【作者】 刘兴斌；

【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， 2011， 博士

【摘要】 本研究应用基因免疫、酶联免疫测定、活体成像和石蜡切片等技术,将新型生长抑素DNA疫苗——减毒鼠伤寒沙门氏菌株CS022(pGM-CSF/SS)口服和鼻腔免疫仔猪,探讨其免疫效力及影响因素；另将减毒猪霍乱沙门氏菌株C500(pGS/2SS-M4GFP-asd)肌肉注射小鼠,探讨外源目的基因在组织器官中表达与分布的时空变化规律,为优化新型DNA疫苗的免疫程序,提高其对仔猪的促生长效果,加快其临床应用奠定基础。1.仔猪口服新型生长抑素DNA疫苗的免疫应答及其促生长效果为了探索疫苗能否诱导仔猪产生免疫应答反应及其促生长效果,将40只9周龄的杜-长-大三元杂交猪随机分为4组,每组10只,公母各半。第1-3组(T1-T3)经口灌服低(5×108CFU/只)、中(5×109CFU/只)、高(5×1010CFU/只)三个剂量的减毒鼠伤寒沙门氏菌株CS022(pGM-CSF/SS)生长抑素DNA疫苗,第4组灌服5ml PBS溶液,用做对照(C1)。1W后以相同方式和剂量加强免疫1次。分别在免疫前、初免后4W、初免后8W称重3次；于免疫前及免疫后5W2次前腔静脉采集血样,用间接ELISA法检测血浆IgG抗体水平,用放射免疫法检测血浆中的SS浓度。结果显示,免疫后仔猪行为正常,受试猪血浆中均能检测到SS抗体,以T3组抗体水平最高；与C1组相比,T1组(P>0.05)差异不显著,T2组(p<0.01)差异极显著,T3组(p<0.01)差异极显著。T3组与T1组相比(p<0.01),差异极显著。试验各组出现的免疫阳性猪比例,其中T1、T2组均为10.0%,T3组为40.0%。试验各组血浆SS浓度与免疫前和C1组相比均大大降低(P<0.01),差异极显著,但组间差异不显著。免疫后0-4W,仔猪日增重T3组比C1组提高20.69%(P<0.05),差异显著,比T1组提高27.59%(P<0.01),差异极显著,比T2提高13.79%(P>0.05),差异不显著；T2组分别比C1组、T1组提高6.90%、13.80%(P>0.05),差异不显著。免疫后5-8W,仔猪日增重T1组比C1组提高11.11%(P>0.05),T2组和T3组比C1组均提高2.47%(P>0.05),差异不显著。免疫后0～8W,仔猪平均日增T3组比C1、T2、T1组分别提高10.00%、5.71%、7.14%(P>0.05),差异不显著。以上结果表明,新型生长抑素DNA疫苗可诱导仔猪产生高水平免疫应答反应,有效促进了仔猪生长；免疫后仔猪SS抗体水平、抗体阳性猪所占比率和平均日增重均与免疫剂量存在正相关关系。2.仔猪鼻腔免疫新型生长抑素DNA疫苗的促生长效果为了探索鼻腔免疫新型生长抑素DNA疫苗对仔猪的促生长效果,将75只遗传背景、年龄一致,体重21.0±2.0Kg杜长大三元杂交阉公猪分成4组,前3组(T4-T6)每组20只,鼻腔免疫3次,每次间隔4W,剂量分别为低(2×108CFU/只)、中(2×109CFU/只)、高(2×1010CFU/只),第4组(C2)15只,鼻腔给药PBS溶液,剂量为2ml,用做对照。分别在免疫前、免疫后12W称量体重。结果显示,3个免疫组(T4-T6)平均日增重较对照组(C2)分别提高11.11%、6.17%、1.23%(P>0.05),差异不显著。随着免疫剂量的升高,平均日增重呈下降趋势,表明鼻腔免疫效果与剂量之间存在负相关关系。在日增重提升幅度一致情况下,鼻腔免疫需要的疫苗剂量远远少于口服免疫。3.新型生长抑素DNA疫苗肌肉注射小鼠后目的基因表达与蛋白分布的时空变化规律为了探索肌肉注射新型生长抑素DNA疫苗小鼠器官组织SS融合蛋白的时空变化规律,将78只7周龄昆明小鼠随机分为13组,每组6只,雌雄各半,采用肌肉纵向两点注射法在小鼠2个后肢股四头肌接种,剂量为150μL×109CFU/只。6个免疫组(A1～A6)免疫减毒猪霍乱沙门氏菌C500(pGS/2SS-M4GFP-asd)生长抑素DNA疫苗,6个阴性对照组(B1-B6)免疫不含质粒的C500空菌疫苗,1个组注射150μLPBS溶液,用于空白对照(C3)。分别于免疫后24h、48h、96h、144h、192h、240h6个时间点依次用乙醚熏晕处死A1～A6和B1-B6组的6只小鼠,C4组小鼠于注射24h处死,采集心脏、肝脏、脾脏、肺脏、肾脏和后腿肌肉样品制作石蜡切片,并用免疫组化法染色切片。结果显示,仅在注射处肌肉、脾脏、肝脏组织中的巨噬细胞依次表达SS蛋白,并在144h到达峰值。肌肉组织最先(24h)表达,持续时间最长,240h仍有大量SS蛋白；脾脏次之,免疫48h开始表达,持续时间居中,240h只有少量SS蛋白；肝脏最晚(144h)表达,持续时间最短,240h已观察不到SS蛋白。结果表明肌肉注射免疫是可行的,外源DNA质粒在动物体内存留和目的蛋白持续表达的时间短暂,在家畜生产中可以应用肌肉注射方式免疫生长抑素DNA疫苗。更多还原

【Abstract】 In this study, the immune efficacy and influence factors of a novel somatostatin DNA vaccine-Attenuated Mouse Typhus Salmonella strains CS022(pGM-CSF/SS) immunized against piglets with oral administration and nasal feeding were investigated, using methods of genetic immunization, ELISA, and paraffin section. Mice were administered attenuated Cholera Swine Salmonella strains C500(pGS/2SS-M4GFP-asd) through intramuscular injection to detect the temporal and spatial patterns of the exogenous gene expression in various tissues and organs, which will contribute to immunization program optimization of novel growth-promoting DNA vaccine, elevation of their growth-promoting effects on the piglets and to accelerate its clinical application.1. Immune responses and Growth-promoting effect of piglets to the novelsomatostatin DNA vaccine by oral vaccination. So as to study the immune responses and the growth-promoting effect of the novel somatostatin DNA vaccine by oral way. a total of40three-ways crossing commercial piglets at9weeks of age were randomly divided into4groups, and each group was evenly composed of5males and5females. T1, T2and T3groups were administered with attenuated Salmonella strain CS022(pGM-CSF/SS) somatostatin DNA vaccine orally with low doses (5×108CFU/each), moderate doses (5×109CFU/each), and high doses (5×1010CFU/each) respectively. Control group C1was administered with5ml PBS solution. A booster was performed with the same doses and same treatment at1week after primary immunization. Body weight gains of the piglets were measured three times (before immunization, after immunization4and8weeks). The blood samples were collected two times at former vena cava (before immunization and after immunization5weeks). IgG antibody levels in plasma was detected using indirect ELISA and SS concentration in plasma was detected using radioimmunoassay.The results showed that the piglets had normal behavior after vaccination. Plasma SS antibodies were detected in all experimental groups, T3group had the highest level. Compared with C2group, T2group did not have significant difference (P>0.05) but both T2and T3had very significant difference (P<0.01). The immunoreactive pigs occurred in each experimental group, and the ratio of T1and T2group were10.0%and T3group was40.0%. Compared with those of pre-immunization and the Cl group, plasma SS concentration declined significantly for each group (p<0.01), but there was no significant difference among experimental groups (P>0.05).T3group pigs average daily gain increased by20.69%compared with Cl group pigs (P<0.05), it had significant difference, and increasing by27.59%compared with T1group pigs (P<0.01) which it had significantly difference after immunization0-4weeks, but it increased only by13.79%compared with T2that had no significant difference. There was no significant difference (P>0.05) among all groups though T1group pigs average daily gain increased by11.11%compared with C1control group after immunization5-8weeks, and also T3group pigs average daily gain increased only by10.00%compared with C1control group after immunization0-8weeks.These results demonstrated that the vaccine could induce the immune response in piglets and the immune effects were significant. There was positive correlation-dependent among antibody levels and the ratio of antibody postive piglets and average daily gain and dose amount.2. Growth-promoting effect of novel somatostatin DNA vaccine by nasal injectionSo as to study the growth-promoting effect of the novel somatostatin DNA vaccine by nasal injection, a total of75three-ways crossing commercial castrated male piglets with the same genetic background and weight21.0±2.0Kg, were divided into four groups. There were20pigs for each group of T4, T5and T6, and15for C2group. The pigs were administered vaccine3times at intervals of4weeks through nasal injection, with low dose (2×108CFU/each), moderate dose (2×109CFU/each) and high doses (2×1010CFU/each) respectively. C2group was treated with PBS solution with a dose of2ml. Body weight gains of the piglets were measured twice (before immunization and after immunization12weeks). The results showed that T4(low dose) group pigs average daily gain increased by11.11%compared with C2control group after immunization, T5(middle dose) and T6(high dose) group increased by6.17%and1.23%respectively. There was no significant difference (P>0.05) between groups. Average daily gain declined with the increasing dose of vaccine, which implied that immune efficacy had negative correlation with vaccine dose. Nasal immunization needed vaccine doses far less than oral immunization for the same daily gain. 3. The temporal and spatial patterns of the target gene and the SS fusion protein of immunized mice through intramuscular injection with novel somatostatin DNA vaccineSo as to study the temporal and spatial patterns of the target gene and the SS fusion protein of immunized mice through intramuscular injection with novel somatostatin DNA vaccine, a total of78Kunming mice at7weeks old were randomly divided into13groups, and each group was evenly composed of3males and3females. Longitudinal muscle injection with dose of150μL×109CFU/each at two hind quadriceps in mice was implemented. The immunized groups A1-A6were inoculated attenuated Salmonella choleraesuis C500(pGS/2SS-M4GFP-asd) somatostatin DNA vaccine, and negative control group B1-B6were inoculated with plasmid-free C500bacteria vaccine, and the control group C3inoculated with150μL PBS saline. Six mice of each group were killed after Ethyl ether smoked dizzy in turn with ether A1-A6and B1-B6at immunization24h,48h,96h,144h,192h and240h, and C3group mice was killed by the same way at immunization24h. Paraffin sections were made after collecting their heart, liver, spleen, lung, kidney and hindlimb skeletal muscle, and to deal with by Immunohistochemical staining method.The results showed that SS fusion protein had been expressed in turn with at macrophage of spleen, liver, muscle with injection site, and its expression reaches the maximum all of them at immunization144h. It occurred firstly at muscle tissue (immunization24h), the second at spleen (immunization48h), the last at liver (immunization144h). Muscle tissue had sustained more time than spleen and liver, and had more quantity until immunization240h. Liver had the shortest time of continuous expression, and there was no SS fusion protein at immunization240h.It demonstrated that it was viable to immune livestock the way of intramuscular injection with novel somatostatin DNA vaccine. It was short time that the exogenous plasmid DNA and purpose protein had continuous expression in mice by intramuscular injection.更多还原