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Results for “Recombinant expression”

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At least 199 records · Page 11Linked to original sources

PubMed1991

Vaccinia virus recombinants expressing either the measles virus fusion or hemagglutinin glycoprotein protect dogs against canine distemper virus challenge.

cDNA clones of the genes encoding either the hemagglutinin (HA) or fusion (F) proteins of the Edmonston strain of measles virus (MV) were expressed in vaccinia virus recombinants. Immunofluorescence analysis detected both proteins on the plasma membranes of unfixed cells as well as internally in fixed cells. Immunoprecipitation of metabolically radiolabeled infected-cell extracts by using specific sera demonstrated a 76-kDa HA polypeptide and gene products of 60, 44, and 23 kDa which correspond to a MV F precursor and cleavage products F0, F1, and F2, respectively. Neither recombinant induced cell fusion of Vero cells when inoculated individually, but efficient cell fusion was readily observed upon coinfection of cells with both recombinants. Inoculation of dogs with the vaccinia virus-MV F recombinant (VV-MVF) did not give rise to detectable MV-neutralizing antibody. Inoculation of dogs with the vaccinia virus-MV HA recombinant (VV-MVHA) or coinoculation with both recombinants (VV-MVF and VV-MVHA) induced significant MV-neutralizing titers that were increased following a booster inoculation. Inoculation of dogs with the vaccinia virus recombinants or with MV failed to induce canine distemper virus (CDV)-neutralizing antibodies. Upon challenge with a lethal dose of virulent CDV, signs of infection were observed in dogs inoculated with (VV-MVF). No symptoms of disease were observed in dogs that had been vaccinated with VV-MVHA or with VV-MVHA and VV-MVF and then challenged with CDV. All dogs vaccinated with the recombinant viruses as well as those inoculated with MV or a vaccine strain of CDV survived CDV challenge.

Animals↗

PubMed1998

Human keratinocyte growth factor recombinantly expressed in Chinese hamster ovary cells: isolation of isoforms and characterization of post-translational modifications.

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor family that acts specifically on epithelial cells in a paracrine mode. We employed a mammalian expression system to synthesize recombinant human KGF and isolated two preparations, KGF-a and KGF-b, from medium conditioned by Chinese hamster ovary cells. On an SDS-PAGE gel, KGF-a migrates as two bands near 25-29 kDa and contains both N- and O-linked sugar moieties attached near the N-terminus. Detailed structural characterization confirms that KGF-a contains a single amino acid sequence predicted from cDNA sequence and the molecule has two intramolecular disulfide bridges, Cys1-Cys15 and Cys102-Cys106. An additional Cys at position 40 is free and resides in a solvent-inaccessible environment. Mass spectrometric analyses of KGF-a peptides verify the occurrence of several post-translational modifications in the molecule, including partial oxidation at Met28, partial sulfation at Tyr27, and glycosylation at Asn14 and Thr22. The Asn-linked carbohydrate structures are heterogeneous, which include biantennary, triantennary, and tetraantennary structures with none or up to four sialic acids attached to various structures, while the Thr-linked carbohydrates contain typical mucin-type structures. KGF-b is an N-terminally truncated form of KGF-a posttranslationally processed at Arg23 and is not glycosylated. Both KGF-a and KGF-b forms are capable of stimulating DNA synthesis in quiescent Balb/MK mouse epidermal keratinocytes.

Amidohydrolases↗

PubMedDate not supplied

Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes.

Following the transfer into fertilized mouse eggs of recombinant genes composed of the upstream region of the rat insulin II gene linked to sequences coding for the large-T antigen of simian virus 40, large-T antigen is detected exclusively in the beta-cells of the endocrine pancreas of transgenic mice. The alpha- and delta-cells normally found in the islets of Langerhans are rare and disordered. Well-vascularized beta-cell tumours arise in mice harbouring and inheriting these hybrid oncogenes.

Adenoma, Islet Cell↗

PubMed1998

Cloning and recombinant expression of mouse coagulation factor X.

Engineering of recombinant coagulation factor X variants, which can be activated by tumor-associated proteinases may lead to the development of new therapeutic molecules. However, the evaluation of such variants requires an appropriate animal model. Therefore, we isolated the complete coding sequence of mouse coagulation factor X from mouse liver cDNA by polymerase chain reaction. The deduced amino acid sequence codes for a prepro protein of 481 amino acids homologous to factor X sequences from various species. Recombinant mouse factor X was expressed in human embryonic kidney cells and secreted into cell culture supernatant as zymogen, which could be converted to catalytically active factor Xa by Russell's viper venom. Purified recombinant mouse factor X restored coagulation in human factor X deficient plasma, demonstrating that mouse factor X is able to functionally interact with the human blood coagulation system. Recombinant mouse factor X opens the possibility to analyze therapeutically useful variants in the mouse system.

Amino Acid Sequence↗

PubMed1995

Recombinant expression of vaccinia virus-encoded TAP1 and TAP2 promotes MHC class I-restricted antigen presentation in a Syrian hamster cell line.

Transporter associated with antigen presentation (TAP) molecules supply small peptides to the lumen of the endoplasmic reticulum for assembly with MHC class I glycoproteins. Here we report that a Syrian hamster cell line, BHK, which is commonly used in cell biological and virological investigations, is deficient in antigen presentation in a xenogeneic model. Thus when the mouse MHC class I restriction element, Kd, is expressed in BHK cells via a vaccinia virus (VV) recombinant, no significant recognition by VV-immune and Kd-restricted cytotoxic T cells is induced. This defect is apparently a consequence of a deficiency in TAP-dependent peptide transport and can be repaired when rat TAP1 and TAP2 are provided by recombinant VV expression.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1987

Protection of guinea pigs from primary and recurrent herpes simplex virus (HSV) type 2 cutaneous disease with vaccinia virus recombinants expressing HSV glycoprotein D.

Vaccinia virus recombinants containing herpes simplex virus (HSV) type 1 (VP176) or type 2 (VP221) glycoprotein D (gD) genes were studied for their protective potential in the guinea pig model of recurrent HSV type 2 disease. Cells infected with these recombinants synthesized at least one protein (precursor, mature form, or both) that was precipitated with monoclonal antibody to HSV type-common determinants on gD. These determinants were detected on the surface of cells infected with the recombinants at 2 hr after infection. VP176 immunization protected against primary (P much less than .001) and recurrent (P much less than .001) cutaneous HSV type 2 lesions and ganglionic latency (62% protection). VP221 immunization protected against recurrent disease (P less than .05), although HSV type 2 ganglionic infection was established. Protection, first observed at two weeks after immunization, apparently did not involve HSV-specific neutralizing antibody because seroconversion was detected at 35-45 days after immunization. Protection was correlated with HSV-specific lymphoproliferation and the elaboration of lymphokines that enhance natural killer cell cytolysis.

Animals↗

PubMed1992

Avian retroviral recombinant expressing foreign envelope delays tumour formation of ASV-A-induced sarcoma.

An avian recombinant retrovirus expressing subgroup A envelope glycoprotein antigenicity, RAV-0-A1, significantly delayed sarcoma induction in chickens after challenge with ASV-A. When tumours were formed, significantly smaller tumours resulted. The mechanism of protection does not appear to be interference mediated, since virus was not detected in the chickens. Protection was correlated with both neutralizing and complement-dependent cytotoxic antibodies (CDCA).

Aging↗

PubMed2006

Prediction of solubility on recombinant expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli.

BACKGROUND: Cellular interactions elicited by Plasmodium falciparum erythrocyte membrane protein antigen 1 (PfEMP1) are brought about by multiple DBL (Duffy binding like), CIDR (cysteine-rich interdomain region) and C2 domain types. Elucidation of the functional and structural characteristics of these domains is contingent on the abundant availability of recombinant protein in a soluble form. A priori prediction of PfEMP1 domains of the 3D7 genome strain, most likely to be expressed in the soluble form in Escherichia coli was computed and proven experimentally. METHODS: A computational analysis correlating sequence-dependent features to likelihood for expression in soluble form was computed and predictions were validated by the colony filtration blot method for rapid identification of soluble protein expression in E. coli. RESULTS: Solubility predictions for all constituent PfEMP1 domains in the decreasing order of their probability to be expressed in a soluble form (% mean solubility) are as follows: ATS (56.7%) > CIDR1alpha (46.8%) > CIDR2beta (42.9%) > DBL2-4gamma (31.7%) > DBL2beta + C2 (30.6%) > DBL1alpha (24.9%) > DBL2-7epsilon (23.1%) > DBL2-5delta (14.8%). The length of the domains does not correlate to their probability for successful expression in the soluble form. Immunoblot analysis probing for soluble protein confirmed the differential in solubility predictions. CONCLUSION: The acidic terminal segment (ATS) and CIDR alpha/beta domain types are suitable for recombinant expression in E. coli while all DBL subtypes (alpha, beta, gamma, delta, epsilon) are a poor choice for obtaining soluble protein on recombinant expression in E. coli. This study has relevance for researchers pursuing functional and structural studies on PfEMP1 domains.

Animals↗

PubMed1993

Serologic and molecular studies of two kindreds expressing recombinant HLA DR1/DR2 haplotypes.

We have recently identified two unrelated kindreds in which DR1 and DR2 co-segregate as a single haplotype spanning several generations. Serology, PCR-RFLP and sequence-specific oligonucleotide probes were used to characterize the presence and segregation of the DRB1 and DRB5 loci in these two kindreds. In both families, the recombination resulted in co-expression of a DRB1 locus that encoded a DR1 serologic phenotype and a DRB5 locus that encoded a "short" DR2 serologic phenotype. One of these kindreds co-expressed the DR1 and DR15 (DRB5*01). The other kindred co-expressed the DR1 and DR16 (DRB5*02). Our data indicate that the recombination event occurred in the region between the DRB1 locus and the DRB5 locus. This recombinant haplotype produces a DR2 phenotype which lacks the epitopes normally encoded by the DRB1 locus, resulting in a serologic "short" antigen. The clinical significance of such a recombinational event becomes evident when patients with such a genotype require allogeneic bone marrow transplantation.

Alleles↗

PubMed1993

Fowlpox virus recombinants expressing the envelope glycoprotein of an avian reticuloendotheliosis retrovirus induce neutralizing antibodies and reduce viremia in chickens.

Eight stable fowlpox virus (FPV) recombinants which express the envelope glycoprotein of the spleen necrosis virus (SNV) strain of reticuloendotheliosis virus (REV), an avian retrovirus, were constructed. These recombinants differ in the genomic location of the inserted genes, in the orientation of the insert relative to flanking viral sequences, and in the promoter used to drive expression of the env gene. Of these variables, promoter strength seems to be the most crucial. The P7.5 promoter of vaccinia virus, which is commonly used in the construction of both vaccinia virus and FPV recombinants, resulted in lower levels of expression of the envelope antigen in infected chicken cells compared with a strong synthetic promoter, as determined by immunofluorescence and enzyme-linked immunosorbent assay. Two peptides encoded by the env gene, the 21-kDa transmembrane peptide and a 62-kDa precursor, were detected by immunoprecipitation of labeled proteins from cells infected with recombinant FPVs, using monoclonal antibodies against REV. These peptides comigrated with those precipitated from REV-infected cells. One of the recombinants (f29R-SNenv) was used for vaccination of 1-day-old chickens. Vaccinated chicks developed neutralizing antibodies to SNV more rapidly than did unvaccinated controls following SNV challenge and were protected against both viremia and the SNV-induced runting syndrome.

Animals↗

PubMed1987

Vaccinia virus recombinants expressing rabiesvirus glycoprotein protect against rabies.

Six recombinants of New York Board of Health (NYBH) vaccinia virus containing cDNA for Challenge Virus Standard (CVS) rabiesvirus glycoprotein (G) were produced by directing gene insertion into the vaccinia thymidine kinase (TK) locus. To regulate expression of G the promoter P7.5 (functions at early and late times postinfection) from the gene for the vaccinia 7.5 kilodalton (kD) protein was used in two of the recombinants; late promoter P11 of the vaccinia 11 kD protein was used in four recombinants. The six differed in nucleotide sequences flanking the translation start codon; in two constructs the encoded signal peptide of G was fused to several additional amino acids. Cells infected with each recombinant made G that reacted with G-specific antibodies, comigrated with authentic G, and was transported to the plasma membrane. The highest amounts of G were made with fusion or standard versions of G with P11 provided that the mRNA leader sequences were identical to the natural gene. Each recombinant in mice and one in dogs induced rabiesvirus neutralizing antibodies and protection against lethal rabiesvirus challenge.

Amino Acid Sequence↗

PubMed2005

Baculovirus-expressed recombinant human zona pellucida glycoprotein-B induces acrosomal exocytosis in capacitated spermatozoa in addition to zona pellucida glycoprotein-C.

To facilitate our understanding of the role of zona pellucida glycoproteins during fertilization in humans, recombinant human zona pellucida glycoprotein-A (hZPA), -B (hZPB) and -C (hZPC) were obtained by using Escherichia coli and baculovirus expression systems. Analysis by SDS-PAGE and Western blot of the Ni-NTA affinity purified recombinant proteins revealed that the baculovirus-expressed hZPA, hZPB and hZPC have an apparent molecular weight of approximately 110, approximately 70-75 and approximately 65 kDa, respectively, as compared to approximately 80, approximately 65 and approximately 50 kDa of the respective E. coli-expressed proteins. Lectin binding studies revealed that the baculovirus-expressed recombinant zona proteins were glycosylated. Major oligosaccharides were represented by strong reactivity with Concanavalin A (mannose alpha 1-3 or mannose alpha 1-6 residues) and Jacalin (alpha-O glycosides of Gal or GalNAc moieties). A significant increase in acrosomal exocytosis was observed when capacitated human sperm were incubated in vitro with baculovirus-expressed hZPB (P=0.0005) and hZPC (P=0.0005) The E. coli-expressed hZPB, hZPC and baculovirus-expressed hZPA failed to induce any significant increase (P>0.05) in acrosome reaction. In contrast to hZPC, the acrosome reaction induced by recombinant hZPB was not inhibited by pertussis toxin. These studies, for the first time, have demonstrated that in humans, ZPB also induces acrosomal exocytosis through a Gi independent pathway.

Acrosome Reaction↗

PubMed1999

Observation of measles virus cell-to-cell spread in astrocytoma cells by using a green fluorescent protein-expressing recombinant virus.

A recombinant measles virus (MV) which expresses enhanced green fluorescent protein (EGFP) has been rescued. This virus, MVeGFP, expresses the reporter gene from an additional transcription unit which is located prior to the gene encoding the measles virus nucleocapsid protein. The recombinant virus was used to infect human astrocytoma cells (GCCM). Immunocytochemistry (ICC) together with EGFP autofluorescence showed that EGFP is both an early and very sensitive indicator of cell infection. Cells that were EGFP-positive and ICC-negative were frequently observed. Confocal microscopy was used to indirectly visualize MV infection of GCCM cells and to subsequently follow cell-to-cell spread in real time. These astrocytoma cells have extended processes, which in many cases are intimately associated. The processes appear to have an important role in cell-to-cell spread, and MVeGFP was observed to utilize them in the infection of surrounding cells. Heterogeneity was seen in cell-to-cell spread in what was expected to be a homogeneous monolayer. In tissue culture, physical constraints govern the integrity of the syncytia which are formed upon extensive cell fusion. When around 50 cells were fused, the syncytia rapidly disintegrated and many of the infected cells detached. Residual adherent EGFP-positive cells were seen to either continue to be involved in the infection of surrounding cells or to remain EGFP positive but no longer participate in the transmission of MV infection to neighboring cells.

Animals↗

PubMed1998

Recombinant expression of alliin lyase from garlic (Allium sativum) in bacteria and yeasts.

Recombinant garlic alliin lyase was produced in Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. A cDNA clone was obtained from garlic bulbs by PCR and introduced into suitable bacterial and yeast expression vectors. The recombinant alliin lyase forms inclusion bodies in all three host organisms, which are deposited in the cytoplasm. After cell lysis and harvesting by centrifugation, the inclusion bodies were solubilized in Zwittergent 3-14 solution and refolded by stepwise dilution. Specific alliin lyase activity could be recovered by this procedure.

Blotting, Western↗

PubMed1987

Vaccinia recombinant expressing Lassa-virus internal nucleocapsid protein protects guineapigs against Lassa fever.

The Lister strain of vaccinia virus was used to construct a recombinant that expressed the nucleocapsid gene of Lassa virus. Guineapigs immunised with the recombinant virus were protected against challenge with Lassa virus, whereas control animals showed the usual disease course, including pyrexia, anorexia, viraemia, and death. These data indicate not only that protective immunity to Lassa fever can be evoked by an experimental vaccine, but also that this response can be mediated by an internal protein component of the virus.

Animals↗

PubMed1996

Heparinase III from Flavobacterium heparinum: cloning and recombinant expression in Escherichia coli.

Heparinase III (E.C. 4.2.2.8), formerly heparinase I, produced by Flavobacterium heparinum is an enzyme that specifically cleaves heparan sulfate-rich regions of acidic polysaccharides. In this study, we report the cloning of the heparinase III gene using polymerase chain reaction (PCR). Two degenerate oligonucleotides, based on amino acid sequences derived from tryptic peptides of purified heparinase III were used to generate a approximately 1100-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. The PCR-derived probe was used to screen a Flavobacterium genomic DNA library in lambda ZAP II. The open reading frame of the heparinase III gene is 1980 bp in length, encoding a precursor protein of 75,950 Da; 10 of the tryptic peptides mapped onto the open reading frame which corresponded to approximately 18% of the protein. Recombinant heparinase III was expressed in Escherichia coli using the T7 polymerase pET expression system. This is the first report of the cloning and recombinant expression of an enzyme primarily degrading heparan sulfate.

Amino Acid Sequence↗

PubMed1998

Isolation and recombinant expression of an MHV-JHM neutralising monoclonal antibody.

The monoclonal antibody A1 (mab A1) efficiently neutralises the infection of susceptible cells by the murine hepatitis virus MHV-JHM in vitro and in vivo (Wege et al., 1984). The variable regions of mab A1 were amplified from mRNA of the respective hybridoma cell line by RT-PCR and integrated into different eukaryotic expression vectors. The biological function of the recombinant antibody constructs was verified by virus neutralisation assays. Whereas a complete recombinant antibody (mab A1rec.) expressed in transfected murine myeloma cells inhibited the MHV-JHM infection as well as the parental antibody, a single-chain Fv derived from mab A1 did not show any neutralising activity.

Animals↗

PubMed2002

Bovine herpesvirus-1 (BHV-1) recombinant expressing pseudorabies virus (PrV) glycoproteins B and C induces type 1 immune response in BALB/c mice.

Bovine herpesvirus 1 (BHV-1) attached poorly and penetrated into a mouse cell line, BALB 3T3/A31, but a recombinant BHV-1/TF7-6, which expresses pseudorabies virus (PrV) gB and gC genes, did attach and penetrated into cells more efficiently. In this study the gene green fluorescent protein (GFP) has been integrated into genome of BHV-1/TF7-6 and its parental line of BHV-1. When the mouse mesenteries were incubated in vitro and infected with BHV-1/TF7-6/GFP, strong fluorescence was observed while BHV-1/GFP infection hardly demonstrated fluorescence, suggesting that BHV-1 recombinant expressing PrV gB and gC can infect mouse tissue cells more efficiently than the parental BHV-1 does. When BALB/c mice were inoculated with purified BHV-1/TF7-6 or its parental BHV-1, the former induced lower level of anti-BHV-1 immunoglobulin G (IgG) than the latter did. When sub-classes of anti-BHV-1 IgG were analyzed, it was found that mice immunized with BHV-1/TF7-6 or the parental BHV-1 demonstrated the same level of IgG2a. Since anti-BHV-1 IgG1 level was lower in mice inoculated with BHV-1/TF7-6, the IgG2a:IgG1 ratio was higher in BHV-1/TF7-6 inoculated mice than in the parental BHV-1 inoculated ones. These results indicate that BHV-1/TF7-6 induces type 1 predominant immune to BALB/c mice.

Animals↗