SEARCH · PubMed Health

Results for “Recombinant expression”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources

PubMed1991

Mapping of the epitopes of Epstein-Barr virus gp350 using monoclonal antibodies and recombinant proteins expressed in Escherichia coli defines three antigenic determinants.

The Epstein-Barr virus (EBV) major surface membrane antigen (MA), gp350/220, induces antibodies that neutralize virus infectivity in vitro. The MA glycoprotein is encoded by nucleotides 1784 to 4504 of the BamHI L fragment of the EBV genome. To define the antigenic epitopes on gp350, sequences encoding portions of the protein were cloned into an Escherichia coli expression system and eight recombinant clones were generated, two overlapping clones representing the C terminus and six overlapping clones representing the N terminus. The epitopes expressed by the recombinant proteins were mapped using 14 anti-MA monoclonal antibodies (MAbs) in a dot blot immunoassay. One of the MAbs reacted with clones that express the C terminus of gp350 and three others reacted with clones expressing the N terminal portion of the protein; the remaining MAbs tested were not reactive with the cloned proteins. The data identify three antigenic determinants on gp350. DNA sequences encoding these epitopes are located between nucleotides 1980 and 2307, 3186 and 3528, and 3528 and 3576 of the BamHI L fragment. In an attempt to elicit neutralizing antibodies, rabbits were immunized with gel-purified recombinant proteins from four of the clones. Neutralization assays indicate that the proteins expressed by these clones do not induce in vitro virus-neutralizing antibodies.

Animals↗

PubMed1990

Synthesis of bluetongue virus (BTV) corelike particles by a recombinant baculovirus expressing the two major structural core proteins of BTV.

The L3 and M7 genes of bluetongue virus (BTV), which encode the two major core proteins of the virus (VP3 and VP7, respectively), were inserted into a baculovirus dual-expression transfer vector and a recombinant baculovirus expressing both foreign genes isolated following in vivo recombination with wild-type Autographa californica nuclear polyhedrosis virus DNA. Spodoptera frugiperda insect cells infected with the recombinant synthesized large amounts of BTV corelike particles. These particles have been shown to be similar to authentic BTV cores in terms of size, appearance, stoichiometric arrangement of VP3 to VP7 (ratio, 2:15), and the predominance of VP7 on the surface of the particles. In infected insect cells, the corelike particles were observed in paracrystalline arrays. The formation of these structures indicates that neither the BTV double-stranded viral RNA species nor the associated minor core proteins are necessary for assembly of cores in insect cells. Furthermore, the three BTV nonstructural proteins NS1, NS2, and NS3, are not required to assist or direct the formation of empty corelike particles from VP3 and VP7.

Animals↗

PubMed2002

[Construction of recombinant vectors expressing antisense RNA to CCR5 and expression in eukaryotic cells].

BACKGROUND: To construct recombinant vector expressing antisense RNA to CCR5 in eukaryotic cells and obtain recombinant pseudovirus, which will be used to block HIV-1 infection. METHODS: The DNA fragment targeted against the initional part of CCR 5 mRNA translation was amplified by using RT-PCR from peripheral blood mononuclear cells (PBMCs) and cloned into retroviral vector pLXSN, then transfected into packaging cell (PA317) with lipofectAMINE. After 2-3 weeks selecting with G418, the pseudovirion in the survival cell's supernatant was detected with RT-PCR (FQ),then was used to infect NIH/3T3 cell. RESULTS: The psuedovirion packed from expression vector of sense/antisense RNA to CCR5 had infected NIH/3T3 cell successfully. The vector had incorporated into its genome and transcripted into RNA. CONCLUSIONS: The gene fragment of antisense RNA to CCR5 could be obtained from PBMCs and transfected into eukaryotic cell with retroviral vector. The results made a great foundation for studying its inhibiting effect on HIV-1 infection.

3T3 Cells↗

PubMed2003

Functional expression of recombinant tumstatin in stably transformed Drosophila melanogaster S2 cells.

Recombinant tumstatin was expressed in stably transformed Drosophila melanogaster S2 cells and secreted into the medium with a molecular size of 29 kDa. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation. Purified recombinant tumstatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition for recombinant tumstatin was approx. 0.7 microg ml(-1). A maximum production of 4.6 microg recombinant tumstatin (10(7) cells)(-1) was obtained in a T-flask culture of S2 cells, 7 d after induction with 0.5 mM CuSO4.

Animals↗

PubMed2005

Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron.

We have generated transgenic maize plants expressing Aspergillus phytase either alone or in combination with the iron-binding protein ferritin. Our aim was to produce grains with increased amounts of bioavailable iron in the endosperm. Maize seeds expressing recombinant phytase showed enzymatic activities of up to 3 IU per gram of seed. In flour paste prepared from these seeds, up to 95% of the endogenous phytic acid was degraded, with a concomitant increase in the amount of available phosphate. In seeds expressing ferritin in addition to phytase, the total iron content was significantly increased. To evaluate the impact of the recombinant proteins on iron absorption in the human gut, we used an in vitro digestion/Caco-2 cell model. We found that phytase in the maize seeds was associated with increased cellular iron uptake, and that the rate of iron uptake correlated with the level of phytase expression regardless of the total iron content of the seeds. We also investigated iron bioavailability under more complex meal conditions by adding ascorbic acid, which promotes iron uptake, to all samples. This resulted in a further increase in iron absorption, but the effects of phytase and ascorbic acid were not additive. We conclude that the expression of recombinant ferritin and phytase could help to increase iron availability and enhance the absorption of iron, particularly in cereal-based diets that lack other nutritional components.

6-Phytase↗

PubMed1994

Type 1 fimbrial shafts of Escherichia coli and Klebsiella pneumoniae influence sugar-binding specificities of their FimH adhesins.

The type 1 fimbriae of enterobacteria comprise FimA, which constitutes most of the fimbrial shaft, and a cassette of three minor ancillary subunits including FimH, the mannose-binding moiety. The sugar-binding specificities of Escherichia coli and Klebsiella pneumoniae type 1 fimbriae were examined by determining the relative activities of two aromatic mannosides in inhibiting the yeast aggregation caused by the fimbriated bacteria. 4-Methylumbelliferyl alpha-mannoside (MeUmb alpha Man) was approximately 10-fold more effective than p-nitrophenyl alpha-mannoside (p-NP alpha Man) in inhibiting the yeast aggregation caused by the recombinant expressing native E. coli type 1 fimbriae. In contrast, MeUmb alpha Man was only fourfold more effective than p-NP alpha Man in assays employing the recombinant expressing native K. pneumoniae type 1 fimbriae. In order to elucidate the molecular mechanisms underlying the sugar-binding specificities of type 1 fimbriae in the two species, transcomplementation studies were performed and resulted in the creation of recombinants expressing two types of hybrid fimbriae: one consisting of a cassette of minor subunits of E. coli fimbriae borne on a filamentous shaft of K. pneumoniae FimA subunits and the other consisting of a cassette of K. pneumoniae minor fimbrial subunits borne on a shaft of E. coli FimA subunits. Although the heterologous FimH was incorporated into the fimbrial filaments in amounts comparable to those observed in native fimbriae, the hemagglutination activities of recombinants expressing hybrid fimbriae were significantly lower than those of their counterparts bearing native fimbriae. The sugar-binding specificity of the recombinant expressing hybrid fimbriae consisting of an E. coli shaft bearing K. pneumoniae FimH was different from those of recombinants expressing native K. pneumoniae fimbriae in its affinity for the two aromatic sugars but was remarkably similar to the specificities exhibited by recombinants expressing native E. coli fimbriae. Conversely, the sugar-binding specificity of the recombinant expressing hybrid fimbriae consisting of a K. pneumoniae shaft bearing E. coli FimH was different from that of the recombinant expressing native E. coli fimbriae but was very similar to those of recombinants expressing native K. pneumoniae fimbriae. We conclude that the differences in the sugar-binding specificity between E. coli and K. pneumoniae FimH fimbrial subunits is influenced by the fimbrial shafts which carry the adhesin molecules in a functionally competent form at the distal tips.

Adhesins, Escherichia coli↗

PubMed1998

[Early diagnosis of nasopharyngeal carcinoma using recombinant antigens expressed in bacteria].

An enzyme-linked immunosorbent assay(ELISA) was developed by using recombinant purified early antigens, EA-D and EA-R to detect IgA antibodies in sera from nasopharyngeal carcinoma(NPC) patients. 30 sera from NPC patients and 49 sera from normal persons were detected by the developed ELISA. The serum positive rate and antibody titer were compared with immunoenzymatic method(IE) on slide with Raji cells mears. ELISA is more sensitive and specific. The serum positive rate of NPC patients was increased from 70% by IE to 100% by ELISA and 77% of the serum antibody titer reached > or = 1:200. The results show that the expressed recombinant p138 and p54 antigens are effective for detection of EA/IgA antibody in sera from NPC patients and ELISA will be a sensitive, specific and convenient method for early diagnosis of NPC.

Antibodies, Viral↗

PubMed2003

Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients.

A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Immunogenicity of viral B- and T-cell epitopes expressed in recombinant bacterial proteins.

Foreign polypeptides can be expressed as genetic inserts in several permissive sites of MalE and LamB, two Escherichia coli envelope proteins. Several viral B and T-cell epitopes have been inserted in these proteins and we analyzed the role of the molecular environment on the immunogenicity of the foreign epitopes. These studies demonstrated that the antigenicity and immunogenicity of B-cell epitopes depend on their site of insertion in the carrier protein. Using bacteria expressing B-cell epitopes either at the cell surface or in the periplasm, it was also shown that the cellular location of a foreign B-cell epitope expressed by recombinant bacteria determines its T-cell dependent or independent characteristics. Analysis of in vivo immunogenicity of purified LamB or MalE hybrid proteins expressing two different T-cell epitopes established that the immunogenicity of recombinant T-cell epitopes may be strongly affected by both the insertion site and inserted adjacent residues. The in vitro analysis of specific T-cell hybridoma response to hybrid MalE proteins also showed that the molecular context of a T-cell determinant alters the diversity of its T-cell recognition.

ATP-Binding Cassette Transporters↗

PubMed1993

A method for producing recombinant baculovirus expression vectors at high frequency.

A system has been developed that can generate recombinant baculovirus expression vectors at frequencies approaching 100%. This system provides a selection for recombinant viruses by using the essential gene downstream of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin expression locus. Two AcMNPV derivatives were constructed in which the expression locus and part of the downstream gene are flanked by restriction sites. The parental viruses are viable; however, restriction of the viral DNAs removes an essential piece of the viral genome. Transfer vectors carry a copy of the missing sequences downstream from the site into which foreign genes are inserted for expression; hence, recombination between a transfer vector and the restricted viral DNA can restore the integrity of the essential gene. Such recombination events also transfer any foreign gene present in the expression locus of the transfer vector to the viral genome. Recombinant viruses therefore have a selective advantage over nonrecombinant viral DNAs. Consequently, a high proportion of the viruses obtained by co-transfecting transfer vector DNA and restricted viral DNA of one of these new viruses expresses the target gene from the transfer vector. This system greatly reduces the time needed to make recombinant baculovirus expression vectors.

Animals↗

PubMed1991

Synthesis and immunogenicity of hepatitis B virus envelope antigen expressed by recombinant vaccinia virus. Finding of retention signal in the C-terminal portion of the preS1 domain of subtype adyw.

Five different recombinant vaccinia viruses expressing the envelope antigen of hepatitis B virus (HBsAg) under the control of the P7.5 promoter were constructed. Cell cultures infected with some of the recombinant viruses synthesized both middle (M) and major surface (S) protein of HBsAg. It was shown that the length of the nontranslated sequence preceding preS2-ATG influenced the extracellular or intracellular HBV antigen distribution and the preS2:S antigen ratio. Some recombinants synthesized an M protein that was enlarged by additional 35 amino acids of preS1 domain and was entirely retained within the infected cells. Antibody responses to the S and preS2 antigens in mice revealed significant differences in the immunogenicity of individual recombinants.

Animals↗

PubMed1991

In vivo effects of recombinant human granulocyte colony-stimulating factor on normal neutrophil function and membrane effector molecule expression.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is now undergoing clinical trials. We investigated the effects of rhG-CSF on the function of neutrophils in vivo in healthy volunteers. rhG-CSF (0.5 micrograms/kg) was injected subcutaneously for 6 consecutive days. The number of neutrophils in peripheral blood decreased transiently within an hour, and thereafter increased 2-10-fold compared to the control 6 to 8 h after injection. The circulating neutrophils remaining during this early neutropenic period showed increases in such functions as random motility, chemotaxis, phagocytosis and superoxide anion production. On the other hand, the function of neutrophils which increased 6 to 8 h after rhG-CSF injection was normal. No decrease of neutrophil function was observed following the use of rhG-CSF. CD33-positive cells increased 3 days after rhG-CSF administration. CD11a (LFA-1) expression on the membranes circulating neutrophils decreased 6 h after rhG-CSF administration. This phenomenon suggested that neutrophils adhered to the reticuloendothelial system during neutropenia, and that there was an influx of CD11a-negative mature cells into the circulatory pool thereafter. All our findings suggest that rhG-CSF enhances the function of normal neutrophils in vivo, and that it is effective against microbial infection very soon after administration.

Adult↗

PubMed1992

Trypanosoma cruzi flagellar repetitive antigen expression by recombinant baculovirus: towards an improved diagnostic reagent for Chagas' disease.

We constructed a recombinant baculovirus that expressed part of a Trypanosoma cruzi flagellar repetitive antigen (FRA). Both cell- associated and secreted forms of recombinant FRA were detected in cultures of virus-infected Spodoptera frugiperda (Sf9) cells. These forms show a complex pattern after polyacrylamide gel electrophoresis and Western blot analysis using either an anti-FRA rabbit serum or human Chagasic sera. Competitive Western-blot experiments revealed that all bands react with the same antibodies as a bacterially-derived FRA. Polymerase chain reaction and Southern blots of the recombinant viral DNA also showed a complex pattern, suggesting the presence of more than one repeat unit in the viral genome. When tested against a panel of human sera from an endemic area for Chagas' disease, FRA recombinant-Sf9 culture supernatant showed the same reactivity as purified FRA produced in bacteria.

Animals↗

PubMed1992

Improvement of recombinant gene expression in Escherichia coli for glucose-controlled continuous and fed-batch cultures.

Escherichia coli TG1 (pHRW500) permanently expressed the human interferon alpha 1 gene (ifn alpha 1) directed by the tryptophan promoter (trpP.O) during continuous and fed-batch cultivation with a limited supply of glucose. The expression of ifn alpha 1 could be improved after insertion of the catabolite activator region (cap) upstream to trpP.O during cultivation of the modified E. coli TG1(pHRW500cap) in glucose-controlled continuous and fed-batch cultures. The cap-mediated stimulatory effect on the expression of cap-trpP.O-ifn alpha 1 increased with decreasing dilution rate. These results are in line with the increase in the level of cAMP with declining dilution rate and the well-known positive effects of cAMP-catabolite gene activator protein (CAP) at the transcriptional level. In addition, expression of the galactokinase gene (trpP.O-galK) in E. coli TG1(pDR720) could be improved in the same way with cap-trpP.O-galK in E. coli TG1(pDR720cap). Determinations of plasmid copy numbers, cellular amounts of galactokinase-mRNA, activity of galactokinase (AGalK) and the concentration of galactokinase at various dilution rates (D) strengthen the conclusion that the increase in AGalK with decreasing D was indeed due to the cap-mediated enhancement of transcription of the galK gene. We suggest that expression of other recombinant genes directed by various promoters that allow permanent transcription during growth with limited glucose supply in chemostat and fed-batch fermentors can be improved by appropriate insertion of the cap region.

Base Sequence↗

PubMed1993

Expression of the La Crosse M segment proteins in a recombinant vaccinia expression system mediates pH-dependent cellular fusion.

To study the expression of La Crosse virus (LAC) glycoproteins, G1 and G2, we constructed a cDNA copy of the open reading frame (ORF) of the middle RNA segment and expressed it in a recombinant vaccinia virus (VV.ORF). Cells infected with VV.ORF expressed G1 and G2 at the cell surface and formed syncytia with a pH profile similar to that of LAC. These experiments provide a system of studying the biological functions of the LAC glycoproteins, including processing, targeting, fusion, receptor binding, and antigenicity.

Animals↗

PubMed1989

HIV-1 core proteins expressed from recombinant vaccinia viruses.

The sequences encoding the core proteins p55, p25, and p18 of the human immunodeficiency virus (HIV-1) have been inserted into the vaccinia virus genome. Infection of cultured cells with the live recombinant viruses led to the expression of proteins that were recognized by sera from HIV-seropositive individuals. Immunization of mice with the recombinant virus expressing the HIV p25 protein and the p55 precursor yielded high levels of antibodies directed against the corresponding HIV antigens. The data obtained are discussed in terms of the possible use of these live recombinant viruses in the development of a strategy toward an AIDS vaccine.

Animals↗

PubMed1993

Application of a recombinant baculovirus expressing hantavirus nucleocapsid protein as a diagnostic antigen in IFA test: cross reactivities among 3 serotypes of hantavirus which causes hemorrhagic fever with renal syndrome (HFRS).

Recombinant baculovirus-infected insect cells which expressed recombinant protein, analogous to the nucleocapsid protein (NP) of Seoul type hantavirus, strain SR-11 (rNP-SR-Sf9) were applied to the indirect immunofluorescent antibody (IFA) test. The rNP-SR-Sf9 reacted with anti NP MAb clones which recognized strain specific or hantavirus common epitopes in the IFA test. The recombinant antigen was detected by antibodies to 3 serotypes of hantaviruses (Hantaan 76-118, SR-11, Puumala). Antibody titers of a group of experimentally infected mouse and urban rat sera using rNP-SR-Sf9 and strain SR-11-infected Vero cell antigens were mutual correlative. These results indicated that the rNP-SR-Sf9 were an effective and safety substitute for Vero E6 cells in the IFA test for serosurveys of hantavirus infection.

Animals↗

PubMed1995

Atypical porcine type I interferon. Biochemical and biological characterization of the recombinant protein expressed in insect cells.

A recombinant baculovirus was designed to express short porcine type I interferon (spI interferon), a novel and atypical type I interferon that was recently described as the product of a gene transcribed in pig trophoblast at the time of implantation in the uterus [Lefèvre, F. & Boulay, V.C. (1993) J. Biol. Chem. 268, 19,760-19,768]. The recombinant protein, secreted into the culture medium of Sf9 cells at 3 days post infection (60,000 IU/ml), was purified by ion-exchange and reverse-phase HPLC. N-terminal sequencing confirmed the predicted signal peptide cleavage site and therefore the size of the mature protein (149 amino acids), the shortest of all reported type I interferons. Purified spI interferon, with a specific antiviral activity using Madin-Darby bovine kidney cells of 3.7 x 10(7) IU/mg, is an N-glycosylated monomer of 19 kDa that possesses several physicochemical characteristics of interferons: (a) disulfide bonds are necessary for bioactivity; spI interferon is thermolabile, stable at pH 2, and able to renature after complete denaturation (1% 2-mercaptoethanol, 1% SDS, and 5 M urea); (b) the carbohydrate chain is not essential for bioactivity since no loss of antiviral activity is observed following complete deglycosylation. In this study, antiviral and anti-proliferation activities of spI interferon in cell culture were compared with those of other interferons, especially with porcine type 1 interferon-alpha. A major difference with porcine type 1 interferon-alpha was that spI interferon was not active on human cells in either test, and it was relatively more active on pig cells compared to bovine cells than porcine type 1 interferon-alpha. Serological cross-neutralization results obtained with anti-(spI interferon) serum confirmed that several members of interferon families are not antigenically related to spI interferon, in agreement with previous observations; this provides further evidence that spI interferon could represent a new family of type I interferon.

Amino Acid Sequence↗