Zhang Jie, Li Lin, Cai Yinfeng, Xu Xujun, Chen Juan, Wu Youbing, Yu Huiqing, Yu Guohua, Liu Siguo, Zhang Aimin, Chen Jianquan, Cheng Guoxiang
Rui Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
Protein Expr Purif. 2008 Feb;57(2):127-35. doi: 10.1016/j.pep.2007.10.015. Epub 2007 Nov 20.
This report details the establishment of a transgenic goat model in order to produce human lactoferrin (hLf) in the mammary gland for large-scale application and research. Two transgenic male goats were generated by microinjecting sequence encoding hLf cDNA to the pronuclear. In the two lines, derived from the two founders, eight lactating female goats could secrete recombinant human lactoferrin (rhLf) at concentrations of up to 0.765 mg/ml. The method of purifying the rhLf from the milk was achieved using ion-exchange chromatography and resulted in 97% purity. Biochemical and physicochemical characteristics of rhLf were similar to native lactoferrin (nhLf); this included N-terminal sequence, isoelectric point, molecular mass, glycosylation, iron-binding/releasing ability, thermal stability, and proteolysis. The rhLf showed broad spectrum antibacterial activity inhibiting the growth of several pathogenic bacterial strains. Also investigated, although to a lesser degree, was a practicable pasteurization method for the downstream processing of rhLf and, further, a method for the oral administration of rhLf. On the basis of these results, our studies show an optimistic and promising approach for the large-scale production and therapeutic application of rhLf expressed in transgenic goats.
本报告详细介绍了转基因山羊模型的建立，以便在乳腺中生产人乳铁蛋白（hLf）用于大规模应用和研究。通过将编码hLf cDNA的序列显微注射到原核中，产生了两只转基因雄山羊。在源自两位奠基者的两个品系中，八只泌乳母山羊能够分泌浓度高达0.765 mg/ml的重组人乳铁蛋白（rhLf）。采用离子交换色谱法从牛奶中纯化rhLf，纯度达到97%。rhLf的生化和物理化学特性与天然乳铁蛋白（nhLf）相似；这包括N端序列、等电点、分子量、糖基化、铁结合/释放能力、热稳定性和蛋白水解。rhLf显示出广谱抗菌活性，可抑制几种致病细菌菌株的生长。还研究了（尽管程度较小）一种用于rhLf下游加工的可行巴氏杀菌方法，以及进一步的rhLf口服给药方法。基于这些结果，我们的研究为转基因山羊中表达的rhLf的大规模生产和治疗应用展示了一种乐观且有前景的方法。
