Novel growth factor of Bombyx mori granulocyte

昆虫血细胞被认为是由造血器官中的造血或循环血细胞的有丝分裂提供的。我们之前的研究表明家蚕的粒细胞是由造血原细胞分化和粒细胞分裂产生的。此外，家蚕素与血淋巴因子协同作用，刺激粒细胞有丝分裂。牛胰岛素也有活性，可以替代蚕豆素。本研究旨在鉴定家蚕粒细胞生长因子(s)。具体而言，(1)对幼虫血淋巴因子进行分析；(2)利用果蝇体内与胰岛素协同作用的DmIDGF基因数据库进行BmIDGF的体外生物测定。先前的研究表明，该因子可能是具有热稳定性的高分子量蛋白，并且从L5D2幼虫中采集的血淋巴最活跃。采用超滤-硫酸铵分馏- DEAE离子交换柱-凝胶过滤柱的高效液相色谱系统对该因子进行纯化。粒细胞生长因子由两个分子组成，大约20kDa的蛋白与胰岛素协同作用，大约95kda的蛋白不与其他分子协同作用。氨基酸分析显示两者都是新的生长因子。利用家蚕脂肪体mRNA cDNA文库和DmIDGF DNA序列数据库设计的引物，确定家蚕影像光盘生长因子（BmIDGF）的DNA序列。用杆状病毒表达系统与Sf9细胞合成的BmIDGF肽未表现出促生长活性，提示粒细胞生长因子为上述分子。

Insect hemocytes are thought to be supplied by hemopoiesis in the hematopoietic organs or by mitotic division of circulating hemocytes. Our previous study demonstrated that Bombyx mori granulocytes are produced by prohemocyte differentiation and granulocyte division. Moreover, bombyxin acts cooperatively with hemolymph factor(s) to stimulate mitotic division of granulocytes. Bovine insulin is also active and substitutes for bombyxin. This research was carried out to identify the Bombyx mori granulocyte growth factor(s). In the concrete, (1) analysis of the factor(s) from larval hemolymph and (2) determination of BmIDGF based on the genetic data base of DmIDGF which acts cooperatively with insulin in Drosophila are carried out with in vitro biological assay.A previous study showed that the factor(s) may be high molecular weight protein with thermal stability and hemolymph collected from L5D2 larvae is most active. The factor(s) has been purified by a combination of ultrafiltration, ammonium sulfate fractionation and the HPLC system of DEAE ion-exchange column and then gel filtration column. The granulocyte growth factor was composed of two molecules, approximately 20kDa protein which acts in collaboration with insulin and approximately 95 kDa protein acting without other molecules. The amino acid analysis revealed that both were novel growth factors.The DNA sequence of Bombyx mori imaginal disc growth factor (BmIDGF) was determined by using cDNA library made from B. mori fat body mRNA and primers designed based on the DNA sequence database of DmIDGF. BmIDGF peptide made by using Baculovirus expression system with Sf9 cells did not show growth promoting activity, suggesting that the granulocyte growth factor(s) are the molecules described above.