本文選題：生長抑素 + 基因疫苗��； 參考：《華中農(nóng)業(yè)大學》2015年碩士論文

【摘要】：前期研究證明,重組菌C500(p VGS/2SS-asd)基因疫苗免疫可以促進小鼠生長,并且對小動物無不良影響。為了進一步了解該疫苗對大動物的免疫效果及其機制和安全性,本研究選擇78頭80日齡的杜長大育肥豬,隨機分成6組,用同一劑量(5×1011CFU/頭)三種免疫方式(口腔+鼻腔、口腔、鼻腔)、同一免疫方式(口腔+鼻腔)三種劑量(高5×1011 CFU/頭、中5×1010 CFU/頭、低5×109 CFU/頭)進行臨床試驗,試驗期90 d,探討最適免疫方法和免疫劑量。采用美國奧飼本(OSBORNE)公司生產(chǎn)的投料器和種豬生產(chǎn)性能WINFIRE測定系統(tǒng)每天自動記錄實驗豬的飼料消耗量、測定豬體重,飼料報酬以評定基因疫苗免疫效果;從免疫后豬的日常行為及體溫是否正常、應用目的基因擴增等方法從基因整合、環(huán)境擴散等方面評價疫苗的安全性;應用ELISA方法測定抗體和激素水平,以探討其作用機制。結果如下:1.促生長效果及飼料報酬采用美國奧飼本公司生產(chǎn)的種豬生產(chǎn)性能WINFIRE測定系統(tǒng)每天全自動記錄豬采食量和體重數(shù)據(jù),免疫前期育肥豬生長速度較快,后期增重減緩;探究免疫方式對育肥豬的促生長效果,結果表明,各時期試驗組日增重均高于對照組,免疫后4-7 w,噴鼻組日增重顯著高于對照組(P0.05);探究免疫劑量對育肥豬的促生長效果,結果表明,與對照組相比,初免疫后7 w,中劑量、低劑量組日增重均提高19.94%,差異極顯著(P0.01),整個試驗階段,各免疫組的平均日增重均高于對照組,而低劑量組的日增重比對照組提高9.41%,差異顯著(P0.05)。免疫方式組、免疫劑量組的日采食量相比對照組均有所增加,但無明顯差別;不同免疫方式組間,鼻腔組的料肉比低于對照組(P0.05),不同免疫劑量組間,低劑量組的料肉比低于對照組(P0.05)。2.安全性研究本研究從免疫后豬的日常行為、基因整合情況、環(huán)境污染三個方面評估重組菌C500(p VGS/2SS-asd)的安全性,結果顯示,所有試驗豬免疫后采食、飲水、糞便情況、精神狀態(tài)等日常行為和體溫均正常,初步證明該疫苗的安全性;免疫后12 w(即出欄時)采集每頭豬的心、肝、肺、肌肉、脂肪組織并提取DNA,經(jīng)PCR方法檢測,所有組織中均未檢測到目的基因GS/2SS,結果提示,該質(zhì)粒沒有整合至動物的基因組中;在每次免疫后第1、3、5、7、10、15、20、30 d采集所有試驗豬的糞樣、水樣、豬舍前后5 m的土壤樣本,用麥康凱選擇培養(yǎng)基培養(yǎng),挑選可疑菌落進行PCR檢測,所有樣本均未擴增出目的基因GS/2SS,表明生長抑素基因疫苗沒有造成環(huán)境污染。3.免疫應答反應探究免疫方式(口腔+鼻腔、口腔、鼻腔)對育肥豬的免疫效果,研究表明,三種免疫方式均誘導機體產(chǎn)生免疫應答,口腔+鼻腔組和口腔組的抗體水平顯著高于對照組(P0.05),且口腔+鼻腔組顯著高于口腔組,提示口腔+鼻腔的免疫方式可以誘導更高水平的免疫應答;探究免疫劑量(高5×1011 CFU/頭、中5×1010 CFU/頭、低5×109 CFU/頭)對育肥豬免疫效果的結果顯示,低劑量組體現(xiàn)了更好的免疫效果,其抗體水平顯著(P0.05)高于對照組,生長激素水平高于對照組,但差異不顯著(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).
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S828

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