人纤溶酶原激活剂抑制物2型(PAI-2)在大肠杆菌中的表达和分离纯化

Expression and Purification of Human Plasminogen Activator Inhibitor Type 2 in E.coli

用ＰＣＲ方法从ｐＰＡＩＪ．７中扩增人纤溶酶原激活剂抑制物２型（ＰＡＩ－２）ｃＤＮＡ，与ｐＵＣ１８重组，经限制性内切酶片段分析与核苷酸序列分析，获得全长人ＰＡＩ－２ｃＤＮＡ．ＰＡＩ－２ｃＤＮＡ与原核表达载体重组，构建原核表达质粒并转化大肠杆菌．经温度诱导表达，重组ＰＡＩ－２占全菌总蛋白的１４％，以可溶性形式存在，具纤溶抑制活性．工程菌发酵、压榨后，用硫酸铵分级沉淀，沉淀物经分子筛、离子交换和疏水性色谱的分离，每升菌液可获得约３０ｍｇ纯度达９０％的蛋白质，比活性为１１８６６ＡＩＵ／ｍｇ蛋白质，得率为１９．２％．

To express human plasminogen activator inhibitor type 2(PAI 2)in E.coli and establish a method for the purificaton of the recombinant protein (rhPAI 2),PAI 2 cDNA was amplified by PCR,using pPAIJ.7 containing human PAI 2 cDNA as a templete. After restriction enzyme analysis and DNA sequencing,the PCR product was ligated with prokaryotic expression vector and transfected a special strain of E.coli. The expression product was identified by Western blot.The biological activity was measured by chromogenic assay.The solube rhPAI 2 was purified by a protocal which included ammonium sulfate precipitation,Sephadex G 75 gel filtration,Q sepharose ion exchange chromatography and hydrophobic interaction chromatography.The expressed recombinant PAI 2 was about 14% of total bacteria protein,and was identical to PAI 2 with respect to interaction with polyclonal antibody.The purity of rhPAI 2 was up to 90%,the protein yielding 30 mg per liter of culture,the specific activity of rhPAI 2 was 11 866 AIU/ mg,and rhPAI 2 was found to inhibit u PA by forming SDS resistant complex.