【作者】 王可洲；

【导师】 佘锐萍；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2003， 博士

【摘要】 成年健康家兔，经腹腔注射猪大肠杆菌刺激后，取圆小囊组织，组织捣碎机捣碎，沸水浴处理后，以5％乙酸提取，调整pH值后，经Sephadex G100凝胶柱过滤层析，收集各组分并用琼脂糖弥散法检测各组分的抗菌活性，获得纯化的兔小囊肽，纯化物经Tricine SDS-PAGE，银染后分析为一条蛋白带，分子量约为7000Da左右，经超滤分析，纯化物的分子量大于3000Da。采用Dot-ELISA方法，用抗菌肽抗体检测兔小囊肽中的抗菌肽抗原，结果为阳性，说明兔小囊肽为抗菌肽类物质。 采用敏感的琼脂糖弥散实验和活菌计数实验检测了兔小囊肽对10株细菌(大肠杆菌（O₁）、大肠杆菌（O₂）、大肠杆菌(O₇₈)、猪致病性大肠杆菌自然分离株、鸡致病性大肠杆菌自然分离株、白色葡萄球菌、金黄色葡萄球菌ATCC25923株、猪致病性沙门氏菌自然分离株、绿脓杆菌ATCC27853株、鱼致病性嗜水气单胞菌)的作用。结果表明，兔小囊肽对以上10种细菌均有不同程度的抑制作用，杀菌率由49.3％-100％不等。初步揭示了该物质强大的抗菌活性和较广泛的抗菌谱。 将用兔小囊肽处理过的绿脓杆菌固定后用醋酸双氧铀染色，透射电镜观察，发现与对照组相比，抗菌肽对绿脓杆菌的主要作用是使菌体皱缩，染色变深，鞭毛损伤，使细菌表面形成囊泡样结构等。 首次采用鸡胚接种法观察了兔小囊肽对鸡新城疫病毒强毒株以及鸡传染性支气管炎病毒的灭活作用。将NDV及IBV用小囊肽处理后再接种9日龄鸡胚，测定鸡胚尿囊液中的NDV血凝效价，并观察鸡胚病变。结果发现，与对照组比较，兔小囊肽使鸡胚尿囊液中新城疫病毒的血凝效价降低，同时还能抑制因以上两种病毒所引起的鸡胚病变。 通过注射方法给予蛋雏鸡兔小囊肽，并测量其体重，胸腺、脾脏、法氏囊的器官指数，测量各肠段肠绒毛长度，测定禽流感灭活疫苗及鸡新城疫活疫苗的抗体水平，肠粘膜上皮内淋巴细胞数量以及肠道组织和法氏囊组织IgA阳性物面积系数，以研究兔小囊肽对以上指标的影响。结果表明，发现其对鸡体重增加无明显影响；对胸腺和脾脏指数无明显影响；对法氏囊指数有显著影响。兔小囊肽可显著提高十二指肠、空回肠结合部肠绒毛长度，对回盲肠结合部肠绒毛长度无明显影响；可提高禽流感灭活疫苗和鸡新城疫活疫苗的抗体水平。使肠粘膜上皮内淋巴细胞显著增加；使主要肠道组织及法氏囊组织IgA阳性物面积系数增加。提示兔小囊肽对鸡特异性免疫及粘膜免疫水平均有提高作用。 综上所述，本研究首次由兔圆小囊组织中分离纯化了兔小囊肽，并证明其为抗菌肽类物质。同时证实此小囊肽具有抗菌、抗病毒作用。首次揭示了小囊肽具有提高全身特异性免疫抗体水平及肠道粘膜免疫功能的作用。进一步揭示了作为肠相关淋巴组织的兔圆小囊在机体全身免疫反应和肠道局部粘膜免疫反应中的功能作用的物质基础——小囊肽的生物学活性。展示了兔小囊肽作为抗菌、抗病毒药物，作为免疫调节剂的可能性和应用前景。更多还原

【Abstract】 Stimulated the adult rabbits with bacteria ,10 hours later, killed them and take the sacculus rotundus out, triturated it and boiled for 15 minutes, mixed it with 5% acetic acid for 10 hours in 4 ℃, then centrifuged it and collected the supernate. Adjust the supernate’s pH value, centrifuged it again , the supernate is the crude extract. With Sephadex G100 chromatography and collect the antibacterial components, the crude extract was purified. Tricine-SDS-PAGE analysis show that the purified sacculus rotundus peptide is only 1 protein strip. Its molecular weight is lower than 14400Da,and about 7000Da.Ultrafiltration test showed that its molecular weight is higher than 3000Da, so its molecular weight was between 3000Da to 7000Da. With Dot-ELISA assay, used antibacterial peptide antibody to detect the antibacterial peptide in purified substances. The result was positive and showed that the purified substance was antibacterial peptide.Used agarose diffusion assay and live bacteria counting assay to reveal its antibacterial activity to" 10 bacteria strains.The result showed the sacculus rutundus peptide have strong antibacterial activity to each bacteria strain.In agarose diffusion assay,7 strains appeared distinct antibacterial loop,the bacteria killing units were 0 to 104.1n live bacteria counting assay.the rate of killed bacteria of each strain was from 100% to 49.3%.This study preliminarily indicate strong antibacterial activity and wide antibacterial range of this ABP.Treated the Pseudomonas aeruginosa with ABP, use transmission electron microscope (TEM), to observe the action of ABP to bacteria. The result showed that the bacteria became smaller and thiner, the color was heavier, and vesicle-like substances appeared on the membrane of bacteria .Treated the Newcastle disease virus(NDV) and Infectious bronchitis virus(IBV) with ABP, then inoculate them into chicken embryo ,then measured the erythrocyte agglutination titer of NDV in chicken embryo allantoic fluids, and observe the pathological changes of chicken embryo. The result showed that the erythrocyte agglutination titer of NDV was lower than controlled series, and the pathological changes were less serious than controlled series, indicate that ABP can inhibit the pathological changes of chicken embryo caused by NDV and IBV.Injected the ABP to chicken, then measure the body weight; calculate the index number of thymus, spleen and bursa Fabricius; measure the intestinal villus lengh of duodenum, jejunum and ileum; measure the antibody titers of live vaccine and inactivated vaccine; count the number of intraepithelial lymphocytes of duodenum, jejunum and ileum; count the IgA positive substances of duodenum, jejunum , ileum, caecum and bursa Fabricius.to study the effect of ABP to the above target. The result showed that ABP had no significant effect to the body weight and index number of thymus and spleen; could significantly effect the index number of bursa Fabricius; could significantly inhance the lengh of intestinal villus of duodenum, jejunum ;had no significant effect to the inhancement of the lengh ofileum intestinal villus; could significantly inhance the antibody liters of live vaccine and inactivated vaccine; could significantly inhance the number of intraepithelial lymphocytes of duodenum, jejunum and ileum.At last ,the ABP could significantly inhance the number of IgA positive substances of duodenum, jejunum, caecum and bursa Fabricius ,and have no significant effect on number of IgA positive substances of ileum.On the whole, through these studies, we isolated and purified the ABP from rabbit sacculus rotundus, revealed its antibacterial and antivirus activity, and its effect on immune function of chicken. Indicated its prospect as antibacterial and antivirus drugs or as immunomodulator.更多还原