【作者】 宋毓民；

【导师】 王建华；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2009， 博士

【摘要】 疯草是全球危害最严重的一类有毒植物,常引起大量的家畜死亡,孕畜流产、早产、死胎以及胎儿发育不良,空怀率高,公畜不育等,给畜牧业造成巨大的损失。为此,人们通过挖除疯草、生物防除、化学灭除、添加解毒剂和轮牧等许多措施,以期降低疯草的危害,然而直到目前为止,这一问题一直没有得到彻底解决。研究证明,苦马豆素(Swainsonine, SW)是疯草中的主要有毒成分。人们对SW的理化性质、中毒机理、药理活性和毒理学等进行了大量研究,而如何通过免疫的方法预防动物疯草中毒已成为目前研究的热点。本研究选择对氯甲基苯甲酸作为SW半抗原和牛血清白蛋白(BSA)载体之间的间隔臂,化学合成了SW-BSA人工抗原,并进行了人工合成抗原中SW与BSA最佳结合比的筛选、免疫佐剂的选择以及对家兔和山羊的免疫学研究,获得以下结果:1.根据液液萃取时不同物质的分散原理,优化了从疯草有毒植物中提取SW的生产工艺,使SW的提取率显著提高,从变异黄芪和甘肃棘豆的提取率分别达到50 mg/kg和64 mg/kg以上,为SW的进一步研究提供了首要条件。2.引入对氯甲基苯甲酸作为SW与BSA之间偶联的间隔臂,通过化学合成的方法,先合成SW的季铵盐化合物,波普分析各步反应为目标化合物后,再用EDC法与BSA偶联得到SW-BSA人工抗原,紫外光谱检测偶联成功。3.紫外光谱法测定了4种SW-BSA人工合成抗原中SW与BSA的结合比分别为14.7、20.2、25.3和32.6。并对小鼠进行免疫试验,间接EILSA检测结果表明,4种偶联率的SW-BSA人工合成抗原对小鼠都产生了较高效价的抗体,其中以偶联比为20.2的免疫效价最高,由此说明SW-BSA人工合成抗原的最佳偶联率为20.2。4.以偶联比为20.2的SW-BSA为免疫原,分别制备弗氏佐剂疫苗、蜂胶佐剂疫苗和白油佐剂疫苗,并对家兔进行免疫试验,间接ELISA检测血清效价表明弗氏佐剂疫苗和白油佐剂疫苗都产生了较高的免疫效价。由于弗氏佐剂疫苗的副作用大,一般仅用于试验研究,所以本试验选择白油佐剂疫苗作进一步的免疫学研究,以为生产实践提供依据。同时,间接ELISA阻断试验和琼脂双向扩散试验表明,家兔体内产生了针对SW的特异性抗体。5.家兔血清加入丙酮,经过超声和离心净化处理,冷冻干燥后硅烷化试剂衍生化,建立了气相色谱分析家兔血清中SW的方法。本方法的线性范围为0.000 08 g/L～2.5 g/L,相关系数γ= 0.999 6,在0.001 g/L～1.0 g/L 4个水平上加标回收率为89.81%～96.23%,相对标准偏差为2.46%～4.35%。该方法适用于家兔血清中苦马豆素含量的测定。6.SW-BSA白油佐剂疫苗免疫家兔2次,间接ELISA法检测血清抗体效价的变化,抗体效价达到较高后进行甘肃棘豆(10 g/kg/d)攻毒试验,同时检测血清各项生化指标。试验结果表明,免疫攻毒组家兔的临床中毒症状比攻毒对照组出现时间延迟30 d,血清中SW浓度比攻毒对照组延缓21 d达到较高水平,血清中AST、ALP、LDH、BUN活性比攻毒对照组延缓31 d达到较高值,血清α-mannosidase的活性比攻毒对照组延缓28 d下降到较低值,家兔各个组织器官的病理变化主要是以细胞呈现急性中毒性缺血缺氧和空泡变性为特点。7.SW-BSA疫苗免疫山羊2次,间接ELISA法检测血清抗体效价的变化,抗体效价达到较高后进行甘肃棘豆(10 g/kg/d)攻毒饲喂试验,同时检测血清各项生化指标。试验结果表明,攻毒组山羊于攻毒后15 d～20 d出现典型的中毒症状,免疫攻毒组于50 d～55 d出现中毒症状,比攻毒对照组延缓30 d～40 d;攻毒后,攻毒对照组山羊血液中生化指标迅速改变,而免疫攻毒组表现渐进性变化,其中SW浓度、AST、ALP、LDH、BUN、α-mannosidase活性分别比攻毒对照组延缓27 d、24 d、27 d、30 d、24 d、30 d达到较高或较低点;中毒山羊组织器官出现以急性中毒性缺血缺氧和空泡变性为特点的病理变化,与家兔的基本一致。以上结果表明,SW-BSA人工合成抗原疫苗对家兔和山羊具有一定的保护作用,能够延缓疯草中毒时间30 d～40 d,为SW-BSA疫苗的临床应用提供了试验依据。更多还原

【Abstract】 Locoweed is one of the most serious toxic plants in the word, often caused a large number of livestock deaths, fetation miscarriage, premature birth, stillbirth and fetal dysplasia, high rate of empty fetation, Sire infertility etc, resulting in great loss of animal husbandry. So, many measures were taken to reduce the locoweed’s harm, such as excavation, biological control, chemical elimination, adding antidotes, rotation grazing and so on. So far, however, this problem has never been completely resolved. Research has shown that swainsonine (SW) is the main Locoweed toxic ingredients. People carried out a lot of study on the physical and chemical properties of the SW, poisoning mechanism, pharmacological activity and toxicological. So how to prevent the locoweed poisoning of animals by the method of immunization has become a research hotspot. In this study, chloromethyl benzoic acid was choiced as spacerarm between the SW hapten and the bovine serum albumin (BSA) carrier. Synthetizing the artificial antigen SW-BSA, selecting the best combination of it and the immunological adjuvant was carried to do some immunological studies on rabbits and goats. The results are as follows:1. Acording to the principle of liquid-liquid extraction of various substances dispersed, the SW extracting from Locoweed toxic plants was optimized so that the extraction rate of SW was significantly improved to 50 mg/kg and 64 mg/kg or more from Astragalus Variabilis Bunge and Oxytropis kansuensis Bunge, which faciliate the further study of SW.2. The chloromethyl benzoate was first introduced as spacerarm between SW and BSA to synthesize SW quaternary ammonium salt compound , after every compounds were identified by poppen analysis, then to get SW-BSA by the methord of EDC. It was success by ultraviolet spectra.3. The four kinds of combination ratios between SW and BSA in artificial synthetic antigen SW-BSA were determined by UV method, and they were 14.7, 20.2, 25.3 and 32.6 respectively. Then the immunity test was carried out with SW-BSA in mice, the results of which showed that the four kinds of SW-BSA in mice produced quite high titers of antibodies by indirect EILSA, among of them the titer highest ratio was 20.2. So it indicated that the best coupling rate of SW-BSA was 20.2.4. Freund’s, propolis and white oil adjuvant vaccine were made of coupling ratio of 20.2 in the SW-BSA, then immunized rabbits and the titers of rabbits serum were detected by indirect EILSA. The test results showed that both Freund’s adjuvant vaccine and oil adjuvant vaccines had a higher titer. Because Freund’s adjuvant vaccine, side effects, it was generally only used for the pilot study, This experiment use white oil adjuvant vaccine trial for further immunological studies, which provide the basis for the production practice.At the same time, indirect blocking ELISA test and agar double diffusion tests showed that specific antibodies of SW in the rabbit body was produced.5. The gas chromatography analysismethord was established to detect SW in rabbit serum, after rabbit serums were dealed with adding acetone, ultrasound and centrifugal purification, freeze-drying and silylation derivatization. The linear range of this method was 0.000 08 g/L ~ 2.5 g/L. The correlation coefficient wasγ= 0.999 6, the recovery of standard addition was 89.81% ~ 96.23% in the 0.001 g/L ~ 1.0 g/L 4 levels. The relative standard deviation was 2.46% ~ 4.35%. This method is suitable for detecting swainsonine in rabbit serum.6. Rabbits were immuned with SW-BSA vaccined twice, the serum antibody titer changes were tested by indirect ELISA assay and attacking toxic with Oxytropis kansuensis (10 g/kg/d) when the antibody titers were in higher. At the same time, the serum biochemical indicators were detected.The results showed that the time of clinical toxic symptom of the infection immune rabbits was delayed 30 d than the control group, the concentrations of SW in serum were delayed for 21 d to rise at higher level in the immunized and poisoning group compared with poisoning control group, the activity level of AST, ALP, LDH, BUN in serum were delayed for 31 d, the descending activity ofα-mannosidase was delayed for 28 d compared with poisoning control group. The pathological changes of tissues and organs was acute toxicity, hypoxia-ischemia and vacuolar degeneration.7. SW-BSA vaccine was used twice to rabbits, the serum antibody titer changes were tested by indirect ELISA assay and attacking toxic with Oxytropis kansuensis (10 g/kg/d) when the antibody titers were in higher level. At the same time, the serum biochemical indicators were detected. The results showed that the goats of infection control group presented the typical toxic symptoms in the 15th~20th day after attacking toxin, while the goats of immunized and poisoning in the 50th ~ 55th day, the time was delayed 30 d~40 d. The biochemical indicators in serum of control group changed rapidly after attacking toxic while immune group were progressive. In which SW concentration, the activity of AST, ALP, LDH, BUN,α-mannosidase respectively were delayed for 27 d, 24 d, 27 d, 30 d, 24 d, 30 d to reach a higher or lower point. The pathological changes of tissues and organs was the same acute toxicity, hypoxia-ischemia and vacuolar degeneration as rabbits.These results show that the artificial synthetic antigen SW-BSA vaccine has a protective effect to rabbits and goats. It can delay the locoweed poisoning time of animals 30 d ~ 40 d and provide a basis for the SW-BSA vaccine clinical trial.更多还原