Characterization of recombinant human lactoferrin secreted in milk of transgenic mice
Characterization of recombinant human lactoferrin secreted in milk of transgenic mice
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DOI：
10.1074/jbc.272.13.8802
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发表时间：
1997-03-28
影响因子：
4.8
通讯作者：
Pieper, FR
中科院分区：
文献类型：
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作者：
Nuijens, JH;vanBerkel, PHC;Pieper, FR
人乳铁蛋白（hLF）是一种铁结合蛋白，参与宿主防御感染和严重炎症。转基因小鼠要么含有hLF cDNA，要么含有与牛α - S-1酪蛋白基因调控元件融合的hLF基因组序列。将转基因小鼠乳中表达的重组hLF（转基因hLF）与天然（人乳源性）hLF进行比较。两种形式的免疫特性通过双抗体免疫测定和转基因小鼠对天然hLF高免疫不产生抗hLF抗体反应得到证实。转基因和天然hLF的单S阳离子交换层析和n端蛋白测序结果显示，转基因和天然hLF的阳离子性和n端序列相同。sds -聚丙烯酰胺凝胶电泳和吸光度测定表明，纯化的转基因hLF蛋白的铁饱和度为90%，而天然hLF蛋白的铁饱和度仅为3%。在ph介导下，转基因hLF对铁的释放与含铁饱和的天然hLF没有什么不同，不饱和转基因hLF在加入铁后可以完全再饱和。通过酶解糖基化消除了转基因和天然hLF在sds -聚丙烯酰胺凝胶电泳上迁移率的细微差异，转基因和天然hLF与一系列配体的结合没有差异，包括细菌脂多糖、肝素、单链DNA、Cibacron蓝FG 3A和凝集素。基于这些观察结果，我们预计（不饱和）rhLF和天然hLF在体内会发挥相似的抗菌和抗炎活性，如果不是完全相同的话。
Human lactoferrin (hLF) is an iron-binding protein involved in host defense against infection and severe inflammation. Transgenic mice were produced harboring either hLF cDNA or genomic hLF sequences fused to regulatory elements of the bovine alpha S-1 casein gene. Recombinant hLF expressed in the milk of transgenic mice (transgenic hLF) was compared with natural (human milk-derived) hLF. Immunological identity of the two forms was shown by double antibody immunoassays and the absence of an anti-hLF antibody response in transgenic mice on hyperimmunization with natural hLF. Mono S cation-exchange chromatography and N-terminal protein sequencing of transgenic and natural hLF revealed identical cationicity and N-terminal sequences. SDS-polyacrylamide gel electrophoresis and absorbance measurements of purified transgenic hLF showed this protein was 90% saturated with iron, whereas natural hLF is only 3% saturated. The pH-mediated release of iron from transgenic hLF was not different from that of iron-saturated natural hLF, Unsaturated transgenic hLF could be completely resaturated upon addition of iron. Slight differences in mobility between transgenic and natural hLF on SDS-polyacrylamide gel electrophoresis were abolished by enzymatic deglycosylation, Binding of transgenic and natural hLF to a range of ligands, including bacterial lipopolysaccharide, heparin, single-stranded DNA, Cibacron blue FG 3A, and lectins, was not different.Based on these observations, we anticipate that (unsaturated) rhLF and natural hLF will exert similar, if not identical, antibacterial and anti inflammatory activity in vivo.