Purification of IGF-I in Bovine Colostrum and its Application for Animal Production Research.

胰岛素样生长因子- i （IGF-I）是一种具有生物活性的肽，由70个氨基酸组成（mol wt. 7,649）。其化学结构与胰岛素原相似。igf - 1的初级结构在测试的哺乳动物物种（人类、大鼠、小鼠、猪、牛、羊）中几乎相同。igf - 1的生物学作用最初是作为硫酸化因子被发现的，近年来的研究表明，igf - 1可以刺激多种细胞类型的增殖和分化。生长激素刺激IGF-I的分泌，然后IGF-I通过加速骨骼伸长和蛋白质积累来刺激幼龄动物的生长。在这一点上，igf - 1已被认为对动物生产很重要。我们发现牛初乳中IGF-I的浓度（0.5mg/l）超过了血浆中IGF-I的浓度，血浆已被用作提取IGF-I的生物材料，表明牛初乳可作为纯化IGF-I的有用材料。初乳是在分娩当天收集的。初乳中添加凝乳酶。37℃孵育后，离心得到不含脂质和酪蛋白的上清。加入酸-乙醇混合物（12.5% HC1, 87.5% EtOH）使IGF-I与结合蛋白分离，再加入冷丙酮得到丙酮粉。我们用丙酮粉作为起始原料。将粉末溶解于1M醋酸溶液中，应用于sephadex G-50柱。IGF-I免疫反应部分洗脱液，含0.7 ug IGF-I/mg蛋白，冻干后用TSKG3000SW进行凝胶过滤。用1M醋酸洗脱TSK柱，igf - 1纯化至1.6 ug/mg蛋白。冻干后的样品用20%乙腈在0.05% TFA中溶解，采用第一次ODP-50反相高效液相色谱法。IGF-I免疫反应性用两步线性梯度洗脱至80%乙腈。洗脱液IGF-I浓度为32 ug/mg蛋白。第二ODP-50反相高效液相色谱进一步纯化igf - 1。用大鼠成肌细胞传代培养细胞的生物测定清楚地显示了从Sephadex和TSK凝胶柱洗脱的样品中IGF-I的生物活性。少

Insulin-like growth factor-I (IGF-I) is a biologically active peptide consisting of 70 amino acids (mol wt. 7,649). Its chemical structure is homologous to proinsulin. The primary structure of IGF-I are almost identical in mammalian species tested (human, rat, mouse, porcine, bovine, ovine).Biological action of IGF-I, firstly found as sulfation factor, has been shown by recent investigations to stimulate proliferation and differentiation of various cell types. Growth hormone stimulates the secretion of IGF-I, then IGF-I stimulates the growth of young animals through accelerating bone elongation and protein accumulation. On this point IGF-I has been recognized to be of importance for animal production. We showed that the concentration (0.5mg/l) of IGF-I in bovine colostrum exceeded that in blood plasma which has been used as a biological material to extract IGF-I, suggesting availability of colostrum to be an useful material for purifying IGF-I. Colostrum was collected on the day of par … More turition. Chymosin was added to colostrum. After incubation at 37 C, the lipid- and casein-free supernatant was obtained by centrifugation. Acid-ethanol mixture (12.5% HC1, 87.5% EtOH) was added to dissociate IGF-I from its binding protein, followed by further addition of cold acetone to get acetone powder. We used the acetone powder as starting material. The powder was dissolved in 1M acetic acid solution to apply to sephadex G-50 column. Eluate of IGF-I immunoreactive fractions, containing 0.7 ug IGF-I/mg protein, was lyophilized and gel filtration procedure by TSKG3000SW was performed. The TSK column was eluted with 1M acetic acid, IGF-I being purified to 1.6 ug/mg protein. The lyophilized sample was dissolved with 20% acetonitrile in 0.05% TFA, and applied to the first ODP-50 reverse-phase HPLC. IGF-I immunoreactivity was eluted by a two-step linear gradient to 80% acetonitrile. IGF-I concentration of the eluate was 32 ug/mg protein. IGF-I was further purified by the second ODP-50 reverse phase HPLC. The bioassay using rat myoblast subculture cells clearly showed IGF-I bioactivity in the samples eluted from Sephadex and TSK gel column. Less