Neuroprotection and Mechanism of Gas-miR36-5p from Gastrodia elata in an Alzheimer's Disease Model by Regulating Glycogen Synthase Kinase-3ß.

Alzheimer's disease (AD) is currently the most common neurodegenerative disease. Glycogen synthase kinase 3ß (GSK-3ß) is a pivotal factor in AD pathogenesis. Recent research has demonstrated that plant miRNAs exert cross-kingdom regulation on the target genes in animals. Gastrodia elata (G. elata) is a valuable traditional Chinese medicine that has significant pharmacological activity against diseases of the central nervous system (CNS). Our previous studies have indicated that G. elata-specific miRNA plays a cross-kingdom regulatory role for the NF-κB signaling pathway in mice. In this study, further bioinformatics analysis suggested that Gas-miR36-5p targets GSK-3ß. Through western blot, RT-qPCR, and assessments of T-AOC, SOD, and MDA levels, Gas-miR36-5p demonstrated its neuroprotective effects in an AD cell model. Furthermore, Gas-miR36-5p was detected in the murine brain tissues. The results of the Morris water maze test and western blot analysis provided positive evidence for reversing the learning deficits and hyperphosphorylation of Tau in AD mice, elucidating significant neuroprotective effects in an AD model following G. elata RNA administration. Our research emphasizes Gas-miR36-5p as a novel G. elata-specific miRNA with neuroprotective properties in Alzheimer's disease by targeting GSK-3ß. Consequently, our findings provide valuable insights into the cross-kingdom regulatory mechanisms underlying G. elata-specific miRNA, presenting a novel perspective for the treatment of Alzheimer's disease.

Biochemical Characterization and Functional Analysis of Glucose Regulated Protein 78 from the Silkworm Bombyx mori.

The glucose regulated protein (GRP78) is an important chaperone for various environmental and physiological stimulations. Despite the importance of GRP78 in cell survival and tumor progression, the information regarding GRP78 in silkworm Bombyx mori L. is poorly explored. We previously identified that GRP78 expression was significantly upregulated in the silkworm Nd mutation proteome database. Herein, we characterized the GRP78 protein from silkworm B. mori (hereafter, BmGRP78). The identified BmGRP78 protein encoded a 658 amino acid residues protein with a predicted molecular weight of approximately 73 kDa and comprised of two structural domains, a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). BmGRP78 was ubiquitously expressed in all examined tissues and developmental stages by quantitative RT-PCR and Western blotting analysis. The purified recombinant BmGRP78 (rBmGRP78) exhibited ATPase activity and could inhibit the aggregating thermolabile model substrates. Heat-induction or Pb/Hg-exposure strongly stimulated the upregulation expression at the translation levels of BmGRP78 in BmN cells, whereas no significant change resulting from BmNPV infection was found. Additionally, heat, Pb, Hg, and BmNPV exposure resulted in the translocation of BmGRP78 into the nucleus. These results lay a foundation for the future identification of the molecular mechanisms related to GRP78 in silkworms.

The Acetylation Modification of SP1 Regulates the Protein Stability in Silkworm.

Acetylation is a highly conservative and reversible post-translational modification. Acetylation modification can regulate gene expression by altering protein function and is widely identified in an increasing number of species. Previously, the acetylated proteome of silkworm was identified by combining acetylated polypeptide enrichment with nano-HPLC/MS/MS; the identification revealed that the SP proteins (SPs) were high acetylated. In this study, the acetylation of SP1, one of the SPs, was further confirmed using immunoprecipitation (IP) and Western blotting. Then, we found the acetylation could upregulate SP1 protein expression by enhancing the protein stability. Further research found that the acetylation of SP1 protein can competitively inhibit its ubiquitination and thus improve the stability and cell accumulation of SP1 protein by inhibiting the ubiquitin-mediated proteasome degradation pathway. This result provides a basis for acetylation to regulate the nutrient storage and utilization of silkworm.

Activated tyrosine kinases in gastrointestinal stromal tumor with loss of KIT oncoprotein expression.

Oncogenic KIT or PDGFRA receptor tyrosine kinase (TK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GISTs), and the KIT/PDGFRA kinase inhibitor, imatinib, is the standard of care for patients with metastatic GIST. However, approximately 10% of KIT-positive GIST metastases lose KIT expression at the time of clinical progression during imatinib therapy. In the present report, we performed TK-activation screens, using phosphotyrosine-TK double immunoaffinity purification and mass spectrometry, in GIST in vitro models lacking KIT expression. These studies demonstrated tyrosine-phosphorylated EGFR, AXL, and EPHA2 in four of six KIT-negative GIST lines (GIST62, GIST522, GIST54, GIST226, GIST48B, and GIST430B), and tyrosine-phosphorylated focal adhesion kinase (FAK) in each of the six KIT-negative lines. AXL expression was strong in KIT-negative or -weak clinical GIST samples that were obtained from progressing metastases during imatinib therapy. AXL knockdown inhibited viability in three KIT-negative GIST cell lines (GIST62, GIST54, and GIST522), but not in an AXL-negative, KIT-positive GIST control cell line (GIST430). AXL inhibition by R428, a specific AXL kinase inhibitor, reduced viability in AXL-activated GIST54. AXL knockdown in GIST62, GIST522, and GIST54 was accompanied by an increase in p21, p27, and p53 expression. By contrast, gefitinib-mediated EGFR inhibition, PF562271-mediated FAK inactivation, and shRNA-mediated knockdowns of EPHA2 and FAK had no effect on viability or colony formation of the KIT-negative GISTs. These findings highlight the potential relevance of AXL/p53 signaling as a therapeutic target in a subset of GISTs that have lost KIT oncoprotein expression.

Detection of piRNAs in whitespotted bamboo shark liver.

Piwi-interacting RNAs (piRNAs) are 26 to 31-nt small non-coding RNAs that have been reported mostly in germ-line cells and cancer cells. However, the presence of piRNAs in the whitespotted bamboo shark liver has not yet been reported. In a previous study of microRNAs in shark liver, some piRNAs were detected from small RNAs sequenced by Solexa technology. A total of 4857 piRNAs were predicted and found in shark liver. We further selected 17 piRNAs with high and significantly differential expression between normal and regenerative liver tissues for subsequent verification by Northern blotting. Ten piRNAs were further identified, and six of these were matched to known piRNAs in piRNABank. The actual expression of six known and four novel piRNAs was validated by qRT-PCR. In addition, a total of 401 target genes of the 10 piRNAs were predicted by miRanda. Through GO and pathway function analyses, only five piRNAs could be annotated with eighteen GO annotations. The results indicated that the identified piRNAs are involved in many important biological responses, including immune inflammation, cell-specific differentiation and development, and angiogenesis. This manuscript provides the first identification of piRNAs in the liver of whitespotted bamboo shark using Solexa technology as well as further elucidation of the regulatory role of piRNAs in whitespotted bamboo shark liver. These findings may provide a useful resource and may facilitate the development of therapeutic strategies against liver damage.

Synergetic Protein Factors That Improve rhGM-CSF Absorption via an Oral Route Exist in Silkworm Pupae.

Recent studies have demonstrated that recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) produced by the silkworm pupae bioreactor is absorbed into blood through oral administration and functions as an active cytokine. The aim of this study was to further examine and identify synergetic protein factors in silkworm pupae that improve rhGM-CSF absorption via an oral route. The concentrations of rhGM-CSF in serum were evaluated in mice after oral administration of rhGM-CSF using different chemical compositions of silkworm pupae as pharmaceutical excipients. The experimental data revealed that the supernatant lyophilized powder (SLP) of a homogenized slurry of silkworm pupae caused a significant increase in the rhGM-CSF level in blood when rhGM-CSF was orally administered with SLP, suggesting that synergetic protein factors that improve the oral absorption of rhGM-CSF primarily exist in SLP. As shown by scanning electron microscopy, microspheres were formed when rhGM-CSF was coated with SLP. Animal experimental data showed that the absorption of orally administered rhGM-CSF through the gastrointestinal (GI) tract primarily resulted from protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration. Surface plasmon resonance spectroscopy analysis demonstrated that several protein factors present in the SLP retentate obtained after 10 kDa ultrafiltration were bound to rhGM-CSF. Proteins bound to rhGM-CSF by liquid chromatography-mass spectrometry were identified as chymotrypsin inhibitor SCI-II precursor, cationic peptide CP8 precursor, Kazal-type proteinase inhibitor, and chymotrypsin inhibitor SCI-I. These findings indicate that these proteinase inhibitors play an important role in improving rhGM-CSF absorption in the GI tract.

Anti-diabetic effects of CTB-APSL fusion protein in type 2 diabetic mice.

To determine whether cholera toxin B subunit and active peptide from shark liver (CTB-APSL) fusion protein plays a role in treatment of type 2 diabetic mice, the CTB-APSL gene was cloned and expressed in silkworm (Bombyx mori) baculovirus expression vector system (BEVS), then the fusion protein was orally administrated at a dose of 100 mg/kg for five weeks in diabetic mice. The results demonstrated that the oral administration of CTB-APSL fusion protein can effectively reduce the levels of both fasting blood glucose (FBG) and glycosylated hemoglobin (GHb), promote insulin secretion and improve insulin resistance, significantly improve lipid metabolism, reduce triglycerides (TG), total cholesterol (TC) and low density lipoprotein (LDL) levels and increase high density lipoprotein (HDL) levels, as well as effectively improve the inflammatory response of type 2 diabetic mice through the reduction of the levels of inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Histopathology shows that the fusion protein can significantly repair damaged pancreatic tissue in type 2 diabetic mice, significantly improve hepatic steatosis and hepatic cell cloudy swelling, reduce the content of lipid droplets in type 2 diabetic mice, effectively inhibit renal interstitial inflammatory cells invasion and improve renal tubular epithelial cell nucleus pyknosis, thus providing an experimental basis for the development of a new type of oral therapy for type 2 diabetes.

MicroRNA-452 promotes tumorigenesis in hepatocellular carcinoma by targeting cyclin-dependent kinase inhibitor 1B.

Dysregulation of miR-452 has been observed in many tumors, but its biological function in hepatocellular carcinoma (HCC) is still unknown. Our results showed that miR-452 expression is significantly increased in HCC tissues and HCC cell lines. We also found that overexpression of miR-452 dramatically accelerated proliferation, induced cell cycle from G1 to S transition, and blocked apoptosis of HCC cells. Migration and matrigel invasion assays indicated that miR-452 significantly promotes HepG2 and QGY-7703 cells migration and invasion in vitro. Further studies showed that miR-452 directly targets the 3'-untranslated region of cyclin-dependent kinase inhibitor 1B (CDKN1B), ectopic miR-452 expression suppressed CDKN1B expression on mRNA and protein level. Silencing CDKN1B by small interfering RNA resembled the phenotype resulting from ectopic miR-452 expression. This study provides new insights into the potential molecular mechanisms that miRNA-452 contributed to HCC.

The Expression, Purification, and Characterization of a Ras Oncogene (Bras2) in Silkworm (Bombyx mori).

The Ras oncogene of silkworm pupae (Bras2) may belong to the Ras superfamily. It shares 77% of its amino acid identity with teratocarcinoma oncogene 21 (TC21) related ras viral oncogene homolog-2 (R-Ras2) and possesses an identical core effector region. The mRNA of Bombyx mori Bras2 has 1412 bp. The open reading frame contains 603 bp, which encodes 200 amino acid residues. This recombinant BmBras2 protein was subsequently used as an antigen to raise a rabbit polyclonal antibody. Western blotting and real-time PCR analyses showed that BmBras2 was expressed during four developmental stages. The BmBras2 expression level was the highest in the pupae and was low in other life cycle stages. BmBras2 was expressed in all eight tested tissues, and it was highly expressed in the head, intestine, and epidermis. Subcellular localization studies indicated that BmBras2 was predominantly localized in the nuclei of Bm5 cells, although cytoplasmic staining was also observed to a lesser extent. A cell proliferation assay showed that rBmBras2 could stimulate the proliferation of hepatoma cells. The higher BmBras2 expression levels in the pupal stage, tissue expression patterns, and a cell proliferation assay indicated that BmBras2 promotes cell division and proliferation, most likely by influencing cell signal transduction.

Construction of the ie1-Bacmid expression system and its use to express EGFP and BmAGO2 in BmN cells.

The presently available expression tools and vectors (e.g., eukaryotic expression vectors and the adenovirus expression system) for studying the functional genes in Bombyx mori are insufficient. The baculovirus expression system is only used as a protein production tool; therefore, recombinant proteins expressed by B. mori using the baculovirus expression system equipped with a polyhedrin promoter cannot be used for in vivo research applications. In this work, we constructed and screened a eukaryotic expression vector for silkworm cells The EGFP and B. mori Argonaute2 proteins were found to be efficiently expressed using the screened pIEx-1 vector with the FuGENE 6 transfection reagent. Additionally, we constructed a novel nucleopolyhedrovirus ie1-Bacmid expression system for the production of recombinant protein; we then used the system to highly express the EGFP and B. mori Argonaute2 proteins. In this system, the protein of interest can be efficiently expressed 13 h after infection by controlling the B. mori nucleopolyhedrovirus immediate early ie1 promoter. The ie1-Bacmid system provides a powerful "adenovirus-like" expression tool; not only can the tool be used to study baculovirus molecular biology for the silkworm but it is also useful in other research applications as well, such as the study of gene functions involved in cellular physiological processes.

Expression analysis of miRNAs in BmN cells.

MicroRNAs (miRNAs) are the family of noncoding single-strand RNA molecules of 21-25 nucleotides in length and play a broad and key regulation role in various physiological and pathological processes including differentiation, apoptosis, proliferation, and tumorigenesis. In Bombyx mori, a total of 487 pre-miRNAs and 562 mature miRNAs were identified by experimental or computational approaches, but their functions remain unknown. To carry out the research of gain-of-function of miRNAs in BmN cells, we firstly identified the endogenous expression of miRNAs in BmN cells by microarray and found that only 73 miRNAs could be detected by miRNA microarray. Then three low abundance or undetected miRNAs, pri-mir-1a, pri-mir-8 and pri-mir-133, were selected to express in BmN cells. The eukaryotic expression vector pIEx-1 harboring baculovirus ie1 promoter and hr5 enhancer was screened and used for expressing miRNA in BmN cells. Three miRNA expression vectors pIEx-1-EGFP-pri-mir-1a/8/133 were constructed, which contained the three corresponding pri-miRNA sequences, respectively. The constructed miRNA vectors were successfully transfected into BmN cells and the qRT-PCR analysis showed that relative abundance of bmo-mir-1a, bmo-mir-8 and bmo-mir-133 in BmN cells transfected with the pIEx-1-EGFP-pri-mir-1a/8/133 is as 32, 4.4 and 904 times as that in BmN cells transfected with the control vector pIEx-1-EGFP, respectively. The present work lays a foundation for the further functional studies of miRNAs in silkworm.

The screening and functional study of proteins binding with the BmNPV polyhedrin promoter.

BACKGROUND: The polyhedrin gene promoter has an essential role in regulating foreign gene expression in baculovirus expression vector systems (BEVS); however, the high-level transcription mechanism is still unknown. One-hybrid screening in yeast is a powerful way of identifying rapidly heterologous transcription factors that can interact with the polyhedrin promoter DNA sequence. In the current study, total RNA was extracted from the fat bodies of fifth-instar silkworm larvae that had been infected with Bombyx mori nuclear polyhedrosis virus (BmNPV) for 5 days; complementary DNA (cDNA) was then generated using reverse-transcription (RT)-PCR to construct a silkworm gene expression library. Key polyhedrin promoter bait sequences were synthesized to generate a bait yeast strain, which was used to screen the one-hybrid cDNA library. RESULTS: In total, 12 positive yeast colonies were obtained from the SD/-Leu/AbA plates; sequencing analysis showed that they belong to two different protein cDNA colonies. Positive colonies underwent bioinformatics analysis, which revealed one colony to be ribosomal proteins [B. mori ribosomal protein SA (BmRPSA)] and the other to be NPV DNA-binding proteins (DBP). To further verify the regulatory function of these two protein groups, transient expression vectors (pSK-IE-dbp and pSK-IE-BmRPSA) were constructed. The recombinant plasmids were then transfected into cultured B. mori N (BmN) cells, which had been infected with a recombinant bacmid containing the gene encoding luciferase (luc). The results showed that overexpression of either dbp or BmRPSA upregulated the polh promoter-driven transcription of luc in BmN cells. In addition, dbp or BmRPSA RNA interference (RNAi) resulted in the downregulation of luciferase reporter expression in BmN cells, demonstrating that DBP and BmRPSA are important for luc transcription. EMSA results further confirmed that DBP could directly bind to the conserved single-stranded polh promoter region in intro. However, EMSA assay also showed that BmRPSA did not bind to this region, precluding a direct DNA association. CONCLUSIONS: Both DBP and BmRPSA are important for polh transcription. DBP can regulate polh promoter activity by direct binding to the conserved single-stranded polh promoter region, BmRPSA may regulate polh promoter activity by indirect binding to this region.

In vivo bioassay of recombinant human growth hormone synthesized in B. mori pupae.

The human growth hormone (hGH) has been expressed in prokaryotic expression system with low bioactivity previously. Then the effective B. mori baculovirus system was employed to express hGH identical to mature hGH successfully in larvae, but the expression level was still limited. In this work, the hGH was expressed in B. mori pupae by baculovirus system. Quantification of recombinant hGH protein (BmrhGH) showed that the expression of BmrhGH reached the level of approximately 890 microg/mL pupae supernatant solution, which was five times more than the level using larvae. Furthermore, Animals were gavaged with BmrhGH at the dose of 4.5 mg/rat.day, and the body weight gain (BWG) of treated group had a significant difference (P < .01) compared with the control group. The other two parameters of liver weight and epiphyseal width were also found to be different between the two groups (P < .05). The results suggested that BmrhGH might be used as a protein drug by oral administration.

Cloning, expression, and cell localization of a novel small heat shock protein gene: BmHSP25.4.

Using molecular approaches, a new member of the Bombyx mori small heat shock protein family was cloned and characterized. The isolated gene contains an open reading frame of 672 bp, encodes a polypeptide of 223 amino acid residues with a predicted molecular mass of 25.4 kDa, and is therefore named BmHSP25.4. The gene codes for a protein homologous to the previously characterized HSP20.4 and HSP19.9. Western blotting analysis revealed that BmHSP25.4 existed in the fifth-instar larva's fatty body and blood tissues. Immunohistochemistry assay also showed that BmHSP25.4 was located in the fifth-instar larva's fatty body. The results of above studies have indicated constitutive expression of BmHSP25.4 in fatty body, blood tissues, and Bm5 cells. Finally, we examined the effect of heat stress on localization of BmHSP25.4 using anti-BmHSP25.4 polyclonal antibody by immunofluorescence. Under normal conditions, BmHSP25.4 was mostly found in the cytoplasm. However, after heat treatment, most of BmHSP25.4 distributed in the cell membrane. After 3 h of recovery following the heat shock treatment, the localization of BmHSP25.4 was the same as that under normal conditions.

Molecular cloning and expression characterization of translationally controlled tumor protein in silkworm pupae.

A Bombyx mori (B. mori) cDNA was isolated from silkworm pupae cDNA library encoding a homologue of translationally controlled tumor protein (BmTCTPk). BmTCTPk was expressed in E. coli; SDS-PAGE and Western blot showed the molecular weight of recombinant and native BmTCTPk is approximately 28 and 25 kDa, respectively; they are larger than the theoretical molecular weight. Immunohistochemical studies showed that BmTCTPk is uniformly distributed throughout the cytoplasm of BmN cells. In silkworm pupae, BmTCTPk is expressed in the midgut wall, the midgut cavity, and some fat body tissues lying between the midgut wall and body wall. Western blot and ELISAs performed on total protein extracts isolated from silkworm pupae at different development stages showed that, although BmTCTPk is expressed during all pupae stages, its expression level increases dramatically during late pupae stages, suggesting that BmTCTPk may play an important role during the developmental transition from pupa to imago.

Bioavailability of orally administered rhGM-CSF: a single-dose, randomized, open-label, two-period crossover trial.

BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is usually administered by injection, and its oral administration in a clinical setting has been not yet reported. Here we demonstrate the bioavailability of orally administered rhGM-CSF in healthy volunteers. The rhGM-CSF was expressed in Bombyx mori expression system (BmrhGM-CSF). METHODS AND FINDINGS: Using a single-dose, randomized, open-label, two-period crossover clinical trial design, 19 healthy volunteers were orally administered with BmrhGM-CSF (8 microg/kg) and subcutaneously injected with rhGM-CSF (3.75 microg/kg) respectively. Serum samples were drawn at 0.0h, 0.5h ,0.75h,1.0h,1.5h,2.0h ,3.0h,4.0h,5.0h,6.0h,8.0h,10.0h and 12.0h after administrations. The hGM-CSF serum concentrations were determined by ELISA. The AUC was calculated using the trapezoid method. The relative bioavailability of BmrhGM-CSF was determined according to the AUC ratio of both orally administered and subcutaneously injected rhGM-CSF. Three volunteers were randomly selected from 15 orally administrated subjects with ELISA detectable values. Their serum samples at the 0.0h, 1.0h, 2.0h, 3.0h and 4.0h after the administrations were analyzed by Q-Trap MS/MS TOF. The different peaks were revealed by the spectrogram profile comparison of the 1.0h, 2.0h, 3.0h and 4.0h samples with that of the 0.0h sample, and further analyzed using both Enhanced Product Ion (EPI) scanning and Peptide Mass Fingerprinting Analysis. The rhGM-CSF was detected in the serum samples from 15 of 19 volunteers administrated with BmrhGM-CSF. Its bioavailability was observed at an average of 1.0%, with the highest of 3.1%. The rhGM-CSF peptide sequences in the serum samples were detected by MS analysis, and their sizes ranging from 2,039 to 7,336 Da. CONCLUSIONS: The results demonstrated that the oral administered BmrhGM-CSF was absorbed into the blood. This study provides an approach for an oral administration of rhGM-CSF protein in clinical settings. TRIAL REGISTRATION: www.chictr.orgChiCTR-TRC-00000107.

A novel method for isolation of membrane proteins: a baculovirus surface display system.

We have developed a novel baculovirus surface display (BVSD) system for the isolation of membrane proteins. We expressed a reporter gene that encoded hemagglutinin gene fused in frame with the signal peptide and transmembrane domain of the baculovirus gp64 protein, which is displayed on the surface of BmNPV virions. The expression of this fusion protein on the virion envelope allowed us to develop two methods for isolating membrane proteins. In the first method, we isolated proteins directly from the envelope of budding BmNPV virions. In the second method, we isolated proteins from cellular membranes that had disintegrated due to viral egress. We isolated 6756 proteins. Of these, 1883 have sequence similarities to membrane proteins and 1550 proteins are homologous to known membrane proteins. This study indicates that membrane proteins can be effectively isolated using our BVSD system. Using an analogous method, membrane proteins can be isolated from other eukaryotic organisms, including human beings, by employing a host cell-specific budding virus.

Subcellular localization and RNA interference of an RNA methyltransferase gene from silkworm, Bombyx mori.

RNA methylation, which is a form of posttranscriptional modification, is catalyzed by S-adenosyl-L-methionone-dependent RNA methyltransterases (RNA MTases). We have identified a novel silkworm gene, BmRNAMTase, containing a 369-bp open reading frame that encodes a putative protein containing 122 amino acid residues and having a molecular weight of 13.88 kd. We expressed a recombinant His-tagged BmRNAMTase in E. coli BL21 (DE3), purified the fusion protein by metal-chelation affinity chromatography, and injected a New Zealand rabbit with the purified protein to generate anti-BmRNAMTase polyclonal antibodies. Immunohistochemistry revealed that BmRNAMTase is abundant in the cytoplasm of Bm5 cells. In addition, using RNA interference to reduce the intracellular activity and content of BmRNAMTase, we determined that this cytoplasmic RNA methyltransferase may be involved in preventing cell death in the silkworm.

Large-scale purification of human granulocyte-macrophage colony-stimulating factor expressed in Bombyx mori pupae.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) acts on many different kinds of cells, including monocytes, macrophages, granulocytes, eosinophils, and multipotential stem cells. To explore further explore pharmaceutical action, we expressed hGM-CSF by the Bombyx mori nucleopolyhedrovirus expression system in silkworm pupae. However, purifying recombinant proteins from silkworm pupae on a large scale has been a big challenge. To establish purification methods suitable for mass production, we tried two crude preparation methods: (NH4)2SO4 fractional precipitation and isoelectric precipitation with a combination of gel filtration and ion-exchange chromatography. The isoelectric precipitation method was found to be more efficient. With this method, we eventually obtained approx 11.7 mg of 95% pure product from 1000 g of infected silkworm pupae. The recovery of purified protein was greatly increased, by approx 40%, compared with the other method. The biologic activity of this protein was determined up to 9.0 x 106 colony-forming units/mg in the final purified product.

Can 29kDa rhGM-CSF expressed by silkworm pupae bioreactor bring into effect as active cytokine through orally administration?

In order to study the effect of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) as active cytokine through orally administration, we expressed hGM-CSF within silkworm pupae bioreactor. The purified rhGM-CSF named as BmrhGM-CSF is characterized as 29kDa glycoprotein, and its biological activity was measured both in vitro and in vivo. We found out BmrhGM-CSF could stimulate the colony formation of human bone marrow cells in a dose-dependent manner whether which were treated with or without gamma-ray 24h before. The ability of colony formation induced by BmrhGM-CSF is negatively correlated with gamma-ray intensity. As soon as 15min post oral administration with BmrhGM-CSF labeled with (125)I, an approximately 20kDa protein fragment was detected within mice blood by SDS-PAGE followed by autoradiography. In blood sample of test mice, a protein was also recognized by anti-hGM-CSF antibody using ELISA. The immunohistochemical analysis showed that BmrhGM-CSF was detected within intestinal histiocyte. This indicated it might be absorbed into blood via intestinal microvillus. Pharmacokinetics analysis after orally administered BmrhGM-CSF in animal model of leucopenia including mice, Beagle dogs and macaques showed that: (1) BmrhGM-CSF promoted the CFU-S formation in mice spleen and the synthesis of DNA in bone marrow cells of mice; (2) BmrhGM-CSF induced bone marrow karyocyta granulocyte growth significantly in both macaques and Beagle dogs compared to the negative control group. On the 9th day of orally administration, the animal WBC significantly increased in a dose-dependant manner, in which neutrophilic granulocyte was predominant. The WBC level of dogs in high dose group was about 1.5x10(9)cells/L more than that in the negative control. And the bone marrow smear revealed that the percents of both myloblast and progranulocyte in WBC in the hGM-CSF group were obviously higher than those in the negative control. These results proved that BmrhGM-CSF, a 29kDa glycoprotein expressed by Silkworm pupae bioreactor, could bring into the effect as active cytokine through oral administration.