Optimized Expression And Antitumor Mechanism Of Antimicrobial Peptide Cecropinxj Of Bombyx Mori(Bombycidae,Lepidoptera)

Antimicrobial peptides(antimicrobial,peptides,AMPs)are a class of small molecular polypeptide that protect the host from invading pathogens,and play an important role in the biological innate and acquired immunity systems.At present,chemotherapy may damage normal cells,causing serious side effects,and even lead to drug resistance.It is urgent to develop a new design potential for antitumor drugs.Compared with traditional antitumor drugs,antimicrobial peptides could kill tumor cells selectively with little toxic and side effect,and may be a promising candidate for use as a new antitumor agent.However,to-date,no method has been developed without intrinsic limitations of poor expression,protein instability,or host toxicity.The need to produce large quantities of antimicrobial peptide has prompted investigators to develop biologically efficient production methods.This project has laid the foundation to for further study the biological functions of CecropinXJ,and to explore ways to design genetically engineering antimicrobial drugs.The antimicrobial peptide gene CecropinXJ was isolated from the larvae of Bombyx mori in previous work was constructed with different expressive plasmids and the corresponding mature peptide has been expressed in Escherichia coli and Saccharomyces cerevisiae,respectively.The inhibition zone assay was used to detected minimal inhibitory concentration of CecropinXJ,and antimicrobial peptide exerts its action by directly disrupting membranes of microorganisms was observed.The study focused on the heat stability,pH stability and the Hemolytic activity of the CecropinXJ was performed.In this study,human esophageal carcinoma cell line Eca 109 cells were used to investigate the antitumor potential of CecropinXJ.The effect of CecropinXJ on cytoskeleton of human esophageal cancer Eca109 cells were detected by scaning electron microscopy.The change of gene expression which related to cytoskeleton was detected by RT-PCR and western blot.The inhibitory effect on the proliferation of Eca109 cells induced by incubation with CecropinXJ was detected by MTT assay.The changes of the morphology of cell membrane induced by CecropinXJ were investigated using electron microscopy.Moreover,Hoechst 33258 immunofluorescence staining,LDH activity-based assay,flow cytometry was used to investigate the cell cycle distribution and apoptosis.Expression of apoptosis and cell cycle-related protein in Eca109 cells was analyzed by western blot.The tumor bearing nude mice model were used to evaluate the anti-tumor activity of CecropinXJ in vivo.The change of cytokines was observed by pathological histology method.The antitumor effects of CecropinXJ on tumor bearing mice were evaluated by tumor volume and weight on mice model.Effect of CecropinXJ and it's combination with chemotherapeutic agents on the proliferation and apoptosis of Eca109 cells were detected by flow cytometry.The polyclonal anti-serum of CecropinXJ was prepare by DNA immunity,and location of fusion protein CecropinXJ in tumor cells were observed under confocal microscopy,which is beneficial to further study on antitumor mechanism.The main results are as follows:(1)pET32a-CecropinXJ was proved to be optimized for induction,and further,the optimal condition of the prokaryotic expression was determined at 0.8 mmol/L IPTG under 37? for 5 h,the amount of target protein expression can be up to 10 mg/L in a form of soluble expression,35%of the total bacterial proteins.Using metal affinity chromatography,90%of the soluble protein can be further purified,which has strong antibacterial activity against Staphylococcus aureus,Escherichia coli with a yield of lOmg/L culture medium after purification on nickel-nitrilotriacetic acid(Ni-NTA)metal affinity chromatography matrices.Purified recombinant antibacterial peptide,CecropinXJ,retained a high stability against Staphylococcus aureus over a temperature range from 4? to 1008 and a pH range from pH 3.0 to 12.0.The minimum inhibitory concentration(MIC)of the fusion protein against Staphylococcus aureus was 0.4 ?M.The recombinant CecropinXJ is also cytotoxic in many human cancer cells.(2)An antibacterial peptide gene of CecropinXJ was cloned into the pYES2/CT/a Factor expression vector and expressed in the S.cerevisiae INVScl strain.Following an induction of recombinant protein expression in yeast for 120 h,the maximum amount of total secreted protein was 1.437 g/L.The percentage of recombinant CecropinXJ was estimated to be 79.45%of the total protein.After purification with Ni-NTA agarose column,recombinant CecropinXJ was noted to exert strong antimicrobial activities against a broad-spectrum of microorganisms,including Gram-negative and Gram-positive bacteria.Its minimal inhibitory concentration(MIC)against E.coli ATCC 25922 was 0.81 p.M.In addition,transmission electron microscopy(TEM)analysis indicated that the surfaces of the treated pathogens underwent obvious morphological changes compared with the untreated controls,suggesting that this antimicrobial peptide exerts its action by directly disrupting membranes of microorganisms.CecropinXJ had a small hemolytic effect on red blood cells even with a peptide concentration of 200?M.Thus,CecropinXJ acts selectively on bacterial membranes.Purified recombinant antibacterial peptide,CecropinXJ,retained a high stability against E.coli ATCC 25922 over a temperature range from 4? to 100? and a pH range from pH 2.0 to 12.0.(3)Cecropin XJ could induce damage to microtubules,actin and microvilli on surface of Eca109 cells in a dose dependent manner,and inhibit migration and invasion of tumor cells.There was obvious correlation between microtubule depolymerization and actin polymerization,which induced by CecropinXJ.Meanwhile,CecropinXJ could decrease gene expression of ?-actin,?-actin,?-actin,?-tubulin and ?-tubulin in a concentration-dependent and time-dependent manner.(4)The inhibitory effect on the proliferation of Eca109 cells induced by incubation with CecropinXJ was detected by MTT assay.The changes of the morphology of cell membrane induced by CecropinXJ were investigated using electron microscopy.Moreover,Hoechst 33258 immunofluorescence staining,LDH activity-based assay,flow cytometry was used to investigate the cell cycle distribution and apoptosis.Expression of apoptosis and cell cycle-related protein in Eca109 cells was analyzed by western blot.The results showed CecropinXJ could inhibit the proliferation of Eca109 cells in time and dose-dependent manners.Under electron microscope,typical apoptotic morphological and ultrastructural changes could be observed.Flow cytometry results showed that CecropinXJ could induce apoptosis of Ecal 09 cells,and CecropinXJ-mediated apoptosis was accompanied by the loss of mitochondrial membrane potential,the release of cytochrome c,and induced cleavage of poly(ADP-ribose)polymerase(PARP)and the activation of caspases-3.CecropinXJ regulated expression of cell cycle and apoptosis related proteins.(5)CecropinXJ was used in the Balb/c Ecal 09 cells-bearing mice model.To investigate whether CecropinXJ exert anti-tumor efficiency through regulating immune response when treated with tumor-bearing mice,which provided experimental basis and theoretical foundation for the anti-tumor mechanism and clinical application.The experiment of mice showed that CecropinXJ could significantly inhibit the proliferation of transplanted tumors in dose-dependent manner.There were significant difference between control group and 10 mg/kg CecropinXJ group on the volume and weight of tumor(p<0.05).HE staining showed that CecropinXJ inhibited Ecal 09 cells infiltrating growth in tumor tissue without affecting viscera.The immunohistochemistry results showed that the expressions of VEGF and bFGF proteins were higher than those in the control group.The proliferation of T cell in vitro showed that CecropinXJ could not stimulate the proliferation of mouse spleen lymphocytes(p>0.05).Flow cytometry was used to detect the expression of cytokines and DC surface molecules,the results that,compared with the control group,CecropinXJ could not significantly promote the expression of CD4,IL-4 and IFN-?,while not involving the expression of surface molecules on DCs in spleen(p>0.05).In cecropinXJ treatment group,TNF-a level in serum was significantly higher than the control group(p<0.01).In summary,CecropinXJ could significantly inhibit tumor growth in Ecal 09 cells-bearing mice,and might not be affected anti-tumor efficiency through regulating immune response.(6)The proliferation of Ecal 09 cells was inhibited by CecropinXJ and it's combination with chemotherapeutic agents in dose-dependent manner.Rates of apoptosis,the level of reactive oxygen species and mitochondrial membrane potential were significant differences in all combined-drug groups,which compared with that in the single-drug groups(p<0.05).CecropinXJ is an agent for anti-human esophageal carcinoma Eca109 cells by inhibition of proliferation and induction of apoptosis in vitro.CecropinXJ can enhance the effect of ADM,DDP,CTX and reduce the effect of CBP.(7)After Eca109 cells were treated with CecropinXJ for different times,antimicrobial peptide antibody and fluorescent-labeled secondary antibody were used to fluorescent stains.The results showed that CecropinXJ could destroy the cell surface micro-villus structure firstly,arid penetrate the cell membrane to the cytoplasm by cleavage gradually,then resulting in leakage of the cell contents,cell atrophy,and finally into the nucleus,killing tumor cells.