Seow H F, Rothel J S, Wood P R
CSIRO Division of Animal Health, Animal Health Research Laboratory, Parkville, Vic., Australia.
Vet Immunol Immunopathol. 1994 Jun;41(3-4):229-39. doi: 10.1016/0165-2427(94)90099-x.
An expression vector bearing the gene segment encoding the mature form of ovine interleukin-1 beta (OvIL-1 beta) was constructed. This vector provided a rapid method for obtaining Escherichia coli derived recombinant OvIL-1 beta (rOvIL-1 beta) using the expression plasmid pGEX-2T. The level of expression of fusion protein in the soluble fraction was approximately 20% of the total accumulated proteins. Affinity purification by glutathione-Sepharose yielded a fusion protein and subsequent thrombin cleavage of this material yielded rOvIL-1 beta. The specific activity of the purified recombinant protein was 10(3)-10(4) times higher than the fusion protein. The rOvIL-1 beta was 10-100 times more potent than human interleukin-1 beta (HuIL-1 beta) in an ovine thymocyte proliferation assay, although they were of equal potency in the NOB-1/CTLL assay. This simple purification method, which produces purified rOvIL-1 beta with a high specific activity (approximately 10(8) U mg-1), will now make it possible to evaluate the in vivo effects of IL-1 beta in sheep.
构建了一个携带编码绵羊白细胞介素-1β（OvIL-1β）成熟形式基因片段的表达载体。该载体提供了一种利用表达质粒pGEX-2T快速获得大肠杆菌衍生重组OvIL-1β（rOvIL-1β）的方法。可溶性部分中融合蛋白的表达水平约占总积累蛋白的20%。用谷胱甘肽-琼脂糖亲和纯化得到一种融合蛋白，随后用凝血酶切割该物质得到rOvIL-1β。纯化的重组蛋白的比活性比融合蛋白高10³-10⁴倍。在绵羊胸腺细胞增殖试验中，rOvIL-1β的效力比人白细胞介素-1β（HuIL-1β）高10-100倍，尽管它们在NOB-1/CTLL试验中的效力相当。这种简单的纯化方法可产生具有高比活性（约10⁸ U mg⁻¹）的纯化rOvIL-1β，现在将有可能评估IL-1β在绵羊体内的作用。
