Development of a Monoclonal Antibody Specific for the Gamma Chain of the T-Cell Antigen Receptor Using an Open Reading Frame Expression Vector

Abstract

To develop an anti-framework monoclonal antibody (mab) specific for the gamma (γ)-chain of the T-cell antigen receptor (TCR), we expressed a part of the constant region of the γ-chain (Cγ2 gene segment) in E. coli using the pWR590 vector. This plasmid contains the E. coli lac promoter, operator, a truncated β-galactosidase (β-gal) gene (coding for the first 590 of the 1,007 amino acids of the β-gal) and a polylinker region (at the 3′end of the β-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 pTγ1 γ-chain cDNA probe, which like the vast majority of the γ-chain specific probes is aberrant and contains an in-frame stop codon at the junction of V and J regions. Computer analysis of the pTγ1 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 Cγ2. Successful insertion of this fragment to the pWR590 vector was confirmed using restriction enzyme analysis. The Cγ insert was 12% of the construct. Expression of the pWR590-HpTγ1 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-HpTγ1 hybrid protein by fusing spleen lymphocytes from BALB/c mice immunized with the pWR590-HpTγ1 protein, with cells of the NSl mouse myeloma cell line. Screening of the mabs was carried out by ELISA against the pWR590-HpTγ1 hybrid protein and the control pWR590 β-gal protein (β-gal 590), derived by expressing in E. coli the pWR590 vector without γ-chain insert. Two groups of mabs were obtained, those reacting with the pWR590-HpTγ1 hybrid protein only and those reacting with both the hybrid and the control β-gal 590 proteins. The specificity of these mabs was further studied by Western blotting with similar results. A mab (clone 3D5; IgG1,κ) which was specific for the pWR590-HpT7l hybrid protein, and exhibited strong