乳腺特异高表达人促红细胞生成素转基因奶山羊的制备

Preparation of Transgenic Goat Highly Expressing Human Erythropoietin in Mammary Gland

目的制备乳腺特异性高表达人促红细胞生成素(hEPO)转基因奶山羊。方法采用牛β-乳球蛋白基因(BLG)调控元件和hEPO全长编码序列基因组DNA构建真核表达载体,应用受精卵原核注射的方法制备hEPO转基因山羊。结果在原核注射获得的188头羔羊中,经Southern blot法检测有4头羊含有hEPO基因,其中3头为母羊,1头公羊于出生后20d死亡;3头转基因母羊hEPO基因的拷贝数分别为1、10、2;Western blot检测结果显示转基因羊乳中的hEPO分子质量为32kDa;MTT法检测结果表明,在泌乳10d的3只转基因羊乳汁中,每毫升乳汁中hEPO活性分别达到1.17×102IU、1.90×104IU、1.91×104IU。结论牛BLG能够调控hEPO基因在山羊乳腺中高表达,为实现其他药用蛋白在山羊乳腺中表达奠定了基础。

Objective To prepare transgenic goat highly expressing human erythropoietin in mammary gland. Methods Regulation region of bovine β-lactoglobulin gene（BLG） and full-length genomic DNA encoding hEPO were used to construct eukaryotic expression vector, and the transgenic goats were produced by pronuclear microinjection. Results The Southern blot result showed that 4 among 188 goats produced by pronuclear microinjection had integrated hEPO gene. 3 of them were female and 1 of them was male which died after 20 d. The hEPO gene copy numbers in the three females were 1,10,2, respectively. The Western blot result showed that the molecular weight of hEPO expressed in transgenic goat milk was 32 kDa. The activity of hEPO in milk of the 3 transgenic goats at tenth day of milking was 1.17 × 10^5 ,1.90 × 10^4 ,1.91 × 10^4 IU/mL, respectively. Conclusion The results confirmed that BLG is able to direct hEPO to be highly expressed in goat mammary gland, thus lays a foundation for production of transgenic goats carrying other pharmaceutical protein genes.