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Results for “Recombinant expression”

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At least 19 recordsLinked to original sources

PubMed1991

Recombinant expression of human follistatin with 315 and 288 amino acids: chemical and biological comparison with native porcine follistatin.

Follistatin is a glycosylated monomeric protein originally isolated from ovarian follicular fluid based on its ability to specifically inhibit pituitary FSH release. To further explore the physiological role of follistatin, we have expressed recombinant human follistatins with 315 (rhFS-315) and 288 (rhFS-288) amino acids in Chinese hamster ovary cells under the control of the simian virus-40 promoter. The two types of FS originated from alternatively spliced mRNAs and rhFS-315 differed from rhFS-288 by having an extra 27-amino acid sequence at the carboxyl-terminal. The yield of the purified rhFS-315 and rhFS-288 after a single step of affinity chromatography on an activin-coupled Affi-Gel column was 3-5 mg/liter conditioned medium. Using the rhFS-315 and rhFS-288 as molecular mass markers, Western blotting with FS carboxyl-terminal-specific antibodies demonstrated that the majority of native FS isolated from porcine ovarian follicular fluid was neither FS-315 nor FS-288, but was composed of 300 amino acids in various forms of glycosylation. This finding is consistent with our earlier results obtained from tryptic peptide fragment analysis of native FS. Only a very small percentage (less than 1%) of native porcine FS was FS-288. In cultures of rat anterior pituitary cells, rhFS-315 (ED50, 115.2 +/- 16.2 pM) is equipotent to native porcine FS (ED50, 86.7 +/- 14.1 pM) on the suppression of FSH release, but, surprisingly, rhFS-288 (ED50, 9.6 +/- 2.2 pM) is 8-10 times more potent than the native protein, similar to the potency of inhibin-A (ED50, 8.6 +/- 0.9 pM). Interestingly, when the in vivo FSH-suppressing activity of rhFS-288 was compared to that of inhibin-A in 1-week ovariectomized adult rats, it was found that rhFS-288 was more potent and longer acting than inhibin-A. Hence, these results indicate that FS-288 is probably one of the most potent natural FSH suppressors.

Amino Acid Sequence↗

PubMed1991

Immunization with baculovirus-expressed recombinant rotavirus proteins VP1, VP4, VP6, and VP7 induces CD8+ T lymphocytes that mediate clearance of chronic rotavirus infection in SCID mice.

Clearance of chronic murine rotavirus infection in SCID mice can be demonstrated by adoptive transfer of immune CD8+ T lymphocytes from histocompatible donor mice immunized with a murine homotypic rotavirus (T. Dharakul, L. Rott, and H.B. Greenberg, J. Virol 64:4375-4382, 1990). The present study focuses on the protein specificity and heterotypic nature of cell-mediated clearance of chronic murine rotavirus infection in SCID mice. Heterotypic cell-mediated clearance was demonstrated in SCID mice infected with EDIM (murine) rotavirus after adoptive transfer of CD8+ T lymphocytes from BALB/c mice that were immunized with a variety of heterologous (nonmurine) rotaviruses including Wa (human, serotype 1), SA11 and RRV (simian, serotype 3), and NCDV and RF (bovine, serotype 6). This finding indicates the serotypic independence of T-cell-mediated rotavirus clearance. To further identify the rotavirus proteins that are capable of generating CD8+ T cells that mediate virus clearance, donor mice were immunized with SF-9 cells infected with a baculovirus recombinant expressing one of the following rotavirus proteins: VP1, VP2, NS53 (from RF), VP4, VP7, NS35 (from RRV), VP6, and NS28 (from SA11). SCID mice stopped shedding rotavirus after receiving CD8+ T cells from mice immunized with VP1, VP4, VP6, and VP7 but not with VP2, NS53, NS35, NS28, or wild-type baculovirus. These results suggest that heterotypic cell-mediated clearance of rotavirus in SCID mice is mediated by three of the major rotavirus structural proteins and by a putative polymerase protein.

Animals↗

PubMed1991

Two vaccinia virus recombinants expressing HBsAg with different concentration of A- and pre-S2 antigenic determinants.

Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M-protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L-pre-S2/15 induced anti-HBsAg a-determinant antibody as well.

Animals↗

PubMedDate not supplied

Live vaccinia virus recombinants expressing herpes simplex virus genes.

Vaccinia virus recombinants expressing antigens from herpes simplex virus (HSV) have been tested as potential live virus vaccines for prevention of HSV infection. We describe three vaccinia virus/HSV recombinants. The first expresses the HSV-1 glycoprotein D (vaccinia/gD), the second expresses the HSV-1 glycoprotein B (vaccinia/gB), and the third expresses both the HSV-1 glycoprotein D and the influenza A hemagglutinin (vaccinia/HSVgD/influenza). Mice immunized with vaccinia/gD or vaccinia/gB developed antibodies capable of neutralizing HSV in vitro and were protected against both lethal and latent infection with HSV. Protection against HSV challenge persisted for greater than 1 year in mice immunized with vaccinia/gD. The immune response to HSV in mice immunized with vaccinia/gD could be increased by a booster vaccination with vaccinia/gD. However, the immune response to HSV was decreased in animals immunized with a vaccinia recombinant that expressed non-HSV genes before vaccination with vaccinia/gD. In separate experiments, a bivalent vaccinia recombinant, vaccinia/HSVgD/influenza, was constructed and was found to be comparable to the vaccinia/gD single recombinant in immunogenicity and protective efficacy against lethal HSV challenge. We conclude that vaccinia/HSV recombinants can provide protection against HSV infection in mice and that these recombinants may provide an alternative approach in the development of a live virus vaccine against HSV.

Animals↗

PubMed1992

Toxicological significance of dog liver cytochrome P-450: examination with the enzyme expressed in Saccharomyces cerevisiae using recombinant expression plasmid.

A complementary DNA (cDNA) coding for a form of beagle dog cytochrome P-450 (Dah1), which is the orthologue to the CYP1A1 cDNA of rat, mouse and human, was inserted between the alcohol dehydrogenase (ADH) promoter and terminator regions of the yeast expression vector pAAH5. On introduction of the resulting recombinant plasmid pDC-1, Saccharomyces cerevisiae strain AH22 cells synthesized up to 1.5 x 10(5) molecules per cell of cytochrome P-450 protein (P-450(Dah1)). The carbon monoxide-bound reduced form of P-450(Dah1) showed an absorption peak at 447 nm and specific content of P-450(Dah1) was about 0.1 nmole P-450 per mg of microsomal protein. P-450(Dah1) cross-reacted with antibodies to rat P-448-H (CYP1A2) and dog P-450-D2 (CYP1A2). P-450(Dah1) activated 2-amino-3-methyl-imidazo[4,5-f]quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) most efficiently in the umu test and exhibited a high activity of aryl hydrocarbon hydroxylase toward benzo[a]pyrene.

Animals↗

PubMed1987

The mechanisms of antiviral immunity induced by a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: clearance of local infection.

We have shown that immunization of mice with a vaccinia virus recombinant expressing glycoprotein D of Herpes simplex virus (HSV)-1 will induce a variety of L3T4+ T cell responses. These included a HSV-specific delayed-type hypersensitivity response, T cell help for the induction of antiviral antibodies, and the ability to eliminate a challenge dose of HSV from the pinna. This protection against a subcutaneous virus challenge was not mediated by the delayed-type hypersensitivity response because intravenous inoculation of the vaccinia virus recombinant expressing HSV-1-gD induced a state of split tolerance. Thus, mice could still clear a HSV challenge inoculum from the pinna yet were unable to mount a HSV-specific delayed-type hypersensitivity response. Evidence is presented that suggests the protective response was, at least, in part mediated by a T cell-dependent induction of virus-neutralizing antibodies. Evidence is also presented that may suggest the failure of a vaccinia virus recombinant expressing HSV-1-gD to induce HSV-specific cytotoxic T cell responses appears to minimize the protective response to only efficiently clearing low 10(4) 50% tissue culture infective dose) challenge populations of virus. These findings are discussed with relevance to the immune control of HSV infections and to the future development of anti-HSV vaccines.

Animals↗

PubMed1992

Protection against Marek's disease by a fowlpox virus recombinant expressing the glycoprotein B of Marek's disease virus.

Fowlpox virus (FPV) recombinants expressing the glycoprotein B and the phosphorylated protein (pp38) of the GA strain of Marek's disease virus (MDV) were assayed for their ability to protect chickens against challenge with virulent MDV. The recombinant FPV expressing the glycoprotein B gene elicited neutralizing antibodies against MDV, significantly reduced the level of cell-associated viremia, and, similar to the conventional herpesvirus of turkeys, protected chickens against challenge with the GA strain and the highly virulent RB1B and Md5 strains of MDV. The recombinant FPV expressing the pp38 gene failed to either elicit neutralizing antibodies against MDV or protect the vaccinated chickens against challenge with MDV.

Animals↗

PubMed1991

Survival and differentiation within the adult mouse striatum of grafted rat pheochromocytoma cells (PC12) genetically modified to express recombinant beta-NGF.

Rat pheochromocytoma PC12 cells were genetically modified in vitro to express recombinant beta-nerve growth factor (beta-NGF) using a replication-deficient retroviral vector carrying the mouse beta-NGF gene and subsequently implanted into the striatum of a mouse model of Parkinson's disease. The fate of the genetically modified PC12 cells (PC12N.8) was assessed at varying times postimplantation by studying immunoreactivity (IR) to tyrosine hydroxylase (TH) or the rat NGF receptor (NGFR). In vitro, the genetically modified PC12 cells displayed a neuronal morphology in the absence of exogenous NGF which was characterized by extensive neurite outgrowth. In addition, the genetically modified PC12 displayed a catecholaminergic phenotype in vitro as assessed by TH-IR. Following implantation into the striatum, the survival of PC12N.8 cells was limited. Surviving cells could be identified by NGFR-IR, but not by TH-IR. In addition, PC12N.8 cells with a neuronal morphology similar to that observed in vitro were only rarely observed in vivo. No tumors were observed in PC12N.8 graft recipients up to 30 days postimplantation. In contrast, intrastriatal tumors were observed in 50% of the PC12 cell recipients. These data demonstrate that PC12 cells genetically modified in vitro to synthesize beta-NGF do not revert to the mitotic phenotype of the parent PC12 cell line following implantation into the adult striatum, an observation that suggests that these cells may continue to express recombinant beta-NGF in vivo. The data further suggest that the genetically modified PC12 cells lose the catecholaminergic phenotype following implantation into the striatal parenchyma.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Gland Neoplasms↗

PubMed1991

Oral vaccination of skunks with raccoon poxvirus recombinants expressing the rabies glycoprotein or the nucleoprotein.

Twenty nine skunks (Mephitis mephitis) were vaccinated orally with raccoon poxvirus (RCN) recombinants: 10 with a recombinant expressing the rabies virus glycoprotein (RCNRG), 10 with RCNRG mixed with a recombinant expressing the rabies virus nucleoprotein (RCNRN) and nine with RCN alone. Rabies virus neutralizing antibodies were detected in six of the 20 skunks; five skunks (three given RCNRG, two given a mixture of recombinants) survived a rabies challenge that was lethal for nine skunks vaccinated with RCN alone.

Administration, Oral↗

PubMed1991

Raccoon poxvirus recombinants expressing the rabies virus nucleoprotein protect mice against lethal rabies virus infection.

Raccoon poxvirus (RCN) recombinants expressing the rabies virus internal structural nucleoprotein (RCN-N) protected A/WySnJ mice against a lethal challenge with street rabies virus (SRV). Maximum survival was achieved following vaccination by tail scratch and footpad (FP) SRV challenge. RCN-N-vaccinated mice inoculated in the FP with SRV were resistant to infection for at least 54 weeks postvaccination. Protection was also elicited by RCN recombinants expressing the rabies virus glycoprotein (RCN-G). Vaccination with RCN-G evoked rabies virus neutralizing antibody. Rabies virus neutralizing antibody was not detected in RCN-N-vaccinated mice prior to or following SRV infection. Radioimmunoprecipitation assays showed that sera from RCN-N-vaccinated mice which survived SRV infection did not contain antibody to SRV structural protein G, M, or NS. The mechanism(s) of N-induced resistance appears to correlate with the failure of peripherally inoculated SRV to enter the central nervous system (CNS). Support for this correlation with resistance was documented by the observations that SRV-inoculated RCN-N-vaccinated mice did not develop clinical signs of CNS rabies virus infection, infectious SRV was not detected in the spinal cord or brain following FP challenge, and all RCN-N-vaccinated mice died following direct intracranial infection of the CNS with SRV. These results suggest that factors other than anti-G neutralizing antibody are important in resistance to rabies virus and that the N protein should be considered for incorporation with the G protein in recombinant vaccines.

Animals↗

PubMed1988

Successful oral rabies vaccination of raccoons with raccoon poxvirus recombinants expressing rabies virus glycoprotein.

Two infectious raccoon poxvirus (RCN) recombinants for expressing rabies virus surface spike glycoprotein (G) were produced by homologous recombination between raccoon poxvirus DNA and chimeric plasmids previously used for production of vaccinia virus recombinants. Expression of G protein was controlled by vaccinia virus promoter P7.5 (early/late class) or by P11 (late class). Immunoprecipitation of infected cell extracts indicated that both of the RCN recombinants directed faithful expression of G protein. Raccoons that were fed polyurethane baits loaded with either recombinant quickly developed high levels of rabies virus neutralizing antibodies and were protected when challenged with lethal raccoon rabies street virus.

Administration, Oral↗

PubMed1990

Vaccinia recombinants expressing early bovine papilloma virus (BPV1) proteins: retardation of BPV1 tumour development.

Papillomaviruses are aetiological agents of epithelial proliferative diseases in animals and in man. It was previously demonstrated that animals inoculated with live vaccinia recombinants expressing early proteins of polyoma virus resist challenge with polyoma-tumour cells, and this approach has been extended to the development of a vaccine against papillomavirus-transformed cells. Bovine papillomavirus type 1 (BPV1), a virus responsible for dermal lesions in cattle, is a prototype virus of the papillomavirus group. Independent vaccinia recombinant viruses expressing the early E1, E2, E5, E6 or E7 open reading frames of BPV1 were tested for their ability to direct the expression of the corresponding protein in cultured cells. Recombinants were then assessed for their ability to elicit anti-tumour immunity in Fischer rats seeded with BPV1-transformed syngeneic FR3T3 cells. Retardation of tumour growth was observed in animals vaccinated with recombinants expressing E5, E6 or E7.

Animals↗

PubMed1992

Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein.

Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.

Animals↗

PubMed1992

Resistance to respiratory syncytial virus (RSV) challenge induced by infection with a vaccinia virus recombinant expressing the RSV M2 protein (Vac-M2) is mediated by CD8+ T cells, while that induced by Vac-F or Vac-G recombinants is mediated by antibodies.

It was previously demonstrated that the vaccinia virus recombinants expressing the respiratory syncytial virus (RSV) F, G, or M2 (also designated as 22K) protein (Vac-F, Vac-G, or Vac-M2, respectively) induced almost complete resistance to RSV challenge in BALB/c mice. In the present study, we sought to identify the humoral and/or cellular mediators of this resistance. Mice were immunized by infection with a single recombinant vaccinia virus and were subsequently given a monoclonal antibody directed against CD4+ or CD8+ T cells or gamma interferon (IFN-gamma) to cause depletion of effector T cells or IFN-gamma, respectively, at the time of RSV challenge (10 days after immunization). Mice immunized with Vac-F or Vac-G were completely or almost completely resistant to RSV challenge after depletion of both CD4+ and CD8+ T cells prior to challenge, indicating that these cells were not required at the time of virus challenge for expression of resistance to RSV infection induced by the recombinants. In contrast, the high level of protection of mice immunized with Vac-M2 was completely abrogated by depletion of CD8+ T cells, whereas depletion of CD4+ T cells or IFN-gamma resulted in intermediate levels of resistance. These results demonstrate that antibodies are sufficient to mediate the resistance to RSV induced by the F and G proteins, whereas the resistance induced by the M2 protein is mediated primarily by CD8+ T cells, with CD4+ T cells and IFN-gamma also contributing to resistance.

Animals↗

PubMed1992

Generation of monoclonal antibodies to human lymphocyte cell surface antigens using insect cells expressing recombinant proteins.

We have expressed human CD40 and human B7 in insect cells using the baculovirus expression system and have used these insect cells to immunize mice for the generation of monoclonal antibodies. We demonstrate here that specific monoclonal antibodies to human CD40 and human B7 were obtained using this approach. One significant advantage of this method is that immunizing mice with insect cells did not evoke an immune response to human cells and, therefore, EBV-transformed human B cells could be used to screen for specific antibody production by the hybridoma clones.

Amino Acid Sequence↗

PubMed1991

Introduction of vascular smooth muscle cells expressing recombinant genes in vivo.

Vascular smooth muscle cells contribute to the formation of atherosclerotic plaques by proliferating in response to vascular injury and releasing growth-promoting factors. Because their autocrine and paracrine effects are not fully understood, expression of such growth factor genes in specific cell types in vivo would help to determine their mechanism of action. We describe a method to transfer vascular smooth muscle cells expressing recombinant gene products to localized segments of the arterial wall. Vascular smooth muscle cells from the inbred Yucatan minipig were infected in vitro with an amphotropic, replication-defective retrovirus transducing the gene for Escherichia coli beta-galactosidase. Vascular smooth muscle cells expressing this recombinant gene were implanted, using a catheter, into denuded iliofemoral artery segments of pigs in vivo. These arteries subsequently demonstrated beta-galactosidase activity in cells of the intima and media. This method, which provides for the introduction of genetically modified smooth muscle cells, can be used to define the biological effects of recombinant genes in the vessel wall and potentially to provide alternative treatments of vascular diseases.

Animals↗

PubMed1987

Immune response in skunks to a vaccinia virus recombinant expressing the rabies virus glycoprotein.

Striped skunks (Mephitis mephitis) were vaccinated with a vaccinia virus recombinant expressing the rabies virus glycoprotein. Virus neutralizing antibodies to rabies virus were present at 14 days postvaccination by the following routes: scarification (6/6), intramuscular (4/4) and intestinal (5/8). Six out of seven skunks that ate vaccine filled baits had virus neutralizing antibodies at 28 days. When challenged intramuscularly with street virus, the survival rates were 5/7 for the bait-fed group, 4/8 for the intestinal group, 3/4 for the intramuscular group, 5/6 for the animals that were scarified, and 0/8 for controls. This is the first report of a high rate of immunization of skunks with a rabies vaccine administered orally.

Animals↗

PubMed1989

Evaluation of antiviral immunity using vaccinia virus recombinants expressing cloned genes for herpes simplex virus type 1 glycoproteins.

Immunization of mice with vaccinia virus recombinants expressing the glycoproteins B or D of herpes simplex virus type 1 (HSV-1) induced humoral antibody as well as multiple aspects of HSV-1-specific T lymphocyte-mediated responses. However, vaccinated mice were not completely resistant to HSV-1 challenge and were unable to eliminate an epithelial infection rapidly. Evidence is presented which indicates that immunization with either vaccinia virus recombinant, while inducing the necessary protective populations of CD4+ T lymphocytes, fails to induce the complementing CD8+ cytotoxic T lymphocytes necessary for high levels of protection against a primary HSV-1 infection. These findings are discussed with relevance to the future development of anti-HSV vaccines.

Animals↗