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  本文關鍵詞：CpG寡核苷酸和霍亂毒素聯(lián)合STAg滴鼻免疫小鼠誘導的弓形蟲特異性免疫應答　出處：《山西醫(yī)科大學》2006年碩士論文　論文類型：學位論文

  更多相關文章： STAg CpG ODN CT 剛地弓形蟲 黏膜免疫 黏膜佐劑 IgG抗體 IgA抗體

【摘要】： 目的確定CpG寡核苷酸(CpG oligonucleotide,CpG ODN)作為可溶性速殖子抗原(soluble tachyzoite antigen,STAg)的最佳劑量,觀察最佳劑量的CpG ODN和STAg鼻內免疫小鼠所誘導的免疫應答的持續(xù)時間,以及CpG ODN和霍亂毒素(cholera toxin,CT)聯(lián)合佐劑的協(xié)同作用,弓形蟲復合黏膜疫苗研制提供理論和實驗依據(jù)。 方法第一部分:5～6周齡BALB/c小鼠50只隨機分為5組,每組10只,實驗組以0、1、5、10μg的CpG ODN聯(lián)合20μg STAg滴鼻免疫小鼠,對照組以PBS滴鼻,間隔2周免疫2次。末次免疫后30d處死全部小鼠,觀察脾、Peyer’s patch(PP)、腸系膜淋巴結(MLN)和IEL淋巴細胞增殖情況。檢測血清IgG和糞便IgA含量。 第二部分:雌性BALB/c小鼠72只隨機分為實驗組和對照組,每組36只。實驗組以10μg CpG ODN聯(lián)合20μg STAg滴鼻免疫,對照組以PBS滴鼻。間隔2周免疫2次,于末次免疫后第1、2、3、4、5、6周分別隨機處死小鼠(6只/組)。測定小腸沖洗液、鼻咽沖洗液、肺沖洗液、陰道沖洗液IgA和血清IgG。分離脾和IEL進行淋巴細胞計數(shù)。 第三部分:5～6周齡BALB/c小鼠30只,雌雄各半,隨機分為4個實驗組和PBS對照組。實驗組分別用20μg STAg、20μg STAg+10μg CpG ODN、20μg STAg+ 1μgCT、20μg STAg+ 1μgCT +10μg CpG ODN滴鼻免疫,間隔2周免疫2次。對照組用PBS以相同程序免疫。末次免疫后第20d處死小鼠。收集血清和腸沖洗液,用ELISA法檢測血清弓形蟲特異性IgG和腸液IgA含量。分離脾淋巴細胞,用MTT法檢測淋巴細胞增殖活性。 結果不同劑量的CpG ODN聯(lián)合STAg滴鼻免疫小鼠后,10μg CpG ODN組小鼠腸相關淋巴組織PP、MLN和IEL以及脾淋巴細胞增殖明顯(P0.05)。10μg組血清IgG遠高于對照組(P0.05),糞便IgA高于對照組(P0.05)。 10μg CpG ODN聯(lián)合STAg滴鼻免疫小鼠后脾淋巴細胞和IEL增生明顯。脾淋巴細胞在第2、3、4、5周顯著高于對照組(P 0.001)。IEL數(shù)量分別在第2、4周出現(xiàn)兩個峰值,第2、3、4周顯著高于對照組(P 0.001)。實驗組小鼠各沖洗液中IgA平均水平高于對照組。小腸沖洗液中弓形蟲特異性IgA抗體水平在各個時間點均高于對照組。與對照組相比,第2、3周(P 0.001),4周(P 0.05)有顯著差異。鼻咽沖洗液中IgA水平在第1周時最高,各時間點雖高于對照組,
[Abstract]:Objective to determine the CpG oligonucleotide CpG oligonucleotide. CpG ODN was the best dosage of soluble tachyzoite antigen STAG. The duration of immune response induced by intranasal immunization with CpG ODN and STAg, and the duration of CpG ODN and cholera toxin were observed. CT) combined with adjuvant can provide theoretical and experimental basis for the development of Toxoplasma gondii complex mucosal vaccine. Methods in the first part, 50 BALB/c mice aged 6 weeks were randomly divided into 5 groups with 10 mice in each group. Mice were immunized with 10 渭 g CpG ODN combined with 20 渭 g STAg. The control group was immunized with PBS twice every 2 weeks. All the mice were killed 30 days after the last immunization and the spleen was observed. The proliferation of Peyer's, mesenteric lymph node (MLN) and IEL lymphocytes were detected. The levels of serum IgG and stool IgA were measured. The second part: 72 female BALB/c mice were randomly divided into experimental group (n = 36) and control group (n = 36). The experimental group was immunized with 10 渭 g CpG ODN and 20 渭 g STAg. The control group was immunized with PBS nasal drip twice every 2 weeks. The mice were killed at random for 6 weeks at the first week after the last immunization. The small intestine flushing fluid and nasopharyngeal lavage fluid were measured. Lung lavage fluid, vaginal lavage fluid IgA and serum IgG. spleen and IEL were separated for lymphocyte count. Part three: 30 BALB/c mice, half male and half female, were randomly divided into 4 experimental groups and PBS control groups. The experimental groups were given 20 渭 g STAg respectively. 20 渭 g STAg, 10 渭 g CpG, 20 渭 g STAg, 1 渭 gCT. 20 渭 g STAg 1 渭 gCT 10 渭 g CpG ODN were intranasal immunized. The control group was immunized with PBS in the same procedure. The mice were killed on the 20th day after the last immunization. Serum and intestinal irrigation fluid were collected. Serum Toxoplasma gondii specific IgG and intestinal fluid IgA were detected by ELISA method, spleen lymphocytes were isolated and lymphocyte proliferative activity was detected by MTT method. Results 10 渭 g CpG ODN mice were immunized with different doses of CpG ODN combined with STAg nasal drip. The levels of serum IgG in MLN and IEL and splenic lymphocyte proliferation were significantly higher in P0.05 渭 g group than in control group (P0.05 渭 g), and in fecal IgA was higher than that in control group (P0.05 渭 g). The splenic lymphocytes and IEL proliferation of mice immunized with 10 渭 g CpG ODN combined with STAg nasal drip were obvious. At 5 weeks, the number of IEL was significantly higher than that of the control group (P 0.001). The number of IEL had two peaks and two peaks at the 4th week and the third peak value at the 2nd week, respectively. At 4 weeks, it was significantly higher than that in control group (P 0.001). The average level of IgA in each flushing fluid of experimental group was higher than that of control group, and the level of Toxoplasma gondii specific IgA antibody in small intestinal lavage fluid was higher than that in control group at all time points. The level of IgA in nasopharyngeal lavage fluid was the highest at the first week, although it was higher than that in the control group at each time point.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R392

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本文編號：1422527