Development of a monoclonal antibody specific for the gamma chain of the T-cell antigen receptor using an open reading frame expression vector.
To develop an anti-framework monoclonal antibody (mab) specific for the gamma (gamma)-chain of the T-cell antigen receptor (TCR), we expressed a part of the constant region of the gamma-chain (C gamma 2 gene segment) in E. coli using the pWR590 vector. This plasmid contains the E. coli lac promoter, operator, a truncated beta-galactosidase (beta-gal) gene (coding for the first 590 of the 1,007 amino acids of the beta-gal) and a polylinker region (at the 3' end of the beta-gal) containing nine restriction sites. These can be cleaved by any one of eight common restriction enzymes, permitting the introduction of the DNA fragment of interest. We employed the pT gamma 1 gamma-chain cDNA probe, which like the vast majority of the gamma-chain specific probes is aberrant and contains an in-frame stop codon at the junction of V and J regions. Computer analysis of the pT gamma 1 sequence revealed several MaeIII restriction sites that could result in a number of fragments. One of these fragments consisted of 245 base pairs (nucleotides 404-648) and contained most of the CI exon of the C gamma 2. Successful insertion of this fragment to the pWR590 vector was confirmed using restriction enzyme analysis. The C gamma insert was 12% of the construct. Expression of the pWR590-HpT gamma 1 recombinant plasmid in E. coli followed by SDS-PAGE analysis revealed a hybrid protein with a molecular weight of 85 kd which constituted at least 25% of the total E. coli insoluble protein. In contrast, cells transformed with the control pWR590 vector without insert expressed a 78 kd polypeptide chain. We developed several mabs against the pWR590-HpT gamma 1 hybrid protein by fusing spleen lymphocytes from BALB/c mice immunized with the pWR590-HpT gamma 1 protein, with cells of the NS1 mouse myeloma cell line. Screening of the mabs was carried out by ELISA against the pWR590-HpT gamma 1 hybrid protein and the control pWR590 beta-gal protein (beta-gal 590), derived by expressing in E. coli the pWR590 vector without gamma-chain insert. Two groups of mabs were obtained, those reacting with the pWR590-HpT gamma 1 hybrid protein only and those reacting with both the hybrid and the control beta-gal 590 proteins. The specificity of these mabs was further studied by Western blotting with similar results.(ABSTRACT TRUNCATED AT 400 WORDS)
Nanno
M
M
Department of Immunology, University of Texas, M.D. Anderson Cancer Center, Houston 77030.
Seki
H
H
Bao
Y D
YD
Ioannides
C D
CD
Morkowski
J
J
Platsoucas
C D
CD
D000818:Animals; D000911:Antibodies, Monoclonal; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D010957:Plasmids; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D011948:Receptors, Antigen, T-Cell; D016692:Receptors, Antigen, T-Cell, gamma-delta; D011994:Recombinant Proteins