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1 Chinese Journal of Applied Entomology 2015, 52(3): DOI: /j.issn * 黄粉虫抗菌肽的提取及其生化特性的研究 ** 朱小奇谢彩云聂荷敏赵肖陈小娜何晓辉 *** 宋欢王俊刚申红摘要目的 Tenebrio molitor Linnaeus 方法 CFU/mL Escherichia coli h ph 结果 P h P 0.05 结论关键词,,,, The biological properties of an antimicrobial peptide extracted from Tenebrio molitor ZHU Xiao-Qi ** XIE Cai-Yun NIE He-Min ZHAO Xiao CHEN Xiao-Na HE Xiao-Hui SONG Huan WANG Jun-Gang SHEN Hong *** (College of Animal Science and Technology, Shihezi University, Shihezi , China) Abstract [Objectives] To determine the concentration and antibacterial activity of an antimicrobial peptide (AMPs) extracted from Tenebrio molitor Linnaeus larvae, and to explore its biological properties. [Methods] AMP production was induced in five-day old Tenebrio molitor larvae by Escherichia coli at a concentration of CFU/mL. AMPs were extracted from the larvae at 12, 24, 36, 48, 60, and 72 h respectively, their concentration detected by coomassie brilliant blue stain and their antibacterial activity measured using the filter paper method. In addition, other features, such as thermal stability, stability after repeated freezing and thawing, proteinase stability, effect of different ph and coagulation, were also measured. [Results] The results show that inducing the production of AMPs with E. coli resulted in a significantly higher concentration of AMPs being extracted from T.molitor (P < 0.01). AMP levels were highest at 48 h. Moreover, the diameter of bacterial inhibition caused by AMPs induced by E. coli was significantly larger (P < 0.05). AMPs extracted from T. molitor were stable with respect to variation in temperature, proteinase and ph. Repeated freezing and thawing had little effect on the biological properties of AMPs, including coagulation. Therefore, the immune system of T. * Supported projects SRP ** First author @qq.com *** Corresponding author shenhong98@163.com Received Accepted

2 3 : 713 molitor produces a higher concentration of AMPs with better biological stability and antibacterial activity, and no coagulation, following induction by E. coli. [Conclusion] These results have laid a theoretical basis for further utilization of antibacterial peptides extracted from T. molitor. Key words Tenebrio molitor, antimicrobial peptide, concentration, antibacterial activity, biological characters Orsolya ~50 (Hancock and Lehrer 1998 Bulet et al Zasloff Zasloff 2002 Auvynet and Rosenstein Zimmerman et al Tenebrio molitor Linnaeus Escherichia coli 43 Tenecin 1 (Moon et al Tenecin 1 C- β- Lee et al Rahnamaeian et al Shin et al 材料与方法 1.1 材料试虫 Tenebrio molitor Linnaeus

3 714 Chinese Journal of Applied Entomology 菌种 Escherichia coli ATCC 方法黄粉虫抗菌肽诱导 CFU/mL 1 µl % 70% h 黄粉虫抗菌肽的提取 75 / mol/l ph=5 35 µg/ml PMSF r/min 30 min min r/min 4 30 min 黄粉虫抗菌肽粗提液蛋白浓度测定 (Bradford ) mg/ml OD 595 OD 黄粉虫抗菌肽粗提液抑菌活性测定 20 μl CFU/mL 6 cm ( 64 图 1 标准蛋白曲线 Fig. 1 Standard protein cure µg/ml 2 5 min 5 min 24 mm 15 mm h 黄粉虫抗菌肽粗提液部分生化特性的研究热稳定性 min r/min 4 15 min 3 l 反复冻融的稳定性 -20 l r/min 4 15 min 3 l 不同 ph 处理对抗菌肽活性的影响 ph l h 3 ph l00

4 3 : 对蛋白酶稳定性 37 l mg/ml K 30 min 3 l 凝血试验 1 ml 10 ml 4 ml r/min 3 min ml Eppendorf (EP 2% 2% 100 μl 24 U 50 μl 数据处理 Excel 2003 SPSS 17.0 t- One-way ANOVA 2 结果与分析 2.1 黄粉虫抗菌肽粗提液蛋白浓度的测定结果 h OD 595 ) h mg/ml P< h 48 h 2.2 黄粉虫抗菌肽粗提液对大肠杆菌抑菌活性的测定结果 1 48 h 48 h mm P 0.05 P 黄粉虫抗菌肽粗提液的部分生化特性测定结果热稳定性 10 min 50~70 P Table 1 表 1 大肠杆菌诱导黄粉虫幼虫抗菌肽粗提液蛋白浓度测定 (mg/ml) Protein concentration assay of antimicrobial peptide extracted from Tenebrio molitor larvae induced by Escherichia coli mg/ml Time after induction (h) Group Control group Induction group B B B B B B A A A A A A SPSS 17.0 t- (P <0.01) The data in the table are mean ± SE, and followed by different capital letters in the same column indicate extremely significant different at 0.01 level by t-test.

5 716 Chinese Journal of Applied Entomology 52 表 2 黄粉虫幼虫抗菌肽对大肠杆菌的抑菌圈直径的测定结果 Table 2 The antibacterial circle diameter produced by the AMPS extracted from Tenebrio molitor larvae induced by Escherichia coli Group Induction group Antibiotic group Control group Diameter of antibacterial circle ( mm ) b a c SPSS 17.0 One-Way ANOVA (P<0.05) The data in the table are mean ± SE, and followed by different small letters in the same column indicate significant different at 0.05 level (One-way ANOVA procedure of SPSS 17.0). The same below. 表 3 黄粉虫幼虫抗菌肽经不同温度处理后的抑菌活性 Table 3 Antibacterial activity of antibacterial peptides from Tenebrio molitor larvae heated at different temperature ( ) Temperature Diameter of antibacterial circle ( ) Rate of bacteriostasis a a a b c c CK a P ~80 90~100 P 反复冻融性 -20 l EP, 6 P ~10 P 表 4 不同冻融次数对黄粉虫幼虫抗菌肽抑菌活性的影响 Table 4 Effect of times of freezing and dissolving on antibacterial activity of antibacterial peptides from Tenebrio molitor larvae Times of freezing and dissolving Diameter of antibacterial circle ( ) Rate of bacteriostasis a a a a b c CK a ph 稳定性 ph ph 3.0~ ph= ph<3 ph>9 50

6 3 : 717 表 5 不同 ph 溶液对黄粉虫幼虫抗菌肽抑菌活性的影响 Table 5 Effect of different ph values on antibacterial activity of antibacterial peptides from Tenebrio molitor larvae ph ph values Diameter of antibacterial circle ( ) Rate of bacteriostasis d c a b c d CK a 蛋白酶稳定性 6 作用显著 P 蛋白酶 K 1.98 K 表 6 蛋白酶对黄粉虫幼虫抗菌肽抑菌活性的影响 Table 6 Effect of proteinase on antibacterial activity of antibacterial peptides from Tenebrio molitor larvae Proteinase Trypsin Pepsin K Proteinase K Diameter of antibacterial circle ( ) Rate of bacteriostasis c c ab CK a 凝血活性检测 2% 100 μl 24 U 50 μl 2~3 min 3 讨论 Hoffmann et al Hancock 2001 Koczulla and Bals 2003 Johnston et al Lipopolysaccharide LPS Zhao et al Cecropins A B D 35~37 cdna Cecropins A B D A B 2 h 48 h

7 718 Chinese Journal of Applied Entomology 52 D 48~96 h 114 h h A B 48 h D Gudmundsson et al Musca domestica h h h Haine et al Li et al Capillary electrophoresis CE h P 0.05 P 0.05 Zn 2+ Brogden 2005 Garcia-Olmedo et al ~ Urich et al Andersons 1991 Fehlbaum et al ph 3.0~ ph= K 98 Cornet et al. 1995

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Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇