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Gonorrhea, an important sexually transmitted disease, is becoming a growing public health problem around the globe. Vaccination is considered the best long-term approach for control of infection. In this study, we designed a novel Neisseria gonorrhoeae (N. gonorrhoeae) DNA vaccine delivered by bacterial ghosts and characterized its immune responses in vitro and in vivo. Our results demonstrate that bacterial ghosts greatly promoted BMDCs maturation and activation. Bacterial ghosts loaded with N. gonorrhoeae DNA vaccine were efficiently taken up by mouse macrophage RAW264.7 cells. Furthermore, oral immunization with the ghost vaccine candidate elicited greater CD4+ and CD8+ T cell responses and induced higher IgG responses than N. gonorrhoeae DNA vaccine alone. In addition, mice immunized with the vaccine candidate responded with a significant rise in bactericidal antibody titer. These results suggest that bacterial ghosts may function as a vaccine adjuvant by promoting BMDCs maturation, which in turn enhances the immune responses to the vaccine antigens. This study also highlights the potential of using bacterial ghosts as antigen delivery system in the development of an efficacious gonorrhea vaccine.

The present study was designed to isolate and purify chemical constituents from solid culture of endophyte Aspergillus terreus LQ, using silica gel column chromatography, gel filtration with Sephadex LH-20, and HPLC. Fumigaclavine I (1), a new alkaloid, was obtained, along with seven known compounds, including fumigaclavine C (2), rhizoctonic acid (3), monomethylsulochrin (4), chaetominine (5), spirotryprostatin A (6), asperfumoid (7), and lumichrome (8). The structure of compound 1 was elucidated by various spectroscopic analyses (UV, MS, 1D and 2D NMR). The in vitro cytotoxicity of compound 1 was determined by MTT assay in human hepatocarcinoma cell line SMMC-7721, showing weaker cytotoxicity, compared with cisplatin, a clinically used cancer chemotherapeutic agent.

Isolation and purification of chemical constituents of liquid culture of symbiotic Chaetomium globosum ML-4 of oyster was performed through silica gel column chromatography, gel filtration over Sephadex LH-20, preparative TLC and HPLC. Five compounds were obtained and their structures were determined as chaetoglobosin V(1), chaetoglobosin Vb(2), tyrosol(3), 5-methyluracil(4)and uracil(5), respectively, based on HR-MS and NMR data and comparison with literatures. In vitro cytotoxicity of compounds against human hepatocellular carcinoma cell line SMMC-7721 were measured byMTT method, and results showed that compound 1 could obviously inhibit the proliferation of SMMC-7721 cells with an IC₅₀ value of 60.5 mg•L⁻¹, while the IC₅₀ value of positive control cisplatin was 19.96 mg•L⁻¹. Further studies discovered that compound 1 could lead to G2 phase arrest in SMMC-7721 cells and induce SMMC-7721 cells apoptosis. The ratio of Bcl-2/Bax in SMMC-7721 cells was decreased. The expression of protein Caspases-3,-8,-9 was improved and the expression and phosphorylation level of Akt were reduced. Aforementioned results revealed that in vitro antitumor activity of compound 1 against SMMC-7721 cells were related to G2 phase cell cycle arrest and induced-apoptosis. The induced-apoptosis was involved in both the mitochondrial pathway and the death receptor pathway and connected with activity decline of PI3K/Akt signaling pathway.

To study the regulation of luteolin on spleen cells and sarcoma S180 cells in normal ICR mice.Spleen cells and S180 cells were incubated with different concentrations of luteolin (50, 100, 200, and 400 μmol/L). The effect of luteolin on spleen cells and sarcoma S180 cells was determined by MTT assay. The apoptosis was detected using propidium iodide staining flow cytometry. Intracellular reactive oxygen species (ROS) was determined by flow cytometric analysis. Activities of free radicals scavenging were determined by hydroxyl radical and DPPH tests.Compared with the solvent control group, 200 and 400 μmol/L luteolin increased the spleen cells viability (P0.05). Luteolin at 100, 200, and 400 μmol/L decreased activities of S180 cells (P0.01). The proportion of sub-G1 phase spleen cells was reduced after treated with 200 and 400 μmol/L luteolin (P0.05). The proportion of sub-G1 phase S180 cells was elevated after treated with 200 and 400 μmol/L luteolin (P0.05). Compared with the solvent control group, levels of intracellular ROS in spleen cells of ICR mice all increased; levels of intracellular ROS in S180 cells all decreased after treated with 50, 100, 200, and 400 μmol/L luteolin (P0.05). Luteolin scavenged hydroxyl radical and DPPH in a dose dependent manner.Luteolin had bilateral regulation on viability and apoptosis of spleen cells and S180 cells (promoting the viability of spleen cells, inhibiting apoptosis of spleen cells, inhibiting the viability of S180 cells, and promoting apoptosis of S180 cells), which was worth further study and exploration.

Melittin, a toxic peptide found in bee venom, has been widely reported to have antimicrobial and anti-proliferative activity in many cell lines. While high-purity natural melittin is rare and expensive, the demand for melittin has increased, making it urgent to find a cheap, suitable method of mass production. In this study, the melittin gene of the Chinese bee (honeybee) was synthesized according to the sequence published in GenBank and cloned into the prokaryotic expression vector pGEX-6P-1. Recombinant melittin was then successfully expressed in Escherichia coli. The activity of affinity-purified recombinant melittin was determined in human leukemic U937 cells. Results show that the recombinant melittin had the same anti-proliferative activity in human leukemic U937 cells in vitro as natural one. This shows the promise of recombinant melittin as a replacement for the natural bee toxins used in drug development and other applications. Key words : Escherichia coli, melittin, expression, anti-proliferative activity.

To prepare a monoclonal antibody against human vascular endothelial growth factor (VEGF165), for further study the VEGF165 in the tumorigenesis, tumor cell migration and the tumor cells escape from the immune response.VEGF165 gene was cloned from the human umbilical vein endothelial cells (HUVEC) by RT-PCR, and then cloned into the pGEX-6P1, constructed the prokaryotic expression of pGEX-6P1-VEGF165. The fusion -protein of VEGF165 was expressed in E.coli (BL21) induced by the 1.0 mmol/L IPTG at 37DegreesCelsius after 4 h. The fusion-protein was purified by the MicroSpin GST purification kit for immunized the BALB/c mouse. The monoclonal antibodies (mAbs) against the VEGF165 were prepared by hybridoma technique, and ELISA and Western blot identified their immunoglobulin subclass and specificity. And we used the inhibition the embryo angiogenesis assay, inhibition the HUVEC migration assay and inhibition the HUVEC tubule information assay to study the bioactivity of the mAbs of VEGF165.The sequence of the VEGF165 is agreed to the GenBank, and we obtained five species VEGF165 mAbs, and the titer of the antibody is high, and we named, they are 5A6, 3F5, 6H3, 7D10 and 7A10. Our study showed that the 5A6, 3F5, 6H3, 7D10 were classified to IgG2a, 7A10 was classified to IgG2b, and the light chain is k.Meanwhile the purified mAbs inhibited formation of chicken embryo blood vessels, and inhibited tubule formation of the HUVEC and inhibited migration of the HUVEC.mAbs against human VEGF165 have the effective bioactivity, which would play a significant role for further study the mechanism of VEGF165.

The recombinant transfer vector pFastBacl-ChIFN-y was constructed by plasmid pcDNA-ChIFN-gamma digested with EcoR I and Not I enzymes and cloned into pFastbacl. Then the transfer vector was transformed into E. coli competent cells DH10Bac which contained the bacmid with amini-attTn7 target site and the helper plasmid. The recombinant bacmid-ChIFN-gamma was generated by transposing themini-Tn7 element located in pFastBacl-ChIFN-gamma to themini-attTn7 attachment site on the Bacmid. Subsequently the recombinant Bacmid-ChIFN-gamma was transfected into the Sf9 insect cells mediated by lipofectin to produce recombinant baculovirus and express recombinant ChIFN-gamma (rChIFN-gamma) products. The result showed that the rChIFN-gamma was successfully expressed in Sf9 cells infected with the recombinant virus by indirect immunofluorescence assay (IFA) at 5 days post-transfection. The biological activity of rChIFN-gamma was identified by its inhibition to Vesicular stomatitis virus-induced cytotoxicity of chicken embryonic fibroblasts (CEF) in vitro. The results showed that the most efficient expression of rChIFN-gamma could be obtained at 96h post-infection with multiplicity of infection (MOI) equal to 1. It is interesting that the viruses such as Avian influenza virus H5N1 or Marek's disease virus (GA strain) could not grow in CEF pre-treated with rChIFN-gamma. Cell pathogenic efficient (CPE) in the CEF infected with H5N1 and GA strain is apparently inhibited by the rChIFN-gamma. However only difference between the HA titres of the supernatant of the pre-treated cells is observed without any obvious inhibition effect in CEF infected with Newcastle disease virus (F48E8 strain).