人抗凝血酶Ⅲ的表达及其抗血清的制备

发布时间：2018-01-04 13:02

本文关键词：人抗凝血酶Ⅲ的表达及其抗血清的制备　出处：《细胞与分子免疫学杂志》2013年02期 　论文类型：期刊论文

【摘要】：目的用大肠杆菌系统表达重组的抗凝血酶Ⅲ(AT-Ⅲ),制备其抗血清并测定效价。方法利用基因克隆技术获得AT-Ⅲ基因片段,构建原核表达质粒pET32a(+)-AT-Ⅲ,转化大肠杆菌BL21感受态细胞,IPTG诱导表达、蛋白纯化并免疫兔,间接ELISA、Western blot法检测抗血清。结果 SDS-PAGE、Western blot法检测原核表达蛋白结果显示,在相对分子质量(Mr)77 000附近出现特异性条带。对该融合蛋白进行分离纯化后免疫新西兰兔,制备AT-Ⅲ抗血清,间接ELISA方法检测抗血清的最高效价可达到1∶12 800,Western blot分析表明抗血清可与293T、CHO细胞表达的AT-Ⅲ蛋白及AT-Ⅲ蛋白纯品特异性结合。结论成功制备了人抗凝血酶Ⅲ抗血清。
[Abstract]:Objective to express recombinant Escherichia coli with antithrombin III (AT- III) system, the preparation of its antiserum titer. And determination method for obtaining AT- III gene fragment by gene cloning and construction of prokaryotic expression plasmid pET32a (+) -AT- III, transformed into Escherichia coli BL21 competent cells, IPTG expression, protein purification and immune rabbit. Indirect ELISA detection method. Results SDS-PAGE blot Western antiserum, Western blot assay showed that the prokaryotic expression protein, with a relative molecular mass (Mr) near 77000 specific bands. The fusion protein was isolated and purified by New Zealand rabbits, preparation of AT- III antiserum, the highest titer of indirect ELISA for the detection of antiserum can reach 1 to 12800, Western blot analysis showed that the antiserum with 293T, combined with AT- and AT- 3 protein 3 protein purified specific CHO cells. Conclusion the successful preparation of human antithrombin III anti blood Clear.

【作者单位】：东北农业大学生命科学学院;山东省农业科学院奶牛研究中心;
【基金】：国家自然科学基金(31272586) 国家转基因重大专项(2009ZX08007-006B,2011ZX08007-002,2011ZX08008-004) 山东省青年自然科学基金(ZR2010ZR029) 山东省自然基金(ZR2010CM012) 济南市高校院所自主创新计划(201004027)
【分类号】：R346
【正文快照】： 人抗凝血酶Ⅲ(antithrombin-Ⅲ,AT-Ⅲ)是一类相对分子质量(Mr)约为58 000的糖蛋白,由432个氨基酸残基组成,含糖量约10%。天然AT-Ⅲ以α、β两种形式存在,其中90%为α形式(即完全糖基化)[1-3]。AT-Ⅲ是由肝细胞和血管内皮细胞分泌的丝氨酸蛋白酶抑制剂(serine protease inhibit

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