1996
A cDNA clone encoding a larval-pupal cuticular protein, named TMLPCP-22, has been isolated by screening a library in expression vector with a monoclonal antibody made against pupal cuticular proteins of Tenebrio molitor. Northern-blot and in situ hybridization analyses showed that the expression of TMLPCP-22 is regulated in a stage-specific and tissue-specific manner; the transcript was present during the secretion of preecdysial larval and pupal cuticles and was restricted to epidermal cells. No expression was observed during adult cuticle deposition. In supernumerary pupae obtained after application of a juvenile hormone analogue, which is known to inhibit the adult programme, TMLPCP-22 m…
Protection of HeLa‐T4 + cells against human immunodeficiency virus (HIV) infection after stable transfection with HIV LTR‐2‘,5‘‐oligoadenylate synthe…
1990
An expression vector (pU3R-III/2-5AS) of human 2',5'-oligoadenylate (2-5A) synthetase was constructed in which a cDNA encoding an active form of the enzyme was located 3' to a 3'-long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1). The LTR-directed expression of this hybrid DNA could be activated in trans by the HIV tat gene product. This vector was used for transfection of HeLa-T4+ cells, which are permissive to HIV infection, as well as of normal HeLa cells. HIV replication after infection of the CD4-receptor-bearing HeLa-T4+ cells with HIV-1 was found to be strongly reduced when drug-selected cells cotransfected with pU3R-III/2-5AS and a hygromycin B resistance gene…
Cloning and characterization of a cDNA coding forCandida albicanspolyubiquitin
1996
Immunoscreening of a Candida albicans cDNA library in the expression vector lambda gt11 with rabbit polyclonal antibodies against the 37 kDa cell surface laminin receptor of C albicans resulted in the isolation of a cDNA clone of 0.9 kb. Sequencing of this clone demonstrated a full length open reading frame encoding the polyubiquitin, which contains three tandem copies, head-to-tail spacerless repeats, of the 228 nucleotides coding for the 76 amino acids of the ubiquitin protein, which is identical to that of Saccharomyces cerevisiae. The third copy possesses an extra C-terminal amino acid which is distinct to that found in S. cerevisiae. Northern blot analysis revealed a single mRNA popula…
p53 Gene Therapy
2002
Based on the frequent inactivation of the p53 gene product in human malignancy, and its functional involvement in tumor suppression, cell cycle control and apoptosis, p53 was identified as an attractive target for somatic gene therapy strategies in cancer. Several pilot and phase I studies explored the intratumoral injection of viral expression vectors encoding the p53 cDNA in patients with advanced cancer. These studies confirmed the safety and feasibility of this approach. Further, vector-specific transgene expression and surrogate markers for biological activity of the transgene were demonstrated. Local tumor regression, or stabilization of tumor growth, were observed in some studies, an…
Strong immunogenic potential of a B7 retroviral expression vector: generation of HLA-B7-restricted CTL response against selectable marker genes.
1998
The stimulation of a specific immune response is an attractive goal in cancer therapy. Gene transfer of co-stimulatory molecules and/or cytokine genes into tumor cells and the injection of these genetically modified cells leads to tumor rejection by syngeneic hosts and the induction of tumor immunity. However, the development of host immune response could be either due to the introduced immunomodulatory genes or due to vector components. In this study, human renal cell carcinoma cell lines were modified by a retrovirus to express the co-stimulatory molecule B7-1 together with the hygromycin/thymidine kinase fusion protein (HygTk) as positive and negative selection markers. These B7-1-transd…
Developments in the use of baculoviruses for the surface display of complex eukaryotic proteins
2001
The ability to couple genotype to phenotype has proven to be of immense value in systems such as phage display and has allowed genes encoding novel functions to be selected directly from complex libraries. However, the complexity of many eukaryotic proteins places a severe constraint on successful display in Escherichia coli. This restriction could be resolved if a eukaryotic virus could be similarly engineered for display purposes. Preliminary data have suggested that the baculovirus Autographa californica, a multiple nuclear polyhedrosis virus (AcMNPV) is a candidate for eukaryotic virus display because the insertion of peptides into the native virus coat protein, or the expression of for…
Encapsulation of the Bacillus thuringiensis secretable toxins Vip3Aa and Cry1Ia in Pseudomonas fluorescens
2013
Vip3A and Cry1I toxins are secreted during the vegetative growth of Bacillus thuringiensis. Vip3A toxins do not share homology to the crystal (Cry) proteins and are active against a different spectrum of lepidopteran species. Cry1I toxins share similarity with the Cry1 protein group but do not accumulate in the parasporal crystal. Since Vip3A and Cry1I toxins are released from the cell, they are excluded from biological formulates based on spores and crystals of B. thuringiensis. As an approach to obtain novel sprayable insecticides containing Vip3 or Cry1I toxins, Vip3Aa and Cry1Ia proteins were expressed in Pseudomonas fluorescens. This bacterium, non-pathogenic to animals or plants, can …