Generation of m11 chimeric Fc protein and m11-specific antibodies
The MCMV m11–Fc (also referred to as vCD44BP–Fc) fusion protein was generated using Sew-PCR to attach the Fc portion of human IgG1 to the part of the m11 gene that encodes its extracellular domain (amino acids 1–212). The construct was transiently transfected into CV-1/EBNA cells and the soluble fusion protein purified using protein-A-Sepharose (Amersham Pharmacia Biotech). A human TRAIL–Fc fusion protein was used as a negative control. A monoclonal antibody (M-627) to m11 was generated by immunizing mice with the m11–Fc protein and fusing splenocytes with a myeloma partner using standard methodologies. The specificity of M-627 was verified using m11 transfected cells and by comparing cells infected with wild-type MCMV and a mutant lacking m11, referred to as ΔvCD44BP—see below. The anti-m11 monoclonal antibody (7G5) was generated by immunizing rats with the m11 ectodomain (amino acids 28–164) and fusing splenocytes with X63 myeloma cells using standard methodologies. Supernatants from hybridoma clones were initially screened by ELISA using plate bound m11–Fc protein and a secondary screen performed by flow cytometry using fibroblasts infected with either MCMV or ΔvCD44BP virus.
Expression cloning of the m11 cognate from a CD40 ligand-stimulated B cell cDNA library
A cDNA library was generated from mouse B cells stimulated with CD40 ligand and cloned into the pDC409 vector using previously described methods53. Approximately 200 pools, each containing ~2,000 clones, were transfected into CV-1/EBNA cells and 2 days later the transfected cells screened using the m11–Fc protein as described54. One positive pool was identified, and this was subdivided into smaller pools until a single positive cDNA was obtained from the original pool. Individual clones were then sequenced, and sequences were compared to public DNA databases.
Construction and cloning of m11 for transfection
The MCMV m11 open reading frame (ORF) was amplified by PCR based on published sequences (accession number AM886412) using purified MCMV DNA templates. The m11 PCR product was cloned into the pDC409 mammalian expression vector54 to generate p409-m11.
Cell lines
COS-7 and EL4 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Gibco); IC-21 cells were cultured in Roswell Park Memorial Institute (RPMI-1640, Gibco); M2-10B4 and mouse embryonic fibroblasts (MEFs) were cultured in minimal essential medium (MEM, Gibco). All culture media were supplemented with 10% fetal calf serum (FCS) (Gibco) (COS-7, EL4 and IC-21) or 10% newborn calf serum (NCS) (M2-10B4 and MEF), and antibiotics (penicillin 100 µg ml−1, CSL; gentamycin 40 µg ml−1, Pharmacia & Upjohn).
The fibroblastic reticular cell line (FRC2) was generated by A.L.F. by isolating FRCs from the lymph nodes of C57BL/6J mice as described55 and FRC enriched via CD45 and CD31 depletion (Miltenyi Biotec). Stromal cells were plated overnight in alpha-MEM with 10% FBS (Gibco) and then co-transfected using Lipofectamine 3000 with a piggyBac expression vector expressing SV40LT with GFP and a piggyBac transposase expression vector, hyPBase (The Sanger Centre, pCM-hyPBase).
CD44–hyaluronic acid binding assay
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