黄鳝抗菌肽的制备及其粗提物对黄鳝出血病保护作用的初步研究
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摘要
抗菌肽是近年来研究发现的新一代抗菌剂,在先天性宿主防御系统中起重要作用。但水生动物抗菌肽的相关研究起步较晚,黄鳝抗菌肽的提取仅本实验室有部分研究,而其余目前尚未见报道。本文研究黄鳝黏液、皮肤等组织粗提物的抗菌活性,在此基础上进一步分离纯化黄鳝黏液抗菌肽,并观察黄鳝黏液抗菌肽粗提物对半致死剂量嗜水气单胞菌(A.hydrophila)感染黄鳝的保护作用。共分三个试验,研究内容如下。
试验一、黄鳝各组织抗菌肽的粗提
本试验采用Tris-HCl提取法和乙酸提取法从诱导后黄鳝黏液、血液、皮肤、肝脏、脾脏、肠道和肾脏组织中分离抗菌肽粗提物。结果表明,从黄鳝黏液、皮肤和肠道成功分离出具有抗菌活性的粗提物。用嗜水气单胞菌和沙门氏菌作为指示菌,平皿法检测,结果显示黄鳝黏液和肠道提取物的抗菌效果明显。在其血液、肝脏、脾脏、肾脏提取物中未观察到抑菌圈。
试验二、抗菌肽粗提物对出血性败血症病理模型黄鳝的保护作用
本试验分别观察不同浓度黄鳝黏液抗菌肽粗提物(160mg/mL、80mg/mL、40mg/mL)对LD_(50)(1×10~(10)CFU/mL)嗜水气单胞菌感染黄鳝的保护作用。用浓度为160mg/mL、80mg/mL和40mg/mL的黏液抗菌肽粗提物对黄鳝进行腹腔注射,黄鳝的死亡数分别降低到0、2尾和3尾。通过病理组织学观察发现,腹腔注射160mg/mL黏液抗菌肽粗提物能有效地减轻嗜水气单胞菌引起的黄鳝肝脏、脾脏、肾脏、肠道等重要脏器的损伤。提示,黄鳝黏液抗菌肽粗提物对嗜水气单胞菌引起的出血性败血症的黄鳝具有保护作用。
试验三、黄鳝黏液抗菌肽的分离纯化
本试验利用Sephadex G-50凝胶对黄鳝黏液抗菌肽粗提物进行初步纯化,再用Macro-Prep High S阳离子交换进行离子交换层析对其进一步纯化。经Sephadex G-50凝胶过滤色谱分析,收集到三个紫外吸收峰的组分,通过抗菌活性筛选,第3个吸收峰有抗菌活性;其抗菌活性样品经Macro Prep High S阳离子交换层析进一步分离纯化,并对收集到的两个紫外吸收峰进行抗菌活性筛选,发现第2个吸收峰有抗菌活性。将该活性组分进行SDS-PAGE电泳,电泳结果显示为单一条带,说明分离到的黄鳝抗菌肽已达到电泳纯,分子量为8400Da。
Antimicrobial peptides(AMPs)are a recently discovered type of antimicrobial activity peptide and play an important role in the innate host defense mechanisms of most living organisms.But related researches on aquatic animal have few reported before.Now research of Monopterus albus AMPs hasn't reported besides our laboratory.Three experiments were studied the antibacterial activity of the AMPs crude sample from Monopterus albus's skin,skin mucus,then were purification AMPs of the skin mucus,the effect of crude antibacterial peptide from Monopterus albus skin mucus Protecting mice from lethal dosage 50%(LD_(50))A.hydrophila challenge were observed.
Experiment 1.antibacterial peptides Raw Extraction of differ tissue from Monopterus albus
This experiment was used methods of Tris-HCl extraction and acetic acid extraction on antibacterial peptide crude sample from induced Monopterus albus's skin mucus,blood,skin,liver,spleen,intestine,kidney.The methods was succeed in separating antibacterial peptide crude sample of skin mucus,skin,intestine.This showed that the antibacterial activity of the raw sample from skin mucus,skin and intestine can be detected inhibition zone by A.hydrophila and salmonelladerby,but the raw sample from blood,liver,spleen,kidney can not.
Experiment 2.The crude extract protecting Monopterus albus from A.hydrophila challenge
The effect of crude peptide from Monopterus albus skin mucus(160mg/mL、80mg/mL、40 mg/mL)protecting Monopterus albus from LD_(50)A.hydrophila(1×10~(10)CFU/kg)challenge were observed.(160mg/mL)could survival all Monopterus albus from LD_(50)of A.hydrophila challenge,crude peptide from Monopterus albus skin mucus also protected Monopterus albus from tissue damage by LD_(50)of A.hydrophila challenge.The experiment had prompted:crude peptide from Monopterus albus skin mucuscould protecting Monopterus albus from LD_(50)A.hydrophila(1×10~(10)CFU/kg)challenge.
Experiment 3.Separating and purification of antibacterial peptide from Monopterus albus skin mucus
Different protein components were separated by using Sephadex G50 gel filtration sequencely,last purification antibacterial peptides was completed by Macro-Prep High S in this experiment.Three ultraviolet absorption peak of antibacterial peptide were collected in Sephadex G50 filtration chromatography,and rough screening of antibacterial activity was investigated.The antibacterial activity was detected from peak 3.The antibacterial samples that collected by Sephadex G50 were separated by Macro-Prep High S.Two ultraviolet absorption peak of antibacterial peptide were collected in Macro-Prep High S.rough screening of antibacterial activity was investigated.The antibacterial activity was detected from peak 2.After SDS-PAGE,the Monopterus albus antibacterial peptide showed a single protein band,and the molecular weight was about 8400Da.
试验一、黄鳝各组织抗菌肽的粗提
本试验采用Tris-HCl提取法和乙酸提取法从诱导后黄鳝黏液、血液、皮肤、肝脏、脾脏、肠道和肾脏组织中分离抗菌肽粗提物。结果表明,从黄鳝黏液、皮肤和肠道成功分离出具有抗菌活性的粗提物。用嗜水气单胞菌和沙门氏菌作为指示菌,平皿法检测,结果显示黄鳝黏液和肠道提取物的抗菌效果明显。在其血液、肝脏、脾脏、肾脏提取物中未观察到抑菌圈。
试验二、抗菌肽粗提物对出血性败血症病理模型黄鳝的保护作用
本试验分别观察不同浓度黄鳝黏液抗菌肽粗提物(160mg/mL、80mg/mL、40mg/mL)对LD_(50)(1×10~(10)CFU/mL)嗜水气单胞菌感染黄鳝的保护作用。用浓度为160mg/mL、80mg/mL和40mg/mL的黏液抗菌肽粗提物对黄鳝进行腹腔注射,黄鳝的死亡数分别降低到0、2尾和3尾。通过病理组织学观察发现,腹腔注射160mg/mL黏液抗菌肽粗提物能有效地减轻嗜水气单胞菌引起的黄鳝肝脏、脾脏、肾脏、肠道等重要脏器的损伤。提示,黄鳝黏液抗菌肽粗提物对嗜水气单胞菌引起的出血性败血症的黄鳝具有保护作用。
试验三、黄鳝黏液抗菌肽的分离纯化
本试验利用Sephadex G-50凝胶对黄鳝黏液抗菌肽粗提物进行初步纯化,再用Macro-Prep High S阳离子交换进行离子交换层析对其进一步纯化。经Sephadex G-50凝胶过滤色谱分析,收集到三个紫外吸收峰的组分,通过抗菌活性筛选,第3个吸收峰有抗菌活性;其抗菌活性样品经Macro Prep High S阳离子交换层析进一步分离纯化,并对收集到的两个紫外吸收峰进行抗菌活性筛选,发现第2个吸收峰有抗菌活性。将该活性组分进行SDS-PAGE电泳,电泳结果显示为单一条带,说明分离到的黄鳝抗菌肽已达到电泳纯,分子量为8400Da。
Antimicrobial peptides(AMPs)are a recently discovered type of antimicrobial activity peptide and play an important role in the innate host defense mechanisms of most living organisms.But related researches on aquatic animal have few reported before.Now research of Monopterus albus AMPs hasn't reported besides our laboratory.Three experiments were studied the antibacterial activity of the AMPs crude sample from Monopterus albus's skin,skin mucus,then were purification AMPs of the skin mucus,the effect of crude antibacterial peptide from Monopterus albus skin mucus Protecting mice from lethal dosage 50%(LD_(50))A.hydrophila challenge were observed.
Experiment 1.antibacterial peptides Raw Extraction of differ tissue from Monopterus albus
This experiment was used methods of Tris-HCl extraction and acetic acid extraction on antibacterial peptide crude sample from induced Monopterus albus's skin mucus,blood,skin,liver,spleen,intestine,kidney.The methods was succeed in separating antibacterial peptide crude sample of skin mucus,skin,intestine.This showed that the antibacterial activity of the raw sample from skin mucus,skin and intestine can be detected inhibition zone by A.hydrophila and salmonelladerby,but the raw sample from blood,liver,spleen,kidney can not.
Experiment 2.The crude extract protecting Monopterus albus from A.hydrophila challenge
The effect of crude peptide from Monopterus albus skin mucus(160mg/mL、80mg/mL、40 mg/mL)protecting Monopterus albus from LD_(50)A.hydrophila(1×10~(10)CFU/kg)challenge were observed.(160mg/mL)could survival all Monopterus albus from LD_(50)of A.hydrophila challenge,crude peptide from Monopterus albus skin mucus also protected Monopterus albus from tissue damage by LD_(50)of A.hydrophila challenge.The experiment had prompted:crude peptide from Monopterus albus skin mucuscould protecting Monopterus albus from LD_(50)A.hydrophila(1×10~(10)CFU/kg)challenge.
Experiment 3.Separating and purification of antibacterial peptide from Monopterus albus skin mucus
Different protein components were separated by using Sephadex G50 gel filtration sequencely,last purification antibacterial peptides was completed by Macro-Prep High S in this experiment.Three ultraviolet absorption peak of antibacterial peptide were collected in Sephadex G50 filtration chromatography,and rough screening of antibacterial activity was investigated.The antibacterial activity was detected from peak 3.The antibacterial samples that collected by Sephadex G50 were separated by Macro-Prep High S.Two ultraviolet absorption peak of antibacterial peptide were collected in Macro-Prep High S.rough screening of antibacterial activity was investigated.The antibacterial activity was detected from peak 2.After SDS-PAGE,the Monopterus albus antibacterial peptide showed a single protein band,and the molecular weight was about 8400Da.
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