Objective:To investigate the effect of utilizing a molecular partner on high-level expression of Mxisca domestica(M.domestica) cecropin in Escherichia coli(E.coli) and to identify the expressed products.Methods:The ge...Objective:To investigate the effect of utilizing a molecular partner on high-level expression of Mxisca domestica(M.domestica) cecropin in Escherichia coli(E.coli) and to identify the expressed products.Methods:The genomic sequence of M.domestica cecropin A(MC) and M. domestica ubiquitin(UBI) were searched from Cenbank and amplified by reverse transcriptase polymerase chain reaction(RT-PCR).Two expression plasmids,pET32a-MC and pET32a-UBI-MC, were constructed and transferred into E.coli and were then induced by Isopropylβ-D-1- Thiogalactopyranoside(IPTG).The expression of the fusion proteins Trx-MC and Trx-UBI-MC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Fusion protein Trx-MC was verified by Western blot analysis.The bactericidal activity of the purified MC was quantitatively determined using E.coli BL21(DE3).Results:The result showed that the fusion proteins were successively expressed in E.coli BL21 cells.A band at the expected position of 24 kDa representing the Trx-MC target protein was positivelystained,and the band at 4 kDa representing the hydrolysis of mature MC protein was also observed at the expected position. The expression levels of Trx-UBI-MC were higher than that of Trx-MC in E.coli.MC exhibited antimicrobial activity.Conclusions:With high-level expression of housefly cecropin A in E.coli using a fusion protein,MC exhibited antimicrobial activity.展开更多
[ Objective ] This study was to investigate the preventive effect of cecropin and lysozyme on skin fester disease in Trionyx sinensis. [ Method ] By employing intramuscular injection method, the preventive effect of m...[ Objective ] This study was to investigate the preventive effect of cecropin and lysozyme on skin fester disease in Trionyx sinensis. [ Method ] By employing intramuscular injection method, the preventive effect of mixtures of cecropin （10 mg/kg） and lysozyme（ 10 mg/kg） and of cecropin（6 mg/kg） and lysozyme（ 5 mg/kg） were challenged on Aeromonas encheleia-caused skin fester disease in T. sinensis. The mortality and relative percent survival were used evaluate the preventive effect of different drugs. The serum agglutination titer, cytophagocytosis and bactericidal activity were detected to compare the effect of drugs on the immunity and disease resistance of T. sinensis. [ Result] The residue concentrations of gentamicin and lysozyme in serum were compared to evaluate the side effect of drugs used challenged. The results showed that combined use of cecropin and lysozyme gave the best preventive effect and immunity in T. sinensis, with an extremely significant difference from other treatments （ P 〈 0.01 ）. Analysis of drug residue in serum indicated that lysozyme was completely metabolized 6 days after medication, while gentamicin residue was still observed 12 days later. [ Conclusion] The combined application of cecropin and lysozyme showed better preventive effect than antibiotics in fester disease of T. sinensis. Moreover, no drug residue and side effect were observed in the combined use treatment. This method avoids the side effect of antibiotics, worthy of extension at commercial scale.展开更多
The aim of the present in vitro study was to assess the tumoricidal potential of the following natural peptides belonging to the Cecropin family, namely Cecropin A and B, on a series of tumour cell lines: MDA-MB-231 (...The aim of the present in vitro study was to assess the tumoricidal potential of the following natural peptides belonging to the Cecropin family, namely Cecropin A and B, on a series of tumour cell lines: MDA-MB-231 (breast adenocarcinoma) and M14K (human mesothelioma). The experimental results reveal that the cytotoxic effects of the two peptides depend on their concentration. Their efficiency is significant at 120 μM concentrations and it persists even at 60 μM concentrations. The effects were insignificant at 30 μM concentrations. On the other hand, the cytotoxic potential was not significantly dependant on the type of peptide but more on the type of tumour cell line used. The MDA MB 231 line cells were much more sensitive to the action of Cecropins A and B than the M14K line cells. The prospects brought about by this experimental research consist of the collection of in vitro experimental data on the tumoricidal potential of these natural cytotoxic peptides on tumour cells. This will enable specialists to develop future in vivo experimental models in order to test the antitumor effect of these cytotoxic peptides. The ultimate goal would be the discovery of agents with efficient antitumor properties, i.e. with maximum tumoricidal effects and minimum toxic side effects.展开更多
Cecropin A1 （CeeA1） promoter from Bombyx mori was cloned and character- ized to provide insight into the transcriptional control of this antimicrobial peptide gene upon immune challenges. Reporter gene assays demons...Cecropin A1 （CeeA1） promoter from Bombyx mori was cloned and character- ized to provide insight into the transcriptional control of this antimicrobial peptide gene upon immune challenges. Reporter gene assays demonstrated that both Escherichia coli and lipopolysaccharide could induce expression in BmE cells but B. bombyseptieus or peptidoglycan failed, and the induction pattern of the reporter gene was coincident with the endogenous CecAl. Analysis of deletion and mutation constructs revealed that the regulatory region was the κB motif located between -176 and -166, and no other pre- dicted elements on CecAl promoter affected its inducibility. Insertion of additional κB motifs increased the activity of CecAl promoter. Furthermore, binding of Relish to lob motif was confirmed by electrophoretic mobility shift assay. These findings indicate the regulatory mechanism of CecAl expression in IMD pathway and suggest an approach of engineering antimicrobial peptide promoter with enhanced activities that may lead to broad applications.展开更多
Nasopharyngeal carcinoma （NPC） is one of the highly prevalent malignancies in Southeast Asia. Chemotherapy and radiotherapy are commonly administrated to treat nasopharyngeal carcinoma, but these therapies can not p...Nasopharyngeal carcinoma （NPC） is one of the highly prevalent malignancies in Southeast Asia. Chemotherapy and radiotherapy are commonly administrated to treat nasopharyngeal carcinoma, but these therapies can not prevent the recurrence and metastasis of tumor cells. Furthermore, these therapies have severe side effects. Therefore, it is important to find a more effective therapy, e. g., a gene therapy for treatment of nasopharyngeal carcinoma. Cecropin AD （Cad） is a hybrid peptide that exhibits higher antibacterial activity than natural cecropin. It consists of 1-11 amino acid residues in the N-end of cecropin A and 12-37 amino acid residues in the c-end of cecropin D. The role of Cad in the malignant cells remains unclear. In this study, the Cad gene was cloned and transferred into NPC CNE2 cells and its pro-apoptotic effect were investigated.展开更多
试验旨在研究抗菌肽 cecropin AD 对肉鸡免疫功能和肠道菌群的影响。试验选用540只1日龄的健康肉公雏,随机分为5个处理,每个处理6个重复,每个重复18只鸡。日粮处理分别为:①空白对照:基础日粮(玉米-豆粕型);②抗生素组:基础日粮+200mg/k...试验旨在研究抗菌肽 cecropin AD 对肉鸡免疫功能和肠道菌群的影响。试验选用540只1日龄的健康肉公雏,随机分为5个处理,每个处理6个重复,每个重复18只鸡。日粮处理分别为:①空白对照:基础日粮(玉米-豆粕型);②抗生素组:基础日粮+200mg/kg硫酸粘杆菌素;③低剂量抗菌肽组:基础日粮+150mg/kg抗菌肽;④高剂量抗菌肽组:基础日粮+300mg/kg抗菌肽;⑤协同组:基础日粮+150mg/kg抗菌肽+200mg/kg硫酸粘杆菌素。在试验期内,肉鸡自由采食和饮水。于试验的第21d和第42d测定肉鸡的免疫指标和肠道菌群。试验结果发现,从整个试验期来看,添加抗菌肽组对免疫器官发育均没有显著影响。第21d时,抗菌肽组的血浆IL-6含量显著低于空白组(P<0.05),且添加抗菌肽300mg/kg组含量最低。第21d和第42d时,抗菌肽显著降低了盲肠大肠杆菌和总厌氧菌(P<0.05),但不及抗生素组抑制作用强;而各处理间的乳酸杆菌均没有显著差异。因此本试验制备的抗菌肽 cecropin AD 能很好的抑制有害菌,构成动物免疫屏障。日粮中添加抗菌肽 cecropin AD 能够起到调节肠道菌群,调控免疫反应,提高动物生长性能的作用。展开更多
[ Objective ] This study aimed to investigate the antimicrobial activity of hybrid antimicrobial peptide buforin II-cecropin B. [ Method ] Gene fragment BC encoding the hybrid antimicrobial peptide Buforin II-Cecropin...[ Objective ] This study aimed to investigate the antimicrobial activity of hybrid antimicrobial peptide buforin II-cecropin B. [ Method ] Gene fragment BC encoding the hybrid antimicrobial peptide Buforin II-Cecropin B was synthesized by SOE-PCR with six primers designed according to the published amino acid sequences. Then, the BC gene fragment was ligated into pET32a vector and expressed in BI21. Antimierobial ability of the crude hybrid antimicrobial peptide was detected with the Oxford cup method. [ Result] The BC gene fragment was correctly amplified by SOE-PCR and ligated into pET32a vector, to construct the expression vector pET32-BC. The hybrid antimicrobial peptide buforin II-cecropin B was expressed in BI21 after induction by IPTG. The optimal induction time was 2 h. Antimicrobial activity assay suggested that the hybrid antimicrobial peptide possessed antimicrobial activity to Escherichia coli and Bacillus subtilis. [ Conclusion] The hybrid antimicrobial peptide buforin II-cecropin B exhibited antimicrobial activity to Gram-negative E. coli and Gram-positive B. subtilis strains.展开更多
基金supported by grants from National Natural Science Foundation of China(No.30972566,3087198)
文摘Objective:To investigate the effect of utilizing a molecular partner on high-level expression of Mxisca domestica(M.domestica) cecropin in Escherichia coli(E.coli) and to identify the expressed products.Methods:The genomic sequence of M.domestica cecropin A(MC) and M. domestica ubiquitin(UBI) were searched from Cenbank and amplified by reverse transcriptase polymerase chain reaction(RT-PCR).Two expression plasmids,pET32a-MC and pET32a-UBI-MC, were constructed and transferred into E.coli and were then induced by Isopropylβ-D-1- Thiogalactopyranoside(IPTG).The expression of the fusion proteins Trx-MC and Trx-UBI-MC was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).Fusion protein Trx-MC was verified by Western blot analysis.The bactericidal activity of the purified MC was quantitatively determined using E.coli BL21(DE3).Results:The result showed that the fusion proteins were successively expressed in E.coli BL21 cells.A band at the expected position of 24 kDa representing the Trx-MC target protein was positivelystained,and the band at 4 kDa representing the hydrolysis of mature MC protein was also observed at the expected position. The expression levels of Trx-UBI-MC were higher than that of Trx-MC in E.coli.MC exhibited antimicrobial activity.Conclusions:With high-level expression of housefly cecropin A in E.coli using a fusion protein,MC exhibited antimicrobial activity.
文摘[ Objective ] This study was to investigate the preventive effect of cecropin and lysozyme on skin fester disease in Trionyx sinensis. [ Method ] By employing intramuscular injection method, the preventive effect of mixtures of cecropin （10 mg/kg） and lysozyme（ 10 mg/kg） and of cecropin（6 mg/kg） and lysozyme（ 5 mg/kg） were challenged on Aeromonas encheleia-caused skin fester disease in T. sinensis. The mortality and relative percent survival were used evaluate the preventive effect of different drugs. The serum agglutination titer, cytophagocytosis and bactericidal activity were detected to compare the effect of drugs on the immunity and disease resistance of T. sinensis. [ Result] The residue concentrations of gentamicin and lysozyme in serum were compared to evaluate the side effect of drugs used challenged. The results showed that combined use of cecropin and lysozyme gave the best preventive effect and immunity in T. sinensis, with an extremely significant difference from other treatments （ P 〈 0.01 ）. Analysis of drug residue in serum indicated that lysozyme was completely metabolized 6 days after medication, while gentamicin residue was still observed 12 days later. [ Conclusion] The combined application of cecropin and lysozyme showed better preventive effect than antibiotics in fester disease of T. sinensis. Moreover, no drug residue and side effect were observed in the combined use treatment. This method avoids the side effect of antibiotics, worthy of extension at commercial scale.
文摘The aim of the present in vitro study was to assess the tumoricidal potential of the following natural peptides belonging to the Cecropin family, namely Cecropin A and B, on a series of tumour cell lines: MDA-MB-231 (breast adenocarcinoma) and M14K (human mesothelioma). The experimental results reveal that the cytotoxic effects of the two peptides depend on their concentration. Their efficiency is significant at 120 μM concentrations and it persists even at 60 μM concentrations. The effects were insignificant at 30 μM concentrations. On the other hand, the cytotoxic potential was not significantly dependant on the type of peptide but more on the type of tumour cell line used. The MDA MB 231 line cells were much more sensitive to the action of Cecropins A and B than the M14K line cells. The prospects brought about by this experimental research consist of the collection of in vitro experimental data on the tumoricidal potential of these natural cytotoxic peptides on tumour cells. This will enable specialists to develop future in vivo experimental models in order to test the antitumor effect of these cytotoxic peptides. The ultimate goal would be the discovery of agents with efficient antitumor properties, i.e. with maximum tumoricidal effects and minimum toxic side effects.
基金This work was supported by grants from the National Basic Research Program of China （No. 2012CB 114600） and the National Natural Science Foundation of China （No. 31201854）.
文摘Cecropin A1 （CeeA1） promoter from Bombyx mori was cloned and character- ized to provide insight into the transcriptional control of this antimicrobial peptide gene upon immune challenges. Reporter gene assays demonstrated that both Escherichia coli and lipopolysaccharide could induce expression in BmE cells but B. bombyseptieus or peptidoglycan failed, and the induction pattern of the reporter gene was coincident with the endogenous CecAl. Analysis of deletion and mutation constructs revealed that the regulatory region was the κB motif located between -176 and -166, and no other pre- dicted elements on CecAl promoter affected its inducibility. Insertion of additional κB motifs increased the activity of CecAl promoter. Furthermore, binding of Relish to lob motif was confirmed by electrophoretic mobility shift assay. These findings indicate the regulatory mechanism of CecAl expression in IMD pathway and suggest an approach of engineering antimicrobial peptide promoter with enhanced activities that may lead to broad applications.
基金This work was supported by a grant from the National 973 Project of China (No. G1999054204).
文摘Nasopharyngeal carcinoma （NPC） is one of the highly prevalent malignancies in Southeast Asia. Chemotherapy and radiotherapy are commonly administrated to treat nasopharyngeal carcinoma, but these therapies can not prevent the recurrence and metastasis of tumor cells. Furthermore, these therapies have severe side effects. Therefore, it is important to find a more effective therapy, e. g., a gene therapy for treatment of nasopharyngeal carcinoma. Cecropin AD （Cad） is a hybrid peptide that exhibits higher antibacterial activity than natural cecropin. It consists of 1-11 amino acid residues in the N-end of cecropin A and 12-37 amino acid residues in the c-end of cecropin D. The role of Cad in the malignant cells remains unclear. In this study, the Cad gene was cloned and transferred into NPC CNE2 cells and its pro-apoptotic effect were investigated.
文摘试验旨在研究抗菌肽 cecropin AD 对肉鸡免疫功能和肠道菌群的影响。试验选用540只1日龄的健康肉公雏,随机分为5个处理,每个处理6个重复,每个重复18只鸡。日粮处理分别为:①空白对照:基础日粮(玉米-豆粕型);②抗生素组:基础日粮+200mg/kg硫酸粘杆菌素;③低剂量抗菌肽组:基础日粮+150mg/kg抗菌肽;④高剂量抗菌肽组:基础日粮+300mg/kg抗菌肽;⑤协同组:基础日粮+150mg/kg抗菌肽+200mg/kg硫酸粘杆菌素。在试验期内,肉鸡自由采食和饮水。于试验的第21d和第42d测定肉鸡的免疫指标和肠道菌群。试验结果发现,从整个试验期来看,添加抗菌肽组对免疫器官发育均没有显著影响。第21d时,抗菌肽组的血浆IL-6含量显著低于空白组(P<0.05),且添加抗菌肽300mg/kg组含量最低。第21d和第42d时,抗菌肽显著降低了盲肠大肠杆菌和总厌氧菌(P<0.05),但不及抗生素组抑制作用强;而各处理间的乳酸杆菌均没有显著差异。因此本试验制备的抗菌肽 cecropin AD 能很好的抑制有害菌,构成动物免疫屏障。日粮中添加抗菌肽 cecropin AD 能够起到调节肠道菌群,调控免疫反应,提高动物生长性能的作用。
基金Supported by the Research Project of Sichuan University of Science&Engineering(No.2011RC12,2014KY02)Scientific Research Foundation of the Education Department of Sichuan Province(NO.15ZA0222)
文摘[ Objective ] This study aimed to investigate the antimicrobial activity of hybrid antimicrobial peptide buforin II-cecropin B. [ Method ] Gene fragment BC encoding the hybrid antimicrobial peptide Buforin II-Cecropin B was synthesized by SOE-PCR with six primers designed according to the published amino acid sequences. Then, the BC gene fragment was ligated into pET32a vector and expressed in BI21. Antimierobial ability of the crude hybrid antimicrobial peptide was detected with the Oxford cup method. [ Result] The BC gene fragment was correctly amplified by SOE-PCR and ligated into pET32a vector, to construct the expression vector pET32-BC. The hybrid antimicrobial peptide buforin II-cecropin B was expressed in BI21 after induction by IPTG. The optimal induction time was 2 h. Antimicrobial activity assay suggested that the hybrid antimicrobial peptide possessed antimicrobial activity to Escherichia coli and Bacillus subtilis. [ Conclusion] The hybrid antimicrobial peptide buforin II-cecropin B exhibited antimicrobial activity to Gram-negative E. coli and Gram-positive B. subtilis strains.
