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Results for “Recombinant expression”

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PubMed1991

Quantitative immunocytochemical staining for recombinant tissue-type plasminogen activator in transfected Chinese hamster ovary cells.

Tissue-type plasminogen activator (tPA) is a serine protease which cleaves plasminogen to its active form, plasmin. tPA plays a physiologic role in hemostasis, wound healing, and embryogenesis. Therapeutically, recombinant human tPA is used as a thrombolytic in myocardial infarction. Although production of therapeutic quantities of tPA in Chinese hamster ovary (CHO) cells transfected with the human gene for tPA is practical, production costs remain high. One important factor which determines the ultimate cost of tPA (or any other recombinant protein expressed in mammalian cells) is its production level on a per cell basis. We have used postembedding immunocytochemical staining with colloidal gold to study the subcellular localization of tPA in CHO cells expressing recombinant tPA (rCHO) in an effort to understand the factor(s) which might limit secretion. Staining for tPA was evaluated visually and by morphometric analysis and was specific and reproducible. Serially passaged rCHO showed no significant change in staining density over 31 serial passages. Staining density was greatest over dilated cisternae of the rough endoplasmic reticulum and nuclear envelope. Golgi stacks and large acid phosphatase-positive vacuoles (probably lysosomes) were also heavily stained. Staining of lysosomal vacuoles suggested that rCHO might be degrading nascent tPA. Incubation of rCHO with 125I-tPA showed that the cells were not internalizing tPA from the media. These results suggest that rCHO fail to secrete a portion of the tPA they synthesize and that it is degraded in lysosomes. This observation may have important implications on the choice of expression systems for efficient production of large quantities of recombinant proteins.

Animals↗

PubMed1996

Memory cytotoxic T lymphocyte responses in human immunodeficiency virus type 1 (HIV-1)-negative volunteers immunized with a recombinant canarypox expressing gp 160 of HIV-1 and boosted with a recombinant gp160.

A vaccine against human immunodeficiency virus (HIV) should induce virus-specific cytotoxic T lymphocyte (CTL) activity. Immunization of uninfected volunteers with a canarypox virus expressing HIV envelope was carried out in a phase I trial. Two injections of canarypox expressing HIV-1MN gp 160 (months 0 and 1) were followed by two boosts of recombinant envelope protein (months 3 and 6). HIV envelope-specific CTL were detected in peripheral blood mononuclear cells stimulated with autologous HIV-1-infected blast cells. T cell lines were obtained from 18 of 20 donors: CTL were detected at least once following immunization in 7 (39%) of these 18. This activity was mediated by major histocompatibility complex class I-restricted CD3+CD8+ T cells. For two subjects, this activity was still present 2 years after the initial immunization. The CTL responses with this prime-boost regimen are the best observed with any HIV vaccine tested in humans.

AIDS Vaccines↗

PubMed2002

Biological consequences of antigen and cytokine co-expression by recombinant Streptococcus gordonii vaccine vectors.

To test the effect of co-expression of immunomodulatory molecules, together with target antigen, two recombinant Streptococcus gordonii strains were constructed which secreted either murine interleukin-2 (IL-2) or interferon-gamma (IFN-gamma) in addition to a surface anchored test antigen (the conserved C-repeat region (CRR) of the M6 protein of Streptococcus pyogenes). The secretion of functional cytokines by S. gordonii was achieved by in-frame fusion of sequences encoding mature IL-2 or IFN-gamma to the sequences encoding the leader signal of the M6 protein. Expression of the M protein CRR region from a separate chromosomal site produced double recombinants expressing a secreted cytokine and the M protein CRR region anchored to the surface. Protein expression was verified by streak blot, immunoblot, and ELISA on both the single and double recombinants. A cytokine bioassay using HT-2 cells verified biological activity of recombinant IL-2 secreted from S. gordonii. When mice were immunized subcutaneously with the different S. gordonii expression strains, cytokine co-expression apparently modulated the systemic immune response. These results show that streptococci can deliver biologically active molecules such as cytokines along with antigens to the immune system. These results demonstrate that a cytokine-secreting, noninvasive, bacterial vaccine vector can be used to modulate immune responses to a co-expressed antigen.

Animals↗

PubMed1990

Vaccinia recombinant virus expressing the rabies virus glycoprotein: safety and efficacy trials in Canadian wildlife.

Twenty-six meadow voles (Microtus pennsylvanicus), ten woodchucks (Marmota monax), thirteen grey squirrels (Sciurus carolinensis), thirteen ring-billed gulls (Larus delawarensis), six red-tailed hawks (Buteo jamaicensis) and eight great horned owls (Bubo virginianus) received vaccinia virus recombinant expressing the rabies virus glycoprotein (V-RG) by direct instillation into the oral cavity. Each of ten coyotes (Canis latrans) received the virus in two vaccine-laden baits. Several voles and most of the gulls died from diseases unrelated to vaccination during the observation period, but all other animals remained healthy and survived. These deaths from causes other than vaccination and the absence of any lesions suggestive of vaccinia infection indicate that it is unlikely that any animal suffered or died as a result of V-RG administration. In addition several animals showed an unexpected high level of rabies neutralizing antibodies.

Administration, Oral↗

PubMed1999

Adventitial expression of recombinant endothelial nitric oxide synthase gene reverses vasoconstrictor effect of endothelin-1.

The present study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on reactivity of canine basilar arteries to endothelin-1 (ET-1). Experiments were performed ex vivo. The arteries were exposed (30 minutes at 37 degrees C) to adenoviral vectors encoding eNOS gene (AdCMVeNOS) or beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the vascular adventitia. Rings of control (nontransduced), AdCMVbeta-Gal- and AdCMVeNOS-transduced arteries with and without endothelium were suspended for isometric tension recording. Levels of guanosine 3',5'-cyclic monophosphate (cGMP) were measured by radioimmunoassay. During contractions to uridine 5'-triphosphate, ET-1 (10(-10) to 3x10(-9) mol/L) caused further increase in tension in control and AdCMVbeta-Gal-transduced arteries. In contrast, ET-1 caused concentration-dependent relaxations of AdCMVeNOS-transduced arteries. The relaxations to ET-1 in AdCMVeNOS-transduced arteries were endothelium-independent. They were abolished by N(G)-nitro-L-arginine methyl ester or by chemical treatment of adventitia with paraformaldehyde before gene transfer. ET-1 (10(-9) mol/L) significantly increased intracellular cGMP levels in AdCMVeNOS-transduced arteries without endothelium. In arteries transduced with AdCMVeNOS, higher concentrations (10(-9) to 3x10(-8) mol/L) of ET-2 also caused relaxations, whereas ET-3 and sarafotoxin, a selective ET(B) receptor agonist, did not produce any relaxations. The relaxations to ET-1 in AdCMVeNOS-transduced arteries were strongly reduced by BQ-123 (10(-7) mol/L), an ET(A) receptor antagonist, but were not affected by BQ-788 (3x10(-7) mol/L), an ET(B) receptor antagonist. These results suggest that genetically modified adventitia can produce nitric oxide and cause relaxations in response to ET-1 via activation of ET(A) receptors. Our findings support a novel concept that successful transfer and expression of recombinant eNOS gene can lead to a qualitative change in responsiveness to vasoconstrictor substances.

Adenoviridae↗

PubMed2000

Expression of recombinant Norwalk-like virus capsid proteins using a bacterial system and the development of its immunologic detection.

The capsid protein of Norwalk-like virus (NLV) isolates NLV-36 (Mexico virus type, genogroup II [GII]), NLV-21 (Lordsdale virus type, GII), NLV-114 (untyped GII virus), and NLV-96-908 (KY89 virus type, GI) have been expressed in an Escherichia coli system. The expressed recombinant NLV capsid proteins, fused with maltose binding protein (MBP-rV) and thioredoxin (TRX-rV) in E. coli lysate, were analyzed using sodium dodecyl sulfate-polyacrylamide gel eletrophoresis. Rabbit IgG (R-IgG) in hyperimmune serum has been raised against MBP-rV-36 capsid protein and was purified before further study. Detection of TRX-rVs using an enzyme-linked immunosorbent assay (ELISA) showed that R-IgG had immunologic reactivity to GII as well as to the GI rV capsid proteins TRX-rV-36, TRX-rV-21, TRX-rV-114, and TRX-rV-96-908. Results of Western immunoblot (WB) analysis showed the same broad recognition of R-IgG when using the same samples. The results of the ELISA tests on serum samples obtained from patients involved in confirmed outbreaks of NLV proved that expressed NLV capsid proteins in E. coli can be detected by NLV-infected human serum. In addition, purified NLVs (LD virus types) derived from patients' stool could be detected using anti-NLV R-IgG, whereas normal R-IgG did not react when using WB. Our results strongly suggest that the immunologic detection of NLV antigens using anti-rV R-IgG is possible and seems a significant step toward simplification of an NLV detection test.

ATP-Binding Cassette Transporters↗

PubMed2004

[Preliminary study of immunity and safety of recombinant adenovirus expressing rotavirus structural proteins in rhesus monkeys].

OBJECTIVE: To preliminarily evaluate the immunity and safety of the recombinant adenoviruses expressing rotavirus structural proteins VP7 and VP6 in rhesus monkeys to lay a foundation for the development of novel genetic engineering vaccine against rotavirus. METHODS: Baby monkeys were immunized with the recombinant adenoviruses intranasally or orally. Serum IgG against rotavirus was measured with ELISA. During the course of the immunization, besides the daily monitoring of body temperature, weight and clinical symptoms, the routine blood and urine tests and liver and kidney function tests were also conducted. RESULTS: Monkeys immunized via intranasal or oral routes could both generate serum IgG against rotavirus. During the immunization, the temperature of monkeys was normal and body weight raise stably. Both routine blood and urine tests and liver and kidney function tests showed no significant alteration compared with the control group. CONCLUSION: The immunization with the recombinant adenoviruses expressing rotavirus antigens is able to induce rotavirus specific efficient immune responses and is safe to baby rhesus monkeys. The preliminary results implied that the recombinant adenoviruses could be an ideal vaccine for rotavirus and lay a foundation for further studies.

Adenoviridae↗

PubMed2000

Recombinant polioviruses expressing hepatitis B virus-specific cytotoxic T-lymphocyte epitopes.

T-cell immune response to recombinant poliovirus expressing foreign antigens has not been elucidated well. In order to investigate the potential use of poliovirus as a T-cell vaccine vector, we constructed recombinant polioviruses expressing HBV-derived CTL epitopes, HBsAg(28-39) (L(d)-restricted; IPQSLDSWWTSL) and HBc(93-100) (K(b)-restricted; MGLKFRQL) at the junction between the P2 and P3 regions, designated V3CDs and V3CDc, respectively. The V3CDs and V3CDc recombinant viruses replicated efficiently in HeLa cells and showed a similar infection profile to that of the wild-type Mahoney strain in one-step growth kinetics. Genetic stability analysis showed that V3CDc retained the foreign insert over twelve successive passages examined, whereas V3CDs lost part of the foreign insert after four passages. Our results indicated that the stability of the inserted foreign sequences was rather affected by their nucleotide sequence than by their length when located between the P2 and P3 regions. The junction between these nonstructural protein-coding regions is a novel site for the construction of replication-competent recombinant poliovirus. Immunization of BALB/c (H-2(d)) and C57BL/6 mice (H-2(b)) with V3CDs and V3CDc, respectively, elicited significant antigen-specific CTL responses to HBsAg(28-39) but not to HBcAg(93-100).

Amino Acid Sequence↗

PubMed1999

CTL epitopes identified with a defective recombinant adenovirus expressing measles virus nucleoprotein and evaluation of their protective capacity in mice.

Cytotoxic T-lymphocyte (CTL) responses to measles virus (MV) play an important role in recovery from infection, with one of the major target proteins for CTL activity being the nucleoprotein (Np). In this report, a replication-deficient adenovirus-5 recombinant, expressing for MV Np (Rad68) was tested for in vivo priming of MV Np-specific CTL responses in BALB/c and CBA mice. In both strains of mice strong Np-specific CTL responses were induced and these responses were shown to be MHC class I restricted. Using overlapping 15mer peptides spanning residues 1-505 of MV Np a single epitope comprising residues 281-295 was identified in BALB/c mice whereas, in CBA mice two epitopes comprising residues 51-65 and 81-95, were identified. These epitopes were found to contain class I motifs for H-2L(d) and H-2K(k) MHC molecules, respectively. Immunization of BALB/c and CBA mice with the respective CTL epitopes resulted in the in vivo induction of peptide-and MV Np-specific CTL responses. In addition, the identified H-2K(k) restricted CTL epitopes conferred some protection against encephalitis induced following intracerebral challenge with a lethal dose of canine distemper virus (the Np of which shares 70% sequence homology with MV Np). These findings highlight the potential of using well-defined CTL epitopes to control virus infection.

Adenoviridae↗

PubMed2001

An efficient homologous recombination vector pTV(I) contains a hot spot for increased recombinant protein expression in Chinese hamster ovary cells.

We employed reverse genetics to clone a 5.0 kb genomic DNA hot spot HIRPE (hot spot for increased recombinant protein expression) flanking the plasmid integration site from a recombinant Chinese hamster ovary (CHO) cell line. DNA sequence analysis of the 5.0 kb fragment revealed that HIRPE is enriched for repetitive elements, Alu-like sequences and matrix-associated regions that are known to be linked with transcriptionally active regions in a number of mammalian systems. The construction of a homologous recombination vector, pTV1, containing the 5.0 kb HIRPE genomic DNA, a recombinant gene human CTLA4-Ig, and the dhfr gene as a positive selection marker is described. It was observed that the pTV1 vector targeted the CTLA4Ig gene to a preferred locus in the CHO genome contributing to high recombinant gene expression in transfected CHO cells. Preliminary studies suggest that similar to the observation with the parental cell line, pTV1-generated transfectomas that were analyzed appear to harbor an inverted duplication of the genomic DNA at the plasmid integration site.

Abatacept↗

PubMed2003

High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture.

Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system.

Cells, Cultured↗

PubMed2006

Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1.

The coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS), SARS-CoV, encodes two large polyproteins (pp1a and pp1ab) that are processed by two viral proteases to yield mature non-structural proteins (nsps). Many of these nsps have essential roles in viral replication, but several have no assigned function and possess amino acid sequences that are unique to the CoV family. One such protein is SARS-CoV nsp1, which is processed from the N-terminus of both pp1a and pp1ab. The mature SARS-CoV protein is present in cells several hours post-infection and co-localizes to the viral replication complex, but its function in the viral life cycle remains unknown. Furthermore, nsp1 sequences are highly divergent across the CoV family, and it has been suggested that this is due to nsp1 possessing a function specific to viral interactions with its host cell or acting as a host specific virulence factor. In order to initiate structural and biophysical studies of SARS-CoV nsp1, a recombinant expression system and a purification protocol have been developed, yielding milligram quantities of highly purified SARS-CoV nsp1. The purified protein was characterized using circular dichroism, size exclusion chromatography, and multi-angle light scattering.

Chromatography, Gel↗

PubMed2002

Expression of recombinant trichosanthin, a ribosome-inactivating protein, in transgenic tobacco.

Trichosanthin (TCS) is an antiviral plant defense protein, classified as a type-I ribosome-inactivating protein, found in the root tuber and leaves of the medicinal plant Trichosanthes kirilowii. It is processed from a larger precursor protein, containing a 23 amino acid amino (N)-terminal sequence (pre sequence) and a 19 amino acid carboxy (C)-terminal extension (pro sequence). Various constructs of the TCS gene were expressed in transgenic tobacco plants to determine the effects of the amino- and carboxy-coding gene sequences on TCS expression and host toxicity in plants. The maximum TCS expression levels of 2.7% of total soluble protein (0.05% of total dry weight) were obtained in transgenic tobacco plants carrying the complete prepro-TCS gene sequence under the Cauliflower mosaic virus 35S RNA promoter. The N-terminal sequence matched the native TCS sequence indicating that the T. kirilowii signal sequence was properly processed in tobacco and the protein translation inhibitory activity of purified rTCS was similar to native TCS. One hundred-fold lower expression levels and phenotypic aberrations were evident in plants expressing the gene constructs without the C-terminal coding sequence. Transgenic tobacco plants expressing recombinant TCS exhibited delayed symptoms of systemic infection following exposure to Cucumber mosaic virus and Tobacco mosaic virus (TMV). Local lesion assays using extracts from the infected transgenic plants indicated reduced levels of TMV compared with nontransgenic controls.

Cucumovirus↗

PubMed2004

[Construction and prokaryotic expression of a recombinant immunotoxin fused with mouse interleukin 18 and truncated Pseudomonas exotoxin].

OBJECTIVE: To construct a new recombinant immunotoxin expression vector by fusion of mouse interleukin 18 (mIL-18) gene and a truncated form of A (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli. METHODS: mIL-18 cDNA was cloned from mouse liver tissue through reverse transcription-polymerase chain reaction (RT-PCR). The mIL-18 cDNA was subcloned into a PE38 gene inserted fusion protein expression plasmid. The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing. After transformed into E. coli BL21 and induced by IPTG, the expressed product was obtained and detected the molecular weight and specificity by SDS-PAGE and Western-blotting. RESULTS: The new recombinant immunotoxin expression vector was constructed successfully. The IL-18-PE38 fusion protein was expressed in E. coli BL21, and the molecular weight of the expression product was identical to the expected value. In addition, the protein expressed could react with the specific antibody against mIL-18. CONCLUSION: IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cell and may have some potential value in the treatment of autoimmune disease and T cell leukemias.

Animals↗

PubMed2004

Protection of weaned pigs by vaccination with human adenovirus 5 recombinant viruses expressing the hemagglutinin and the nucleoprotein of H3N2 swine influenza virus.

Swine influenza virus (SIV), subtype H3N2, is a recent reassortant virus that emerged in 1998 in North American swine causing severe respiratory and reproductive disease. In this study, two replication-defective adenovirus recombinants were developed as potential vaccines against H3N2 influenza viruses. Three groups of 3-week-old pigs (10 pigs per group) were vaccinated intramuscularly (IM) with the recombinants; one group was vaccinated with the recombinant adenovirus expressing the influenza virus H3 hemagglutinin (HA) protein, one group was vaccinated with the recombinant adenovirus expressing the nucleoprotein (NP), and one group was vaccinated with both recombinants in a mixture. Two additional control groups (10 pigs per group) were included in the animal trial. One control group was challenged with a virulent H3N2 field strain and one control group remained unchallenged. The results showed that pigs in the groups given the recombinant adenovirus expressing HA alone and HA plus NP developed high levels of virus-specific hemagglutination-inhibition (HI) antibody by 4 weeks post vaccination. Pigs in the group vaccinated with both recombinant viruses in a mixture were completely protected. Complete protection was shown by the lack of nasal shedding of virus following challenge and by the lack of lung lesions at 1 week following the challenge infection. Thus, replication-incompetent adenovirus vaccines given simultaneously to pigs are efficacious for SIV and have the additional advantage over commercial vaccines that suckling piglets have no pre-existing maternally-derived antibody to block early life vaccination.

Adenoviridae↗

PubMed1997

Expression of recombinant human glutamic acid decarboxylase (GAD) in myeloma cells and enzyme-linked immunosorbent assay (ELISA) for autoantibodies to GAD.

Detection of serum autoantibodies to glutamic acid decarboxylase (GAD) is a new method to differentiate insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM). We established a transformed mouse myeloma cell line, SPG14, which expresses recombinant human GAD65, a major isomer of 65 kDa, inside the cells. GAD65 was partially purified by affinity chromatography using the mouse anti-GAD antibody (Ab). We also established a sandwich ELISA for anti-GAD Ab of the IgG class using GAD65 for coating and the anti-human IgG for detection and examined 54 sera of the IDDM patients and 45 sera of normal individuals. When the mean +2 SD of the color development of the sera of normal individuals was used as a cut-off level, 59.2% of patients with IDDM were positive. This indicates that the ELISA was effective to differentiate IDDM and NIDDM. The result also indicates that the autoantibody to GAD does not dissociate from the recombinant GAD rapidly, even when unbound anti-GAD Ab was fully removed. By using a perfusion culture system, we obtained as many as 4.2 x 10(10) SPG14 cells, from which 5 mg of GAD65 could be obtained; this is sufficient for 5,000 assays. This system could be clinically useful for large-scale diagnosis of IDDM.

Animals↗

PubMed2001

Construction of recombinant eukaryotic expression plasmid containing hepatitis B virus genome with partial deletion of the core promoter.

OBJECTIVE: To construct an eukaryotic expression vector containing the full-length genome with partially deleted core promoter of hepatitis B virus (HBV). METHODS: A linearized genome containing the entire HBV 3.5 kb mRNA transcriptional units (P3.8 I plasmid) was used, which initiated from the upstream sequences of the basic core promoter. The objective eukaryotic expression vector was constructed by molecular cloning and PCR-based site-directed mutagensis in vitro, and identifcation was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by cloning and sequencing analysis. RESULTS: The eukaryotic expression vectors containing HBV genomes with 20/21 bp deletion (position 1 748/1 747 to 1 767) in the core promoter or with precore stop mutation at nucleotide 1896 as well were constructed successfully as confirmed by sequence analysis with RFLP. CONCLUSION: The recombinant expression vector may lay the foundation for further studies into the biological significance of the above mentioned mutations in vitro.

Journal Article↗

PubMed1993

Viral liposomes released from insect cells infected with recombinant baculovirus expressing the matrix protein of vesicular stomatitis virus.

The matrix (M) protein of vesicular stomatitis virus (VSV) has been found to promote assembly and budding of virions as well as down-regulating of VSV transcription. Large quantities of M protein can be produced in insect cells infected with recombinant baculovirus expressing the VSV M gene under control of the polyhedrin promoter. Analysis by pulse-chase experiments and density gradient centrifugation revealed that the [35S]methionine-labeled M protein synthesized in insect cells is released into the extracellular medium in association with lipid vesicles (liposomes). Electron microscopy and immunogold labeling showed that M protein expressed in insect cells induced the formation on plasma membrane of vesicles containing M protein, which are released from the cell surface in the form of liposomes. The baculovirus vector itself or recombinants expressing VSV glycoprotein (G) or nucleocapsid (N) protein did not produce the formation of vesicles in infected cells. The baculovirus-expressed M protein retains biological activity as demonstrated by its capacity to inhibit transcription when reconstituted with VSV nucleocapsids in vitro. These data suggest that M protein has the capacity to associate with the plasma membrane of infected cells and, in so doing, causes evagination of the membrane to form a vesicle which is released from the cell. This observation leads to the postulate, which requires further proof, that the VSV M protein can induce the formation and budding of liposomes from the cell membrane surface.

Animals↗