为了在枯草芽孢杆菌中分泌表达具有生物活性的Abaecin,采用重叠延伸PCR方法拼接合成含有编码Abaecin的基因及其相关调控元件的重组表达质粒pGplat.将构建的表达质粒pGplat电击转化至枯草杆菌,筛选正常生长的菌体,摇瓶发酵,取上清液冻干作为样品.通过Western blot试验检测Abaecin在枯草芽孢杆菌中的分泌表达情况.采用琼脂扩散法检测表达产物Abaecin的抑菌活性.Western blot试验结果证实在3.9 ku处有表达产物的目的条带.琼脂扩散法结果证明表达产物Abaecin对大肠杆菌、鸡沙门氏菌等革兰氏阴性菌有抑制活性.试验表明在枯草杆菌中能有效分泌表达具有生物活性的抗菌肽Abaecin.本研究结果为开发全新高效抗生素肽类药物和饲料添加剂提供了理论依据.%A recombinant plasmid pGplat containing the coding gene of abaecin was constructed, and it was transformed into B. subtilis 1A751 to express antimicrobial peptide abaecin. The overlap-extension PCR method was used to splice the recombinant plasmid pGplat. The construc-ted recombinant plasmid with multi-copy expression cassette was transformed into Bacillus subti-lis strain by electroporation, positive clones were screened and inoculated to LB medium for ex-pression under induction of maltose. The sample of culture supernatant of B. subtilis 1A751 was treated by vacuum freeze-drying. Western blot assays were applied to investigate the expression of the abaecin gene in B. subtilis 1A751. Bioactivity of abaecin in culture supernatant of B. subtilis 1A751 was tested with plate diffusion method. The results showed that the secretory-expressed abaecin was identified by Western blot, and there is a strap at 3.9 kD. Bioactivity of abaecin was tested with plate diffusion method, and it had high bacteriostatic activity against Gram-negative bacteria as E. coli and Salmonella pullorum. These results indicated that bioactive abaecin could be expressed in B. subtilis 1A751, which lays a foundation for further study of new efficient pep-tide antibiotics and feed additives.
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