Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology · 2003
[The expression and application of human Fas ligand in E.coli].
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Li Ning-li, Nie Hong, Yu Qi-wen, Bo Jun, Ma An-lun, Zhang Ji-ying, Shen Bai-hua, Wang Li, Zhang Dong-qing
Аннотация
AIM: To express recombinant human FasL molecule in E.coli.
METHODS: RT-PCR was applied to amplify FasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. The DNA fragment was cloned into PCR2.1 vector. After sequencing, the FasL gene was inserted into pQE-31 vector and expressed in E.coli M15. The FasL protein was purified through Ni-ATA affinity chromatography column and identified by SDS-PAGE and Western blot. The mice were immunized with the FasL protein and the specific anti-serum was harvested 6 weeks after immunization. The serum level of FasL from with different kinds of diseases patients were detected using the anti-FasL antibodies from the immunized mice.
RESULTS: The expressed protein could be recognized by anti-human FasL antibody in Western-blot analysis with M(r)40 000. This protein could induce Jurket cells apoptosis. anti-FasL serum prepared from mouse could detect the serum FasL as sensitive as commercial ELISA kits.
CONCLUSION: The human FasL protein is obtained. It lays the foundation for the further detecting the concentration of FasL and sFasL of patients.
METHODS: RT-PCR was applied to amplify FasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. The DNA fragment was cloned into PCR2.1 vector. After sequencing, the FasL gene was inserted into pQE-31 vector and expressed in E.coli M15. The FasL protein was purified through Ni-ATA affinity chromatography column and identified by SDS-PAGE and Western blot. The mice were immunized with the FasL protein and the specific anti-serum was harvested 6 weeks after immunization. The serum level of FasL from with different kinds of diseases patients were detected using the anti-FasL antibodies from the immunized mice.
RESULTS: The expressed protein could be recognized by anti-human FasL antibody in Western-blot analysis with M(r)40 000. This protein could induce Jurket cells apoptosis. anti-FasL serum prepared from mouse could detect the serum FasL as sensitive as commercial ELISA kits.
CONCLUSION: The human FasL protein is obtained. It lays the foundation for the further detecting the concentration of FasL and sFasL of patients.