论文部分内容阅读
[目的]为探索HD-5作为抗菌生物制剂替代传统抗生素的研究提供实验依据。[方法]构建毕赤酵母重组表达质粒pGAPZαA-HD5,转化毕赤酵母菌GS115,于Zeocin平板筛选阳性重组菌。振荡培养重组菌约48h,离心取培养上清原液进行冷冻干燥,再稀释获得含有HD-5的原液(I液)、5倍浓缩液(Ⅱ液)和10倍浓缩液(Ⅲ液),以不含HD-5的培养上清(O液)为对照,采用平板打孔药敏法,观察它们对大肠埃希菌、金黄色葡萄球菌和铜绿假单胞菌的抑菌环直径大小。[结果]随着HD-5浓度的逐渐增加,O、I、Ⅱ、Ⅲ液对大肠埃希菌的抑菌环直径分别为0mm、(16.56±0.45)mm、(20.09±0.67)mm、(26.72±0.54)mm;对金黄色葡萄球菌的抑菌环直径分别为0mm、(11.45±0.13)mm、(18.09±0.32)mm、(25.50±0.21)mm;仅Ⅲ液对铜绿假单胞菌产生了抑菌环[(17.45±0.56)mm]。[结论]体外实验证实HD-5的真核表达产物对革兰阳性和革兰阴性细菌有较显著的抑菌作用,提示其作为抗菌生物制剂替代传统抗生素具有潜在的临床应用价值。
[Objective] The research aimed to explore the research of HD-5 as an alternative to traditional antibiotics as antibacterial biological agents. [Method] Pichia pastoris recombinant plasmid pGAPZαA-HD5 was constructed and transformed into Pichia pastoris GS115. Positive recombinant bacteria were screened on Zeocin plates. The recombinant bacteria were cultured by shaking for about 48 hours. The original supernatant of the culture supernatant was centrifuged and lyophilized to obtain a stock solution of HD-5 (liquid I), 5 times concentrated liquid (liquid Ⅱ) and 10 times concentrated liquid (liquid Ⅲ) HD-5-free culture supernatant (O solution) was used as a control, and the antibacterial ring diameters of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were observed by the flat-plate method. [Results] With the increasing of HD-5 concentration, the diameters of antibacterial rings of O, I, II and III against Escherichia coli were 0mm, (16.56 ± 0.45) mm, (20.09 ± 0.67) mm, 26.72 ± 0.54) mm. The diameters of antibacterial rings against Staphylococcus aureus were 0 mm, (11.45 ± 0.13) mm, (18.09 ± 0.32) mm and (25.50 ± 0.21) mm respectively. The antibacterial ring [(17.45 ± 0.56) mm] was produced. [Conclusion] The in vitro experiments confirmed that the eukaryotic expression product of HD-5 had significant antibacterial activity against Gram-positive and Gram-negative bacteria, suggesting that it has potential clinical value as an antibacterial biological agent instead of traditional antibiotics.