蚕蛹中天然人生长激素类似蛋白的纯化与功能研究

Human Growth Hormone-like(hGH-L)was first found in the hmolymph of fifthinstar larvae of the Bombyx mori and silkworm pupa which can bind with the antibody of hGH protein in the natural state, its weight is about 30kDa. In this research, we use fresh silkworm pupa as material, discard impurity substance with homogenate, filtration and high speed centrifuge, then collect the supernatant and remove the grease by ammonium sulphate precipitation, resuspend precipitation, depigment and further isolated with gel filtration chromatography. Finally, hGH-L protien was extracted by affinity chromatography. we analysised these protein with 2D electrophoresis, digged the interested protein dots from 2D PAGE and then digested with trypsin. The protein's PMF (Peptide Mass Fingerprinting) was obtained through the MALDI-TOF-MS. At last, we found hGH-L protien consists of two lipoproteins which belong to β-trefoil superfamily and their β-strcuture domains have a close affinity to the structure of hGH. It is indicated that hGH-L protien can promote k562 cells division by in vitro biological assay. In order to further validate the growth-promoting effect of hGH-L, we clone the gene sequence of hGH-L2 from cDNA silkworm pupa, construct the recombinant BmNPV and infect BmN cells as low titer. Flow cytometry assay indicated that hGH-L protein results in shortening of G0/G1 phase, switching cell to G2 phase through G1/S check point quickly and accelerating cell cycle progression and cell proliferation. Beacuse of discovery of hGH-L protein, people will focus on the medicinal value of silkworm again, which will set up a substantial foundation for the developing of novel hormone drugs.