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《Acta Zoologica Sinica》 2005-02
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Recombinant expression in E. coli,purification and polyclonal antibody preparation of a defensin from the housefly

WANG Lai-Cheng,WANG Jin-Xing **,ZHAO Xiao-Fan,CAI YingSchool of Life Sciences,Shandong University,Jinan 250100, China  
Defensin is a kind of cationic, inducible antimicrobial peptides that have been found in large range of living organisms and contribute to host defense by disrupting the cytoplasmic membrane of microorganisms. A novel full-length 430 base pairs cDNA of an insect defensin was cloned using polymerase chain reaction (PCR) from the cDNA library of the housefly Musca domestica. To further study the function and to prepare the anti-Mdde polyclonal antibody, we chose the prokaryotic expression system (pGEX-4T-1) to express the mature Mdde as a foreign protein. Mature defensin gene was amplified from cDNA library using specific primers by PCR. The DNA fragment was modified by adding EcoRⅠsite at 5′ end and Xho Ⅰsite at 3′ end respectively, and then cloned into expression vector pGEX-4T-1. The recombinant mMdde/pGEX-4T-1 was identified by sequencing. The E.coli BL21 was transformed by recombinant mMdde/pGEX-4T-1 and the positive clones (mMdde/pGEX-4T-1/BL21) were screened by PCR. 1 mmol/L IPTG can induce a high level of expression of GST-mdde which consists of 26 kD Glutathione S-Tranferase (GST) and 4 kD of Mdde. The fusion protein GST-Mdde was purified by GST-affinity chromatograph and cleaved by thrombin for getting Mdde. The recombinant Mdde showed low antibacterial activities by liquid growth inhibition method .
【Fund】: 本研究得到高等学校骨干教师资助计划项目 (GG 180 25001 1995);; 博士点基金 (20020422052) ;; 山东省科技厅计划项目(012100104) 资助~~
【CateGory Index】: Q819
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