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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。
Objective To express recombinant antithrombin Ⅲ (AT-Ⅲ) in Escherichia coli system, prepare its antiserum and measure the titer. Methods AT-Ⅲ gene fragment was obtained by gene cloning technique. The prokaryotic expression plasmid pET32a (+) - AT-Ⅲ was constructed and transformed into E. coli BL21 competent cells. The recombinant protein was induced by IPTG. The protein was purified and immunized with rabbit. Antiserum. Results The results of SDS-PAGE and Western blot showed that the specific bands appeared at a molecular weight of 77,000. The fusion protein was isolated and purified before immunization of New Zealand rabbits to prepare AT-III antiserum, indirect ELISA method to detect the highest titer of antiserum reached 1: 1200, Western blot analysis showed that the antiserum can be expressed with 293T, CHO cells AT-III protein and AT-III protein pure product-specific binding. Conclusion Human antithrombin Ⅲ antiserum was successfully prepared.