文档介绍：T细胞CTLA-4、TCRVβ8基因克隆、重组及表达
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作者:王晶,朱江,朱本章,马卫国
【摘要】目的克隆Graves’病患者甲状腺组织T淋巴细胞的CTLA-4胞外段和TCRVβ8目的基因,重组为CTLA-4-TCRVβ8基因并表达出融合蛋白。方法 RT-PCR从Graves’病患者甲状腺组织中克隆T淋巴细胞的CTLA-4胞外段和TCRVβ8基因,依次与表达质粒进行连接,双酶切及测序鉴定获得正确的重组子;原核表达融合蛋白,SDS-PAGE及Western-Blotting检测蛋白表达情况。结果基因克隆扩增出CTLA-4和TCRVβ8目的基因片段,测序结果证实序列与已发表的一致。重组基因原核表达出与预期分子质量大小相符的目的蛋白。结论成功构建和表达了CTLA-4-TCRVβ8基因,为探索Graves’病免疫耐受治疗提供了基因产品
【关键词】 Graves’病;CTLA-4;TCRVβ;基因表达;融合蛋白
ABSTRACT: Objective To clone CTLA-4 and TCRVβ8 gene from T lymphocyte of thyroid of Graves’ disease (GD) patients, bine to form CTLA-4-TCRVβ8 fusion gene and express the fusion protein. Methods CTLA-4 and TCRV
β8 gene was cloned from T lymphocyte of thyroid of GD patients by RT-PCR. Then it was bined with expression plasmid in order. The correct plasmids were obtained after the restriction analysis and DNA sequencing. Prokaryotic expression of the fusion protein in , SDS-PAGE and Western Blotting were used to verify the fusion protein. Results Restriction analysis and DNA sequencing confirmed the correct sequence and insertion site of the binant plasmid. The binant fusion protein was essfully expressed in , which was consistent with the predicted putative calculating molecular weight. Conclusion CTLA-4-TCRVβ8 gene was constructed and expressed essfully, providing gene product and application theory for immune tole