Transfer Of Human Serum Albumin Yanbian Dairy Goat Somatic Cell Cloned Embryo Production System Optimization

Human Serum Albumin (Human serum albumin, HSA) is a protein drugs that needed largely by recently. Now, however, the mode of production is in the person’s blood which is obtained by fractional filter, production is limited, and because of the complexity of the plasma source, easily lead to contamination.Accordingly, the mammary gland expression vector was constructed by transgenic technology, the use of somatic cell cloning techniques to obtain the secretion of HSA-containing milk of transgenic cloned animals, and from animal milk extraction HSA, is an effective method to solve the shortage of supply of HSA. Mammary gland bioreactor production has the advantages of low production cost, high yield, high activity, easy to purify. The main factors restricting transgenic mammary gland bioreactor pharmaceutical somatic cell nuclear transfer is inefficient.In this study, oocyte maturation rate, enucleation, turn HSA the Yanbian milk goat-sheep reconstructed embryos (hereinafter referred to as the reconstructed embryos) integration, activation and in vitro development of the quality of research in order to improve the efficiency of nuclear transfer, transgenic the Yanbian milk goat after expression of HSA to lay a solid foundation.Our study is divided into three parts, the first part is about oocyte maturation in vitro studies of different nuclear maturation inhibitors and antioxidants; Second part is about the construction of turn HSA Yanbian dairy goat-sheep reconstructed embryos; And the third is about the effect of epigallocatechin gallate (Epi-gallocatechin-3-gallate, EGCG) on the quality of transfer HSA goat-sheep reconstruction embryos.The main results are as follows:1. In sheep oocytes matured in vitro nuclear maturation, the most suitable concentrations of the inhibitor HX, IBMX were2mM and0.2mM and the most suitable concentrations of cysteine is0.6mM; EGCG is5μM and the remaining three amount of the β-ME, VC, VE are100mM;2. There is no significant differences between the two methods of nuclear transfer in the cleacage rate of reconstructed embryos and the enucleation rate can reach100%when we used Chemical auxiliary enucleation method(0.2μg/mL, Deme processing0.5h), it is quickly and efficiently, and does not affect the early development of the reconstructed embryos; 3. The most suitable fusion parameters of reconstructed embryos is120-130V,40μs pulses of electricity and the most suitable activation method for reconstructed embryos is Ion pre-active for5minutes,6-DMAP activation2h;4.The most suitable concentration of EGCG for reconstructed embryos culture in vitro is15μM and EGCG can be used in the IVM period and reconstructed embryos were cultured in vitro for1-2days.