The Construction And Characterization Of Human Prourokinase Mutant

Arterial thrombosis is the underlying cause of a wide variety of cardiovascular diseases including myocardial infarction. Most of the currently used thrombolytic agents are plasminogen activators, which are very efficient in restoring the blood flew. They can convert plasminogen into plasmin and thus degrade fibrin. Despite the widespread use of a established thrombolytic agents such as streptokinase, tissue-type plasminogen activators (t-PA), urokinase-type plasminogen activators (u-PA), recombinant tissue-type plasminogen activator(rt-PA),anisoylated plasminogen streptokinase activator complex (APSAC),all these agents suffer from a number of inadequacies including resistance to reperfusion, occurrence of coronary reocclusion and bleeding complications.The quest continues for plasminogen activators with higher potency, specific thrombolytic activity, fibrin selectivity and longer half-life time. Based on the detail understanding of the relationship between structure and function of thrombolytic agents, the new generation of thrombolytic agents showed considerable potential with regard to improving to the efficacy of thrombolytic therapy. Prourokinase(pro-UK) is a single-chain urokinase-type plasminogen activators belonging to serine protease, able to promote the dissolution of fibrin clot through the activation of plasminogen to plasmin which has a broad application in thrombolysis therapy. In-vitro and in-vivo, pro-UK induces fibrin specific clot lysis, when dosage is limited to a range at which activation of pro-UK to urokinase(UK) is restricted to the fibrin surface. At higher doses, systemic conversion to UK takes place,some of the benefits of pro-UK are lose. Characterization of pro-UK showed that it was made up of 411 residues with a plasmin or kallikrein susceptible site at Lys158-Ile159 .Another functionally important cleavage site at Arg156-Phe157 is hydrolyzed by thrombin and generates a two-chain form with little catalytic activity against plasminogen.To prevent systemic activation of plasminogen, the regulation of pro-UK activity involves three plasminogen activator inhibitors(PAIs).PAI-1 is the major PAI of plasma, forming inactive covalent complexes with the activator. A reseach showed that the pro-UK with four modified Arg residues by phenylglyoxal under mild conditions was found to be the most stable derivative in human blood plasma; it causes a more efficient lysis of plasma clots than the native activator. Three of four modified Arg residues are supposed to be within the 178RRHRGGS184 cluster, which was localized in the superficial loop of the pro-UK globule and was shown to interact with the complementary series of negatively charged residues in the molecule of the main plasma inhibitor PAI-1. The neutralization of positively charged Arg residues in this cluster decreases the affinity of pro-UK for PAI-1, which results in an enhancement of the stability in plasma and the fibrinolytic efficiency of the activator. Based on the above researches, three pro-UK mutant (pro-UKm) genes were constructed in this experiment with the PCR point-mutant method. The thrombin cleavage site Arg156 in pro-UK was mutated into His156, and named as pro-UKM1; PAI binding site Arg178, Arg179,Arg181 was mutated into Lys178, Lys179, His181, named as pro-UKM3; The mutant containing His156, Lys178, Lys179, His181 as pro-UKM13. The primers with mutant sites were designed, then the mutant cDNA was constructed through PCR. Because post-translation modification such as glycosylation ,disulfide bond formation, specific proteolysis, and formation of higher-order structure are important for protein activity, eukaryotic cell—-chinese hamster ovary cell—-has been used as a host cell to produce heterologous proteins—pro-UKm. Three mutants were analyzed with fibrin plate assay , mutant antigenic specificity assay ,SDS-PAGE fibrin plate assay, western blot, and Lysis in vitro of human blood clots after inhibition with thrombin and/or PAI. The fibrin plate assay , antigenic specificity assay displayed that the three mutant can dissolute fibrin and combine with pro-UK primary antigen.The SDS-PAGE fibrin plate assay and western blot results showed that the three mutants and the native pro-UK have the same single electrophoresis band indicating most of the pro-UK was single chain. In vitro plasma clot lysis assays indicated that the pro-UKM1 have the ability to resistant against thrombin digestion; pro-UK3 could resist against PAI inhibition; while pro-UK13 improved resistances against both thrombin and PAI. It looks very promising that the pro-UK13 can be a new medicine of dissolving thrombus. To produce transgenic animals which can express pro-UKm in animal mammary gland, two kinds of vector were constrcted. In vectorⅠ, pro-UKM1, pro-UKM3, pro-UKM13 were respectively inserted to the PBCⅠvector by the XhoⅠcloning site. The PBCⅠvector uses the goat ?-casein promoter to drive high-level expression ofpro-UKm.The goat ?-casein promoter is a tissue-specific promoter that target expression of pro-UKm almost exclusively to the lactating mammary gland. The vector Ⅰwere identified by PCR, and can be used to produce transgenic animals by microinjection or sperm-media technology. Rous Sarcoma Virus enhancer/promoter, modified HIV-1 5'and 3'Long Terminal Repeat(LTR), HIV-1 psi(ψ) packageing sequence, HIV Rev response element were amplified by PCR, then partial goat ?-casein promoter, partial goat ?-casein genomic sequence, the PCR products, BGH poly(A), pro-UKM13 cDNA, and pGRM-7ZF were used to construct the vector Ⅱ-another mammary gland expression lentivirus vector, which can be transfected into package 293FT cell to get recombinant lentivirus used to infect zygote to produce transgenic animal. To efficiently construct cloned human type 5 'recombinant adenovirus bearing pro-UK mutant by employing homologous recombination in Escherichia coli, pro-UKM13 cDNA was first subcloned into vector pShuttle -CMV. After identification, the resultant plasmid was linearized by Pme I and subsequently cotransformed in to E. coli BJ 5183 cells by electroporation with adenoviral backbone plasmid pAdEasy-1. To directly confirm recombination, pro-UKM13 cDNA was amplified by PCR with oligonucleotide in pro-UKM13 cDNA . After the cloned recombinant adenovirus plasmid DNA was obtained, it was digested with P ac I for releasing inverted terminal repeat(ITR) and transfected into package 293 cell to get recombinant virus. As a result, PCR products indicated that pro-UKM13 cDNA was successfully integrated into the adenoviral genome. Homogenous recombinant virus could be easily obtained by transfecting 293 cell with this infectious DNA. The resultant recombinant adenovirus could reach 4.6×106 pfu/ml in 293 cells.This experiments suggest that construction of cloned recombinant adenovirus genome by homologous recombination in Escherichia coli is a very efficient method to construct homogeneous recombinant adenovirus. This recombinant adenovirus can be injected into mammary gland of mice or goat directly, allowed for the efficient secretion of pro-UKM13 in the milk. ,...