论文部分内容阅读
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。
Objective To construct PAI-2ΔCD mutant gene and express in E.coli and to establish a method for the isolation and purification of recombinant human PAI-2ΔCD. Methods PAI-2ΔCD gene was amplified by PCR. After restriction fragment analysis and nucleotide sequence analysis, the mutant gene was recombined with prokaryotic expression vector pLY-4 and transformed into protease-deficient strain JF1125. After induced by temperature, the expressed products were identified by SDS-PAGE, Western blot and fibrinolytic activity assay. After the engineering bacteria are fermented, they are broken by high-pressure homogenization and separated by ion exchange, hydrophobic chromatography and molecular sieve. The results of temperature-induced expression, SDS-PAGE confirmed that the expression of the corresponding molecular weight bands, accounting for about 15% of the total bacterial protein. Western blotting confirmed that the expressed product possessed the PAI-2 immunogenicity, and the lysosome inhibition method and the flip-flop method confirmed the fibrinolytic activity of the expressed product. 5L fermentation broth can get wet bacteria about 100g, after multi-step purification, each liter of fermentation broth available purity of 97% of PAI-2ΔCD about 36mg, specific activity of 28640AIU / mg. Conclusion PAI-2ΔCD mutant was successfully constructed and expressed in Escherichia coli. The recombinant human PAI-2ΔCD protein was successfully isolated and purified.