转基因山羊羊乳中重组人乳铁蛋白性质的检定与研究

Identification and characterization of recombinant human lactoferrin in the milk of transgenic goats

目的:检定与研究转基因山羊 CLF123-1羊乳中重组人乳铁蛋白(rhLF)及其分子特性。方法:利用 SDS～PAGE,West～ern-blotting 及 Edman 降解法测定分析转基因山羊羊乳中 rhLF 的相对分子质量、免疫学特性和 N-末端15个氨基酸残基;利用紫外分光光度法比较 rhLF、天然人乳铁蛋白(hLF)与 Fe^(3+)离子结合的动力学过程,测定 Fe^(3+)与 LF 发生结合反应达到平衡状态后在279 nm 和465 nm 的吸收度值,Fe^(3+)与 LF 的物质的量比值分别为:0:1,0.5:1,1:1,2:1,4:1。结果:rhLF 的相对分子质量为(8.06±0.15)万(2次实验,6条电泳谱带计算结果);Western-blotting 结果显示 rhLF 可特异地与兔抗人 LF 抗体发生特异性结合;rhLF 的 N-末端1～15个氨基酸残基的序列为 G R R R R S V Q W X T V S Q P;rhLF 和 hLF 与 Fe^(3+)结合特性与趋势几乎完全一致。结论:本文所研究的转基因山羊羊乳中的乳铁蛋白是与人乳铁蛋白分子特性一致的 rhLF。

Objective ： To identify and characterize the recombinant human lactoferrin （rhLF） in the milk of transgenic goats. Methods：rhLF was characterized and analyzed by SDS -PAGE, Western -blotting and N- terminal sequencing with Edman degradation. The iron- binding properties of natural human lactoferrin （hLF） and rhLF at the mole ratio 0： 1,0.5：1 ,l： 1,2： 1 and 4：1 （Fe^3＋ ：LF） were compared by using UV absorbance at 279 nm and 465 nm. Result：The relative molecular mass of rhLF was （8.06 ± 0. 15） × 10^4（ two tests and n = 6）. The Western - blotting showed that rhLF can specially bind with rabbit anti hLF antibody. N - terminal sequence amino acid res- idues from 1 - 15 were G R R R R S V Q W X T V S Q P. The iron - binding of rhLF and hLF was highly similar. Conclusion：The protein with relative molecular mass （8.06 ± 0. 15 ） ×10^4 in the milk of transgenic goah is rhLF.