新型生长抑素DNA疫苗免疫猪的效果及其影响因素和作用机制研究
发布时间：2018-09-11 08:34
【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(P0.05)差异不显著,T2组(p0.01)差异极显著,T3组(p0.01)差异极显著。T3组与T1组相比(p0.01),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(P0.01),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%(P0.05),差异显著,比T1组提高27.59%(P0.01),差异极显著,比T2提高13.79%(P0.05),差异不显著；T2组分别比C1组、T1组提高6.90%、13.80%(P0.05),差异不显著。免疫后5-8W,仔猪日增重T1组比C1组提高11.11%(P0.05),T2组和T3组比C1组均提高2.47%(P0.05),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%(P0.05),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%(P0.05),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。
[Abstract]:In this study, gene immunization, enzyme-linked immunosorbent assay (in vivo), live imaging and paraffin sections were used to investigate the immune efficacy and influence factors of the new somatostatin DNA Vaccine * attenuated Salmonella typhimurium * CS022 (pGM-CSF/SS) orally and in the nasal cavity. The attenuated Salmonella cholerae C500 (pGS/2SS-M4GFP-asd) muscle was also attenuated. To study the temporal and spatial variation of expression and distribution of foreign target genes in tissues and organs of mice after injection, and to optimize the immune program of new DNA vaccine, improve its growth promoting effect on piglets, and accelerate its clinical application. 1. Immune response and growth promoting effect of new oral somatostatin DNA vaccine on piglets.
In order to explore whether vaccine can induce immune response and * promote growth of piglets, 40 9 week old Duchang * large three hybrid pigs were randomly divided into 4 groups, 10 in each group, 10 in each group. Group 1-3 (T1-T3) was administered by oral administration of low (5 x 108CFU/), medium (5 * 109CFU/), and high (5 x 1010CFU/) three doses of attenuated Salmonella typhimurium C. S022 (pGM-CSF/SS) somatostatin DNA vaccine was administered by 5 ml PBS solution in group 4. The immunization was strengthened once in the same manner and dosage as control (C1). The concentration of SS in plasma was detected by the method.
The results showed that the * * pigs were normal after immunization, and the SS antibody was detected in the plasma of the tested pigs. The antibody level in group T3 was the highest. Compared with the C1 group, there was no significant difference in the T1 group (P0.05), T2 group (P0.01) had a very significant difference, T3 group (P0.01) had a very significant difference (*) compared with the T1 group (P0.01), the difference was very significant. The concentration of SS in plasma of each group was significantly lower than that before immunization (P 0.01), but there was no significant difference between the two groups.
The daily gain of piglets in group T3 was 20.69% (P 0.05) higher than that in group C1 (P 0.05), 27.59% (P 0.01) higher than that in group T1 (P 0.05), and 13.79% (P 0.05) higher than that in group T2 (P 0.05), the difference was insignificant. The daily gain of piglets in group T2 was 6.90% (P 0.05) and 13.80% (P 0.05) higher than that in group C1 (P 0.05), respectively. T3 group was 2.47% (P 0.05) higher than C1 group, the difference was not significant. After immunization, the average daily increase of T3 group was 10.00%, 5.71%, 7.14% (P 0.05) higher than C1, T2 and T1 group, respectively.
The above results showed that the new somatostatin DNA vaccine could induce the * * * pigs to produce a high level of immune response, which effectively promoted the growth of piglets. The SS antibody level, the proportion of antibody positive pigs and the average daily gain were positively correlated with the immunization dose.
2. * growth promoting effect of new somatostatin DNA vaccine on piglets nasal cavity immunity
In order to explore the effect of nasal immunization with a new type of somatostatin DNA vaccine on the growth of piglets, 75 three-way cross-bred male piglets with the same genetic background, age and body weight of 21.0 (+ 2.0 kg) were divided into four groups. The first three groups (T4-T6) were 20 piglets in each group, immunized three times in nasal cavity at intervals of 4 W. The doses were 2 (+ 108 CFU / piglet) and 2 (+ 109 CFU / piglet) respectively. The results showed that the average daily weight gain of the three immunization groups (T4-T6) was 11.11%, 6.17%, and 1.23% higher than that of the control group (C2), respectively. There was a negative correlation between the nasal immune response and the dosage. In the case of the same daily gain, the dosage of vaccine needed for nasal immunization was much less than that for oral immunization.
3. Temporal and spatial variation of target gene expression and protein distribution in mice after intramuscular injection of a novel somatostatin DNA vaccine
In order to explore the temporal and spatial variation of SS fusion protein in organs and tissues of mice inoculated with a new type of somatostatin DNA vaccine, 78 7-week-old Kunming mice were randomly divided into 13 groups, 6 mice in each group, half male and half female. Two quadriceps femoris of hind limbs were inoculated with longitudinal two-point injection of somatostatin DNA vaccine in mice, and the dosage was 150 mu L *109 CFU/mouse. Six immune groups (A1-A6) were immunized. * attenuated Salmonella cholerae C500 (pGS/2SS-M4GFP-asd) somatostatin DNA vaccine, 6 negative control group (B1-B6) immunized with no plasmid C500 empty vaccine, and 1 groups were injected with 150 LPBS solution for blank control (C3). After immunization, 24h, 48h, 96h, 144H, 48h, and time points were used to kill 6 by 6. Mice in the C4 group were sacrificed 24 hours after injection. Samples of heart, liver, spleen, lung, kidney and hind leg muscles were collected to make paraffin sections and stained with immunohistochemistry.
The results showed that SS protein was expressed by macrophages in muscle, spleen and liver at the injection site, and reached its peak at 144 H. The expression of SS protein in muscle tissue was first (24 h) and lasted most for 240 H. The expression of SS protein in spleen began at 48 h and lasted only a small amount at 240 H. The expression of SS protein in liver was the latest (144 h). The results showed that intramuscular immunization was feasible. Exogenous DNA plasmids retained in animals and persisted in the expression of target proteins for a short time, and somatostatin DNA vaccines could be immunized by intramuscular injection in livestock production.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
本文编号：2236168
[Abstract]:In this study, gene immunization, enzyme-linked immunosorbent assay (in vivo), live imaging and paraffin sections were used to investigate the immune efficacy and influence factors of the new somatostatin DNA Vaccine * attenuated Salmonella typhimurium * CS022 (pGM-CSF/SS) orally and in the nasal cavity. The attenuated Salmonella cholerae C500 (pGS/2SS-M4GFP-asd) muscle was also attenuated. To study the temporal and spatial variation of expression and distribution of foreign target genes in tissues and organs of mice after injection, and to optimize the immune program of new DNA vaccine, improve its growth promoting effect on piglets, and accelerate its clinical application. 1. Immune response and growth promoting effect of new oral somatostatin DNA vaccine on piglets.
In order to explore whether vaccine can induce immune response and * promote growth of piglets, 40 9 week old Duchang * large three hybrid pigs were randomly divided into 4 groups, 10 in each group, 10 in each group. Group 1-3 (T1-T3) was administered by oral administration of low (5 x 108CFU/), medium (5 * 109CFU/), and high (5 x 1010CFU/) three doses of attenuated Salmonella typhimurium C. S022 (pGM-CSF/SS) somatostatin DNA vaccine was administered by 5 ml PBS solution in group 4. The immunization was strengthened once in the same manner and dosage as control (C1). The concentration of SS in plasma was detected by the method.
The results showed that the * * pigs were normal after immunization, and the SS antibody was detected in the plasma of the tested pigs. The antibody level in group T3 was the highest. Compared with the C1 group, there was no significant difference in the T1 group (P0.05), T2 group (P0.01) had a very significant difference, T3 group (P0.01) had a very significant difference (*) compared with the T1 group (P0.01), the difference was very significant. The concentration of SS in plasma of each group was significantly lower than that before immunization (P 0.01), but there was no significant difference between the two groups.
The daily gain of piglets in group T3 was 20.69% (P 0.05) higher than that in group C1 (P 0.05), 27.59% (P 0.01) higher than that in group T1 (P 0.05), and 13.79% (P 0.05) higher than that in group T2 (P 0.05), the difference was insignificant. The daily gain of piglets in group T2 was 6.90% (P 0.05) and 13.80% (P 0.05) higher than that in group C1 (P 0.05), respectively. T3 group was 2.47% (P 0.05) higher than C1 group, the difference was not significant. After immunization, the average daily increase of T3 group was 10.00%, 5.71%, 7.14% (P 0.05) higher than C1, T2 and T1 group, respectively.
The above results showed that the new somatostatin DNA vaccine could induce the * * * pigs to produce a high level of immune response, which effectively promoted the growth of piglets. The SS antibody level, the proportion of antibody positive pigs and the average daily gain were positively correlated with the immunization dose.
2. * growth promoting effect of new somatostatin DNA vaccine on piglets nasal cavity immunity
In order to explore the effect of nasal immunization with a new type of somatostatin DNA vaccine on the growth of piglets, 75 three-way cross-bred male piglets with the same genetic background, age and body weight of 21.0 (+ 2.0 kg) were divided into four groups. The first three groups (T4-T6) were 20 piglets in each group, immunized three times in nasal cavity at intervals of 4 W. The doses were 2 (+ 108 CFU / piglet) and 2 (+ 109 CFU / piglet) respectively. The results showed that the average daily weight gain of the three immunization groups (T4-T6) was 11.11%, 6.17%, and 1.23% higher than that of the control group (C2), respectively. There was a negative correlation between the nasal immune response and the dosage. In the case of the same daily gain, the dosage of vaccine needed for nasal immunization was much less than that for oral immunization.
3. Temporal and spatial variation of target gene expression and protein distribution in mice after intramuscular injection of a novel somatostatin DNA vaccine
In order to explore the temporal and spatial variation of SS fusion protein in organs and tissues of mice inoculated with a new type of somatostatin DNA vaccine, 78 7-week-old Kunming mice were randomly divided into 13 groups, 6 mice in each group, half male and half female. Two quadriceps femoris of hind limbs were inoculated with longitudinal two-point injection of somatostatin DNA vaccine in mice, and the dosage was 150 mu L *109 CFU/mouse. Six immune groups (A1-A6) were immunized. * attenuated Salmonella cholerae C500 (pGS/2SS-M4GFP-asd) somatostatin DNA vaccine, 6 negative control group (B1-B6) immunized with no plasmid C500 empty vaccine, and 1 groups were injected with 150 LPBS solution for blank control (C3). After immunization, 24h, 48h, 96h, 144H, 48h, and time points were used to kill 6 by 6. Mice in the C4 group were sacrificed 24 hours after injection. Samples of heart, liver, spleen, lung, kidney and hind leg muscles were collected to make paraffin sections and stained with immunohistochemistry.
The results showed that SS protein was expressed by macrophages in muscle, spleen and liver at the injection site, and reached its peak at 144 H. The expression of SS protein in muscle tissue was first (24 h) and lasted most for 240 H. The expression of SS protein in spleen began at 48 h and lasted only a small amount at 240 H. The expression of SS protein in liver was the latest (144 h). The results showed that intramuscular immunization was feasible. Exogenous DNA plasmids retained in animals and persisted in the expression of target proteins for a short time, and somatostatin DNA vaccines could be immunized by intramuscular injection in livestock production.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392
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