ESA联合STAg滴鼻免疫小鼠诱导的弓形虫特异性黏膜及系统免疫应答

发布时间：2018-11-11 08:07

【摘要】： 目的观察弓形虫排泄分泌抗原(excreted/secreted antigens, ESA)与可溶性速殖子抗原(soluble tachyzoite antigen, STAg)联合滴鼻免疫小鼠诱导的黏膜及系统免疫应答的能力,探讨其诱导免疫应答的机制,为弓形虫黏膜疫苗复合抗原的筛选提供初步的理论和实验依据。方法本实验分三部分:第一部分观察弓形虫感染小鼠不同时间从腹水中获取的ESA滴鼻免疫小鼠诱导黏膜和系统免疫应答的效果,确定获取ESA的适宜时间。分别用弓形虫感染第0h、6h、12h、24h、36h、48h和60h无菌收集的腹腔液中获取的ESA以20μg/只或PBS以20μl/只滴鼻免疫10只小鼠2次,间隔14天。观察小鼠状况并记录体重,末次免疫后第14天颈椎脱臼处死小鼠,ELISA法检测血清弓形虫特异性IgG、小肠冲洗液sIgA抗体水平,分离脾淋巴细胞、肠上皮内淋巴细胞(intestinal intraepithelial lymphocytes, iIEL)及肠系膜淋巴结淋巴细胞(mesenteric lymph node lymphocytes,MLNL)并计数。第二部分观察以不同剂量弓形虫感染小鼠48h获取的ESA滴鼻免疫小鼠诱导黏膜及系统免疫应答的效果,确定合适的ESA鼻内接种剂量。分别用5μg、10μg、20μg和30μg ESA/只或PBS 20μl/只滴鼻免疫8只小鼠2次,间隔14天。观察小鼠状况,于末次免疫后第14天颈椎脱臼处死小鼠,ELISA法检测鼻咽冲洗液弓形虫特异性sIgA、小肠冲洗液sIgA、血清IgG抗体水平,分离iIEL和MLNL及脾淋巴细胞并计数。第三部分观察弓形虫排泄分泌抗原与可溶性速殖子抗原联合或单独滴鼻免疫小鼠诱导黏膜及系统免疫应答的效果,筛选弓形虫复合黏膜疫苗候选抗原成分。分别用30μg ESA、20μg STAg、25μg联合抗原(15μg ESA加10μgSTAg)/只或PBS 20μl/只滴鼻免疫10只小鼠2次,间隔14天。观察小鼠健康状况并记录体重,于末次免疫后第14天颈椎脱臼处死小鼠,ELISA法检测小肠冲洗液弓形虫特异性sIgA、血清IgG抗体水平,分离iIEL和脾淋巴细胞并计数。结果不同时相ESA滴鼻免疫小鼠后,除60h组外,其它实验组及PBS组小鼠健康状况良好,体重变化随时间均呈现上升趋势。60h组小鼠状态欠佳,体重增长较其它实验组及PBS组缓慢(P0.05)。实验组血清和黏膜特异性抗体水平升高,MLNL、iIEL和脾淋巴细胞发生增殖性应答。48h组和60组血清IgG、小肠冲洗液sIgA水平及iIEL、MLNL和脾淋巴细胞数量均明显高于除36h组外的其他实验组及PBS组(P0.01)。36h组小肠冲洗液sIgA水平及MLNL数量明显高于0h组和PBS组(P0.05)。不同剂量ESA滴鼻免疫小鼠后,各组小鼠健康状况良好。随鼻内免疫ESA剂量的增加,实验组特异性抗体水平不同程度的升高,MLNL、iIEL及脾淋巴细胞也发生不同程度
[Abstract]:Objective to observe the ability of excreted/secreted antigens, ESA) and (soluble tachyzoite antigen, STAg) to induce mucosal and systemic immune response in mice and to explore the mechanism of immune response induced by Toxoplasma gondii excretory secretory antigen (excreted/secreted antigens, ESA) and soluble tachyzoite antigen (soluble tachyzoite antigen, STAg). To provide a preliminary theoretical and experimental basis for screening complex antigen of Toxoplasma mucosal vaccine. Methods the experiment was divided into three parts: the first part was to observe the induction of mucosal and systemic immune response in mice inoculated with ESA from ascites at different times, and to determine the appropriate time for obtaining ESA in mice infected with Toxoplasma gondii (Toxoplasma gondii). 10 mice were immunized twice with Toxoplasma gondii (Toxoplasma gondii) infection at 20 渭 g / mouse or PBS with 20 渭 l / a intranasal drip for 48 h and 60 h, respectively, after infection with Toxoplasma gondii (Toxoplasma gondii) for 12 hours and 24 hours for 36 hours and 60 hours, respectively, with an interval of 14 days. The mice were killed by cervical dislocations on the 14th day after the last immunization. ELISA assay was used to detect the level of sIgA antibody to Toxoplasma gondii specific IgG, small intestinal irrigation fluid, and to isolate spleen lymphocytes and intestinal intraepithelial lymphocytes (intestinal intraepithelial lymphocytes,). IIEL) and mesenteric lymph node lymphocyte (mesenteric lymph node lymphocytes,MLNL) were counted. The second part was to observe the induction of mucosal and systemic immune response of mice immunized with ESA nasal drip with different doses of Toxoplasma gondii for 48 h, and to determine the appropriate dose of intranasal ESA inoculation. 8 mice were immunized with 5 渭 g 10 渭 g ESA/ or 30 渭 g ESA/ or 20 渭 l / PBS for 14 days. The mice were killed on the 14th day after the last immunization. The serum IgG antibody levels of Toxoplasma gondii specific sIgA, small intestinal lavage fluid (sIgA,), iIEL and MLNL and splenic lymphocytes were detected by ELISA method. The third part was to observe the effect of Toxoplasma gondii excretory secretory antigen and soluble tachyzoite antigen on mucosal and systemic immune responses of mice immunized with Toxoplasma gondii excretory secretory antigen and soluble tachyzoite antigen alone. 10 mice were immunized twice with 30 渭 g ESA,20 渭 g STAg,25 渭 g combined antigen (15 渭 g ESA plus 10 渭 gSTAg) or PBS 20 渭 l / mouse for 14 days. The mice were killed on the 14th day after the last immunization. The serum IgG antibody level of Toxoplasma gondii specific sIgA, was detected by ELISA method, and the iIEL and spleen lymphocytes were separated and counted. Results after nasal immunization with ESA at different time stages, the mice in the other experimental and PBS groups were in good health condition except for 60 h group, and the body weight change showed an increasing trend with time, and the mice in 60 h group were not in good condition. Weight gain was slower than other experimental groups and PBS group (P0.05). The levels of serum and mucosal specific antibodies increased, and MLNL,iIEL and splenic lymphocytes developed proliferative response in experimental group. 48 h group and 60 group of serum IgG, small bowel lavage fluid sIgA level and iIEL, The number of MLNL and splenic lymphocytes were significantly higher than those of other experimental groups and PBS groups except 36h group (P0.01). The sIgA level and MLNL number of small intestinal lavage fluid in 36h group were significantly higher than those in 0h group and PBS group (P0.05). After nasal immunization with different doses of ESA, the mice in each group were in good health. With the increase of the dose of intranasal immunized ESA, the level of specific antibody in the experimental group increased in different degrees, and MLNL,iIEL and splenic lymphocytes also occurred in different degrees.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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