Purification And Functional Analysis Of Bm8K, A Small Cationic Protein In Silkworm, Bombyx Mori

Insulin and insulin-like growth factors (IGFs) signal system control growth and metabolism in both vertebrates and invertebrates. This system is composed of conserved effective factors. In particular, the N-and C-terminal domain sequences of the insulin-like growth factor binding proteins (IGFBPs) are highly conserved. They play an important role on cell proliferation, differentiation, and apoptosis. Insulin/IGFs signal system is the focus of biological research and any advance in in this field will have broad application prospects in agriculture and in disease treatment.As model organisms of Lepidopteran insects, silkworm not only has important economic value, but also provides an important reference for other insect studies. Currently, there are few researches to report the functional homologue of IGFBPs in invertebrate. Our work studied on the bioinformatics, purification and preliminary functional analysis of a silkworm protein, named as Bm8K protein. The main results are as follows:1. Bioinformatics analysis of Bm8K geneAccording to genome sequence of Bm8K, the gene consists of 2 exons interrupted 1 introns. The length of intron is 578bp and 2 exons are 193bp and 110bp length, respectively. TESS software predicted that it has 34 putative insulin signaling system-related transcirptional elements, in the 2500bp sequence upstream to the transcription initiation site (+1). Analyzed by online ProtParam software indicated that the molecular weight of mature Bm8K peptide is 8694Da with isoelectric point 8.64, including five disulfide bonds and. The alignment of amino acid sequences of the Bm8K protein with insulin-like growth factor binding protein (IGFBPs) found the sequence similarity between two proteins, especially at the N-terminal functional region.2. Purification and identification of Bm8K protein In order to obtain the native Bm8K protein, ammonium sulfate precipitation, gel filtration and cation ion-exchange chromatography, we carried out to purify this protein from the hemolymph of silkworm larvae at the fifth instar day 3. The detection results of Assist matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and western blotting showed that this protein was successfully.3. Effects of glucose feeding on expression of Bm8K geneSilkworm larvae were fed on the artificial diet containing 15% glucose from fourth instar to fifth instar. Total RNAs from fat body and head of larvae at the fifth instar day 3 were extracted and the corresponding cDNAs were reverse-transcripted. The gene expression of Bm8K and BombyxinA3 were investigated using cDNAs as templates in RT-PCR. The results revealed that Bm8K gene and BombyxinA3 gene were upregulated in glucose-treatment compared to the control.4. Effects of Bm8K protein on the growth of silkworm ovarian cellsThe Bm8K protein with different concentrations (25,50,100μg/mL) were incubated with the silkworm ovarian cells in logarithmic growth phase. In this experiment, same amout of PBS was used as the control. After being incubated for 48h, the growth of the ovarian cells were measured using a Cell Counting Kit-8 kit. The results showed that Bm8K protein stimulated the growth of ovarian cells with dose-dependent manner.