【作者】 张晓萍；

【导师】 赵茹茜；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2004， 硕士

【摘要】 为了探讨本实验室自制的CCK卵黄抗体的生物学活性及其作用机理，本实验以SD大鼠和AA肉鸡为动物模型，观察日粮添加不同剂量的CCK卵黄抗体粉对其生长性能的影响，并分析部分血清激素水平变化。然后，采用Western blot和ELISA验证大鼠十二指肠能否吸收CCK卵黄抗体，进一步利用RT-PCR分析肉鸡十二指肠和下丘脑的相关基因表达，探讨CCK卵黄抗体的可能作用机理。 一、CCK卵黄抗体粉的制备、检测和提取纯化 本实验室制备的CCK卵黄抗体应用ELISA检测，抗体效价高达1：12800以上。卵黄液经冷冻干燥和钴-60辐射消毒处理后，效价无影响。表明CCK卵黄抗体通过冻干处理及灭菌消毒后，仍保持较高效价，便于贮存、运输及在生产上的应用。 利用水稀释法和盐析法相结合，提取CCK卵黄抗体。提取后的IgY纯度为83.8％。同时，SDS-PAGE电泳显示，IgY分子量为200kDa。 二、CCK卵黄抗体粉对大鼠生长性能及部分血清激素水平的影响 21日龄雄性SD大鼠120只，随机分成4组，分别在基础日粮中添加0、70、150、300mg／kg的CCK卵黄抗体粉，添加剂量不足300mg／kg的以阴性卵黄粉补足。试验过程中，记录大鼠每7天摄食量、每15天体重变化。饲喂42天后，根据体重变化，将试验Ⅰ组(70mg／kg)和对照组大鼠断头采血，离心得血清，并进行屠宰试验，测定肝脏、肾脂肪囊、腹脂以及腓肠肌湿重。 试验结果显示，70mg／kg添加剂量的CCK卵黄抗体粉处理，能显著促进大鼠的体重和体增重(P＜0.05)，150mg／kg和300mg／kg的CCK卵黄抗体粉对大鼠的生长无显著影响(P＞0.05)；各剂量组大鼠部分器官重量、器官指数及平均日摄食量变化不明显(P＞0.05)；同时，70mg／kg的CCK卵黄抗体粉处理后，大鼠血清胰岛素水平有升高趋势(P=0.068)，血清Leptin水平有下降趋势(P=0.067)。 三、CCK卵黄抗体在大鼠十二指肠的吸收 为了探讨CCK卵黄抗体能否被动物胃肠道所吸收，我们应用ELISA和Western blot方法，检测灌胃CCK卵黄抗体后SD大鼠十二指肠静脉血液中CCK卵黄抗体的张晓萍胆囊收缩素卵黄抗体的生物学活性及其作用机理初探杭体效价及存在形式.试验采用14只雄性成年SD大鼠，随机分成试验组和对照组，分别灌胃1 50mg/ml ccK卵黄杭体粉混悬液和生理盐水(2 mU只)。安装亚急性十二指肠静脉插管，在灌胃后第2、3、4h采集十二指肠静脉血液，分离血浆. ELISA结果显示，试验组大鼠第2、3h血装中，均检测到CCK卵黄抗体效价，效价达1:128以上.同时，Weatem blot分析发现，试验组第2、3h血浆中存在完整分子形式的IgY.然而，第4h和对照组血浆中均未检测到IgY.四、C CK卵黄杭体粉对肉鸡生长性能及部分血清激素水平和相关基因表达的影响 l日龄AA肉鸡200羽，随机分为4组，分别在基础日粮中添加0、50、70、90mg/kg的ccK卵黄抗体粉，添加剂量不足90m叭g的以阴性卵黄粉补足.试验持续42天，记录每周肉鸡体重、耗料量变化.试验结束后，进行屠宰试验，肉鸡断头采血，离心得血清，根据体重变化，采取试验I组(50m叭g)和对照组十二指肠和下丘脑组织样. 结果显示，50、70、90m叭g的CCK卵黄抗体粉处理，均能显著提高肉鸡体重和体增重(P<0.05)，其中50m叭g组差异极显著(P<0 .01);各剂量组肉鸡摄食量、月同体组成无明显变化(P>.05);50、70、90m叭g组肉鸡血清胰岛素水平显著升高，90m叭g剂量组血清LePtin水平显著下降(P<0 .05);50m叭g的CCK卵黄抗体粉处理后，肉鸡十二指肠CCK及CCK一AR基因表达与对照组相比均无明显差异(P>0.05)，然而下丘脑CCK基因表达显著下调，CCK一AR的基因表达显著上调(P<0 .05). 综上所述，CCK卵黄抗体粉既具有较高的抗体效价，也具有生物学活性，能够显著提高SD大鼠(6 .7%)和AA肉鸡(9 .5%)的生长性能;ccK卵黄杭体能在大鼠十二指肠段以完整分子形式吸收或部分吸收进入血液，发挥其免疫学效应和生物学效应;CCK卵黄抗体处理能调节机体内分泌，并对中枢系统的CCK及CCK一AR基因表达有影响.提示，CCK卵黄抗体对动物的促生长作用可能主要是通过调节中枢系统的CCK及其受体而实现的.更多还原

【Abstract】 The objectives of present studies were 1) to detect the immunoreactivity of yolk antibody against CCK by ELISA; 2) to elucidate the effect of CCK yolk antibody on growth performance and growth-related hormones in SD rats and AA broiler chickens; 3) to determine whether CCK yolk antibody can be absorbed intact from duodenum in rats; 4) to reveal the possible mechanisms involved in the effect of CCK yolk antibody on growth performance of broiler chickens.1. Preparation, detection and purification of CCK yolk antibody powder.The specific titer of CCK yolk antibody reached 1:12800 with ELISA. The processes of lyophilization and radicidation had no influence in the titer of CCK yolk antibody.Using water dilution combined with salting out, we isolated the immunoglobulin from the egg yolk. The purity of CCK yolk antibody was 83.8%. SDS-PAGE electrophoresis showed that the moleculer weight of CCK yolk antibody was 200 kDa.2. Effect of CCK yolk antibody powder on growth performance and secretion of related hormones in SD rats.One hundred and twenty male SD rats at 21 days of age were randomly divided into four groups, with dietary supplementation of CCK yolk antibody powder at the dosage of 0, 70, 150, 300 mg/kg, respectively. The body weight and food intake of rats were recorded regularly during the experiment. After 6 weeks, rats in 70 mg/kg treatment and control groups were sacrificed for blood sampling, liver, gastrocnemius muscle, geliac and peirenal fat were weighed.Results showed that 70 mg/kg of CCK yolk antibody powder significantly increased body weight and body weight gain of rats (P<0.05), whereas the other treatment groups at higher doses did not show significant differences in growth performance compared with control group (P>0.05). At the same time, the organ weight, organ index and food intake of rats had no changes (P>0.05). Seventy mg/kg of CCK yolk antibody powder increased serum insulin (P=0.067) and decreased serum leptin (P=0.068) concentrations in rats.3. Absorption of CCK yolk antibody from duodenum in the rats.To clarify whether CCK yolk antibody can be absorbed from duodenum in rats, ELISA and Western blot analysis were employed to detect the immunoreactivity and the chemical form of CCK yolk antibody in duodenal venous blood after gastric infusion of CCK yolk antibody. Fourteen adult male SD rats were divided at random into treatment and control groups. Two ml of CCK yolk antibody (150 mg/ml) or physiological solution (0.9% NaCl) was administered intragastrically to rats in treatment and control group, respectively. Duodenal venous blood was collected from duodenal venous catheters at the 2nd, 3rd and 4th hour after infusion.CCK yolk antibody was detected in the duodenal venous plasma of treatment group by ELISA at the 2nd and 3rd hour after administration with the titer of 1:128. Western blot analysis confirmed the finding of ELISA that intact CCK yolk antibody existed in duodenal venous plasma of treatment group. These results suggest that CCK yolk antibody can be absorbed or partly absorbed from duodenum in rats and play its role in systematic circulation.4. Effect of CCK yolk antibody powder on growth performance and secretion of related hormones and gene expression in broiler chicken.Two hundred AA broiler chickens were randomly divided into four groups, with basal diet supplemented with CCK yolk antibody powder at the dosage of 0, 50, 70, 90 mg/kg, respectively. The body weight and food intake of broiler chicken were recorded weekly during the experiment. After 42 days, broiler chickens were slaughtered for carcass analysis and blood sampling. The duodenums and hypothalamus of chickens in 50 mg/kg treatment and control groups were sampled for further analysis. Serum concentrations of insulin and leptin were determined by RIA. The total RNA was extracted from tissue samples to measure the abundance of CCK, CCK-A receptor (CCK-AR) mRNA expression by RT-PCR with p-actin as an internal standard.The results showed that CCK yolk antibody powder at 50, 70, 90 mg/更多还原