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Results for “Recombinant expression”

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PubMed2001

Identification, cloning, and recombinant expression of procalin, a major triatomine allergen.

Among the most frequent anaphylactic reactions to insects are those attributed to reduviid bugs. We report the purification and identification of the major salivary allergen of these insects. This 20-kDa protein (procalin) is a member of the lipocalin family, which includes salivary allergens from other invertebrates and mammals. An expression system capable of producing reagent quantities of recombinant allergen was developed in Saccharomyces cerevisiae. Antisera produced against recombinant protein cross-reacts with ELISA with salivary allergen. Recombinant Ag is also shown to react with sera from an allergic patient but not with control sera. By immunolocalization, the source of the salivary Ag is the salivary gland epithelium and its secretions.

Allergens↗

PubMed1988

Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination.

Previously we showed that mice immunized with a vaccinia virus vector expressing the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene (vaccinia/gD) were protected against both lethal and latent infections with HSV-1 for at least 6 weeks after immunization (K. J. Cremer, M. Mackett, C. Wohlenberg, A. L. Notkins, and B. Moss, Science 228:737-740, 1985). In the experiments described here, we examined long-term immunity to HSV following vaccinia/gD vaccination, the effect of revaccination with vaccinia/gD, and the impact of previous immunity to vaccinia virus on immunization with the gD recombinant. Mice immunized with vaccinia/gD showed 100, 100, and 80% protection against lethal infection with HSV-1 at 18, 44, and 60 weeks postimmunization, respectively. Protection against latent trigeminal ganglionic infection was 70, 50, and 31% at 6, 41, and 60 weeks postvaccination, respectively. To study the effect of reimmunization on antibody levels, mice vaccinated with vaccinia/gD were given a second immunization (booster dose) 3 months after the first. These mice developed a 10-fold increase in neutralizing-antibody titer (221 to 2,934) and demonstrated a significant increase in protection against lethal HSV-1 challenge compared with animals that received only one dose of vaccinia/gD. To determine whether preexisting immunity to vaccinia virus inhibited the response to vaccination with vaccinia/gD virus, mice were immunized with a recombinant vaccinia virus vector expressing antigens from either influenza A or hepatitis B virus and were then immunized (2 to 3 months later) with vaccinia/gD. These mice showed reduced titers of neutralizing antibody to HSV-1 and decreased protection against both lethal and latent infections with HSV-1 compared with animals vaccinated only with vaccinia/gD. We conclude that vaccination with vaccinia/gD produces immunity against HSV-1 that lasts over 1 year and that this immunity can be increased by a booster but that prior immunization with a vaccinia recombinant virus expressing a non-HSV gene reduces the levels of neutralizing antibody and protective immunity against HSV-1 challenge.

Animals↗

PubMed1988

Functional studies on the p10 gene of Autographa californica nuclear polyhedrosis virus using a recombinant expressing a p10-beta-galactosidase fusion gene.

The beta-galactosidase gene (lacZ) of Escherichia coli was inserted in phase with the coding sequence of the Autographa californica nuclear polyhedrosis virus (AcMNPV) late-expressed Mr 10,000 (p10) gene. The fusion gene was inserted into the AcMNPV genome by cotransfection of a recombinant plasmid pAcR159Z, consisting of the EcoRI P fragment-containing pBR325-derived plasmid pAcR159 and the lacZ insert in the p10 gene, and wild-type AcMNPVDNA. Infection of Spodoptera frugiperda cells by the resulting recombinant AcMNPV/p10Z-2 showed high level expression of a p10-lacZ fusion protein, but no synthesis of p10. Therefore, the p10 gene is dispensable for virus replication and the p10 promoter is effective in driving the expression of foreign genes. Cells infected with AcMNPV/p10Z recombinants resembled those infected with wild-type AcMNPV in the amounts of polyhedrin synthesized and polyhedra formed, although p10 was absent. The nucleus and cytoplasm of AcMNPV/p10Z-2-infected cells lacked the fibrous structures that are associated with p10 in wild-type AcMNPV-infected cells. Instead, large granular structures were observed that were found by immunogold labelling to contain the lacZ gene product. The electron-dense 'spacers', thought to be precursors of the polyhedron membrane, were absent from cells infected by the recombinant virus and the polyhedra did not have a membrane. The recombinant AcMNPV/p10Z-2 was at least twice as virulent for second instar S. exigua larvae than was wild-type AcMNPV. The increased virulence of the recombinant is an important property for the control of insects.

Animals↗

PubMed1999

Cell-mediated immune responses in cattle vaccinated with a vaccinia virus recombinant expressing the nucleocapsid protein of rinderpest virus.

Rinderpest virus (RPV) is a member of the genus Morbillivirus in the family Paramyxoviridae which causes an acute and often fatal disease in large ruminants. To examine the immune response to the virus nucleocapsid (N) protein, a recombinant vaccinia virus expressing RPV nucleocapsid protein (rVV-RPV-N) was used to vaccinate cattle. The recombinant vaccine induced low levels of non-neutralizing anti-N antibodies. RPV-specific cell-mediated immunity induced by the recombinant was assessed by measuring both the lymphocyte proliferation and cytotoxic T-lymphocyte responses. The protective immune response was examined by challenging the vaccinated cattle with either a highly virulent (Saudi 1/81) or a mild (Kenya/eland/96) strain of the virus. The vaccinated cattle were not protected against challenge with the virulent RPV strain, except they showed a slight delay in the onset of disease when compared with the unvaccinated controls. In cattle challenged with the mild strain, apart from a transient fever, no clinical signs of rinderpest infection were seen in the vaccinated cattle. One out of two control cattle showed a similar response but the other died from classic rinderpest disease. Virus-neutralizing antibodies were induced more quickly following challenge with the mild strain in vaccinated cattle compared to the control animals. These data suggested that the cell-mediated immunity induced by rVV-RPV-N could stimulate the rapid production of neutralizing antibodies following RPV challenge but this response was not sufficient to protect against challenge with a virulent strain of the virus. Protection was seen in one of three animals challenged with a mild strain of the virus; however, a greater number of animals would need to be tested to estimate the significance of the protection afforded by the N protein.

Animals↗

PubMed1993

Leukotriene synthesis in U937 cells expressing recombinant 5-lipoxygenase.

The U937 human promyelocytic cell line does not express 5-lipoxygenase, but does express 5-lipoxygenase-activating protein (FLAP). U937 cells do not synthesize leukotrienes after stimulation by calcium ionophore A23187. Dimethyl sulfoxide (DMSO) differentiation of U937 cells, towards a more mature monocyte-macrophage lineage, induces the expression of FLAP but not 5-lipoxygenase. These DMSO-differentiated U937 cells also lack the ability to synthesize leukotrienes. We infected viral RNA coding for 5-lipoxygenase into U937 cells using a retroviral vector and measured the synthesis of 5-lipoxygenase, FLAP, leukotrienes and 5-hydroxyeicosatetraenoic acid (5-HETE) by these cells after stimulation with A23187. Undifferentiated U937 cells infected with 5-lipoxygenase RNA expressed 5-lipoxygenase and FLAP but neither leukotrienes nor 5-HETE were detected after these cells were stimulated with A23187. Exposure of the 5-lipoxygenase-infected U937 cells to DMSO increased the expression of 5-lipoxygenase and FLAP, and these cells produced leukotrienes and 5-HETE in response to A23187. The synthesis of these products was inhibited by MK-886, a compound which specifically binds to FLAP.

5-Lipoxygenase-Activating Proteins↗

PubMed2002

Serodiagnosis of mice minute virus and mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirus-expressed recombinant VP2 proteins.

Mice minute virus (MMV) and mouse parvovirus (MPV) type 1 are the two parvoviruses known to naturally infect laboratory mice and are among the most prevalent infectious agents found in contemporary laboratory mouse colonies. Serologic assays are commonly used to diagnose MMV and MPV infections in laboratory mice; however, highly accurate, high-throughput serologic assays for the detection of MMV- and MPV-infected mice are needed. To this end, the major capsid viral protein (VP2) genes of MMV and MPV were cloned and MMV recombinant VP2 (rVP2) and MPV rVP2 proteins were expressed by using a baculovirus system. MMV rVP2 and MPV rVP2 spontaneously formed virus-like particles that were morphologically similar to empty parvovirus capsids. These proteins were used as antigens in enzyme-linked immunosorbent assays (ELISAs) to detect anti-MMV or anti-MPV antibodies in the sera of infected mice. Sera from mice experimentally infected with MMV (n = 43) or MPV (n = 35) and sera from uninfected mice (n = 30) were used to evaluate the ELISAs. The MMV ELISA was 100% sensitive and 100% specific in detecting MMV-infected mice, and the MPV ELISA was 100% sensitive and 98.6% specific in detecting MPV-infected mice. Both assays outperformed a parvovirus ELISA that uses a recombinant nonstructural protein (NS1) of MMV as antigen. The MMV rVP2 and MPV rVP2 proteins provide a ready source of easily produced antigen, and the ELISAs developed provide highly accurate, high-throughput assays for the serodiagnosis of MMV and MPV infections in laboratory mice.

Animals↗

PubMed2005

Immunization against a low-frequency human platelet alloantigen in fetal alloimmune thrombocytopenia is not a single event: characterization by the combined use of reference DNA and novel allele-specific cell lines expressing recombinant antigens.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal immunization against a fetal platelet (PLT) alloantigen. In cases of FNAIT attributed to low-frequency PLT alloantigens, the laboratory diagnosis is often hampered by the lack of adequate PLTs. STUDY DESIGN AND METHODS: Three families with maternal immunization against fetal PLT antigens were analyzed. In Family 1, previous immunization of another female or woman has been observed. In Families 2 and 3, newborns presented with the typical clinical picture of FNAIT. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing with reference to DNA from Epstein-Barr virus-transformed B-lymphoblastoid cell lines. Antibodies were characterized by glycoprotein (GP)-specific immunoassay with a panel of stable Chinese hamster ovary cell lines expressing low-frequency alloantigens. RESULTS: In three families, maternal immunization associated with the low-frequency alloantigens human PLT antigen (HPA)-8bw (Sra), HPA-11bw (Groa), and HPA-13bw (Sita) was identified. Maternal serum samples showed positive reactions in an antigen capture assay with cell lines carrying recombinant GP IIb/IIIa (HPA-8bw and -11bw) or GPIa/IIa (HPA-13bw), respectively. These results could be confirmed by genotyping analysis of fathers and newborns. CONCLUSION: This study demonstrates that cases of FNAIT attributed to low-frequency PLT alloantigens cannot be regarded as single events. The availability of reference DNA and cell lines expressing recombinant PLT alloantigens can facilitate their identification.

Antigens, Human Platelet↗

PubMed1988

Protection of cattle against rinderpest with vaccinia virus recombinants expressing the HA or F gene.

Rinderpest is a highly contagious ruminant viral disease manifested by a rapid course and greater than 90% mortality. Infectious vaccinia virus recombinants were constructed that express either the hemagglutinin or the fusion gene of rinderpest virus. All cattle vaccinated with either recombinant or with the combined recombinants produced neutralizing antibodies against rinderpest virus and were protected against the disease when challenged with more than 1000 times the lethal dose of the virus.

Animals↗

PubMed1996

Detection of glutamic acid decarboxylase (GAD) autoantibodies by indirect immunofluorescence using CHO cells expressing recombinant human GAD65.

The reaction between human glutamic acid decarboxylase (GAD65) expressed in CHO cells and GAD antibodies was studied by indirect immunofluorescence (IIF). The monoclonal antibodies GAD1 and GAD6, which recognize conformational and continuous GAD epitopes respectively, yielded distinct staining patterns. Twelve of 26 sera from newly-diagnosed insulin-dependent diabetes mellitus (IDDM) patients displayed a variety of anti GAD specific IIF images encompassing the two extremes observed with the monoclonal antibodies. None of 21 normal sera tested positive in this assay. As a control, the sera were tested by a reference immunoprecipitation (IP) assay using in vitro produced, folded 35S-GAD65. Only one of the patient sera reacted by IP using heat- and detergent-denatured 35S-GAD65 indicating that most of the auto-antibodies recognized only a folded antigen. Eleven patient sera were both IIF and IP anti-GAD-positive. The IIF reactivity of these sera was blocked by soluble GAD from brain extracts. One serum was positive only by IIF, and its reactivity was not blocked by soluble GAD. Eight sera were positive only by IP. Our results established differences in anti GAD antibodies in terms of their capacity to recognize human GAD65 in the context of transformed CHO cells compared with conventional IP assays. These differences should be considered in future attempts to improve the available assays for the detection of IDDM autoantibodies.

Adolescent↗

PubMed1997

Recombinant expression and catalytic analysis of rapid and slow acetylator Syrian hamster chimeric NAT2 alleles.

Polymorphic aromatic amine N-acetyltransferase (NAT2) catalyzes the N-acetylation of aromatic amines and the metabolic activation of N-hydroxyarylamines (via O-acetylation) and N-hydroxy-N-acetylarylamines (via N,O-acetylation) to electrophilic intermediates that mutate DNA. Acetylation capacity in humans and other mammalian species such as Syrian hamsters is subject to a genetic polymorphism. NAT2 is regulated by a single gene (NAT2) containing a single coding exon of 870 bp. Syrian hamster slow acetylator differs from the rapid acetylator NAT2 coding region by three nucleotide substitutions at T36C, A633G, and C727T. We measured expression of immunoreactive NAT2 protein and aromatic amine N-acetylation. N-hydroxyarylamine O-acetylation and N-hydroxy-N-acetylarylamine N,O-acetylation by recombinant NAT2 proteins expressed from alleles containing all combinations of the T36C, A633G, and C727T substitutions. The C727T substitution, which creates an opal stop codon in slow acetylator NAT2, was the sole mutation responsible for substantial reduction in expression of a truncated NAT2 protein with reduced capacity for the deactivation of aromatic amines (N-acetylation) and the metabolic activation of N-hydroxyarylamines (O-acetylation) and N-hydroxy-N-acetylarylamines (N,O-acetylation). The reductions in aromatic amine N-acetylation correlated very highly with the reductions in metabolic activation of the corresponding N-hydroxyarylamines and N-hydroxy-N-acetylarylamines.

Acetylation↗

PubMed2001

Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells.

Transient gene expression (TGE) in mammalian cells at the reactor scale is becoming increasingly important for the rapid production of recombinant proteins. We improved a process for transient calcium phosphate-based transfection of HEK293-EBNA cells in a 1-3 L bioreactor volume. Cells were adapted to suspension culture using a commercially available medium (BioWhittaker, Walkersville, MD). Process parameters were optimized using a plasmid reporter vector encoding the enhanced green fluorescent protein (EGFP/CLONTECH, Palo Alto, CA, USA). Using GFP as a marker-protein, we observed by microscopic examination transfection efficiencies between 70-100%. Three different recombinant proteins were synthesized within a timeframe of 7 days from time of transfection to harvest. The first, a human recombinant IgG(1)-type antibody, was secreted into the supernatant of the cell culture and achieved a final concentration of >20 mg/L. An E. coli-derived DNA-binding protein remained intracellular, as expected, but accumulated to such a concentration that the lysate of cells, taken up into the entire culture volume, gave a concentration of 18 mg/L. The third protein, a transmembrane receptor, was expressed at 3-6 x 10(6) molecules/cell.

Bioreactors↗

PubMed1997

Human mast cells expressing recombinant proteinase 3 (PR3) as substrate for clinical testing for anti-neutrophil cytoplasmic antibodies (ANCA).

We have expressed conformationally intact, enzymatically active recombinant PR3 in HMC-1 cells (HMC-1/PR3 cells) that is recognized by C-ANCA. Here we directly compared the clinical utility of C-ANCA testing by indirect immunofluorescence (IIF) using HMC-1/PR3 cell cytospin versus polymorphonuclear neutrophil (PMN) cytospin preparations and commercially available anti-PR3 ELISA kits. Two hundred sera were tested independently by three investigators: 101 previously determined to be C-ANCA-positive by routine clinical laboratory testing using standard IIF on PMN cytospins, and 99 control samples chosen primarily because they contained antibodies against other cytoplasmic target antigens. Discrepant test results between the two cellular substrates were found in seven samples: 2/7 were PMN-positive and HMC-1/PR3 cell-negative (one Sjogren's syndrome, one hand injury); 5/7 were PMN-negative and HMC-1/PR3-positive (all Wegener's granulomatosis (WG)). All C-ANCA-positive WG patients were also positive on HMC-1/PR3 cells. IIF using HMC-1/PR3 cells was as sensitive as the most sensitive anti-PR3 ELISA (79.8% versus 80.7%, P = 0.739), and more sensitive than standard IIF C-ANCA testing using PMN cytospins (79.8% versus 75.2%, P = 0.025) or the anti-PR3 ELISA with the least false-positive test results (79.8% versus 63%, P < 0.01). These findings indicate that HMC-1/PR3 cells are a very sensitive antigen-specific substrate for clinical anti-PR3 ANCA testing which appears superior to standard C-ANCA testing using PMN cytospin substrates and anti-PR3 ELISA. Our results also suggest that in WG the C-ANCA fluorescence pattern is not caused by antibodies against target antigens other than PR3.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed2004

Isolation and characterisation of three moss-derived beta-tubulin promoters suitable for recombinant expression.

The moss Physcomitrella patens is an excellent tool to study plant gene-function relationships due to its high rate of homologous recombination (HR). It has also been shown to be very useful in the production of recombinant proteins which are secreted into a simple medium. Thus, there is a need for suitable promoters functional in this well established model organism. We isolated genomic flanking regions of the beta-tubulin gene family from Physcomitrella, concentrating on those family members showing high transcript abundance integrated over gametophytic tissues. Using a novel, fast and reliable quantification assay based on the transient expression and secretion of a recombinant human protein, three genomic upstream regions were characterised in serial deletion constructs. Expression rates were up to three times higher than those obtained with the 35S cauliflower mosaic virus (35S) promoter, which served as a reference.

5' Untranslated Regions↗

PubMed1992

Recombinant expression, purification and biochemical characterization of a superoxide dismutase from Entamoeba histolytica.

A recombinant iron-containing superoxide dismutase (recFeSOD) of Entamoeba histolytica was produced in a prokaryotic expression system. Purified recFeSOD was found to be enzymatically active as determined by (i) inhibition of ferri-cytochrome c reduction, (ii) dismutation of superoxide anions generated by human neutrophils and (iii) inhibition of nitroblue tetrazolium reduction. The enzymatic properties of recFeSOD were similar to those of the native protein in trophozoite extracts. In an ELISA using recFeSOD as antigen, 96% of sera from patients having invasive amebiasis were reactive whereas none of the healthy controls or of patients suffering from malaria, bacterial or viral infections were reactive. Only sera of Toxoplasma-, Leishmania- or Trypanosoma-infected individuals exhibited partial cross-reactivity to recFeSOD.

Amino Acid Sequence↗

PubMed1993

The cytolytic activity of pulmonary CD8+ lymphocytes, induced by infection with a vaccinia virus recombinant expressing the M2 protein of respiratory syncytial virus (RSV), correlates with resistance to RSV infection in mice.

Previous studies demonstrated that the pulmonary resistance to respiratory syncytial virus (RSV) challenge induced by immunization with a recombinant vaccinia virus expressing the M2 protein of RSV (vac-M2) was significantly greater 9 days after immunization than at 28 days and was mediated predominantly by CD8+ T cells. In this study, we have extended these findings and sought to determine whether the level of CD8+ cytotoxic T-lymphocyte (CTL) activity measured in vitro correlates with the resistance to RSV challenge in vivo. Three lines of evidence documented an association between the presence of pulmonary CTL activity and resistance to RSV challenge. First, vac-M2 immunization induced pulmonary CD8+ CTL activity and pulmonary resistance to RSV infection in BALB/c (H-2d) mice, whereas significant levels of pulmonary CTL activity and resistance to RSV infection were not seen in BALB.K (H-2k) or BALB.B (H-2b) mice. Second, pulmonary CD8+ CTL activity was not induced by infection with other vaccinia virus-RSV recombinants that did not induce resistance to RSV challenge. Third, the peak of pulmonary CTL activity correlated with the peak of resistance to RSV replication (day 6), with little resistance being observed 45 days after immunization. An accelerated clearance of virus was not observed when mice were challenged with RSV 45 days after immunization with vac-M2. The results indicate that resistance to RSV induced by immunization with vac-M2 is mainly mediated by primary pulmonary CTLs and that this resistance decreases to very low levels within 2 months following immunization. The implications for inclusion of CTL epitopes into RSV vaccines are discussed in the context of these observations.

Animals↗

PubMed1999

Function of the propeptide region in recombinant expression of active procathepsin L in Escherichia coli.

In order to determine the functional role of the procathepsin L propeptide region for the preparation of active recombinant rat cathepsin L (CL), cDNAs encoding two short-length propeptides (C-terminal 2 and 27 residues) and the full-length (96 residues) one plus the entire CL were expressed as two soluble fusion proteins with a fragment of maltose-binding protein and an insoluble fusion protein with glutathione-S-transferase in Escherichia coli, respectively. After refolding of the insoluble fusion protein, each gene product was purified to homogeneity by amylose or glutathione-Sepharose-4B affinity column, and digestion with factor Xa and alpha-thrombin under alkaline conditions (pH approximately 8.0) led to the elution of two pure short-length procathepsin Ls (PCLs) and a full-length one, respectively. The enzymatic activity, estimated by hydrolytic assaying of benzoxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide under acidic conditions (pH 5.5), indicated that the two short-length PCLs exhibited in a great loss of the activity, as compared with the full-length PCL. The CD spectra of the short-length PCLs were different from that of the full-length one. The present results clearly show that the full-length propeptide is essential for construction of the active tertiary structure of CL at the stage of recombinant protein expression, although the expression of CL itself in E. coli does not require the propeptide. Based on the tertiary structure of PCL, the propeptide region necessary for the construction of the CL active structure has been discussed.

Animals↗

PubMed2003

Cloning, sequencing and recombinant expression of the open reading frame encoding a novel member of the Sarcocystis muris (Apicomplexa) microneme lectin family.

Micronemes are characteristic secretory organelles located within the apical cell region of apicomplexan parasites. The protein contents are exocytosed during an early phase of host cell invasion and contribute to parasite motility and the invasion of target cells. We report here on the cloning and heterologous expression of a novel member of the Sarcocystis muris microneme lectin family. The deduced amino acid sequence is in total agreement with that obtained after sequencing the native protein and is characterized by two copies of the apple domain motif. The recombinant polypeptide is expressed in a biologically active conformation as demonstrated by its galactose binding properties.

Amino Acid Sequence↗

PubMed1994

Growth hormone (GH) induces tyrosine-phosphorylated proteins in mouse L cells that express recombinant GH receptors.

Porcine and bovine GH receptor (GHR) cDNAs were stably expressed in mouse L cells, which normally do not possess detectable levels of mouse GHR. Expression of the GHR cDNAs resulted in specific binding of 125I-labeled GH by these cell lines. To study GHR-related signaling events in these cells, protein tyrosine phosphorylation was examined. In GH-treated cells, a tyrosine-phosphorylated protein with a molecular mass of approximately 95 kDa (pp95) was increased dramatically (approximately 100-fold) relative to non-GH-treated cells. The amount of pp95 within the cells after GH treatment was positively correlated with the number of GHRs on the cells. Tyrosine phosphorylation of pp95 could not be induced by prolactin, insulin, insulin-like growth factor I, interleukin 2, epidermal growth factor, platelet-derived growth factor, or fibroblast growth factor. Phosphorylation of pp95 was found to be a rapid event that could be observed 60 sec after GH treatment. Also, pp95 appears to exist as a complex of two proteins, i.e., pp95 and pp96. The GH-induced response by these cells may be of use in screening GH analogs for biological activity.

Animals↗