Ⅰ型纤溶酶原激活物抑制剂cDNA表达质粒的构建及其在哺乳动物细胞中的表达

CONSTRUCTION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 cDNA CLONE AND EXPRESSION IN MAMMALLAN CELLS

建立Ⅰ型纤溶酶原激活物抑制剂（ＰＡⅠ－１）ｃＤＮＡ哺乳动物细胞表达体系。用人工合成的６个寡核苷酸连接成编码ＰＡⅠ－１Ｎ－端１０个氨基酸和２３个氨基酸的信号肽顺序；将构成的完整ＰＡⅠ－１ｃＤＮＡ插入真核表达质粒ｐＳＶ２ｄｈｆｒ中，获得真核表达质粒ｐＺＹＳ－１；将ｐＭＡ２１２中的ｈＭＴ－ⅡＡ的起动子再插入ｐＺＹＳ－１，使ＰＡⅠ－１ｃＤＮＡ受ＳＶ４０和ｈＭＴ－ⅡＡ双启动子控制，得ｐＺＹＳ－２。２个表达质粒分别在ＣＯＳ－７细胞中进行表达。结果：获得两个真核表达质粒，在ＣＯＳ－７细胞中得到表达，在培养液中的ＰＡⅠ－１活性和抗原含量分别为２３４．７ＩＵ／ｍｌ和３０μｇ／Ｌ。结论：ｐＺＹＳ－１和ｐＺＹＳ－２两株ＰＡⅠ－１ｃＤＮＡ真核表达质粒在ＣＯＳ－７细胞中能有效表达和分泌。

PURPOSE A mammalian cell expression system was developed that allows the system to be used for the efficient expression of PAⅠ-1 cDN.METHODS The six synthezed oligonucleotides coding for PAI-1 signal peptide and Ntermminal ten amino acid residues were ligated and inserted between SV40 promoter and SV40 poly A signal in pSV2dhfr,producing expression plasmid pZYS-1. The hMT-ⅡA promoter from pMA2.2A was inserted into downstream of SV40 promoter, producing expression plasmid pZYS-2.They were transfected into COS-7 cells and expressed.RESULTS Two expression plsmids,pYZS-1 and pYZS-2,were obtained and expressed in COS-7 cells. The activity and antigen of rPAⅠ-1 in medium were 34.7 IU/ml and 30 μg/L,respectvely.CONCLUSIONS PAⅠ-1 cDNA in both pZYS-1 and pZYS-2 expression plasmids were expressed and efficiently secreted by COS-7 cells.