肺炎克雷伯杆菌特异性卵黄抗体的制备及活性研究

发布时间：2018-03-13 16:39

本文选题：特异性卵黄抗体　切入点：肺炎克雷伯杆菌　出处：《大连医科大学》2011年硕士论文　论文类型：学位论文

【摘要】：目的:制备肺炎克雷伯杆菌特异性卵黄抗体(IgY),考察其体外抑菌活性及对小鼠克雷伯杆菌肺炎的保护作用。方法:以灭活的肺炎克雷伯杆菌为抗原免疫蛋鸡获得特异性IgY。通过水稀释、硫酸铵盐析、硫酸钠盐析和超滤等方法分离纯化抗体,再经冷冻干燥制成冻干粉。利用聚丙烯酰胺凝胶电泳检测抗体的纯度;采用间接酶联免疫吸附试验(ELISA)测定卵黄水溶性组分中的抗体效价。采用液体培养基比浊法考察不同浓度的特异性IgY对抗原菌的生长抑制活性。经鼻滴入肺炎克雷伯杆菌(2×1011cfu/ml)建立小鼠肺炎模型,攻毒剂量为每只100μl。小鼠随机分为空白对照组(不感染肺炎克雷伯杆菌)、阴性对照组(生理盐水)、头孢哌酮组(1 mg/ml)、地塞米松组(0.5 mg/ml)、特异性IgY高剂量组(40 mg/ml)、特异性IgY低剂量组(20 mg/ml)。攻毒前24h给药一次,除地塞米松组为每50μl/10g外,其余各组小鼠为150μl/10g;攻毒后每隔24 h给药一次,连续四天,除地塞米松组为100μl/10g外,其余各组小鼠为250μl/10g。观察小鼠的临床表现、体重、体温、白细胞计数、各组小鼠的死亡率、肺组织细菌培养、肺组织外观及肺病理切片。结果:经水稀释、盐析和超滤得到IgY的纯度与标准IgY相近。ELISA检测表明首次免疫后40～45天效价达到1600。体外抑菌试验表明,特异性IgY浓度在1～20 mg/ml之间能抑制肺炎克雷伯杆菌的生长且呈剂量依赖性,但其活性低于阳性对照头孢哌酮。肺炎克雷伯杆菌攻毒后,除空白对照组外,各组小鼠体温、体重、外周血中WBC数均有不同程度下降,IgY治疗组的下降明显低于阴性对照组,与阳性对照头孢哌酮组和地塞米松相近;除空白对照组外,其余各组小鼠肺组织细菌培养均呈阳性;IgY治疗组和阳性对照组细菌计数低于阴性对照组;肺组织有不同程度的损伤,肺病理切片显示肺泡结构破坏、肺纤维化,IgY治疗组和阳性组损伤程度低于阴性组;各组小鼠四天的死亡率分别为:空白对照组为0,阴性对照组为90%,其余均为80%。结论:实验中采用的分离纯化方法可获得高纯度的IgY。所制备的特异性IgY具有高效价和明显的体外抑菌活性。特异性IgY对小鼠肺炎克雷伯杆菌肺炎有保护作用。
[Abstract]:Aim: to prepare the specific egg yolk antibody of Klebsiella pneumoniae and to investigate its antibacterial activity in vitro and its protective effect on Klebsiella pneumoniae pneumonia in mice. Methods: layer hens were immunized with inactivated Klebsiella pneumoniae as antigen. The antibody was purified by water dilution, ammonium sulfate salting out, sodium sulfate salting out and ultrafiltration. Then freeze-dried powder was prepared. The purity of antibody was detected by polyacrylamide gel electrophoresis. Indirect enzyme-linked immunosorbent assay (Elisa) was used to determine the titer of antibodies in water soluble components of yolk. The growth inhibitory activity of specific IgY at different concentrations against antigenic bacteria was investigated by liquid medium turbidimetry. A mouse model of pneumonia was established by 2 脳 1011cfurml. Mice were randomly divided into blank control group (no infection with Klebsiella pneumoniae), negative control group (normal saline, cefoperazone group 1 mg / ml), dexamethasone group (0.5 mg / ml), specific IgY high dose group (40 mg / ml), specific IgY. In the low dose group, 20 mg / ml of the drug was given once 24 hours before the attack. With the exception of dexamethasone group, the other mice were 150 渭 l / 10 g every 50 渭 l / 10 g, administered every 24 hours for four days, with the exception of dexamethasone group (100 渭 l / 10 g), the other groups were 250 渭 l / 10 g. The clinical manifestations, body weight, body temperature, white blood cell count were observed. Mortality, bacterial culture of lung tissue, appearance of lung tissue and pathological section of lung in each group. Results: the purity of IgY obtained by water dilution, salting out and ultrafiltration was similar to that of standard IgY. Elisa test showed that the titer of IgY reached 160045 days after the first immunization. The bacteriostatic test in vitro showed that the purity of IgY was similar to that of standard IgY. The specific IgY concentration of 20 mg/ml could inhibit the growth of Klebsiella pneumoniae in a dose-dependent manner, but its activity was lower than that of the positive control group cefoperazone. The number of WBC in peripheral blood of IGY group was significantly lower than that of negative control group, which was similar to that of cefoperazone group and dexamethasone group, except for blank control group. The count of bacteria in the other groups was lower than that in the negative control group, and the lung tissue was damaged in different degree, and the alveolar structure was damaged by pathological sections of the lung, and the number of bacteria in the positive control group and the positive control group were lower than those in the negative control group. The degree of injury in IGY group and positive group was lower than that in negative group, and the mortality of four days in each group was 0 in the blank control group, 90 in the negative control group, and 80 in the rest. Conclusion: the method of isolation and purification can obtain high purity IgY. the specific IgY prepared has high titer and obvious antibacterial activity in vitro. The specific IgY has protective effect on Klebsiella pneumoniae pneumonia in mice.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

【相似文献】