【摘要】 目的构建ＰＡＩ－２ΔＣＤ突变体基因，实现在大肠杆菌中的表达并建立重组人ＰＡＩ－２ΔＣＤ的分离纯化方法。方法用ＰＣＲ扩增ＰＡＩ－２ΔＣＤ基因，经限制性内切酶片段分析及核苷酸序列分析后，突变体基因与原核表达载体ｐＬＹ－４重组并转化蛋白酶缺陷型菌株ＪＦ１１２５。经温度诱导表达，表达产物用ＳＤＳ－ＰＡＧＥ，Ｗｅｓｔｅｒｎ印迹法和纤溶抑制活性测定等方法鉴定。工程菌发酵后，经高压匀浆破菌，用离子交换、疏水性色谱和分子筛进行分离。结果经温度诱导表达，ＳＤＳ－ＰＡＧＥ证实在相应相对分子质量处出现表达条带，约占全菌总蛋白的１５％。Ｗｅｓｔｅｒｎ印迹法证实表达产物具有ＰＡＩ－２免疫原性，溶圈抑制法及翻转板法证实表达产物具有纤溶抑制活性。５Ｌ发酵菌液可得湿菌约１００ｇ，经多步纯化后，每升发酵菌液可得纯度达９７％的ＰＡＩ－２ΔＣＤ约３６ｍｇ，比活性为２８６４０ＡＩＵ／ｍｇ。结论成功构建了ＰＡＩ－２ΔＣＤ突变体，实现了在大肠杆菌中的表达，并成功地分离纯化了重组人ＰＡＩ－２ΔＣＤ蛋白。更多还原

【Abstract】 Purpose To construct and express human plasminogen activator inhibitor type-2 (PAI-2) mutant,PAI-2 Δ CD in E.coli and establish a method for the purification of the recombinant protein(rhPAI-2 Δ CD ). Methods PCR was used to generate the mutant,PAI-2 Δ CD.After restriction enzyme analysis and DNA sequencing,the mutant gene was ligated with prokaryotic expression vector pLY-4 and transfeted a special strain of E.coli .The expression product was identified by Western Blot and the PAI activity assay.After fermentation,the bacterial cells were homogenized at the pressure of 50 MPa.The PAI-2 mutant was purified by a protocol including Q-Sepharose ion exchange chromatography,hydrophobic interaction chromatography and Sephadex G-75 gel filtration. Results The expressed product was about 15% of total bacteria proteins,and was identical to the mutant of PAI-2 with respect to interaction with PAI-2 polyclonal antibody.The mutant could inhibit urokinase type plasminogen activitor measured by milk agarose plate assay and reveres milk agarose zymograph.After purification,the purity of rhPAI-2 Δ CD was up to 97%,the protein yielding was 36 mg per liter of culture,the specific activity was 28 640 AIU/mg. Conclusions The construction and expression of PAI-2 Δ CD in E.coli was successful.更多还原