| Project/Area Number |
13670566
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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 |
Gastroenterology
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| Research Institution | THE JIKEI UNIVERSITY SCHOOL OF MEDICINE |
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Principal Investigator |
MATSUFUZI Tamiko FACULTY OF MEDICINE, THE JIKEI UNIVERSITY SCHOOL OF MEDICINE, LECTURER N/A, 医学部, 講師 (00199845)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2001: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | autoimmune hepatitis / selenocystainyl tRNA / soluble liven antigren.(SLA / LP) / autoantibody / expression / immunization |
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| Research Abstract |
1) Construction of recombinant SLA/LP expression system
A partial-length SLA/LP cDNA, in which N-terminal 30 residues are truncated, was prepared and expressed as C-terminal His-tagged form. When the cDNA was expressed in E. coll BL21 (DE3), the product was insoluble forming inclusion and could not be solubilized by changing the induction conditions or culture temperature. However, when it was expressed in the rabbit reticulocyte lysate translation system on T7 RNA polymerase-synthesized mRNA most of the product was yielded in a soluble fraction.
2) Detection of anti SLA/LP autoantibodies in the sera of autoimmune hepatitis (AIH) patients
The recombinant SLA/LP protein was expressed in the rabbit reticulocyte lysate in the presence of [35S] methionine.The labeled product and protein A-Sepharose was used for immunoprecipitation analyses of anti SLA/LP antibody in the sera. The imnmunopreciptated materials were separated on SDS-PAGE followed by radioraetry on a FLA200 scanner. At least six AIH patient out of 29 cases carried a significant activity to precipitate the labeled SLA/LP protein. Of those three, cased also carried direct antibody against Sec-tRNA. However, the immunoprecipitation of SLA/LP was not abolished by Rnase treatment of lysate suggesting that the precitipation of SLA/LP was not mediated by the direct antibody against Sec-tRNA.
3) Experimental autoimmune hepatitis model
We attempted to induce autoantibody to Sec-tRNA by injecting BALB/c mice with Sec-tRNA with Ffreund's adjuvant, but the elevation of serum titer for anti-Sec-tRNA has not been observed so far. We are constructing a bacterial expression system of a full-length SLA/LP, the product of which will be mixed with Sec-tRNA and used to immunize mice.
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