新型生長抑素DNA疫苗免疫豬的效果及其影響因素和作用機制研究
發(fā)布時間：2018-09-11 08:34
【摘要】：本研究應(yīng)用基因免疫、酶聯(lián)免疫測定、活體成像和石蠟切片等技術(shù),將新型生長抑素DNA疫苗——減毒鼠傷寒沙門氏菌株CS022(pGM-CSF/SS)口服和鼻腔免疫仔豬,探討其免疫效力及影響因素；另將減毒豬霍亂沙門氏菌株C500(pGS/2SS-M4GFP-asd)肌肉注射小鼠,探討外源目的基因在組織器官中表達與分布的時空變化規(guī)律,為優(yōu)化新型DNA疫苗的免疫程序,提高其對仔豬的促生長效果,加快其臨床應(yīng)用奠定基礎(chǔ)。1.仔豬口服新型生長抑素DNA疫苗的免疫應(yīng)答及其促生長效果 為了探索疫苗能否誘導(dǎo)仔豬產(chǎn)生免疫應(yīng)答反應(yīng)及其促生長效果,將40只9周齡的杜-長-大三元雜交豬隨機分為4組,每組10只,公母各半。第1-3組(T1-T3)經(jīng)口灌服低(5×108CFU/只)、中(5×109CFU/只)、高(5×1010CFU/只)三個劑量的減毒鼠傷寒沙門氏菌株CS022(pGM-CSF/SS)生長抑素DNA疫苗,第4組灌服5ml PBS溶液,用做對照(C1)。1W后以相同方式和劑量加強免疫1次。分別在免疫前、初免后4W、初免后8W稱重3次；于免疫前及免疫后5W2次前腔靜脈采集血樣,用間接ELISA法檢測血漿IgG抗體水平,用放射免疫法檢測血漿中的SS濃度。 結(jié)果顯示,免疫后仔豬行為正常,受試豬血漿中均能檢測到SS抗體,以T3組抗體水平最高；與C1組相比,T1組(P0.05)差異不顯著,T2組(p0.01)差異極顯著,T3組(p0.01)差異極顯著。T3組與T1組相比(p0.01),差異極顯著。試驗各組出現(xiàn)的免疫陽性豬比例,其中T1、T2組均為10.0%,T3組為40.0%。試驗各組血漿SS濃度與免疫前和C1組相比均大大降低(P0.01),差異極顯著,但組間差異不顯著。 免疫后0-4W,仔豬日增重T3組比C1組提高20.69%(P0.05),差異顯著,比T1組提高27.59%(P0.01),差異極顯著,比T2提高13.79%(P0.05),差異不顯著；T2組分別比C1組、T1組提高6.90%、13.80%(P0.05),差異不顯著。免疫后5-8W,仔豬日增重T1組比C1組提高11.11%(P0.05),T2組和T3組比C1組均提高2.47%(P0.05),差異不顯著。免疫后0～8W,仔豬平均日增T3組比C1、T2、T1組分別提高10.00%、5.71%、7.14%(P0.05),差異不顯著。 以上結(jié)果表明,新型生長抑素DNA疫苗可誘導(dǎo)仔豬產(chǎn)生高水平免疫應(yīng)答反應(yīng),有效促進了仔豬生長；免疫后仔豬SS抗體水平、抗體陽性豬所占比率和平均日增重均與免疫劑量存在正相關(guān)關(guān)系。 2.仔豬鼻腔免疫新型生長抑素DNA疫苗的促生長效果 為了探索鼻腔免疫新型生長抑素DNA疫苗對仔豬的促生長效果,將75只遺傳背景、年齡一致,體重21.0±2.0Kg杜長大三元雜交閹公豬分成4組,前3組(T4-T6)每組20只,鼻腔免疫3次,每次間隔4W,劑量分別為低(2×108CFU/只)、中(2×109CFU/只)、高(2×1010CFU/只),第4組(C2)15只,鼻腔給藥PBS溶液,劑量為2ml,用做對照。分別在免疫前、免疫后12W稱量體重。結(jié)果顯示,3個免疫組(T4-T6)平均日增重較對照組(C2)分別提高11.11%、6.17%、1.23%(P0.05),差異不顯著。隨著免疫劑量的升高,平均日增重呈下降趨勢,表明鼻腔免疫效果與劑量之間存在負相關(guān)關(guān)系。在日增重提升幅度一致情況下,鼻腔免疫需要的疫苗劑量遠遠少于口服免疫。 3.新型生長抑素DNA疫苗肌肉注射小鼠后目的基因表達與蛋白分布的時空變化規(guī)律 為了探索肌肉注射新型生長抑素DNA疫苗小鼠器官組織SS融合蛋白的時空變化規(guī)律,將78只7周齡昆明小鼠隨機分為13組,每組6只,雌雄各半,采用肌肉縱向兩點注射法在小鼠2個后肢股四頭肌接種,劑量為150μL×109CFU/只。6個免疫組(A1～A6)免疫減毒豬霍亂沙門氏菌C500(pGS/2SS-M4GFP-asd)生長抑素DNA疫苗,6個陰性對照組(B1-B6)免疫不含質(zhì)粒的C500空菌疫苗,1個組注射150μLPBS溶液,用于空白對照(C3)。分別于免疫后24h、48h、96h、144h、192h、240h6個時間點依次用乙醚熏暈處死A1～A6和B1-B6組的6只小鼠,C4組小鼠于注射24h處死,采集心臟、肝臟、脾臟、肺臟、腎臟和后腿肌肉樣品制作石蠟切片,并用免疫組化法染色切片。 結(jié)果顯示,僅在注射處肌肉、脾臟、肝臟組織中的巨噬細胞依次表達SS蛋白,并在144h到達峰值。肌肉組織最先(24h)表達,持續(xù)時間最長,240h仍有大量SS蛋白；脾臟次之,免疫48h開始表達,持續(xù)時間居中,240h只有少量SS蛋白；肝臟最晚(144h)表達,持續(xù)時間最短,240h已觀察不到SS蛋白。結(jié)果表明肌肉注射免疫是可行的,外源DNA質(zhì)粒在動物體內(nèi)存留和目的蛋白持續(xù)表達的時間短暫,在家畜生產(chǎn)中可以應(yīng)用肌肉注射方式免疫生長抑素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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392
本文編號：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392
【參考文獻】
相關(guān)期刊論文 前10條
1 孫樹漢;影響核酸疫苗應(yīng)用研究的若干問題[J];第二軍醫(yī)大學(xué)學(xué)報;2002年04期
2 郭麗宏,劉建國;DNA疫苗免疫途徑的研究現(xiàn)狀[J];國外醫(yī)學(xué).病毒學(xué)分冊;1999年01期
3 鄧小玲;DNA疫苗的作用機理以及抗腫瘤的臨床應(yīng)用進展[J];國外醫(yī)學(xué)(免疫學(xué)分冊);2005年01期
4 滕家波,蘇國富;載體—宿主平衡致死系統(tǒng)在減毒傷寒沙門氏活菌苗中的應(yīng)用[J];國外醫(yī)學(xué)(免疫學(xué)分冊);1999年03期
5 崔先利;楊利國;茆達干;;黃牛對抑制素pCISI基因免疫的反應(yīng)性[J];吉林農(nóng)業(yè)大學(xué)學(xué)報;2006年02期
6 曹少先;何曉紅;孫延鳴;劉鐵錚;楊利國;;生長抑素基因疫苗pES/2SS的構(gòu)建與表達[J];揚州大學(xué)學(xué)報(農(nóng)業(yè)與生命科學(xué)版);2007年02期
7 曹少先,楊利國,茆達干,張文偉,邢朝芳;生長抑素DNA疫苗pEGS/2SS在大鼠體內(nèi)的表達與分布[J];南京農(nóng)業(yè)大學(xué)學(xué)報;2005年02期
8 薛春林;茆達干;楊利國;王勇;;生長抑素DNA疫苗對湖羊羔羊生長和相關(guān)激素的作用——佐劑效應(yīng)[J];南京農(nóng)業(yè)大學(xué)學(xué)報;2007年02期
9 曹少先,張文偉,茆達干,管峰,楊利國;生長抑素基因疫苗質(zhì)粒pcS/2SS的構(gòu)建、表達及免疫[J];農(nóng)業(yè)生物技術(shù)學(xué)報;2005年04期
10 舒鄧群;茆達干;曹少先;楊利國;吳志敏;;粒細胞-巨噬細胞集落刺激因子(GM-CSF)與生長抑素(SS)融合表達質(zhì)粒的構(gòu)建及其對小鼠的免疫效果[J];農(nóng)業(yè)生物技術(shù)學(xué)報;2008年01期
相關(guān)博士學(xué)位論文 前2條
1 戴賢君;減毒沙門氏菌為載體的生長抑素DNA疫苗構(gòu)建、表達及對草魚生長影響研究[D];浙江大學(xué);2005年
2 張劍;奶牛產(chǎn)后生殖機能恢復(fù)與生長抑素基因免疫[D];華中農(nóng)業(yè)大學(xué);2010年
相關(guān)碩士學(xué)位論文 前4條
1 趙興華;IL-6與GM-CSF基因融合表達生長抑素基因疫苗對小鼠生長的影響[D];華中農(nóng)業(yè)大學(xué);2010年
2 程寶;基于CpG基序的生長抑素基因新疫苗的構(gòu)建與鑒定及免疫效果與作用機制的研究[D];南京農(nóng)業(yè)大學(xué);2006年
3 白莉雅;pGM-CSF/SS基因免疫對雌鼠生長和繁殖的影響及其安全性研究[D];華中農(nóng)業(yè)大學(xué);2008年
4 張麗娟;堆型艾美耳球蟲pcDNA3-3-1E的免疫效應(yīng)及其安全性研究[D];河北農(nóng)業(yè)大學(xué);2010年
，本文編號：2236168
本文鏈接：http://sikaile.net/xiyixuelunwen/2236168.html
最近更新
教材專著
