Screening And Expression Of Homozygote Of Recombinant Human Plasminogen Activator (rhPA) Transgenic Rabbits

Thrombotic diseases have become one of the major threats to the human life and health, and which can endanger the body’s organs and systems. At present, thrombolytic agents have become the main method of treating such diseases. By continuous research, development of thrombolytic agents experienced three stages to improve its features of thrombolysis. Recombinant human plasminogen activator(r-tPA) is a new type drug for the treatment of thromboembolic disease. Compared with natural human tissue plasminogen activator, r-tPA have the advantages of strong ability to thrombolysis, high affinity to fibrin, small side effects of systemic bleeding, the long half-life in the blood, low total dose, and so on. Therefore, as a representative of the third generation thrombolytic agents, recombinant human plasminogen activator more and more receives the favor by the majorities of patients. It will have broad prospect of application.The copy number of transgene in homozygous transgenic animals is double of hemizygous transgenic animals. The aim of this research was to screen out rhPA homozygous transgenic rabbits with high level of expression. There is no report for the difference of expression level between homozygous and hemizygous transgenic rabbit. Another purpose is to explore the relationship between expression quantity and the copy number of rhPA with the same integration sites. Finally, homozygous transgenic rabbits with high level of expression were screened out and new strains of rhPA homozygous transgenic rabbit for biopharmaceuticals would be established. This research provides a basis for large-scale preparation of biological pharmaceutical, which used for dissolving thrombus in the future. Meanwhile, the biopharmaceuticals can be used for pharmacological research and bioactive analysis. It is advantageous to the development and application of thrombolytic agents in the later stage.In this research, the mammary gland-specific vector PCL25/rhPA was constructed. The founder rhPA transgenic rabbits were obtained by microinjection of a linear gene fragment into embryo pronuclei of fertilized eggs. F1 transgenic rabbits sib mating or backcross to obtain the F2 transgenic rabbits. Then the homozygous rabbits of F2 transgenic rabbits were screened out by testcross. The expression level of rhPA between homozygous rabbits and hemizygous rabbits was compared by ELISA.Sexually mature female New Zealand White rabbits are appropriated for the experiment. Fertilized eggs were microinjected with the PCL25/rhPA vector to generate transgenic rabbits. In this research,64 receptors were transplanted. There were 46 pregnant and 207 offsprings were produced. The result of PCR showed that 56 rabbits were transgenic integrated positive and 37 rabbits were survival. The pregnancy rate is 71.9% and the integration rate is 27.1%.ELISA was used to analyze the whey of founder or F1 generation transgenic rabbits. The result showed that 12 transgenic rabbits mammary gland-specific expressed rhPA.The transgenic rabbit K29 was selected to breeding with normal female New Zealand rabbit. A total of 25 F1 offsprings were obtained. The result showed that 16 F1 rabbits (10 female and 6 male) were transgenic by PCR. F1 transgenic rabbits sib mating to obtain F2 transgenic rabbits. A total of 10 F2 offsprings were obtained. The result showed that 5 F2 rabbits (4 female and 1 male) were transgenic by PCR. F3 rabbits were obtained by F2 transgenic rabbits breeding with normal rabbits. PCR were used to analyze the genomic DNA samples of F3 rabbits. The result of PCR showed that 2 homozygous transgenic rabbit (K29 F2-05 and K29 F2-09) were obtained from the F2 transgenic rabbits. ELISA was used to analyze the whey of the homozygous transgenic rabbit K29 F2-05. The result indicated that the rhPA expressing level of homozygous transgenic rabbit was significantly higher than hemizygous transgenic rabbits.The transgenic rabbits with high level of expression were obtained. New strain (K29) of rhPA transgenic rabbit was established in this research. And 2 homozygous transgenic rabbit (K29 F2-05 and K29 F2-09) were obtained. The result of ELISA indicated that the rhPA expressing level of K29 F2-09 was significantly higher than hemizygous transgenic rabbits. This research proved that the expression level of PCL25/rhPA in homozygous transgenic rabbits is higher than hemizygous transgenic rabbits.This research verified that it is feasible to improve the expression level of foreign genes by cultivating the homozygous transgenic animals. It provides an experimental basis and guiding significance for establishing new strain of rhPA homozygous transgenic rabbit and large-scale preparation of thrombolytic agents.