Novel growth factor of Bombyx mori granulocyte

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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.