ut-PA抗PAI-1突变体的构建及在sf-9细胞中的表达

为使嵌合分子 ut- PA获得抗 PAI- 1抑制作用的性质 ,将删除了编码 u- PA中 R1 78- R1 79-H1 80 - R1 81的 1 2个核苷酸的 u- PA c DNA[u- PA( 1 ) ]的 Bam H - Eco R 部分酶切片段 ,克隆到含嵌合蛋白 ut- PA基因的转移载体 p VL 1 392 - ut- PA的相应位点中 ,构建了一个含有新的嵌合蛋白基因 ut- PA( 1 )的转移表达载体 p VL1 392 - ut- PA( 1 ) .在昆虫病毒表达系统 sf- 9细胞中表达该嵌合蛋白基因 ,表达上清具有纤溶性 ,用血纤维蛋白平板法和 S2 4 44 显色底物法分别测得活力为 2 4 8IU/ml和 380 IU/ml

Construction of a Mutant Chimerical Plasminogen Activator Resisting to Inhibition of PAI-1 and Its Expression in sf-9 Cells

t PA and UK are used widely as thrombolytic agents in curing acute myocardial infarction.To obtain a novel chimeric thrombolytic molecule which can resist the inhibition of plasminogen activatior inhibitor 1(PAI 1),a recombinant was constructed on the base of the genes of chimeric protein ut PA and the mutant u PA in which 12 nucleotide encoding Arg 178 Arg 179 His 180 Arg 181 of u PA were deleted,while the tetra peptide was supposed to be related to the binding of u PA to plasminogen activator inhibitor 1.The mutant cDNA was cloned into the transfer vector pVL1392 and expressed in insect expression system of sf 9 cells.The products showed PA activity with 248 IU/ml (determined by fibrin plate method)and 380 IU/ml (by S 2444 substrate method).