Constructing EGFP Fusion Vectors of Bovine Immune Regulators

In order to construct EGFP fusion expression vectors of bovine CTLA-4, CD80, CD86, and CD28, we designed the primers to amplify the ORF region that did not have a stop codon. The amplified region fragment of bovine CTLA-4, CD80, CD86, and CD28 was inserted into the cloning site of a pEGFP-N2 vector (Clontech, Palo Alto, CA, USA). The complete vectors, pEGFP-N2-CD80, pEGFP-N2-CD86, and pEGFP-N2-CD28 were transfected into Cos-7 cells by Lipofectamine 2000 reagent (Thermo Fisher Scientific) and cultured for 48 h. The complete vector, pEGFP-N2-CTLA-4 was transfected into CHO-DG44 cells (provided by Dr. Suzuki, Hokkaido University, Japan) with the Lipofectamine LTX reagent (Thermo Fisher Scientific). CTLA-4-EGFP expressing CHO-DG44 cells were cloned and established CTLA-4-EGFP highly expressing CHO-DG44 cells. The binding of CTLA-4-Ig to the CD80 or CD86 expressing cells or the binding of CD80-Ig or CD86-Ig to CTLA-4 expressing cells was confirmed using flow cytometry with FACSVerse (BD Biosciences, San Jose, CA, USA) and FCS Express 4 (De Novo Software, Glendale, CA, USA) as previously described with some modifications [22 (link)]. Rabbit IgG (Southern Biotech, Birmingham, AL, USA) was used as a control Ig.