PAI－1糖基化位点突变体的构建及其在哺乳动物细胞中表达

CONSTRUCTION OF MUTANT GLYCOSYLATION SITES OF PAI-1 AND EXPRESSION IN MAMMALIAN CELLS

Ⅰ型纤溶酶原激活物抑制剂（ＰＡＩ－１）是一种Ｍｒ为５０×１０３的单链糖蛋白，有３７９个氨基酸残基，３个Ｎ型糖基化位点。构建ＰＡＩ－１糖基化突变体，以便研究糖链的功能。用寡核苷酸定位突变技术将３个糖基化位点２０９，２６５，３２９位进行突变，把３个糖基化位点都发生了突变的ＰＡＩ－１ｃＤＮＡ组装到真核表达载体ｐＳＶ２中，得到真核表达质粒ｐＺＨ－ｐ１－Ｍ３Ｅ；在二氢叶酸还原酶缺陷型中国仓鼠卵巢细胞（ＣＨＯｄｈｆｒ－）中进行短暂表达，用发色底物法和夹心ＥＬＩＳＡ方法检测培养液中ＰＡＩ－１的活性和含量。结果：糖基化位点突变的ＰＡＩ－１能在ＣＨＯ细胞中表达，但表达水平及活性较低。非糖基化ＰＡＩ－１的活性和抗原分别为４．３４ＩＵ／ｍｌ和３．１５μｇ／Ｌ结论：用寡核苷酸定位突变方法获得了ＰＡＩ－１糖基化突变体，并且在ＣＨＯ细胞中得到表达。

PURPOSE Plasminogen activator inhibitor l(PAI-1) is a single chain glycoprotein with Mr 50×103. The mature protein contains 379 amino acid residues with three potential N-linked glycosylation sites at position 209, 265, 329. The mutant of the three N-linked glycosylation sites of PAI-1 was constructed in order to study functions of oligosaccharide chains of PAI-1.METHODS The mutant was constructed with oligonucleotide site-directed mutagenesis.The PAI-1 cDNA mutant was inserted into the eucaryotic expression vector pSV2, producing pZH-P1-ME and expressed in the dhfr deficient Chinese hamster ovary(CHO) cells. The transient expression products of the mutant PAI-1 by CHO cells was examined by chromogenic substrate and ELISA.RESULTS Low level of mutant PAI-1 was expressed in CHO dhfr deficient cells,The activity and antigen of mutant PAI-1 were 4.34 IU/ml and 3. 15 μg/L,respectvely.CONCLUSIONS The mutant of nonglycosylated PAI-1 cDNA was obtained and expressed in CHO cells.