Epstein-Barr disease (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from overexpressing EBNA1. latency [3] and regulates transcription at multiple viral promoters [4]C[6]. The N-terminus of EBNA1 is made up primarily of a 239-amino acid website comprised of a Glycine-Glycine-Alanine (GGA) repeat region. An EBNA1 derivative (referred to as 1553), encoding only fifteen residues from your GGA repeat region, maintains the ability to support replication and transcription in cell tradition [7] and the ability to immortalize B cells [8]. Amino acids 64C89 comprise a transcriptional activation website [9]. The C-terminus of EBNA1 consists of a dimerization website and a DNA binding website. EBNA1 also contains two linking areas (LR1 and LR2), which allow EBNA1 dimers bound to DNA to associate with additional bound EBNA1 dimers and therefore loop intervening DNA sequences or link Rabbit Polyclonal to MASTL two DNA molecules as explained in [12]. Open in a separate window Number 1 Epitope mapping of the N-terminal anti-EBNA1 mAbs.A) The constructs used to analyze mAb reactivity. Full length EBNA1 is definitely shown at the top, numbered according to the B95-8 EBV strain. LR-1 and LR-2 (linking areas 1 & 2) are designated, as well as the GGA repeat region between amino acids 90 and 325. The C-terminus is largely composed of the DNA-binding and dimerization website. 1553 encodes functionally wildtype EBNA1, but lacks the majority of the GGA stretch. The remaining EBNA1 derivatives are derived from the 1553 construct and thus carry the GGA deletion. B) 1891 was transfected into 293 cells and whole cell extract was analyzed by Western blot. The first lane in each series contains purified 1553, the second lane is untransfected 293 whole cell extract (mock), and the third lane is whole cell extract from cells transfected with the 1891 plasmid. Equal concentrations of purified mAbs were used to probed the Western blots. C) Plasmids 2728 and 2729 were transfected into 293 cells and whole cell extract was analyzed by Western blot. NT, not transfected. Equal concentrations of AG-014699 ic50 purified mAbs were used to probe the Western blots. D) Purified EBNA1 derivatives were analyzed by Western blot for mAb reactivity. Equal concentrations of mAb 1EB12 or mAb 1EB14 were used to probe the Western blots. E) Summary of the anti-EBNA1 mAb epitopes. The schematic shows endogenous EBNA1 protein, numbered according to the B95-8 strain of EBV, and the domain structure. The lines represent the various epitope regions and the mAbs that interact in that area are written below. MW is molecular weight. Cells 293 cells are derived from human embryonic kidney cells [21] and were grown in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum, 200 U/ml penicillin, and 200 g/ml streptomycin. AG-014699 ic50 All cells were grown at 37C in a humidified 5% CO2 atmosphere. Transfections Plasmids 1891, 2728, and 2729 were transiently transfected into 293 cells. 5 g of DNA and 5 g of empty vector DNA had been mixed in 500 l Opti-Mem (Invitrogen) and blended with 40 g polyethyleneimine (PEI) in 500 l Opti-Mem. The perfect solution is was put into 10 ml of cells, and incubated at 23C for 20 min. Cells had been permitted to grow 48 h at 37C AG-014699 ic50 and gathered. MAbs and Hybridomas For the isolation of hybridomas that create EBNA1-particular mAbs, Ni-NTA-purified recombinant EBNA1 was ready as referred to in [12] and injected into Balb/c ByJ mice (Jackson Lab, Bar Harbor, Me personally) based on the pursuing plan: four feminine mice had been each injected on day time 1 with 5 g, on day time 14 with 10 g, and AG-014699 ic50 on day time 28 with 20 g. The 1st injection was within Freund’s full adjuvant, and following injections were within Freund’s imperfect adjuvant. Each shot (100 l) was given subcutaneously (SQ) and intraperitoneally (IP). Pets had been bled on day time 1 to get the pre-immune sera for a poor control and day time 42 to check for reactivity with EBNA1 antigen within an enzyme-linked immunosorbent assay.