Abstract: 【Objective】In this study, the rhPA/gGH double transgenic rabbits were constructed, and the expression level of rhPA,rabbit growth and development features were analyzed, which might provide a new idea for obtain rhPA high level expression transgenic animals.【Method】Two rhPA transgenic rabbits fertilized eggs were microinjected with linearized GH plasmid to obtain the rhPA/gGH rabbits.The integration of rhPA/gGH gene was detected by PCR. The rhPA expression level in transgenic rabbit milk was detected by ELISA and Western blotting,and FAPA was performed to detect the in vitro thrombolytic activity of rhPA.The body weight of transgenic rabbits at different growth stages were measured to test the effect of gGH gene on rhPA/gGH double transgenic rabbits growth and development .【Result】A total of 151 rhPA transgenic rabbits fertilized eggs were obtained through superovulation, 125 of them were microinjected with linearized GH plasmid and transplanted into 8 surrogate mother rabbits.Six surrogate mother rabbits were pregnant, with a pregnancy rate of 75.0% (6/8)，16 rhPA/gGH gene double transgenic rabbits were identified by PCR (10♂,6♀). The rhPA expression levels in rhPA single-transgenic rabbit whey were 0.27–0.63g/L, while the rhPA expression leves were 4.98-12.24 g/L in the rhPA/gGH double-transgenic rabbits whey. The rhPA expression levels of rhPA/gGH double-transgenic rabbit whey were significantly increased by about 17.2–23.8 times, and had higher thrombolytic activity in vitro. There was no significant difference in body weight between rhPA/gGH double transgenic rabbits, rhPA single transgenic or non-transgenic rabbits from birthday to 10 months age(P>0.05). 【Conclusion】The rhPA/gGH double transgenic rabbits were successfully constrected, which was proved that the introduction of gGH gene could significantly increase the rhPA expression level in the milk of transgenic rabbits and without affecting the growth and development of transgenic rabbits, which laid a foundation for the preparation of transgenic rabbits with higher recombinant protein expression level in the future, and also provide new ideas and new methods for the establishment of mammary gland bioreactor.

The human tissue-plasminogen activator (tPA) is a thrombolytic drug widely used in the treatment of stroke, pulmonary thrombosis, acute myocardial infarction, and other thrombotic diseases. The double gene co-integration into the organisms and cells can produce a synergistic effect, which can improve the expression level of the target gene. We selected the mammary gland-specific expression vectors BLC14/tPA and BLC14/GH with the β-lactoglobulin gene as a regulatory sequence in our previous research. The tPA and GH were co-transfected into goat mammary epithelial cells by electro-transfection. The results showed that the expression level of the tPA in single-gene cells was 8.0-64.0 μg/mL, while in double-gene cells was 200-7200 μg/mL, which was significantly higher than that in single-gene cells. In addition, the tPA also had a relatively strong in-vitro thrombolytic activity function determined by FAPA. The results showed that the goat mammary epithelial cell lines with the tPA/GH gene integration were successfully obtained by electro-transfection and it was proved that the expression level of the tPA in the double gene integration cell lines with the tPA/GH genes integration was significantly increased, which provided a new way for the preparation of highly expressed transgenic goat and other animals by somatic cell nuclear transfer, and also laid a foundation for the efficient production of pharmaceutical proteins in transgenic animal mammary gland reactors in the future.