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
This work studied the transcriptional patterns of three antibacterial genes,attacin,defensin and cecropin,during the development of Musca domestica. Quantitative analysis by real-time PCR was performed on mRNA levels ...This work studied the transcriptional patterns of three antibacterial genes,attacin,defensin and cecropin,during the development of Musca domestica. Quantitative analysis by real-time PCR was performed on mRNA levels in different development stages and challenged 3rd-instar larva at different time points after challenge of Musca domestica. The results revealed a predominance of the transcripts of all three genes during the 3rd-instar larvae and the adults. In the meanwhile,it revealed the greatest increase in mRNA. The transcript levels increased to 801 times,1009 times and 2500 times respectively for cecropin,attacin and defensin in 3rd-instar larvae after challenging susceptible bacterium. The results suggested that the transcriptional patterns of Musca domestica antibacterial genes were different during the different growth stages as well as the microbial challenge encountered in 3rd-instar larvae.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.
基金grant of State Natural Science Foundation(30671832)Key Science and Technology Programof Guangdong Province (2003B31602)Key Science and Technology Program of Guangzhou (2005Z3-E0211) .
基金Supported by the National Natural Science Foundation of China (Grant No. 30671832)the Key Science and Technology Program of Guangzhou (Grant No. 2005Z32E0211)
文摘This work studied the transcriptional patterns of three antibacterial genes,attacin,defensin and cecropin,during the development of Musca domestica. Quantitative analysis by real-time PCR was performed on mRNA levels in different development stages and challenged 3rd-instar larva at different time points after challenge of Musca domestica. The results revealed a predominance of the transcripts of all three genes during the 3rd-instar larvae and the adults. In the meanwhile,it revealed the greatest increase in mRNA. The transcript levels increased to 801 times,1009 times and 2500 times respectively for cecropin,attacin and defensin in 3rd-instar larvae after challenging susceptible bacterium. The results suggested that the transcriptional patterns of Musca domestica antibacterial genes were different during the different growth stages as well as the microbial challenge encountered in 3rd-instar larvae.
