非抗性筛选生长抑素DNA疫苗免疫育肥猪的临床试验效果和安全性研究

发布时间：2018-06-20 01:29

本文选题：生长抑素 + 基因疫苗　；参考：《华中农业大学》2015年硕士论文

【摘要】：前期研究证明,重组菌C500(p VGS/2SS-asd)基因疫苗免疫可以促进小鼠生长,并且对小动物无不良影响。为了进一步了解该疫苗对大动物的免疫效果及其机制和安全性,本研究选择78头80日龄的杜长大育肥猪,随机分成6组,用同一剂量(5×1011CFU/头)三种免疫方式(口腔+鼻腔、口腔、鼻腔)、同一免疫方式(口腔+鼻腔)三种剂量(高5×1011 CFU/头、中5×1010 CFU/头、低5×109 CFU/头)进行临床试验,试验期90 d,探讨最适免疫方法和免疫剂量。采用美国奥饲本(OSBORNE)公司生产的投料器和种猪生产性能WINFIRE测定系统每天自动记录实验猪的饲料消耗量、测定猪体重,饲料报酬以评定基因疫苗免疫效果;从免疫后猪的日常行为及体温是否正常、应用目的基因扩增等方法从基因整合、环境扩散等方面评价疫苗的安全性;应用ELISA方法测定抗体和激素水平,以探讨其作用机制。结果如下:1.促生长效果及饲料报酬采用美国奥饲本公司生产的种猪生产性能WINFIRE测定系统每天全自动记录猪采食量和体重数据,免疫前期育肥猪生长速度较快,后期增重减缓;探究免疫方式对育肥猪的促生长效果,结果表明,各时期试验组日增重均高于对照组,免疫后4-7 w,喷鼻组日增重显著高于对照组(P0.05);探究免疫剂量对育肥猪的促生长效果,结果表明,与对照组相比,初免疫后7 w,中剂量、低剂量组日增重均提高19.94%,差异极显著(P0.01),整个试验阶段,各免疫组的平均日增重均高于对照组,而低剂量组的日增重比对照组提高9.41%,差异显著(P0.05)。免疫方式组、免疫剂量组的日采食量相比对照组均有所增加,但无明显差别;不同免疫方式组间,鼻腔组的料肉比低于对照组(P0.05),不同免疫剂量组间,低剂量组的料肉比低于对照组(P0.05)。2.安全性研究本研究从免疫后猪的日常行为、基因整合情况、环境污染三个方面评估重组菌C500(p VGS/2SS-asd)的安全性,结果显示,所有试验猪免疫后采食、饮水、粪便情况、精神状态等日常行为和体温均正常,初步证明该疫苗的安全性;免疫后12 w(即出栏时)采集每头猪的心、肝、肺、肌肉、脂肪组织并提取DNA,经PCR方法检测,所有组织中均未检测到目的基因GS/2SS,结果提示,该质粒没有整合至动物的基因组中;在每次免疫后第1、3、5、7、10、15、20、30 d采集所有试验猪的粪样、水样、猪舍前后5 m的土壤样本,用麦康凯选择培养基培养,挑选可疑菌落进行PCR检测,所有样本均未扩增出目的基因GS/2SS,表明生长抑素基因疫苗没有造成环境污染。3.免疫应答反应探究免疫方式(口腔+鼻腔、口腔、鼻腔)对育肥猪的免疫效果,研究表明,三种免疫方式均诱导机体产生免疫应答,口腔+鼻腔组和口腔组的抗体水平显著高于对照组(P0.05),且口腔+鼻腔组显著高于口腔组,提示口腔+鼻腔的免疫方式可以诱导更高水平的免疫应答;探究免疫剂量(高5×1011 CFU/头、中5×1010 CFU/头、低5×109 CFU/头)对育肥猪免疫效果的结果显示,低剂量组体现了更好的免疫效果,其抗体水平显著(P0.05)高于对照组,生长激素水平高于对照组,但差异不显著(P0.05)。
[Abstract]:Previous studies have shown that the immunization of recombinant C500 (P VGS/2SS-asd) gene vaccine can promote the growth of mice and have no adverse effects on small animals. In order to further understand the immune effect, mechanism and safety of the vaccine to large animals, this study chose 78 80 days old Duchang big pig, randomly divided into 6 groups, using the same dose (5 x 1011CFU). Three kinds of immune methods (oral cavity + nasal cavity, oral cavity, nasal cavity), three kinds of doses (high 5 * 1011 CFU/ head, 5 x 1010 CFU/ head and low 5 * 109 CFU/ head) for the same immune system (5 x 1010 CFU/ head, 5 * 109 CFU/ head) for clinical trial, and the test period 90 d to explore the optimum immunization and immunization dose. The performance WINFIRE measurement system automatically records the feed consumption of experimental pigs every day, determines the weight of pigs and the feed reward to evaluate the immune effect of the gene vaccine, and evaluates the safety of the vaccine from the aspects of gene integration and environmental spread from the daily behavior and body temperature of the pig, and the application of the target gene amplification to evaluate the safety of the vaccine. The ELISA method is applied. Determine the level of antibody and hormone to explore its mechanism of action. The results are as follows: 1. the effect of growth promoting and feed reward is used to record the data of pig feed intake and weight automatically every day by the WINFIRE system of the production performance of the sows produced by the American Austrian forage company. The growth rate of the pre immunization fattening pig is faster and the later weight gain slows down, and the immunization method is explored. The results showed that the growth promoting effect of the fattening pig was higher than that of the control group. The daily weight gain of the group was 4-7 w after immunization, and the daily weight gain of the nasal spray group was significantly higher than that of the control group (P0.05). The results showed that the effect of the immunization dose on the growth of the fattening pigs was 7 W after the initial immunization, and the daily weight gain of the medium dose and low dose group increased by 19.94%. The average daily gain of each immune group was higher than that of the control group, while the average daily gain of each immune group was higher than the control group, while the daily gain of the low dose group was 9.41% higher than that of the control group (P0.05). The daily intake of the immunization group and the immune dose group increased, but there was no significant difference between the immunization group and the control group, but there was no significant difference between the immunization group and the P0.01 group. Compared with the control group (P0.05), the meat meat ratio in the low dose group was lower than that of the control group (P0.05).2. safety study. This study evaluated the safety of the recombinant bacteria C500 (P VGS/2SS-asd) from three aspects of the daily behavior, gene integration and environmental pollution of the immune pigs. The results showed that all the test pigs were fed, drinking, and feces after immunization. The daily behavior and body temperature are normal, and the safety of the vaccine is preliminarily proved. 12 W after immunization (when out of column) collect the heart, liver, lung, muscle, fat tissue and DNA of each pig. The gene GS/2SS is not detected in all tissues by PCR method. The results suggest that the plasmid is not integrated into the gene of the animal. In the group, the feces samples of all test pigs, water samples, 5 m soil samples before and after the pig house were collected after each immunization, and the culture medium was selected by Kang Kai selection medium before and after the pig house, and the suspicious colonies were selected for PCR detection. All the samples did not amplify the target gene GS/2SS, indicating that the vaccine did not cause environmental pollution.3. immune response. In answer to the response, the immunological effects of immunization (oral cavity, nasal cavity, mouth, nasal cavity) on fattening pigs were investigated. The results showed that the immune responses were induced by the three immune modes. The antibody level in oral + nasal group and oral group was significantly higher than that of the control group (P0.05), and the oral + nasal cavity group was significantly higher than that in the oral group, suggesting that the oral + nasal cavity was immune to the oral cavity. In order to induce a higher level of immune response, the immune effect of immunization dose (5 x 1011 CFU/ head, 5 x 1010 CFU/ head and low 5 x 109 CFU/ head) showed that the low dose group showed better immune effect, its antibody level was significantly higher than that of the control group, and the level of growth hormone was higher than that of the control group, but the difference was not significant (P0.05).
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S828

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