High-level expression of housefly cecropin A in Escherichia coli using a fusion protein  

作　　者：Xueli Zheng Wei Wang 

机构地区：[1]Department of Pathogenic Biology,School of Public Health and Tropica Medicine,Southern Medical University

出　　处：《Asian Pacific Journal of Tropical Medicine》2010年第6期421-426,共6页亚太热带医药杂志（英文版）

基　　金：supported by grants from National Natural Science Foundation of China(No.30972566,3087198)

摘　　要：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.Objective: To investigate the effect of utilizing a molecular partner on high-level expression of Masca 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 Genbank and amplified by reverse transcriptase polymerase chain reaction (RT-PCR). Two expression plasmids, pFT32a-MC and pET32a-UBI-MC MC, were constructed and transferred into E. call and were then induced by Isopropyl 13 D l Thiogalactopyranoside (IPTG). The expression of the fusion proteins Trx-MC and Trx-UBI-MC was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SOS 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.

关 键 词：Musca domestica CECROPIN A Molecular PARTNER Fusion expression ANTIMICROBIAL activity 

分 类 号：R378[医药卫生—病原生物学]