Study Of The LMW-uPA And ATF-Fc Fusion Protein

The myocardial infarction, cerebral thrombosis and lung embolism arethe common ailment that harm people health seriously. Cardiovascular andcerebrovascular disease has been the problem with the highest death rate indeveloped country. And in our country the number of people who dead ofcardiovascular and cerebrovascular disease is the second, lower than cancer.Urokinase is the thrombolytic drug that we usually use in our country. But theproduct we use is extract from urina hominis. There are many problems suchas limited resource, pollution by pathogen, and hard quality control. So it's agood chose to study of the gene engineering urokinase to substitute theurinourokinase.It's a perfect choice to study of the gene engineering ukinase to instead ofthe uronoukinase and the product of fetus kidney. The LMW-UK mutantabsent the 1-143aa of the HMW-UK to delete the action site of the plasmin,and turn the 156th Arg to Lys, to delete the action site of the thrombin. Afterthese change, the product we study can abstent being deactivation in blood.We also mutant the Asn302 to Lys, to delet the glycosylation site. And expressthe LMW-uPA mutant through mammalian cell. It has been proved that weget the LMW-uPA mutant without ATF, and the protein we get coulddissolve the fibrin protein effectively.In the study, we construct the non-glycosy and anti-thrombin LMW-uPAmutant. The study shows that the LMW-uPA mutant remains the activity siteof plasmin, and the specific activity to the plasmin is 1.6×105IU/mg. It'shigher 60% than HMW-UK(1.04×105IU/mg).It has the four feature comparewith the HMW-UK: (1) Without the enzyme site of thrombin, the LMW-uPAenhance its stability and uniform. It's good for the quality control. Also ,it canavoid the destroy of the thrombin in the blood. (2) Without the N-glychainstructure, it doesn't influence the stability in vivo and it's good for its uniform.(3) Without the 1-143aa, it avoid being band with the receptor on thecellenvelope, it can enhance the carbinoxamine compound drops, so that thethrombolysis rate higher. (4) The specific activity of the protein improved,reduce the amount of the durg.A attenuated selection marker bicistronic eukaryotic vector for stable andhigh-level expression of recombinant protein in mammalian cells by selectivepressure of methotrexate (MTX), designated pIRES-dhfr, was developed byinserting the dihydrofolate reductase (dhfr) gene into the downstream site ofthe attenuated encephalomyocarditis virus (ECMV) internal ribosome entrysite (IRES) in pIRES vector.A unglycosylated and thrombin-resistant mutant of prourokinase that hasno EGF-like domain and kringle domain (1-143 aa of pro-UK was deleted),and has two amino acid substitutions in active site region (Arg156→Lys,Asn302→Ala) is studied. A attenuated selection marker bicistronic eukaryoticvector for stable and high-level expression of recombinant protein inmammalian cells by selective pressure of methotrexate (MTX), designatedpIRES-dhfr, was used to transfer the aim protein. The dhfr-deficient CHOcells were transfected by pIRES-dhfr/LMW-UK vector withlipofectine-mediated gene transfer technique, and results showed thatfollowing one round of amplification under the selection of MTX andmonoclonal culture, all of monoclonal cell lines were positive clone, and about50% of recombinant cell lines expressed the similar and high levels ofLMW-uPA (500-5000 IU/106cells/d). The recombinant protein was purifiedwith cation-exchange chromatography and gel-filtration from the culturesupernatant, which was harvested from serum-free culture of a recombinantCHO cell line of expression level at about 17.5μg /106cells/d, LB2-UK, inroller bottles. The purity of the recovered recombinant protein was about 99%.Analyzed by S-2444 chromogenic assay, the active two-chain urokinase(tcu-PA) ratio of the LMW-uPA variant was about 98%.A series of impotent function of the uPA have been studied after band to thereceptor. After uPA band to uPAR, many iter actived. For example, active thesignal transduction in the cell, and mediate the degradation of the extracellularmatrix. In this way, the tumour cell could enter the blood vessel through basalmembrane, the tumour transfers. So , it become the new way to inhibite thetumour. ATF is the important section in the progress of uPA banding to theuPAR. We believe, the tumour could be inhibit when the uPA-uPAR beinginhibited.In our study, we construct the ATF-Fc fusion protein, and express it in themammalian cell. After the tumor cell wound study, the ATF-Fc fusion proteinhas the ability to kill the tumour cell. It's a real new step on the way of curecancer.