小皱蝽抗菌物质的诱导及抗菌肽AMP-2的分离纯化和特性

Induced Antibacterial Substances and Characterization of Antibacterial Peptide AMP-2 Purified from Cyclopelta parva Distant

针刺小皱蝽成虫腹部,诱导免疫防御反应,小皱蝽成虫组织经过浸提、浸提液TFA酸化、超速离心、超滤、固相萃取等分离步骤,平板生长抑制试验检测抗菌活性.在C18Sep-Pak固相萃取柱上,40%ACN洗脱组分为初样品,经RP-HPLC、GPC-HPLC及填料孔径更小的反相色谱柱纯化,得到0.452mgAMP2样品.琼脂孔扩散法检测AMP2抗菌活性,回收率为4.04%.抗菌肽AMP2在SuperdexPeptide凝胶过滤柱上测得分子量(Mr)为3.2×103,茚三酮反应紫色.AMP2具有一定热稳定性,在pH3～8的乙酸铵缓冲液中抗菌活性强,对检测的动植物病原菌具有广谱抗菌活性,对金黄色葡萄球菌、大肠杆菌D22、枯草芽孢杆菌的MIC值为2.5μg/mL,对四联球菌的MIC值为5.0μg/mL,AMP2可能为富含脯氨酸的小分子抗菌肽.

Cyclopelta parva Distant were pricked in abdomen with needles, so the innate immunity was induced. The separation steps included extraction, acidification with trifluoroacetic acid （TFA）, uhracentrifugation, uhrafihration and solid - phase extraction. 40% acetonitrile （ACN） fraction eluted on Waters Sep -Pak C18 cartridge was the primary sample and antibacterial activity was analyzed by the growth inhibition of the indicative strains on agar plate. Further purification was performed by consecutive chromatographic steps： RP - HPLC on Aquapore OD300 nm C18 （ 10 μ. 4. 6 ×250 mm） column, GPC - HPLC on Superdex Peptide （7.5 × 300 mm） column and RP - HPLC on PepMap C18 （5 μ, 4.6 × 250 mm） column. All aliquots of samples were guided by a sensitive radial diffusion assay. Consequently 0. 452 mg AMP-2 was obtained and the active recovery rate was 4.04%. Its molecular weight determined by GPC - HPLC was 3.2 × 10^3 and AMP-2 was thermostable to some extent. The antibacterial activity of AMP-2 remained potent in the buffer with pH 3 - 8 . AMP-2 could kill most pathogens from propagation, and its MIC value was 2.5 μg/mL towards E. coli D22, Staphyloccus aureur, Bacillus subtillis and 5.0 μg/mL towards Micrococcus tetragenus. AMP-2 was possibly a small proline - rich antibacterial peptide. Fig 3, Tab 2, Ref 11