CD28Ig 및 CTLA4Ig 융합단백 생산 포유동물 세포주 수립에 관한 연구 = Establishment of Stable Transfectant Cell Lines Producing Human CTLA4Ig and CD28Ig Fusion Proteins

다국어 초록 (Multilingual Abstract)

CTLA-4, which is homologous to CD28, recognizes the same coutner-receptors B7.1 and B7.2. Although initial researches implied that these molecules may act to each other synergistically, recent data suggest that CTLA-4 may acts as a negative regulator of T cell activation. In this study human CD28 and CTLA-4 cDNAs were amplified from PHA-stimulated human peripheral blood lymphocytes mRNAs by using RT-PCR method. IgG1 Fc portion cDNA was amplified from human peripheral blood lymphocytes of patients with febrile illness and fused to C"-terminal parts of CD28 and CTLA-4 extracellular domains. The fusion constructs were subcloned into pCI-neo vector and then transfected into Sp-2/0murine myeloma cell. Stable cell lines were established by geneticin selection and cloning. In the sequence of CTLA-4 cDNA from 3 different healthy persons it was found that 2 sites of bases were different from that published in original paper and submitted to genbank database. The 49th and 331st bases of open reading frame were changed from adenine to guanine and from guanine to adenine respectively. It was confirmed that the 17th and 111th amino acid of human CTLA-4 were alanine and threonine respectively, 5 different human CTLA-4 clones with serial N'-terminal deletion were constructed.
CTLA4/L_1 was intact CTLA-4 gene of native form without any form of mutation. CTLA4/L_2 was a mutatn with deletion in gene coding N'-termial 6 amino acids. CTLA4/L_3 was a mutant with deletion in gene coding N'-termial 11 amino acids. CTLA4/L_4 was a mutant with deletion in gene coding N'-termial 16 amino acids. CTLA4/L_5 was a mutant with deletion in gene coding N'-termial 22 amino acids. Level of surface expression of CTLA4Ig on L_1, L_2, L_3, L_4 and L_5 transfectant cell lines were 5%, 3%, -1.5%, 8%, and 6% respectively. These 2 fusion proteins, CD28Ig and CTLA4Ig, would be useful tools for further researches on role of the costimulations in T cell activation.