Puriifcation And Functional Research Of Natural HCH-like Protein From Silkworm Pupa

Human Growth Hormone-like(hGH-like) protein was found in the hmolymph offifthinstar larvae of the silkworm (Bombyx mori) and silkworm pupa, which can beproked by hGH antibody in the native state, with the weight about30kD.In this article, the fresh silkworm pupa was used as material to research. Thesupernatant was collected by homogenate, filtration and high speed centrifuge. Then,the ammonium sulphate precipitation was proformed to remove the oril and fat fromthe supernatant. After resuspended precipitation, the rude extract was concentrated by10kD millipore. The supernatant was depigmented and further isolated with gelfiltration chromatography. Finally, hGH-like protien was purified from silkworm pupaby affinity chromatography.The result of2D electrophoresis showed that there are two dots in the PAGE.Then, digged these two dots, which were digested with trypsin, and analysed byMALDI-TOF MS. After obtained the PMF(Peptide Mass Fingerprinting), weretrieved the information and performed sequences alignment by NCBI database. Itwas found that these two protiens were kind of lipoprotein, which belong to β-trefoilsuperfamily in the silkworm, named as hGH-like1and hGH-like2, respectively.The in vitro biological assay showed that the mixed protiens can promote k562cells division, with the peak activity at200ng/ml. In order to further validate thegrowth-promoting effect of hGH-like, we used BmN cells to perform the samedetection and obtained the similar result at200μg/mL, which also proved the functionof promoting. Meanwhile, the gene coded hGH-like2protein was successfullycoloned from silkworm cDNA library and constrcuted into the recombinant Bombyxmori baculovirus BmNPV-ph-hGH-like2, which were harvested by Bac-to-Bac system.With the control of BmNPV-ph, the recombinant baculovius was used to infect BmNcells as low titer. After analysis of flow cytometry assay, it was indicated thathGH-like2protein were succeed to express in BmN cells, which results in shorteningof G0/G1phase, switching cell to G2phase through G1/S check point quickly and accelerating cell cycle progression and cell proliferation. The result of tissuelocalization showed that the native hGH-like protein widely distributed in tissues,such as head, midgut, body wal and tail.It was reported that the silkworm pupa powder can promote the absorption oforal administered protein medicines. Thus, the mixed hGH-like protein labeled byFITC was injected in the mouse and be measured during the metabolic process. Theresult showed that the mixed proteins were appeared around the liver and enrichedgradually after8h, with the high level in12h, and excreted after36h.The silkworm pupa was a new nutrient source. In the future, the hGH-likeprotein may instead of hGH, which will set up a substantial foundation for thedeveloping of novel oral hormone drugs through the changing of drug forms.