【作者】 姚艳丰；

【导师】 杨利国；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 生长抑素是由下丘脑分泌的一种神经多肽，能抑制GH释放进而抑制动物生长。DNA疫苗由于能同时诱导机体产生特异性的体液和细胞免疫反应等优点，已经成为研究者关注的焦点之一，但单独使用DNA疫苗在大动物或人体诱导的免疫反应效果不理想，必须改善现有免疫方式来增强其免疫原性，如基因枪的使用或佐剂的开发利用。基因枪尽管有其高效性，但所需装置昂贵，应用成本较高，因而限制其应用。佐剂的使用是比较理想的方式，但其大都需要特殊处理，符合特定标准，并同DNA疫苗混合后方可应用。另外，DNA疫苗也必须从工程菌中大量抽提出来，费时费力，这势必影响了实验的可操作性，也使实验的成本加大，不利于推广。因此，开发新型免疫策略已成为亟待解决的问题。本研究对一种生长抑素基因免疫的新方法进行了评估，即用含目的质粒的灭活菌体直接免疫(不用抽提质粒)来取代现行的质粒DNA免疫方法(质粒)。以纯质粒免疫组为参照组，同用灭活菌体免疫组进行比较，引用四个参数分别从不同的角度来评估，包括抗生长抑素抗体检测、生长测定、细胞免疫检测、脏器指数检测。1抗生长抑素抗体检测灭活菌体免疫组，在实验的各个时期，抗体P／N值均极显著高于质粒组(P＜0.01)。在实验期，灭活菌体组的阳性鼠比例亦均显著高于纯质粒组，最大P／N值也出现在此组。并且灭活菌体组也使机体产生有效的免疫记忆或长期的免疫力。2生长测定即增重检测在增重实验中，灭活菌体组免疫亦表现出比纯质粒组的优势。加免两周时，灭活菌体组增重极显著高于对照组(P＜0.01)，显著高于纯质粒组(P＜0.05)。加免四周后，两实验组增重极显著高于对照组(P＜0.01)，灭活菌体组增重极显著高于纯质粒组(P＜0.01)。实验结束时，两实验组之间差异不显著(P＞0.05)，但灭活菌体组增重高于纯质粒组。3细胞免疫检测细胞免疫方面，进行了T细胞增殖和DTH两个实验。在T细胞增殖实验中，纯质粒免疫组和灭活菌体免疫组在诱导SS的特异性淋巴细胞增殖方面效果相似，二者差异不显著(P＞0.05)，但都优于各自对照组。DTH实验中无论是纯质粒组，还是灭活菌体组，左脚掌厚度与右脚掌厚度的比值P／N均大于1.0，且在第48h时比值达到高峰，但灭活菌体组和纯质粒组小鼠P／N之间差异不显著(P＞0.05)。4脏器指数检测脏器指数是重要的生物学指标之一。灭活菌体免疫组同对照组在心脏指数、肝脏指数、肾脏指数、脾脏脾脏、肺脏指数上差异不显著(P＞0.05)，表明新免疫方法并不影响动物的正常生长发育。灭活菌体组在脾脏指数高于对照组和纯质粒免疫组，但差异不显著(P＞0.05)，表明灭活菌体免疫有免疫增强作用。基于以上实验结果，我们得到一种更为经济、方便、免疫效果更好、拥有独立知识产权的基因免疫方式，即这种免疫方式能刺激机体产生较强的细胞和体液免疫反应，使机体产生有效的免疫记忆或长期的免疫力，产生良好的免疫效果，是一种很有前途的基因免疫的新方式。更多还原

【Abstract】 Somatostatin (SS), consisting of 14 peptides, is one of the most importantneuropeptides secreted from hypothalamus nuclei which inhibits the secretion ofgrowth hormone (GH) and depresses the growth. Many studies demonstrate that theuse of immuno-neutralization against SS can enhance the growth of animals. DNAvaccination is useful for generating immune responses, particularly the cell-mediatedimmune response, in a wide variety of species. However, DNA vaccination generallyinduces only relatively weak responses, particularly in large animals and human.Although, various approaches have been developed recently in order to improve itsefficacy or immunopotency, limited success has been achieved in a practical term.Though the gene gun improves the efficiency of transfection, the cost is very high andthen limits the application. Recently many adjuvants have been used for theirusefulness as in facilitating DNA vaccination. However, most of the adjuvants shouldbe processed so as to comply with special standard. And then they are mixed with theplasmid for future immunization. It is clear that the plasmid should be extracted fromthe engineering bacteria firstly. All of these are unbeneficial to the operability of theexperiment, to increase the cost, adverse to further spread. So it is stringent to developthe new immunizing strategy.In the present study, we evaluated the feasibility of the new strategy thatimmunized with inactivated bacteria containing target plasmid directly. Wedetermined it by four parameters including the level of anti-somatostatin antibody,weight gain, cell mediated immunity and organ index.1 Detection of anti- somatostatin antibodyThe inactivated bacteria groups induced higher levels of IgG during the course ofsampling than from the plasmid groups (P＜0.01), the percent of mice with positive Ab.against somatostatin was always higher than the mice in plasmid groups. As for themaximum P/N value, it was also more excellent in the inactivated bacteria groupsthan in the plasmid groups. 2 Weigh gain of miceIn BM2w, the activated group had a significant difference to plasmid group (P＜0.05). Furthermore, there was high significant difference in BM4w (P＜0.01). InBM8w, there was no difference between them (P＞0.05), but the activated group washigher suggesting that the effect immunized by the new method was good.3 Detection of cell mediated immunityIn the experiment of cell mediated immunity, the inactivated groups were similarto the plasmid group in T cell proliferation. There is no difference in the groups(P＞0.05); As to DTH, in every experimental group, the ratio was higher than thecontrol and reached the peak at 48h. However, there was no significant differenceamong the plasmid groups and the inactivated groups (P＞0.05). It was notpreponderant in inducing the cell mediated immunity.4 Organ index detectionOrgan index was one of the major bionomics indexes suggesting thedevelopment normality of the animals. Comparing the experimental groups withcontrol, there were no difference in heart, liver, spleen, lung and kidney (P＞0.05)suggesting the new strategy was safe. In inactivated bacteria groups, the spleen indexis higher than the plasmid groups suggesting immunological enhancement. It iscoherent to the result of antibody detection.Based on above results, a more improved and potent strategy was tested in theregards to augment the cellular and humoral responses without compromisinglong-term immunity. It is a promising strategy of gene immunization.更多还原