Xuemei Lu

Expression of the antimicrobial peptide cecropin fused with human lysozyme in Escherichia coli.

Lysozyme is an abundant, cationic antimicrobial protein that plays an important role in host defense. It targets the β (1–4) glycosidic bond between N-acetylglucosamine and N-acetylmuramic residues that make up peptidoglycan, making lysozyme highly active against Gram-positive bacteria. However, lysozyme alone is inactive against Gram-negative bacteria because it cannot reach the peptidoglycan layer. Cecropins are cationic molecules with a wide range of antimicrobial activities. The main target for these peptides is the cytoplasmic membrane. We resume that cecopin may disrupt the outer membrane, giving the enzyme access to the peptidoglycan in cell wall. So in the present study, novel hybrid protein combining Musca domestica cecropin (Mdc) with human lysozyme (Hly) was designed. The DNA sequence encoding recombination fusion protein Mdc–hly was cloned into the pET-32a vector for protein expression in Escherichia coli strain BL21 (DE3). The protein was expressed as a His-tagged fusion protein, and the Mdc–hly was released from the fusion by enterokinase cleavage and separated from the carrier thioredoxin. Antimicrobial activity assays showed that the recombinant fusion protein Mdc–hly has improved in vitro antimicrobial activity and action spectrum compared to Mdc and hly. Mdc–hly may have important potential application as a future safely administered human drug and food additive.

IFN-CSP Inhibiting Hepatitis B Virus in HepG2.2.15 Cells Involves JAK-STAT Signal Pathway

Frequent and high-dose administration of interferon to patients with viral hepatitis results in various side effects. In our previous study, a novel liver-targeting interferon (IFN-CSP) combining Plasmodium region I peptide with IFNα2b was successfully designed and expressed in the Escherichia coli expression systems. This targeting would target the IFNα2b specifically to the liver, thus reducing the adverse events. In the present study, we further investigated the anti-HBV effects and molecular mechanisms of recombinant IFN-CSP in HepG2.2.15 cell line. Hepatitis B surface antigen (HBsAg) and HBe antigen (HBeAg) in the culture supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA). HBV-DNA was measured by real-time quantitative PCR. HBV core protein was assayed by immunofluorescent and western blot analysis. The expressions of signal transducers and transactivator 1 (STAT1), STAT2, IFN regulatory factor 9 (IRF-9), and 2′-5′-oligoadenylate synthetase 1 (OAS1) were investigated by the reverse transcription PCR and western blot analysis. Results indicate IFN-CSP efficiently inhibited HBsAg and HBeAg secretion, HBV-DNA replication, and HBV core protein expression in HepG2.2.15 cells. The anti-HBV mechanisms involve activation of JAK-STAT signaling and increase of the anti-HBV protein OAS expression. IFN-CSP could be a good substitute for IFNα2b for anti-HBV treatment.

High-level expression of a novel liver-targeting fusion interferon with preferred Escherichia coli codon preference and its anti-hepatitis B virus activity in vivo

BackgroundIn our previous study, a novel liver-targeting fusion interferon (IFN-CSP) combining IFN α2b with plasmodium region I peptide was successfully constructed. IFN-CSP has significant inhibition effects on HBV-DNA replication in HepG2.2.15 cells. The aim of the present investigation was focused on how to produce high levels of recombinant IFN-CSP and its in vivo anti-hepatitis B virus (HBV) activity.MethodsA modified DNA fragment encoding IFN-CSP was synthesized according to Escherichia coli (E. coli) preferred codon usage and transformed into E. coli BL21 (DE3) for protein expression. The induction conditions were systematically examined by combining one-factor experiments with an orthogonal test (L(9)(3)(4)). The antigenicity of the purified protein was characterized by western blot analysis. The in vivo tissue distribution were assayed and compared with native IFN α2b. HBV-transgenic mice were used as in vivo model to evaluate the anti-HBV effect of the recombinant IFN-CSP.ResultsThe results showed that the E. coli expression system was very efficient to produce target protein.ConclusionOur current research demonstrates for the first time that IFN-CSP gene can be expressed at high levels in E. coli through codon and expression conditions optimization. The purified recombinant IFN-CSP showed liver-targeting potentiality and anti-HBV activity in vivo. The present study further supported the application of IFN-CSP in liver-targeting anti-HBV medicines.

Cecropin Suppresses Human Hepatocellular Carcinoma BEL- 7402 Cell Growth and Survival in vivo without Side-Toxicity

Conventional chemotherapy against hepatocellular carcinoma typically causes various side effects. Our previous study showed that cecropin of Musca domestica can induce apoptosis in human hepatocellular carcinoma BEL-7402 cells in vitro. However, whether cecropin inhibits BEL-7402 cell in vivo and the question of possible side effects remained undentified. The present study confirmed tumor-inhibitory effects of cecropin in vivo, and furthermore strongly suggested that cecropin cytotoxicity in BEL-7402 cells in vivo may be mainly derived from its pro-apoptotic action. Specifically, we found that cecropin exerted no obvious side effects in tumor-bearing mice as it had no significant hematoxicity as well as visceral toxicity. Therefore, cecropin may be a potential candidate for further investigation as an antitumor agent against hepatocellular carcinoma.

Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of a Plasmodium Region I-Plus Peptide

Interferon alpha (IFN α) exerts a multiplicity of biological actions including antiviral, immunomodulatory, and antiproliferative effects. Administration of IFN α is the current treatment for chronic hepatitis B; however, therapy outcome has not been completely satisfactory. The systemic effects of IFN α may account for its low in vivo biological activity and multiple adverse events. The purpose of this study was to design a novel liver-targeting fusion interferon (IFN-CSP) by fusing IFN α2b with a Plasmodium region I-plus peptide, thus targeting the drug specifically to the liver. The DNA sequence encoding IFN-CSP was constructed using improved splicing by overlapping extension-PCR method, and then cloned into the pET-21b vector for protein expression in E. coli BL21 (DE3). The recombinant protein was expressed as a His-tagged protein and purified using a combination of Ni affinity and HiTrap affinity chromatography at a purity of over 95%. The final yield of biologically active IFN-CSP was up to 270 mg/L culture. The purified recombinant protein showed anti-HBV activity and liver-targeting potentiality in vitro. These data suggests that the novel fusion interferon IFN-CSP may be an excellent candidate as a liver-targeting anti-HBV agent.

Oral administration of Grifola frondosa polysaccharides improves memory impairment in aged rats via antioxidant action

SCOPE Grifola frondosa is an edible/medicinal mushroom with great nutritional value and bioactivity. The present study was performed to evaluate the beneficial effect of polysaccharides isolated from Grifola frondosa on memory impairment in aged rats. METHODS AND RESULTS 20-month-old rats were gavaged with Grifola frondosa polysaccharides (GFP) for 8 weeks. Morris Water Maze test revealed that GFP administration significantly improved memory impairment in aged rats. GFP supply was also found to attenuate age-associated changes of brain histology and ultrastructure observed by light microscopy and transmission electron microscopy. Moreover, the increase of total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) activity, superoxide dismutase (SOD) activity, catalase (CAT) activity, as well as the decreased nitric oxide (NO) and malondialdehyde (MDA) levels, were consistent with the behavioral results. CONCLUSION These findings indicated that oral administration of GFP could improve memory impairment via antioxidant action, and dietary supplementation with GFP may provide potential benefits on brain aging.

Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression

A novel liver-targeting interferon (IFN-CSP) was successfully over-expressed in our previous work. The in vitro and in vivo investigation revealed that IFN-CSP has significant anti-hepatitis B virus (HBV) effect and liver-targeting capacity. However, due to the IFN-CSP tends to form inclusion bodies in recombinant Escherichia coli (E. coli), efficient production of the soluble liver-targeting interferon is a challenge. In view of biomedical application, novel strategies for efficiently expressing liver-targeting interferon and overcoming its poor solubility are necessary and important. In the present study, a modified mu-IFN-CSP was designed base on the amino acid mutant of the native IFN-CSP. Meanwhile, the coding sequence of mu-IFN-CSP was optimized for E. coli preferred codon and the induction conditions for expression were optimized by an orthogonal test. After amino acid mutant, codon optimization and induction conditions optimization, the solubility of Mu-IFN-CSP in E. coli was up to 98.4%. The structural comparison and molecular dynamic simulation showed that the Mu-IFN-CSP formed three structure changes and were more stable than the native IFN-CSP. Tissue sections binding assays revealed that Mu-IFN-CSP was also able to specific binding to liver. In vitro anti-HBV activity assays showed that the soluble Mu-IFN-CSP has improved anti-HBV effect in HepG2.2.15 cells compared to the native IFN-CSP. The present study reports for the first time that liver-targeting interferon Mu-IFN-CSP can be expressed as soluble form, and also contributes to further support its application as liver-targeting anti-HBV medicine.

Effect of Musca domestic maggot polypeptide extract on HUVEC dysfunction induced by early-activated macrophages

Abstract Context: Musca domestica Linn. maggot is a traditional Chinese medicine. In our previous studies, Musca domestica maggot protein-enriched fraction and polypeptide extract (molecular weight <30 kD) were found to reverse endothelial cell dysfunction in atherosclerotic lesions. Objective: This study determines whether and how M. domestica maggot polypeptide extract affects the dysfunction of human umbilical vein endothelial cells (HUVEC) induced by macrophages (Mϕ). Materials and methods: HUVEC and early-activated THP-1 Mϕ (incubated with LPS of 1 μg/ml for 2 h) were co-cultured in a Transwell system. The effects of Musca domestica maggot polypeptide extract (with increasing concentrations, i.e., 1.0, 2.5, 5.0, 10.0, 20.0, and 40.0 µg/ml) on the proliferation and migration HUVEC and their secretion of vascular endothelial growth factor (VEGF) were determined by flow cytometry, modified Boyden chamber assay, and enzyme-linked immunosorbent assay (ELISA) after incubation for 24 h. Results: Musca domestica maggot polypeptide extract decreased the proliferation of HUVEC in a concentration-dependent manner, with a 50% effective concentration (EC50) of 22.16 ± 1.48 µg/ml, and effectively inhibited HUVEC migration (EC50 = 35.15 ± 2.03 µg/ml) and VEGF secretion (EC50 = 28.64 ± 1.29 µg/ml). Discussion and conclusion: Musca domestica maggot polypeptide extract can inhibit the dysfunction of HUVEC induced by early-activated THP-1 Mϕ.