【作者】 崔先利；

【导师】 杨利国；

【作者基本信息】 南京农业大学 ， 动物遗传育种与繁殖， 2006， 硕士

【摘要】 本实验应用分子克隆、基因免疫、酶免疫测定、放射免疫等技术，研究抑制素基因免疫对黄牛抗抑制素抗体产生、生殖及生殖内分泌的影响，通过比较不同免疫原纯度、免疫剂量和免疫次数的免疫效果，探索最佳抑制素基因免疫方案，达到有效提高动物生殖力、降低实际成本、推广繁育新技术的目的。本研究的主要内容如下： 1．南阳黄牛对抑制素基因疫苗pCISI的免疲反应性研究 为分析黄牛对抑制素基因免疫的免疫反应性及其影响因素，本实验采用不同剂量、不同纯度的双拷贝抑制素融合质粒pCISI免疫两次免疫黄牛。结果表明：首次免疫10天后即产生了一定的抗抑制素抗体，首次免疫期间整个免疫组的抗体P／N值显著高于对照组（P＜0.05），加强免疫期间抗体P／N值极显著高于对照组（P＜0.01），抗体阳性牛的比例由16.83％提高到52.75％，前后差异显著（P＜0.05）。无论是抗抑制素抗体水平还是抗体阳性率，四个剂量组（0.75mg、1.5mg、2.25mg、3.0mg）组间均无明显差异（P＞0.05），其中，以3.0mg剂量组产生的抗抑制素抗体水平最高（1.97±0.62），2.25mg剂量组的抗体阳性率最高（56.62％，13／23）。高纯度pCISI(OD₂₆₀／OD₂₈₀介于1.8～2.0)的免疫效果略高于低纯度组(OD₂₆₀／OD₂₈₀介于1.7～1.8和2.0～2.1)，但两个纯度组组间无显著性差异（P＞0.05）。结合本次研究涉及的剂量与纯度，低纯度组中0.75mg抑制素融合质粒pCISI引起的免疫应答效果最佳，高纯度组中2.25mg抑制素融合质粒pCISI最佳。2.25mg和3.0mg剂量组中，高纯度组的抗体阳性率在全期显著高于低纯度组（P＜0.05），但平均抗体P／N值无显著性差异（P＞0.05）。无论是抗体阳性率还是抗体P／N值，其余两剂量的高、低纯度组组间在各个免疫阶段均无显著性差异（P＞0.05）。研究表明，融合到乙肝表面抗原上的抑制素基因的表达产物在大型动物黄牛体内同样具有较强的抑制素免疫学活性，免疫效果随着免疫次数的增加而有所提高，但并没有完全随着免疫原剂量的增加、纯度的提高而提高。 2．抑制素pCISI基因免疫对黄牛生殖及生殖内分泌的影响 为探讨抑制素基因免疫对黄牛生殖及生殖内分泌的作用，本实验采用不同剂量（0.75mg、1.5mg、2.25mg、3.0mg）、不同纯度（高、低）的抑制素融合质粒pCISI两次免疫黄牛。结果发现，免疫组平均每头牛的成熟卵泡为1.99±0.99个，胎儿为1.35±0.50头，至少具有两个成熟大卵泡的牛的比例达61.29％，双胎率达35.14％，显更多还原

【Abstract】 Techniques such as molecular clone, gene immunization, enzyme-linked immunoassay （ELISA） and radio immunoassay （RIA） etc were applied to study the immune response to inhibin gene immunization, effects on the reproduction and reproductive endocrinology in cattle. The different immune effects caused by different purity of vaccine, dose and adjuvant were studied to search for the best way to increase the reproduction of cattle, reduce the costs and popularize the new technology. The main content presents as follows:1. The study of immunoreactive character of inhibin pCISI gene immunization on Nanyang cattleIn order to analyze the immunoreactive character of cattle to inhibin gene immunization and its affecting factors, the different dosages, purity of inhibin fusion expressing plasmid pCISI were used to immune the cattle twice. The results showed that there were some sheep with positive antibody against inhibin on 10 days after primary immunization, and the average antibody P/N value of immunization group were higher than the control remarkably in the period of primary immunization （P<0.05）, those in the period of booster immunization were greatly remarkable higher than the control （P<0.01）. After booster immunization the rate of cattle with positive antibody is from 16.83% to 52.75 % 46.7%, which was remarkably different. Both the level of antibody against inhibin and the rate of positive antibody, there was no remarkable difference in the four groups with different doses （0.75 mg, 1.5mg, 2.25mg, and 3.0 mg）. The level of antibody against inhibin of 3.0mg immunization group was the highest （1.97±0.62）, and the rate of positive antibody of 2.25mg immunization group was the highest （56.62%, 13/23）. The immunization effect of highly purified pCISI (OD₂₆₀/ OD₂₈₀ between 1.8 and 2.0) was slightly better than pCISI with lower purity (OD₂₆₀/ OD₂₈₀ between1.7 and 1.8 or between 2.0 and 2.1), but the difference wasn’t remarkable （P>0.05）. Combined the dose with the purity, the effect of immunization response of 0.75mg inhibin fusion expressing plasmid pCISI in groups with lower purity was the best, and the effect of 2.25mg pCISI in groups with high purity was更多还原