2000 Fiscal Year Final Research Report Summary
Effects of a Sjogren's syndrome-associated mutation of the TAP2 gene on the diversity of HLA-bound antigen peptides
| Project/Area Number |
11670446
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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 | Kobe University |
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Principal Investigator |
KUMAGAI Shunichi Kobe University School of Medicine, Professor, 医学部, 教授 (00153346)
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| Co-Investigator(Kenkyū-buntansha) |
MORINOBU Akio Kobe University School of Medicine, Assistant, 医学部, 助手 (10294216)
KONDO Shinichi Kobe University Hospital, Lecturer, 医学部・附属病院, 講師 (50153721)
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| Project Period (FY) |
1999 – 2000
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| Keywords | TAP2 Gene / antigen peptides / Sjogren's syndrome / peptide transporter / HLA |
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| Research Abstract |
1) Preparation of the mutated TAP2 gene.
The A to G mutation was introduced at the 1729^<th> base of TAP2 gene by the method of Shuldier. A.R.et al (Analyt. Biochem. 194, 9-15, 1991), and the mutated (TAP2^*Bky2) and wild (TAP2^*b) type cDNA were prepared.
2) Esablishment of TAP1 and TAP2 genes transfected cell lines.
TAP1 and TAP2 genes (TAP2^*B or TAP2^*Bky2) were transfected to TAP genes-deficient cell line, T2, by electroporation method. Two cell lines. T2/Bky2 and T2/B, which were different only at the codon 577, were established.
3) Collection of HLA-bound peptides from the established cell lines
Antigen peptides bound to HLA-B5 were collected by anti-HLA column using anti-HLA-B5 monoclonal antibody (4D12). After filtration with 5,000 dalton cut-off filter, peptide fraction was collected.
4) Analysis of HLA-bound peptides
The fraction was applied on a reversed-phase HPLC, and some peaks were collected. The repertory and amino acid sequence of HLA-bound peptides are now further analyzed by mass-spectrometry.
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