黑水虻抗菌肽诱导及粗提物活性研究

发布时间：2018-02-07 13:32

  本文关键词： 黑水虻 抗菌肽 诱导 抑菌活性 生物学活性　出处：《遵义医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的：采用不同诱导方法对黑水虻(Hermetia illucens L.)幼虫抗菌肽进行诱导,并对所提取粗提物进行活性研究,探讨黑水虻幼虫抗菌肽粗提物的抑菌活性及生物学活性。期待通过本研究丰富昆虫抗菌肽的种类,并为黑水虻抗菌肽的研发及利用提供有效的实验方法。 方法：本研究利用大肠杆菌菌液针刺浸泡诱导法诱导黑水虻幼虫,通过研磨提取法提取幼虫抗菌肽粗提物确定最佳诱导龄期；采用大肠杆菌菌液、金黄色葡萄球菌菌液注射诱导法,大肠杆菌、金黄色葡萄球菌菌液针刺浸泡诱导法,不同功率、不同时间超声波诱导法对黑水虻5龄幼虫进行抗菌肽诱导。利用研磨提取法提取抗菌肽粗提物,并对粗提物分别进行抑菌活性研究。选用抑菌效果最佳的抗菌肽粗提物进行生物学活性鉴定及利用反相高效液相色谱(RP—HPLC)对粗提物进行初步检测。 结果：研究表明,黑水虻幼虫产生抗菌肽的最佳诱导龄期为5龄；不同诱导方法产生抗菌肽对大肠杆菌抑菌活性不同,大肠杆菌菌液针刺浸泡60s,诱导后饲养24h提取的抗菌肽抑菌活性最好；其次为超声波100W诱导20min产生抗菌肽抑菌活性；其余诱导方法产生抗菌肽也有一定的抑菌效果但与未诱导组相比差异不显著。生物活性研究表明,黑水虻抗菌肽经25℃、37℃、50℃处理10min后仍具有较为理想的抑菌效果；反复冻融次数少于4次时对抗菌肽抑菌活性无显著影响,冻融6次以上抑菌活性减小较为明显；pH值在5.0-9.0范围仍可保持抑菌活性,pH5或pH9的环境中无明显抑菌活性。利用反相高效液相色谱(RP—HPLC)得到抗菌肽独立峰。 结论：1.诱导黑水虻幼虫产生抗菌肽的最佳龄期为5龄。2.不同诱导方法产生抗菌肽对大肠杆菌抑菌活性不同。大肠杆菌菌液针刺浸泡60s,诱导后饲养24h提取的抗菌肽抑菌活性最好；其次抑菌活性较高的为100W超声波诱导20min产生抗菌肽。3.黑水虻抗菌肽粗提物在25℃、37℃、50℃时均有较好的活性,低温反复冻融次数少于4次时可保持较好抑菌活性；pH值的适宜范围为5.0-9.0。4.利用反相高效液相色谱(RP—HPLC)得到抗菌肽粗提物独立峰。
[Abstract]:Objective: to induce the antimicrobial peptides of Hermetia illucens L. by different methods, and to study the activity of the extracted crude extracts. To study the antimicrobial activity and biological activity of the crude extract of antimicrobial peptides from the larvae of Tabanus nigra. It is expected that this study will enrich the species of insect antimicrobial peptides and provide an effective experimental method for the development and utilization of antimicrobial peptides. Methods: in this study, the best inducing age was determined by the extraction of antimicrobial peptides from the larvae by the method of lapping extraction, the best inducing age was determined by the method of needle immersion induction of Escherichia coli bacteria, and the optimum induction age was determined by the method of grinding extraction. Staphylococcus aureus liquid injection induction method, Escherichia coli, Staphylococcus aureus liquid acupuncture immersion induction method, different power, The antimicrobial peptides of the 5th instar larvae of the black water fly were induced by ultrasonic induction at different times, and the crude antimicrobial peptides were extracted by the grinding extraction method. The antimicrobial activity of crude extract was studied respectively. The crude antimicrobial peptide extract with the best antibacterial effect was selected for biological activity identification and RP-HPLC was preliminarily detected by RP-HPLC. Results: the results showed that the best inducing age of antimicrobial peptide production was 5 years old, and the antimicrobial activity of different induction methods was different to Escherichia coli. The bacteriostatic activity of antibacterial peptides extracted from Escherichia coli was the best after incubation for 60 s, and the second was the antibacterial activity of antibacterial peptides induced by ultrasonic wave 100W for 20 minutes. The antimicrobial peptides produced by the other methods also had a certain bacteriostatic effect, but there was no significant difference compared with the uninduced group. The bioactivity study showed that the antimicrobial peptides of the black water gadfly still had ideal bacteriostatic effect after being treated at 25 鈩，

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