The use of melittin to enhance transgene expression mediated by recombinant adeno-associated virus serotype 2 vectors both in vitro and in vivo / 中西医结合学报

OBJECTIVE@#Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy.@*METHODS@#The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression.@*RESULTS@#rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity.@*CONCLUSION@#Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.

A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

Abstract Background: Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer. Methods: Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated. Results: A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed. Conclusion: Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.

A new therapeutic approach for bone metastasis in colorectal cancer: intratumoral melittin

Background: Melittin has shown antiproliferative effects on tumor cells. Therefore, it comprises a valuable compound for studies on cancer treatment. To the best of our knowledge, no studies have reported melittin effects on bone metastasis. Herein, we propose an approach based on intrametastatic melittin injection to treat bone metastases in colorectal cancer. Methods: Following the characterization of melittin and antiproliferative tests in vitro, a single dose was injected through intrametastatic route into the mouse bone metastasis model. Following treatment, metastasis growth was evaluated. Results: A single dose of melittin was able to inhibit metastasis growth. Histological analysis showed necrosis and inflammatory processes in melittin-treated metastasis. Except by mild weight loss, no other systemic effects were observed. Conclusion: Our data suggest that melittin might be a promising agent for the future development of treatment strategies aiming to reduce the bone metastasis skeletal-related impact in colorectal cancer patients with bone metastasis.(AU)

Anaphylactic shock with pulmonary eosinophilic infiltration due to honeybee attack in a donkey: case report / [Choque anafilático com infiltração eosinófila pulmonar devido a ataque de abelhas em um burro: relato de caso]

A case of a donkey attacked by Africanized honeybee is reported here with clinical signs of agitation, dehydration, congestion of the ocular mucous membranes, tongue edema, tachycardia and inspiratory dyspnea, and progression to death. At necropsy, diffuse, severe subcutaneous edema at face and cervical regions and severe diffuse pulmonary hyperemia with abundant edema without parenchymal collapse were observed. Microscopically, marked, diffuse deep dermis and panniculus carnosus edema and marked diffuse alveolar edema, with moderate population of eosinophils predominantly around larger caliber vessels were noted. The final diagnosis of anaphylactic shock was supported by history, clinical signs, and anatomic pathology findings. This is the first report of a honeybee attack with pulmonary eosinophilic infiltration in a mammal.(AU)

Descreve-se um caso de ataque de abelha africanizada em um burro, com sinais clínicos de agitação, desidratação, mucosas oculares congestas, edema de língua, taquicardia e dispneia inspiratória, com progressão e morte. Na necropsia, foram verificados edema subcutâneo difuso grave nas regiões de face e cervical, hiperemia pulmonar difusa grave com edema abundante e sem colapso do parênquima. Microscopicamente, foram observados edema marcado difuso na derme profunda e panículo carnoso e edema alveolar difuso acentuado, com população moderada de eosinófilos predominantemente em torno de vasos de maior calibre. O diagnóstico de choque anafilático foi baseado no histórico, em sinais clínicos e em achados anatomopatológicos. Este é o primeiro relato de ataque de abelhas com infiltração eosinofílica pulmonar em um mamífero.(AU)

Structure modification and antimicrobial activity of novel cationic melittin analogues / 药学学报

Melittin exhibits high antibacterial potency against drug-resistant bacteria. However, the clinical utility of melittin is limited by its serious hemolytic activity. Thus, the need for developing novel melittin analogues with high antimicrobial activity and low hemolytic activity has grown. We designed, synthesized, and evaluated 20 novel melittin analogues with varying hydrophobic, polar or positively charged amino acids. The results showed that 8 compounds had antimicrobial activity (MIC: 1-4 μg·mL-1) against gram-positive pathogens equal to or better than that of melittin, and 16 compounds had low hemolytic activity (HC50 ≥ 11.9 μg·mL-1). Compounds 13 (MIC: 2-4 μg·mL-1) and 15 (MIC: 1-2 μg·mL-1) showed equal or better antimicrobial activity against both susceptible and resistant strains of Staphylococcus aureus and Enterococcus faecium compared to melittin (MIC: 4 μg·mL-1). Compound 13 (HC50: 24.0 ± 4.3 μg·mL-1) displayed noticeably decreased hemolytic activity compared to melittin (HC50: 5.3 ± 0.4 μg·mL-1). This work established a base for further study on the structure-activity relationships and structure-toxicity relationships of melittin.

Melittin suppresses growth and induces apoptosis of non-small-cell lung cancer cells via down-regulation of TGF-ß-mediated ERK signal pathway

The purpose of this study was to investigate the anti-cancer effect of melittin on growth, migration, invasion, and apoptosis of non-small-cell lung cancer (NSCLC) cells. This study also explored the potential anti-cancer mechanism of melittin in NSCLC cells. The results demonstrated that melittin suppressed growth, migration, and invasion, and induced apoptosis of NSCLC cells in vitro. Melittin increased pro-apoptotic caspase-3 and Apaf-1 gene expression. Melittin inhibited tumor growth factor (TGF)-β expression and phosphorylated ERK/total ERK (pERK/tERK) in NSCLC cells. However, TGF-β overexpression (pTGF-β) abolished melittin-decreased TGF-β expression and pERK/tERK in NSCLC cells. Treatment with melittin suppressed tumor growth and prolonged mouse survival during the 120-day observation in vivo. Treatment with melittin increased TUNEL-positive cells and decreased expression levels of TGF-β and ERK in tumor tissue compared to the control group. In conclusion, the findings of this study indicated that melittin inhibited growth, migration, and invasion, and induced apoptosis of NSCLC cells through down-regulation of TGF-β-mediated ERK signaling pathway, suggesting melittin may be a promising anti-cancer agent for NSCLC therapy.

A Review on Bioactivities of Honey Bee Venom

The Honeybee (Apis mellifera) is one of the world’s most beneficial insects, as it plays a critical role in many terrestrial ecosystems. The use of honeybee products has been documented for thousands of years in many cultures for the treatment of human diseases, and their healing properties have been documented in many religious texts. The present study sets out to compile information on the history, chemical composition and scientific evidence concerning bee venom research. The promising bioactivities have the potential to provide practical directions for further investigation. PubMed database, Google Scholar Library, research articles, books, and relevant web pages have been accessed to accumulate data so that the updated information included in this study is as current as possible. At least 18 pharmacologically active components including various enzymes, peptides, and amines are present in bee venom. Medicinal use of bee venom therapy wields significant in vivo and in vitro outcomes to some extent mitigate the effects of Parkinson’s disease, Alzheimer’s disease, HIV, arthritis, liver fibrosis, cancer, tumors, fibrotic diseases, Lyme disease, etc. The effects of bee venom were the first documented in 1888 with the publication of a European clinical study conducted on its impact on rheumatism. According to a study published in the journal, bee venom has been used to treat various conditions for centuries. Such research activities confirm the therapeutic effectiveness of bee venom and as a potential future biomedicine.

Potencial antiviral e virucida da melitina e apamina contra herpesvírus bovino tipo 1 e vírus da diarreia viral bovina / Antiviral and virucidal potential of melittin and apamin against bovine herpesvirus type 1 and bovine viral diarrhea virus

A busca por alternativa aos fármacos sintéticos têm revelado descobertas no campo da farmacologia e, nesse sentido, melitina e apamina, dois constituintes do veneno de abelhas, foram descritas com várias ações farmacológicas. Este estudo objetivou avaliar in vitro as capacidades antiviral e virucida destes componentes. Para tanto, células MDBK (Madin Darby Bovine Kidney), após verificação das respectivas doses tóxicas por ensaio MTT ((3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo), foram cultivadas em microplacas e tratadas com diferentes concentrações de apamina, melitina e sua associação. Esse tratamento ocorreu antes e após a infecção com 0,1 MOI (multiplicidade de infecção) de cepas citopatogênicas de herpesvírus bovino tipo 1 (BoHV-1) cepa Los Angeles e vírus da diarreia viral bovina (BVDV) cepa NADL. Após incubação por 72 horas, 37oC, as células foram submetidas ao ensaio MTT para estimativa da viabilidade celular. Em experimento paralelo, placas que foram submetidas ao mesmo procedimento sofreram ciclo de congelamento e descongelamento das células, para rompimento das mesmas e mensuração dos títulos virais. O ensaio virucida foi realizado incubando-se suspensões de BoHV-1 e BVDV com as soluções de apamina, melitina e associação por 24 horas a 37oC e 22oC. O título viral foi avaliado às 0 horas, 1, 2, 4, 8 e 24 horas de incubação. A concentração citotóxica para 50% das células (CC50) de melitina foi 2,32 μg/ml e apamina não demonstrou toxicidade à maior concentração testada (100μg/ml). Houve efeito antiviral da melitina sobre BoHV-1, especialmente na concentração de 2μg/ml, onde observou-se 85,96% de viabilidade celular quando o tratamento foi realizado antes da infecção e 86,78% de viabilidade quando o tratamento foi realizado após a infecção. Houve ainda redução de 90% das partículas virais de BoHV-1. Em menores concentrações (1 e 1,5μg/ml) de melitina não houve atividade antiviral, pois a viabilidade celular foi baixa, demonstrando efeito citopático do vírus. Na associação das duas substâncias houve queda no título de BVDV e observou-se maior viabilidade celular quando comparados à ação isolada dos composto sobre este vírus. Isso se confirma na atividade virucida, uma vez que houve decréscimo de 90% das partículas virais de BVDV com a associação dos dois compostos do veneno de abelhas. Atuando individualmente, melitina apresentou efeito antiviral e virucida frente ao BoHV-1, zerando seu título em apenas 2 horas a 37oC. Conclui-se que melitina tem ação antiviral e virucida frente ao BoHV-1 e sua associação com apamina potencializou seus efeitos frente ao BVDV.(AU)

The search for an alternative to synthetic drugs have revealed discoveries in the field of pharmacology and, according to melittin and apamin, two components of bee venom which have been described were with various pharmacological actions.This study aimed to evaluate the in vitro antiviral and virucidal capabilities of these components. Therefore, after verification of their toxic doses by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, MDBK cells (Madin Darby Bovine Kidney) have been cultivated in microplates and treated with different concentrations of apamin, melittin and its association. This treatment occurred before and after infection with MOI (multiplicity of infection) 0.1 of cytopathogenic strains of bovine herpesvirus type 1 (BoHV-1) strain Los Angeles and bovine viral diarrhea virus (BVDV) strain NADL. After incubation for 72 hours, 37°C, the cells were submitted to MTT assay to estimate cell viability. In parallel experiments, plates were subjected to the same procedure suffered freezing and thawing cycle the cells to rupture the same and measurement of viral titers. The virucidal assay was performed by incubating suspension of bovine herpesvirus type-1 and BVDV with apamin solutions, melittin and association for 24 hours at 37°C and 22°C. The viral titer was evaluated at 0 hours, 1, 2, 4, 8 and 24 hours of incubation. The cytotoxic concentration to 50% of the cells (CC50) of melittin was 2.32g/mL and apamin did not show toxicity at the greater concentration tested (100μg/mL). There was antiviral effect of melittin on bovine herpesvirus type-1, especially at a concentration of 2μg/mL, where was observed 85.96% cell viability when treatment was performed before the infection and 86.78% viability when the treatment was carried out after infection. There was also a 90% reduction of viral particles of bovine herpesvirus type-1. In lower concentrations (1 and 1.5μg/mL) melittin no antiviral activity because cell viability was low, showing cytopathic effect of the virus. At the association two substances there were a decrease in the title of BVDV and there was higher cell viability when compared to the isolated action of the compounds of this virus. This is confirmed in the virucidal activity, since there was a decrease of 90% of the viral particles of BVDV with the combination of the two compounds of bee venom. Acting individually, melittin showed antiviral effect and virucidal against for BoHV-1, zeroing its title in just 2 hours at 37°C. It is concluded that melittin has antiviral and virucidal action against the BoHV-1 and its association with apamin potentiate its effects against BVDV.(AU)

Preparation and characterization of restructured vesicles of grape-derived multicomponents for active peptides loading / 中草药

Objective To reconstruct the functional vesicles by using the components isolated from grapes, and investigate its properties of the loading of peptides. Methods Grape polyphenols (GP) were extracted by ethanol, and the extraction technology of GP was optimized by single factor and orthogonal experiments. GP was purified by macroporous resin adsorption and the purification technology. The preparation of GP and melittin (Mel) complexes (GPMC) were determined by orthogonal tests, and Sucrose density gradient centrifugation was used to extract grape-derived vesicles (GDVs) for loading complex GPMC to get GPMC-GDVs. The stability of GPMC-GDVs in PBS, DMEM, and 10% FBS were investigated. The cytotoxicity of GPMC and GPMC-GDVs on SMMC-7721 or HepG 2 cells was investigated by MTT method. Results The extraction process of GP was as follows: the extraction solvent was 60% ethanol solution, the heating temperature was 50 ℃, the solid-liquid ratio was 1:10, the extraction time was 50 min and extract 2 times. The purification conditions of GP were as follows: 4 mg/mL GP crude sample volume was 7 times of bed volume (BV), the washing dosage was 5 BV, and the elution volume of 60% ethanol was 5 BV. The preparation of GPMC-GDVs was as follows: 0.4 mg/mL GDVs was slowly dripped into GPMC solution with equal volume of Mel containing 2 mg/mL and incubated at room temperature for 30 min. As-prepared GPMC-GDVs had good stability in PBS, DMEM, and 10% FBS. The results of MTT method showed that GPMC-GDVs had a better tumor inhibitive effect. Conclusion By extracting the components of grape GP and GDVs and reorganizing the structures, the vesicles can be prepared for the loading of active melittin, which has a good application prospect in the field of delivery and anti-tumor effect of polypeptide drugs.

Research on “unification of medicines and excipients” nanosystem comprising tea polyphenol and melittin for antitumor therapy / 中草药

Objective: To prepare nanocomplex comprising tea polyphenol and melittin (EMN) with synergistic effect for antitumor therapy. Methods: Poly-epigallocatechin gallate (polyEGCG) was synthesized by Bayer reaction and characterized by 1H-NMR and MS spectra. The preparation process of EMN was optimized by orthogonal test, and the in vitro release of EMN was conducted at different medium with different pH. The cell uptake of FITC-labeled Mel and EMN was measured by flow cytometry. The synergistic antitumor effect of polyEGCG and Mel was investigated using B16F10 and A549 cells by MTT assay. Results: The structure of polyEGCG was confirmed by 1H-NMR and MS. The optimized preparation process of EMN was as follws: 0.5 mg/mL polyEGCG solution was added dropwise to 1 mg/mL equal volume of Mel solution, and then incubated at room temperature for 30 min. In vitro release studies showed that acidic environment could accelerate the release of Mel. Cell uptake experiments showed that the cell uptake of Mel and EMN was comparable. MTT assay results showed the combination index (CI) of polyEGCG and Mel was less than 1, which intimated the synergistic effect when combined therapy with polyEGCG and Mel. Conclusion: EMN was prepared by a self-assembly method, which has a simple preparation process, a suitable particle size, and synergistic antitumor effect, and it is worthy of further studies.

Mechanism study of the role of rhuPAa-melittin in ovarian cancer cells in vitro and in vivo / 实用医学杂志

Objective To investigate the role of the activated rhuPAa-melittin in ovarian cancer cells and to study the inhibitory effect of rhuPAa-melittinon on ovarian cancer cells. Methods rhuPAa-melittin was used to treat the ovarian cancer cells at different concentrations for 48 hrs. Then flow cytometery was applied to detect the cell cycle and cell apoptosis. rhuPAa-melittin protein was delivered to the mouse mode to investigate the effect of rhuPAa-melittin on the growth of the xenotransplanted tumor. Results rhuPAa-melittin was used to treat ovarian cancer cells at the concentration of 0 ,4 ,8 and 16 μg/mL for 48 hrs ,respectively. The results of cell apoptosis assay was 1.16%,3.83%,6.51% and 10.2%,respectively. Moreover,different concentrations of rhuPAa-melittin had no effects on the cells at G0/G1 phase,rhuPAa-melittin inhibited S phase cells to process into G2/M phase, contributing to suppressing the growth of ovarian cancer cells. Conclusion The in vivo and in vitro studies revealed that rhuPAa-melittin inhibits the growth of ovarian cancer.

Mechanism study of the role of rhuPAa-melittin in ovarian cancer cells in vitro and in vivo / 实用医学杂志

Objective To investigate the role of the activated rhuPAa-melittin in ovarian cancer cells and to study the inhibitory effect of rhuPAa-melittinon on ovarian cancer cells. Methods rhuPAa-melittin was used to treat the ovarian cancer cells at different concentrations for 48 hrs. Then flow cytometery was applied to detect the cell cycle and cell apoptosis. rhuPAa-melittin protein was delivered to the mouse mode to investigate the effect of rhuPAa-melittin on the growth of the xenotransplanted tumor. Results rhuPAa-melittin was used to treat ovarian cancer cells at the concentration of 0 ,4 ,8 and 16 μg/mL for 48 hrs ,respectively. The results of cell apoptosis assay was 1.16%,3.83%,6.51% and 10.2%,respectively. Moreover,different concentrations of rhuPAa-melittin had no effects on the cells at G0/G1 phase,rhuPAa-melittin inhibited S phase cells to process into G2/M phase, contributing to suppressing the growth of ovarian cancer cells. Conclusion The in vivo and in vitro studies revealed that rhuPAa-melittin inhibits the growth of ovarian cancer.

Effects of silencing MALAT1 on proliferation and apoptosis in HepG2 cells induced by Melittin / 中国药理学通报

Aim To investigate the effects of silencing MALAT1 gene on cell proliferation inhibition and apop-tosis induced by Melittin in human hepatocellular car-cinoma HepG2 cells. Methods The inhibitory rate of cell proliferation treated with Melittin in HepG2 cells was examined by MTT assay. Apoptotic rate was detec-ted by flow cytometry. The MALAT1 expression level in HepG2 cells was measured by qPCR. Specific siR-NAs were utilized to silence MALAT1 expression. The rates of cell proliferation inhibition and apoptosis in HepG2 cells treated with siRNA and Melittin were compared with those of Melittin alone. Results Melit-tin significantly suppressed the growth of HepG2 and induced cell apoptosis in a dose-dependent manner. Compared with normal liver cell lines, MALAT1 was highly expressed in HepG2 cells ( P<0. 05 ) . The ex-pression of MALAT1 in HepG2 cells was inhibited by Melittin, and the inhibitory rate increased with the in-crease of concentration. The rates of cell proliferation inhibition and apoptosis in HepG2 cells treated with siRNA and Melittin were significantly higher than those treated merely with Melittin. Conclusion Melittin can reduce the expression of MALAT1 and silencing MALAT1 can effectively promote proliferation inhibi-tion and apoptosis in HepG2 cells induced by Melittin.

Melittin induces in vitro death of Leishmania (Leishmania) infantum by triggering the cellular innate immune response

Abstract Background Apis mellifera venom, which has already been recommended as an alternative anti-inflammatory treatment, may be also considered an important source of candidate molecules for biotechnological and biomedical uses, such as the treatment of parasitic diseases. Methods Africanized honeybee venom from Apis mellifera was fractionated by RP-C18-HPLC and the obtained melittin was incubated with promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Cytotoxicity to mice peritoneal macrophages was evaluated through mitochondrial oxidative activity. The production of anti- and pro-inflammatory cytokines, NO and H2O2 by macrophages was determined. Results Promastigotes and intracellular amastigotes were susceptible to melittin (IC50 28.3 μg.mL−1 and 1.4 μg.mL−1, respectively), but also showed mammalian cell cytotoxicity with an IC50 value of 5.7 μg.mL−1. Uninfected macrophages treated with melittin increased the production of IL-10, TNF-α, NO and H2O2. Infected melittin-treated macrophages increased IL-12 production, but decreased the levels of IL-10, TNF-α, NO and H2O2. Conclusions The results showed that melittin acts in vitro against promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Furthermore, they can act indirectly on intracellular amastigotes through a macrophage immunomodulatory effect.

Effect of melittin on proliferation and apoptosis of human HepG2 cells / 中国药理学通报

Aim To observe the effect of melittin on human hepatocelluar carcinoma HepG2 cell prolifera-tion in vitro and its further mechanisms.Methods The capacity of cellular proliferation and apoptosis was measured with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,Hoechst 33258 assay and Annexin V-FITC /PI assay.The mR-NA expression of Shh, PTCH1, SMO, GLi1 and HDAC2 was performed by qRT-PCR.And the protein expression of Shh,PTCH1,SMO,GLi1 and HDAC2 was assessed by western blotting.Results Our study found that melittin effectively inhibited cell prolifera-tion and promoted cell apoptosis in vitro using MTT method and Flow cytometry.The mRNA and protein expression of Shh,PTCH1,SMO,GLi1 and HDAC2 were obviously decreased after treated with various con-centrations of melittin for 48h in HepG2 cells.Conclu-sions Taken together,our data suggest that melittin could inhibit cell proliferation and promote cell apopto-sis,reduce the level of HDAC2 and down-regulate the Hedgehog signaling pathway in this process simultane-ously.

Construction of eukaryotic expression plasmid pEGFP-N3-M-IL-2(88 Arg,125 Ala) and its effects on Glioma cell line U87 / 中国免疫学杂志

Objective:To construct a recombinant eukaryotic expressive vector of pEGFP-N3-M-IL-2(88 Arg,125 Ala),and to study the expression of this gene in the Glioma cell line U 87,and to detect its antitumor activities of the fusion protein M-IL-2(88 Arg,125 Ala).Methods:The target fusion gene M-IL-2 (88 Arg,125 Ala) was amplified by PCR from pPICZαA/M-IL-2 (88 Arg,125 Ala) and cloned into pEGFP-N3 vector after digestion to construct recombinant eukaryotic expressive vector pEGFP -N3-M-IL-2( 88 Arg,125 Ala).And then recombinant plasmid pEGFP-N3-M-IL-2(88Arg,125Ala) was transfected into Glioma cell U87 by LipofectamineTM2000 immediately after it was confirmed by restrictive enzyme analysis and sequencing .RT-PCR and Western blot were used to confirm expression of the fusion gene in the U87.Prohibitory effect of recombinant M-IL-2(88Arg,125Ala) protein on U87 was assessed by CCK-8 assay.Results:Restrictive analysis and sequence analysis revealed that M-IL-2(88Arg,125Ala) fusion gene was cloned into the vector pEGFP-N3 suc-cessfully,fusion gene M-IL-2(88Arg,125Ala) could express in U87 cells and could inhibit the growth of U87 cells.Conclusion:The eu-karyotic expression plasmid pEGFP-N3-M-IL-2( 88 Arg,125 Ala) was constructed and expressed in U 87 cells successfully ,the fusion protein could inhibit the growth of U87 cells.We laid a foundation for further research of gene M-IL-2(88 Arg,125 Ala).

Melittin Ameliorates the Inflammation of Organs in an Amyotrophic Lateral Sclerosis Animal Model

Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disorder characterized by a selective loss of motor neurons in the spinal cord, brainstem, and motor cortex, leading to weakness of the limb and bulbar muscles. Although the immediate cause of death in ALS is the destruction of motor neurons, ALS is a multi-organ disease that also affects the lungs, spleen, and liver. Melittin is one of components of bee venom and has anti-neuroinflammatory effects in the spinal cord, as shown in an ALS animal model. To investigate the effects of melittin on inflammation in the lungs and spleen, we used hSOD1(G93A) transgenic mice that are mimic for ALS. Melittin treatment reduced the expression of inflammatory proteins, including Iba-1 and CD14 by 1.9- and 1.3-fold (p<0.05), respectively, in the lungs of symptomatic hSOD1(G93A) transgenic mice. In the spleen, the expression of CD14 and COX2 that are related to inflammation were decreased by 1.4 fold (p<0.05) and cell survival proteins such as pERK and Bcl2 were increased by 1.3- and 1.5-fold (p<0.05) in the melittin-treated hSOD1G93A transgenic mice. These findings suggest that melittin could be a candidate to regulate the immune system in organs affected by ALS.

Expression and purification of AnxB1-MLT chimera and its inhibitory effect on SMMC7721 and HepG2 cell proliferation / 第二军医大学学报

Objective To obtain a chimera composed of annexin Bl (AnxBl) and melittin (MLT) and to investigate its inhibitory effect on phosphatidylserine liposome activity and SMMC7721 and HepG2 cell proliferation. Methods A fusion gene AnxBl-MLT was constructed by overlap extension gene splicing and then was inserted into plasmid pGEX-5T. The recombinant plasmid was transformed into E. coli strain K802 and induced by IPTG at low temperature. The expression condition was optimized and GST affinity chromatography column was used for purification. Calcium-dependent phospholipid binding assay was used to determine whether the chimera kept the activity of AnxBl. CCK8 analysis was employed to investigate the effect of AnxBl-MLT on SMMC7721 and HepG2 cell proliferation. Results After being inserted into the expression plasmid, AnxBl-MLT protein expressed in K802 cells, with a high level recombinant protein induced by 0. 2 mmol/ L IPTG at 22-24»C for 4 h. The protein AnxBl-MLT was purified by using GST affinity chromatography column (a band at 63 000) and the purification of the final purified protein was >95%. AnxBl-MLT was able to bind to phosphatidylserine liposome in a calcium-dependent manner. CCK8 analysis indicated that AnxBl-MLT inhibited SMMC7721 and HepG2 cell proliferation at a dose-dependent manner. Conclusion We have successfully constructed a chimera AnxBl-MLT, which retains the calcium-dependent phospholipid binding activity of AnxB1, and can inhibit the proliferation of hepatic cancer cell lines SMMC7721 and HepG2.

Effects of Melittin on the growth and G_2/M phase arrest in SGC-7901 cells / 中国药理学通报

Aim To explore the effects of the Melittin on growth and cell cycle of SGC-7901 cells.Methods Growth inhibition effect of Melittin was evaluated using SRB in SGC-7901 cells in vitro;Melittin induced cell cycle arrest was investigated using flow cytometry assay;reverse transcription PCR(RT-PCR)was used to detect the associated protein mRNA of cell cycle.Results Proliferation activity of SGC-7901 cells was inhibited after treatment with Melittin(1,2,4,8,16,32×10~(-3) μg·L~(-1))(P<0.05 or P<0.01)for 24 h;Flowcytometry analysis revealed that SGC-7901 cells accumulated in the G_2/M phase after treatment with Melittin(4,8×10~(-3) μg·L~(-1))for 24 h;the expression of CylinB1,CDK1 and Cdc25c mRNA were decreased.Conclutions Proliferation activity of SGC-7901 cells was inhibited by Melittin,which may be related to the inhibitory effect of Melittin on associated protein transcription in the G_2/M stage of SGC-7901 cells.

Expression, purification and preliminary identification of Melittin in E. Coli / 中国生化药物杂志

Purpose Melittin was expressed in prokaryotic vector pGEX-2T for production. Methods Melittin gene synthesized with enterokinase digested sequence,the gene was cloned into vector pGEX-2T,and constructed a recombinant plasmid of pGEX-MEL. Then the recombinant vector was introduced into E. coli BL21 (DE3)for expression. Fusion protein was purified by affinity chromatography. Hemolytic activity of Melittin was detected. Results Analysis result showed that the expression products accumulate in the cells to about 29.5 % of total cell protein. Detection of western blot using ant-GST as the first antibody showed that a special blot was revealed among the expression products. It certified that we have succeeded in expressing the fusion protein. SDS-PAGE showed that most part of the products is resoluble. The purity of obtained protein is 95% , by through GST affinity chromatography system. Melittin is harvested with a recovery of 80% by EK digestion. Test results showed melittin has good hemolytic activity. Conclusion We have expressed Melittin successfully by prokaryotic expression system.