【作者】 马卫明；

【导师】 佘锐萍；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2004， 博士

【摘要】 新鲜猪小肠经充分破碎后，再经高温处理、乙酸浸提，最后经Sephadex G-100和Sephadex G-25凝胶柱过滤层析，超滤离心管超滤后，收集各组分并用琼脂糖弥散法检测各组分的抗菌活性。具有抗菌活性的组分，经Tricine SDS-PAGE分析为一条蛋白带，分子量约为5972Da左右，得到电泳纯的猪小肠抗菌肽，并进一步对分离纯化的猪小肠抗菌肽的生物学功能进行了如下研究。 采用琼脂糖弥散实验和活菌计数实验检测了猪小肠抗菌肽对鸡大肠杆菌O₁ C83845株、鸡白痢沙门氏菌C79-13株、E.coli O₂、E.coli 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等11株细菌的作用。结果表明，猪小肠抗菌肽除对E.coli O₂和猪致病性沙门氏菌自然分离株作用较弱外，对以上其他9株菌株均有较强的抑制或杀灭作用，杀菌率由59.5％～98.5％不等。揭示了该物质具有较强的抗菌活性和较广的抗菌谱。 将鸡大肠杆菌O₁ C83845株、鸡白痢沙门氏菌C79-13株和金黄色葡萄球菌ATCC25923株与猪小肠抗菌肽在室温下作用15min。扫描电镜下观察猪小肠抗菌肽对这三株细菌形态结构的影响。结果发现，猪小肠抗菌肽可作用于细菌，导致细菌表面出现囊泡样或不规则突起样结构，细菌严重变形、扭曲，表面变得粗糙，皱缩，胞膜破裂，内容物外流而死亡。从而可推断该抗菌肽类物质对细菌具有溶膜性作用，在一定程度上揭示了该物质的抗菌机理。 将IBV用猪小肠抗菌肽处理后再接种9日龄鸡胚，测定鸡胚尿囊液中的IBV血凝效价，并观察鸡胚病变。结果发现，猪小肠抗菌肽可使鸡胚尿囊液中IBV的血凝效价降低，同时还能抑制IBV所引起的鸡胚病变。用猪小肠抗菌肽处理NDV和IBDV后再感染SPF鸡，结果发现，猪小肠抗菌肽可以降低ND和IBD的发病率和死亡率。表明猪小肠抗菌肽对IBV、NDV和IBDV等有一定的灭活作用。 给雏鸡肌肉注射猪小肠抗菌肽后，定期测定雏鸡腹腔巨噬细胞吞噬率和吞噬指数、免疫器官指数、ND弱毒苗及IBD中等毒力苗的血清抗体水平、生长指标与各段肠管肠绒毛长度、黏膜厚度、绒毛长度与隐窝深度比值（V／C），上皮内淋巴细胞（IEL）、肥大细胞、杯状细胞的数量以及肠道组织中IgA阳性物面积系数。结果发现猪小肠抗菌肽可提高鸡腹腔巨噬细胞吞噬率和吞噬指数；提高免疫器官指数；提高ND弱毒苗及IBD中等毒力苗的血清抗体水平；使肠道粘膜IEL、肥大细胞和杯状细胞数量以及肠道组织中的IgA阳性物面积系数显著增加；可提高各段肠管肠绒毛长度和V／C值，但对肠黏膜厚度无显著影响，实验组鸡的生长指标也较对照组明显升高。另外，猪小肠抗菌肽也可以增强小鼠腹腔巨噬细胞LDH活性。这些结果表明猪小肠抗菌肽对鸡特异性免疫与肠道粘膜免疫水平均有不同程度的提高作用；猪小肠抗菌肽还能促进雏鸡生长发育，并从结构上初步揭示了猪小肠抗菌肽促进生长发育的机理是使肠绒毛增长，改善了肠绒毛结构。 本研究还利用化学交联法增强猪小肠抗菌肽免疫原性，以此抗原免疫家兔成功制备了猪小肠抗菌肽多抗，采用免疫组化方法对猪小肠抗菌肽在小肠中的分布定位进行了初步观察。猪小肠抗菌肽多抗的成功制备为进一步深入研究抗菌肽生物学功能及其特性和抗菌肽与肠道黏膜免疫的关系奠定了基础。更多还原

【Abstract】 Antibacterial peptides (ABPs) are important resources from which it may be able to develop novel therapeutic agents against the ever growing number of antibiotics resistant microorganisms. The small intestine of pig may possess one or more antimicrobial peptides that could be developed as excellent therapeutic agents. After the fresh small intestines of pig were treated by grinding, ultrasonic treatment, heating and 5% acetic acid extraction, antibacterial peptides was isolated from small intestines of pig by Sephadex G-100 , Sephadex G-25 gel filtration chromatography and ultrafiltration. The fractions obtained were tested against Escherichia coli O78. The fractions showing the antibacterial activity underwent further characterization. The purity and molecular weight of the antibacterial peptide were estimated using tricine polyacrylamide gel electrophoresis (Tricine SDS-PAGE). The results showed that an electrophoresis-grade antibacterial peptide was purified from small intestine of pig and its molecular weight was estimated to be about 5972 daltons.Using agarose diffusion assay and live bacteria counting assay, its antibacterial activity against 11 bacteria strains {Salmonella pullorum C79-13, Escherichia coli O1 C83845, Staphylococcus aureus ATCC25923, Escherichia coli O2, Escherichia coli O-j%, Pseudomonas aeruginosa ATCC27853, porcine pathogenic Escherichia coli isolate, chicken pathogenic Escherichia coli isolate, porcine pathogenic Salmonella typhisuis isolate, Staphylococcus albus and fish pathogenic Aeromonas hydrophila isolate ) was revealed. The results showed that the small intestine antibacterial peptide was strongly active against all the bacteria strains above except E.coli O2 and porcine pathogenic S.typhisuis isolate, and the bacteria-killing rates varied from 59.5% to 98.5%. The results preliminarily indicated that the small intestine antibacterial peptide was strongly active against bacteria and possessed broad antibacterial or bactericidal spectrum.Morphological observation of S.pullorum C79-13, E.coli Ox C83845 and S.aureus ATCC25923 treated with the small intestine antibacterial peptide, respectively, at 37.0C for 15 minutes under scanning electron microscopy (SEM) showed that the surfaces of the tested bacteria above became considerably rough, crimpled, shrivelled, distorted and blebbed. The results provided conclusive evidence that the small intestine antibacterial peptide was membrane active and how the antibacterial peptide killed bacteria.Infectious bronchitis virus (IBV) was treated with the small intestine antibacterial peptide at room temperature for 15 minutes and the small intestine ABP-treated IBV was inoculated into allantoic cavity of 9-day-old chicken embryos. The hemagglutination titer of IBV in chicken embryo allantoic fluids was determined and the pathological changes of chicken embryos were observed. The results showed that the hemagglutination titer of IBV was lower and the pathological changes were less serious than the chicken embryos inoculated with the ABP-untreated IBV. SPF chicken were challenged by Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) treated with the small intestine antibacterial peptide, respectively, and the results showed that the small intestine antibacterial peptidecould reduce the mortality and incidence of chickens caused by ND and IBD.The small intestine antibacterial peptide was administered intramuscularly into chicken at 10ug per chicken dose weekly since 7 days old. The immune organ index, the phagocytic percentage and phagocytic index of macrophage in the abdominal cavity of chicken, the serum antibody titers against NDV and IBDV, the growth index, the villus length, mucosa thickness and V/C value of duodenum, jejunum and ileum were determined at the age of 7, 21, 35, 49 days, respectively. The number of intraepithelial lymphocytes (IEL), mast cell and goblet cell of duodenum, jejunum, ileum and the IgA positive substances were also counted at above age, respectively. The results showed that the s更多还原