ESA和STAg鼻内免疫小鼠诱导的抗弓形虫感染保护性免疫应答

发布时间：2018-05-01 11:19

  本文选题：弓形虫 + 排泄-分泌抗原　； 参考：《山西医科大学》2007年硕士论文

【摘要】： 目的本研究首先筛选体外制备弓形虫排泄-分泌抗原(excreted/secreted antigen,ESA)的最适条件,再观察两种不同来源的弓形虫ESA(即Vero细胞体外培养弓形虫获取的ESA和感染鼠腹水中分离的ESA)以及可溶性速殖子抗原(soluble tachyzoite antigen,STAg)鼻内免疫小鼠诱导的黏膜及系统免疫应答及其抗弓形虫感染的能力。探讨三种抗原鼻内免疫诱导的免疫应答及抗感染机制,为弓形虫鼻内复合疫苗的研制奠定理论基础。 方法本研究分三部分:第一部分观察弓形虫与Vero细胞不同共培养条件对ESA含量的影响。首先观察不同血清浓度及含血清培养时间对ESA蛋白浓度的影响。按血清浓度0.5%、1%、2%、4%分4组,组内又按含血清培养时间(3h、6h、12h、24h)分4个时间点。弓形虫速殖子与Vero细胞分别在含不同浓度血清的培养基中共培养,并分别于3h、6h、12h或24h后改为无血清培养基继续培养,第7d收集培养上清液并检测蛋白含量。其次观察共培养时间及含血清培养时间对ESA蛋白浓度的影响。按共培养时间(7d、9d、11d、13d)分为4组,组内又按不同的含血清培养时间(12h、24h、48h、72h)分4个时间点。弓形虫速殖子与Vero细胞在含1%血清的培养基中共培养,各组分别于培养12h、24h、48h、72h后改为无血清培养基继续培养,并分别于第7d、9d、11d、13d收集培养上清液,检测蛋白含量。 第二部分观察两种不同来源的ESA及STAg鼻内免疫小鼠诱导的黏膜及系统免疫应答。BALB/c小鼠随机分为4组,分别用PBS 20μl/只、体外ESA、腹腔ESA或STAg各20μg/只鼻内免疫2次,间隔14d。分别于末次免疫后14d和44d处死,计数肠上皮内淋巴细胞(intestinal intraepithelial lymphocytes,IEL)和脾淋巴细胞,检测血清IgG和肠液sIgA。 第三部分观察两种不同来源的ESA及STAg鼻内免疫小鼠诱导的抗弓形虫作用。BALB/c小鼠随机分为4组,分别用PBS 20μl/只、体外ESA、腹腔ESA和STAg各20μg/只鼻内免疫2次,间隔14d。末次免疫后14d用1×10~4个速殖子/只灌胃攻击,观察小鼠健康状况及体重变化,攻击后30d处死小鼠,计数脾、脑内速殖子。 结果弓形虫速殖子与Vero细胞在不同培养条件中共培养,培养基中血清浓度为1%、共培养12h或24h改为无血清培养基所提取的ESA蛋白含量明显高于其它培养条件(P＜0.05)。12h或24h改为无血清培养基并在共培养第13d收集培养上清液获取的ESA蛋白含量显著高于其它时间点(P＜0.001)。 体外ESA、腹腔ESA和STAg鼻内免疫小鼠后,腹腔ESA组小鼠状态欠佳,其它各组小鼠健康状况良好。末次免疫后14d,各抗原组脾淋巴细胞(P＜0.05)及IEL(P＜0.01)均增殖活跃,至免疫后44d,两种ESA组脾淋巴细胞数及IEL仍高于PBS组(P＜0.05)。与PBS组比较,各抗原组血清IgG水平在免疫后14d(P＜0.001)和44d(P＜0.05)均明显增高。肠液sIgA水平,体外ESA(P＜0.05)、腹腔ESA(P＜0.001)和STAg(P＜0.001)组在免疫后14d明显高于PBS组,两种ESA组在免疫后44d仍与PBS组有差异(P＜0.05)。 体外ESA、腹腔ESA和STAg鼻内免疫小鼠后,腹腔ESA组小鼠出现轻微竖毛、倦怠等异常表现,其它各组小鼠健康状况良好。攻虫后,PBS组小鼠体重逐渐降低,15d后逐渐趋于平缓,而体外ESA组和STAg组(P＜0.05)小鼠体重仍呈增高趋势,腹腔ESA组未见明显升高。各抗原组脾、脑组织内虫荷显著低于PBS组(P＜0.01)。 结论弓形虫速殖子与Vero细胞在含1%血清的培养基中共培养12h或24h后,换无血清培养基继续培养,第13d收集培养上清可获得较高含量的弓形虫ESA。体外ESA、腹腔ESA和STAg鼻内免疫均可诱导黏膜及系统的细胞和体液免疫应答,产生抗弓形虫感染的部分保护。体外ESA和腹腔ESA诱导的免疫应答较STAg持久,但腹腔ESA可能对机体有毒副作用,不适宜直接鼻内免疫,体外ESA和STAg符合疫苗设计的安全性及有效性原则,可用于弓形虫黏膜疫苗的研制。
[Abstract]:Objective this study first screened the optimum conditions for the preparation of Toxoplasma excretory antigen (excreted/secreted antigen, ESA) in vitro, and then observed two different sources of Toxoplasma ESA (ESA obtained by Vero cells in vitro culture of Toxoplasma gondii and ESA isolated from infected rat ascites), and soluble tachyonus antigen (soluble tachyzoite antigen, STAg). The immune response and the ability to resist Toxoplasma infection induced by intranasal immunization in mice were studied. The immune response and anti infection mechanism of three antigens in nasal immunization were discussed, which lay a theoretical foundation for the development of the compound vaccine of Toxoplasma gondii.
Methods this study was divided into three parts: the first part observed the effect of different co culture conditions of Toxoplasma and Vero cells on the content of ESA. First, the effects of different serum concentration and serum culture time on the concentration of ESA protein were observed. According to the serum concentration of 0.5%, 1%, 2%, 4% points, 4 groups were divided into 4 time points according to the serum incubation time (3H, 6h, 12h, 24h). The tachytachus and Vero cells were cultured in the medium containing different concentrations of serum, respectively, after 3h, 6h, 12h or 24h, to continue to be cultured in serum-free medium. 7d was used to collect and culture supernatant and to detect protein content. Secondly, the co culture time and the influence of serum culture time on the concentration of ESA protein were observed. Co culture time (7d,) 9D, 11d, 13D) were divided into 4 groups, and the group was divided into 4 time points according to the different serum culture time (12h, 24h, 48h, 72h). The tachygonite and Vero cells were cultured in the medium containing 1% sera. Each group was cultured 12h, 24h, 48h, after 72h, and continued to be cultured in serum-free medium. The content of protein was measured.
In the second part, the.BALB/c mice induced by two different sources of ESA and STAg intranasal immunization were randomly divided into 4 groups, with PBS 20 mu l/, ESA in vitro, 20 micron 20 micron in the abdominal cavity, and 20 micron in the nasal cavity, and the interval 14D. was killed after the last immunization, and the lymphocytes were counted. Nal intraepithelial lymphocytes, IEL) and splenic lymphocytes were used to detect serum IgG and intestinal fluid sIgA..
The third part observed the.BALB/c mice induced by two different sources of ESA and STAg intranasal immunization mice. The mice were randomly divided into 4 groups, with PBS 20 mu l/, ESA in vitro, 20 micron g/ in the abdominal cavity of ESA and STAg for 2 times, and 1 * 10~4 tachyonus / gavage after the last immunization at the end of 14D.. The health status and body of the mice were observed. After the attack, 30d was killed and the spleen and intracerebral tachycardia were counted.
Results the serum concentration of Toxoplasma gondii and Vero cells in different culture conditions was 1%. The content of ESA protein extracted from co culture 12h or 24h to serum-free medium was significantly higher than that of other culture conditions (P < 0.05).12h or 24h was changed into serum-free medium and collected in co culture 13D for culture supernatant. Protein content was significantly higher than other time points (P < 0.001).
In vitro ESA, intraperitoneal ESA and STAg intranasal immunization mice, the state of the abdominal cavity ESA mice was not good, the other mice were in good health condition. After the last immunization, the spleen lymphocyte (P < 0.05) and IEL (P < 0.01) proliferated active after the last immunization, and the number and IEL of the splenic lymphocyte in the two ESA groups were still higher than those in the PBS group (0.05). The levels of serum IgG in each antigen group were significantly higher in 14d (P < 0.001) and 44d (P < 0.05) after immunization. The level of sIgA in the intestinal fluid, ESA (P < 0.05) in vitro, ESA (P < 0.001) in the abdominal cavity and STAg (P < 0.001) groups were significantly higher than those in the group after immunization. The two groups were still different from those of the group after immunization (0.05).
After ESA, intraperitoneal ESA and STAg intranasally immunized mice, the mice in the ESA group of the abdominal cavity showed slight erection and burnout, and the other mice were in good health. After the attack, the body weight of the PBS group gradually decreased and the 15d gradually tended to slow, while the body weight of the ESA group and the STAg group (P < 0.05) in the in vitro ESA group was still higher, and the ESA group in the abdominal cavity was not clear. The weight of spleen and brain in each antigen group was significantly lower than that in group PBS (P < 0.01).
Conclusion the Toxoplasma gondii and Vero cells were cultured for 12h or 24h in the culture medium containing 1% sera. The culture medium without serum was continued to be cultured. The high content of Toxoplasma ESA. in vitro ESA was obtained from the culture supernatant of 13D. The immune response of the mucous and systemic cells and body fluids could be induced in the intraperitoneal and intraperitoneal ESA and STAg, and the anti Toxoplasma gondii could be produced. Partial protection of infection. The immune response induced by ESA and ESA in vitro is longer than that of STAg, but ESA in abdominal cavity may have toxic side effects to the body. It is not suitable for direct intranasal immunity. In vitro ESA and STAg conform to the safety and effectiveness principles of vaccine design. It can be used for the development of Toxoplasma mucous membrane vaccine.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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