Showing papers by "Richard Lathe published in 1984"

Expression of rabies virus glycoprotein from a recombinant vaccinia virus

Abstract: Rabies is one of the oldest diseases know to man, but its successful control has remained elusive. Although effective vaccines of tissue culture origin against rabies do exist, such preparations are expensive. Live vaccinia virus (VV) recombinants expressing influenza or hepatitis B antigens have recently been used to immunize against these diseases. We have now used this approach to produce a novel rabies vaccine. We first altered the rabies glycoprotein cDNA by site-directed mutagenesis and removed the poly(dG) tail. We then aligned the modified cDNA with an early VV promoter sequence inserted within a cloned copy of the vaccinia thymidine kinase gene and transfected this plasmid into VV-infected cells. Recombination between the virus and the plasmid resulted in a recombinant virus harbouring the rabies glycoprotein cDNA. Inoculation of rabbits with the live recombinant virus induced high titres of rabies virus-neutralizing antibodies, and scarification with the recombinant VV protected mice against challenge with street rabies virus.

Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene

Abstract: Inoculation of rabbits and mice with a vaccinia-rabies glycoprotein recombinant (V-RG) virus resulted in rapid induction of high concentrations of rabies virus-neutralizing antibodies and protection from severe intracerebral challenge with several strains of rabies virus. Protection from virus challenge also was achieved against the rabies-related Duvenhage virus but not against the Mokola virus. Effective immunization by V-RG depended on the expression of a rabies glycoprotein that registered proline rather than leucine as the eighth amino acid from its NH2 terminus (V-RGpro8). A minimum dose required for effective immunization of mice was 10(4) plaque-forming units of V-RGpro8 virus. beta-propiolactone-inactivated preparations of V-RGpro8 virus also induced high levels of rabies virus-neutralizing antibody and protected mice against intracerebral challenge with street rabies virus. V-RGpro8 virus was highly effective in priming mice to generate a secondary rabies virus-specific cytotoxic T-lymphocyte response following culture of lymphocytes with either ERA or PM strains of rabies virus.

Linker tailing: unphosphorylated linker oligonucleotides for joining DNA termini

Abstract: Current procedures for inserting linker oligonucleotides between DNA termini involve terminal addition of linker duplexes followed by restriction enzyme cleavage and religation. Such methods have the disadvantage that target DNA molecules containing internal recognition sites are cut during the linker cleavage step. We describe a method for adding linker oligonucleotides to DNA termini which eliminates the requirement for subsequent restriction enzyme treatment. Nonphosphorylated linker duplexes are ligated to target DNA termini on one strand alone. Removal of the unligated strand generates a single-stranded protrusion at each terminus. Two such tailed termini may now be linked by annealing their single-stranded complementary tails, resulting in a hybrid DNA molecule containing a single linker insertion. Each step of the procedure has been studied to optimize the recovery of recombinant molecules, and we present examples of how the method may be applied.

High-level production of biologically active human alpha 1-antitrypsin in Escherichia coli.

Abstract: A cDNA clone containing the complete human alpha 1-antitrypsin sequence was isolated from a human liver cDNA bank by screening with a chemically synthesized oligonucleotide probe DNA sequences encoding the alpha 1-antitrypsin mature polypeptide were inserted into an Escherichia coli expression vector that allows transcription from the efficient leftward promoter of bacteriophage lambda (PL) and initiation of translation at the lambda cII gene ribosome-binding site This construction resulted in the induction of a 45-kilodalton protein at a level of approximately 15% of total cell protein The polypeptide produced was recognized by antisera raised against human alpha 1-antitrypsin protein and displayed normal biological activity in an in vitro antielastase assay

M13 bacteriophage vectors for the expression of foreign proteins in Escherichia coli: the rabies glycoprotein.

Abstract: The expression of a protein-coding DNA fragment in a microorganism such as Escherichia coli requires that the exogenous DNA segment be inserted precisely in phase with bacterial translation initiation signals. We report the construction of derivatives of the M13 vectors M13mp7 and M13mp701 in which a HindIII site has been inserted, within the N-terminal section of the beta-galactosidase gene, in all three phases of translation. These vectors may thus be used for the expression, under the control of the lac promoter and translation initiation signals, of protein coding DNA segments flanked by a HindIII site. In all cases the insertion of a DNA fragment can be recognized by the abolition of beta-galactosidase activity. These vectors have been used to direct the expression, in E. coli, of a cDNA segment coding for the rabies virus surface glycoprotein. The proteins produced have been shown to react with antisera raised against authentic rabies glycoprotein.

Expression vehicles and their use in the preparation of a protein having human alpha-antitrypsin activity

Abstract: A vector for the clonal selection and the expression of a gene determined in a gram-negative bacteria, comprising: the origin of replication of a bacterial plasmid; a promotor of the bacteriophage lambda : PL, PR or P'R; a region coding for the initiation of the translation; a clonal selection region comprising unic restrictions sites, and a plasmid, comprising all or part of the sequence coding for the human antitrypsin- alpha 1 and particularly a vector as described hereabove.

Vectors for the expression of an antigenic protein of rabies in eucaryotic cells and application thereof to the preparation of a vaccine

Abstract: The vectors for the expression of an antigenic protein of rabies in eucaryotic cells are characterized in that they comprise at least a DNA sequence (1) coding for said antigenic protein of rabies, and the expression elements of said sequence in a eucaryotic cell. Said vectors may be used to transform eucaryotic cells in order to make them produce an antigenic rabies glycoprotein.

NEW EXPRESSION VECTORS AND APPLICATION THEREOF TO THE PREPARATION OF A PROTEIN HAVING THE ACTIVITY OF HUMAN ANTITRYPSIN-alpha1

Abstract: A vector for the clonal selection and the expression of a gene determined in a gram-negative bacteria, comprising: the origin of replication of a bacterial plasmid; a promotor of the bacteriophage lambda: PL, PR or P"R; a region coding for the initiation of the translation; a clonal selection region comprising unic restrictions sites, and a plasmid, comprising all or part of the sequence coding for the human antitrypsin-alpha1 and particularly a vector as described hereabove.

Vectors for the expression of an antigenic rabies protein in eukaryotic cells, and their use in the preparation of a vaccine

Abstract: The vectors for the expression of an antigenic protein of rabies in eucaryotic cells are characterized in that they comprise at least a DNA sequence (1) coding for said antigenic protein of rabies, and the expression elements of said sequence in a eucaryotic cell. Said vectors may be used to transform eucaryotic cells in order to make them produce an antigenic rabies glycoprotein.