1997 Fiscal Year Final Research Report Summary
Molecular cloning of autoantibody genes by a phage-display method
| Project/Area Number |
08670509
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
内科学一般
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| Research Institution | University of Tsukuba |
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Principal Investigator |
SUZUKI Hiroshi University of Tsukuba, Institute of Clinical Medicine, Lecturer, 臨床医学系内科, 講師 (00179243)
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| Project Period (FY) |
1996 – 1997
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| Keywords | phage-display method / anti-DNA antibody / SLE / SLE |
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| Research Abstract |
Phage-display method is a powerful tool for isolation of human autoantibody genes from peripheral lymphoid tissues. In addition, production of recombinant monoclonal autoantibodies by the method may be useful for the study of the physiological roles of the pathogenic autoantibodies.
In this study, we reproduced recombinant Fab from an IgM anti-DNA monoclonal antibody (Ab) producing cell line NE-1 by the phage-display method. The recombinant Fab produced by the method revealed identical antigen-specificity as the parental mAb and expressed NE-1 idiotype. The nucleotide sequences of variable regions of heavy and light chain of the Fab-phage clone p4-1 derived from NE-1 was completely identical to those of the parental NE-1 except serveral 5'^1 nucleotides due to the primers used in PCR.Accordingly, identical Fab molecules as the parental mAb was reproduced by the method. Second, we tried to isolate anti-DNA antibody Fab fragments from peripheral blood lymphocytes (PBLs) from patients with active systemic lupus erythematosus (SLE). Eight recombinat Fab clones which showed significant binding to solid-phase DNA were isolated from Fab-phage libraries after 5 times pannings to DNA.Binding to double strand DNA of Fab molecules of these clones was confirmed by Clithidia assay. Three of these Fab clones showed significant binding to dsDNA by Clithidia assay at less than 100 pg/ml. Sequence analysis of variable regions of immunoglobulin genes of the 8 Fab clones demonstrated that VH were all derived from VH26 germline gene, but diverse VL germline genes were used. In addition, CDR3 regions of the 8 Fab clones are similar each other, suggesting that these clones may be derived from a B cell clone.
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