ABSTRACT
High- and low-molecular recombinant peptides of LCM virus nucleoprotein, representing individual immunodominant antigenic sites, were obtained in pJC40 expressing vector and studied in solid-phase enzyme immunoassay. 2-7% proteins resultant from total cellular synthesis are recombinant peptides. Comparative analysis of antigenic properties of recombinant peptides and native viral protein showed that recombinant peptides are virtually not inferior to native viral protein in antigenic properties.
Subject(s)
Antigens, Viral/chemistry , Lymphocytic choriomeningitis virus/immunology , Nucleoproteins/chemistry , Peptide Fragments/chemistry , Base Sequence , DNA Primers , Electrophoresis, Polyacrylamide Gel , Genetic Vectors , Immunoenzyme Techniques , Molecular Weight , Nucleoproteins/genetics , Nucleoproteins/immunology , Peptide Fragments/genetics , Peptide Fragments/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunologySubject(s)
Antigens, CD/genetics , Interleukin-8/genetics , Receptors, Interleukin/genetics , 3T3 Cells , Animals , Antigens, CD/metabolism , Blotting, Northern , DNA, Complementary/genetics , Flow Cytometry , Humans , Interleukin-8/metabolism , Mice , Mice, Inbred BALB C , Molecular Weight , Neutrophils/metabolism , Receptors, Interleukin/metabolism , Receptors, Interleukin-8A , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
Construction and using of retrovirus vectors derived from the Moloney murine leukemia virus (MoMuLV) are described. These vectors, designated minimal vectors, contain the left and right long terminal repeats (LTRs), a binding site for proline tRNA, a polypurine tract (PPT), and a dominant marker for selective introduction of vectors into a packaging cell line, but lack the internal sequences of the virus genome. The experiments showed that the minimal vectors can be replicated and that their titer was approximately 1500-fold lower than that of wild-type vectors. The minimal vectors were shown to contain all the cis-acting sequences necessary for correct reverse transcription. One infectious virion. like wild-type viruses, produced only one provirus. Unlike the avian reticuloendotheliosis virus (REV), psi+ and psi(-)-genomes of MoMuLV did not compete for virion proteins in the psi2 packaging cell line. When an insert was introduced into a central part of the lTR U5 region, the titer of the minimal vector remained the same, while the titer of the wild-type vector decreased approximately 40-fold.
Subject(s)
DNA Replication/genetics , Genome, Viral , Moloney murine leukemia virus/genetics , 3T3 Cells , Animals , Genetic Vectors , Mice , Moloney murine leukemia virus/pathogenicity , Repetitive Sequences, Nucleic Acid , Transcription, Genetic , Viral Proteins/metabolism , Virion/genetics , Virion/pathogenicityABSTRACT
Two different techniques were used for labeling cells for subsequent generation of hybrid hybridomata. One of them was described earlier by De Lau and included isolation of an HATSNeoR double mutants of hybridomata cells. We proposed the second one consisting of introduction of dominant selectable genes conferring resistance to genecetin (G418) and hygromycin B into distinct hybridomata by retroviral vectors. Hybrid hybridomata secreting the bispecific antibodies have been obtained by both methods. However, comparison of the methods has revealed that highly efficient retroviral gene transfer of the selectable genes is a simpler and more convenient method for generation of cellular hybrids.
