Mammary - Specific Expression And Thrombolytic Activity Of Multi - Locus Integrated RhPA Gene In Rabbits

Transgenic animal mammary gland bioreactor pharmaceutical has towards industrialization, in the foreign countries, part of the medical proteins have entered into clinical application. In this method, the medical protein can be processed and modified in the cells, leading to high biological activity. Thrombotic disease is the primary harmful factor for human health and safety forits high incidence and mortality rate. Currently, thrombolytic therapy using thrombolytic drugs for recanalization is the most extensive and effective method of treatment. Issue-type plasminogen activator as the representative of the third-generation thrombolytic drugs, has good thrombolytic effect. We designed a new rhPA recombinant DNA, used for the preparation of unit point and double site integration of transgenic rabbit, to study transgenic rabbits mammary specific expression and the activity of recombinant protein of thrombolysis.Used PCL-25/LTF-Neo plasmid stored in our laboratory, restriction endonuclease digestion by Nhe I, then recovered Neor gene, then we used PCL-25/rhPA plasmid restriction endonuclease digestion by Nhe I to make a linearization and recovered linearized plasmid. T4 DNA ligase was used to combine Neor gene and linearized PCL-25/rhPA plasmid together, to get PCL-25/rhPA-Neo. Restriction endonuclease digestion of PCL-25/rhPA-Neo by Sal I、Not I, recovered 18kb strips was used to make transgenic rabbits.Sexually mature female New Zealand White rabbits are appropriated for the experiment, therefore it was superovulated with FSH and hCG, mating them with transgenic rhPA male rabbits which made in our laboratory. Out of the zygotes through surgery method, injection of PCL-25/rhPA-Neo fragments to zygotes was performed by microinjection. Transplanted zygotes after injection into the recipient females which is estrus synchronized, to make the transgenic Rabbits.Raised the pups until 30-day-old, clipped rabbit ear and extracted genome, used two pairs of primers, Neo-s/Neo-a and CMV-tPA-s/CMV-tPA-a. PCR was subsequently used to identify the integration. We feed the rabbit which integrate genes to 5-month -old, then mated them with normal male rabbits. After accouchement, mike was collected, ELISA and FAPA were applied to detect the expression level and activity of the rhPA in the mike and compare it with single gene integration rabbits.In order to establish mice thrombus model, Divided ICR mice into six, Each group of three mice,3mg、1.5mg、0.3mg、0.15mg、 0.03mg carrageenan via intraperitoneal injection to induce mice tail thrombosis. The thrombus mice were treated with the rhPA of different administration concentrations divided ICR mice into five groups, each group of four mice, 0.1mg/mL,0.4mg/mL,1.6mg/mL, by measuring the mice tail thrombus length change and detecting PT/TT to analyze the rhPA thrombolysis in mice.We made six transgenic Rabbits which integrated both PCL-25/rhPA-Neo and PCL-25/rhPA(4♀,2♂). One transgenic rabbit which integrated PCL-25/rhPA-Neo(1 ♀). Seven transgenic rabbits which integrated PCL-25/rhPA(4(?),3♂). Through the ELISA and FAPA analysis, we found rhPA expression level and activity is higher than single gene integration rabbit. The results proved that the rhPA activity and expression level which made by transgenic rabbits transgenic is higher than the tPA from single gene integration rabbits.Used different concentrations carrageenan via intraperitoneal injection to induce mice tail thrombosis The first group to the fourth group thrombus(3mg、1.5mg、 0.3mg、0.15mg、0.03mg) average relative percentage of about 75%, five group (0.03mg)thrombus average relative percentage is 11.6% and the 0.15mgwas optimal induced concentration. The thrombus mice were treated with the rhPA of different administrative concentrations, The results showed that the rhPA is significantly superior to alteplase in efficacy and dose. Moreover, the thrombotic disease was completely cured by three administrations of 1.6mg/mL.We made transgenic Rabbits which integrated both PCL-25/rhPA-Neo and PCL-25/rhPA and found the rhPA activity and expression level which made by transgenic rabbits twice transgenic is higher than the rhPA made from single gene integration rabbits. The rhPA is significantly superior to alteplase in efficacy and dose, lying a strong foundation for further clinical trials and post-mass production of novel recombinant thrombolytic drugs. In rabbit breast express restructuring of the original organization fibrinolytic enzyme activator, and animal thrombolysis rhPA test not reported both at home and abroad.