旋毛虫成虫抗原和肌幼虫抗原保护性免疫的比较研究

1、旋毛虫成虫抗原和肌幼虫抗原保护性免疫的比较研究         11-01-28 14:46:00     编辑：studa20            作者：毛立群, 唐洁, 杨静, 李覃, 牛秀珑, 叶路, 孙奕 【摘要】  目的: 比较旋毛虫成虫抗原和肌幼虫抗原诱导小鼠产生保护性免疫效果的异同。方法: 制备旋毛虫成虫抗原和肌幼虫抗原, 分组免疫

2、小鼠, 并于末次免疫后10 d用旋毛虫感染性幼虫对各组小鼠实施攻击感染, 检测攻击感染后各组小鼠肠道成虫和肌肉幼虫数并计算减虫率, 同时采用ELISA法检测各组小鼠血清IgG抗体水平。结果: 成虫抗原免疫组小鼠和肌幼虫抗原免疫组小鼠检获的肠道成虫数和肌幼虫数均显著少于对照组（P0.05), 其中以成虫抗原免疫组小鼠减虫率最高（P0.01）。成虫抗原免疫组和肌幼虫抗原免疫组小鼠于实施旋毛虫感染性幼虫进行攻击感染的当日所检测的血清IgG抗体水平与其免疫前相比均升高（P0.01, P0.05), 与对照组相比亦升高（P0.01, P0.05), 其中也以成虫抗原免疫组小鼠血清IgG抗体升高最为显著（

3、P0.01）。结论: 旋毛虫成虫抗原和肌幼虫抗原均能激发小鼠产生保护性免疫, 其中成虫抗原免疫组小鼠显示出更好的抗感染保护性。 【关键词】  旋毛虫; 成虫抗原; 肌幼虫抗原; 保护性免疫AbstractAIM:  To compare the immune protective effects of the mice immunized by antigens derived from adult worms and muscle larvae of Trichinella spiralis. METHODS:  The adult worm and muscl

4、e larvae antigens of Trichinella spiralis were prepared and two groups of mice (adult worm antigen group and muscle larvae antigen group) were immunized with them,  respectively. After 10 days of the final vaccination,  the mice in each group were challenged with infective larvae of Trichi

5、nella spiralis. The number of intestinal adult worms and muscle larvae were examined and their reduction rate was calculated. Meanwhile,  the level of serum IgG in each group was detected by ELISA. RESULTS:  Compared with control group,  the number of intestinal adult worms and muscle

6、 larvae in adult worm group and muscle larvae antigen group was lower（P0.05) and number of intestinal adult worms and muscle larvae in adult worm antigen group was at the lowest（P0.01). When the mice in the two groups were challenged with infective larvae of Trichinella spiralis,  the titer of

7、serum IgG detected on the same day was higher than that before they were vaccinated with the antigens（P0.01,  P0.05) and it was also higher than that in control group（P0.01,  P0.05). The level of serum IgG of the mice detected on the day when they were challenged with infective larvae of T

8、richinella spiralis was significantly higher than that before the mice were vaccinated and it was also higher than that in adult worm antigen group（P0.01）. CONCLUSION:  Both the adult worm and muscle larvae antigens of Trichinella spiralis can stimulate mice to produce protective immunity and t

9、he mice immunized with adult worm antigens have stronger resistance to the subsequent challenge infection.Keywordstrichinella spiralis;  adult worm antigen;  muscle larvae antigen;  protective immunity旋毛虫病是一种人畜共患病, 对人类及畜牧业危害严重。旋毛虫抗原成分复杂, 各种抗原诱导产生的保护性免疫效果也各有不同1, 给旋毛虫病的预防工作带来很大困难。我们应用旋毛

10、虫成虫抗原和肌幼虫抗原分别免疫小鼠, 再采用相同数量的感染性幼虫对其进行攻击感染, 通过检测减虫率及小鼠血清IgG水平来评价旋毛虫成虫抗原和肌幼虫抗原免疫后小鼠所获得的抗感染能力, 以研究旋毛虫不同发育时期的抗原所诱导产生的保护性免疫效果, 为进一步研究旋毛虫的人工预防提供实验数据。1  材料和方法1.1  材料 昆明种小鼠购自武警医学院动物饲养场。旋毛虫虫种由武警医学院寄生虫学教研室提供, 为小鼠保种传代的猪源旋毛虫。1.2  方法1.2.1  旋毛虫抗原的制备旋毛虫成虫可溶性抗原的制备: 小鼠灌胃感染300条/只旋毛虫感染期幼虫, 7 d

11、后处死, 取小肠纵行剪开并剪成23 cm长小段, 无菌生理盐水漂洗, 加入37预温的无菌生理盐水, 置37恒温培养箱孵育45 h, 使成虫从肠壁钻出, 反复无菌生理盐水沉淀、 漂洗后收集纯净的成虫。将成虫研磨成匀浆, 超声粉碎仪间歇粉碎, 4冰箱过夜, 以10 000 r/min 4离心30 min, 取上清即为成虫可溶性抗原。紫外分光光度计测定蛋白含量, -20冻存, 备用。旋毛虫肌幼虫抗原的制备: 小鼠罐胃感染300条/只旋毛虫感染期幼虫, 40 d 后处死, 取全部肌肉剪碎, 加入胃蛋白酶消化液, 置37温箱812 h, 待肌肉被完全消化后, 铜网过滤, 自然沉淀, 再用生理盐水漂洗3次

12、, 收集脱囊肌幼虫。将肌幼虫中加入无菌生理盐水, 用人工玻璃匀浆器研磨成匀浆, 超声波粉碎仪粉碎3次, 每次10 min, 置4冰箱过夜, 以10 000 r/min 4离心30 min, 收集上清即为肌幼虫可溶性抗原。紫外分光光度计测定蛋白含量, -20 冻存, 备用。1.2.2 小鼠的免疫接种和攻击感染 小鼠80只, 1月龄, 体质量1822 g, 每组20只, 随机分为4组, 即成虫抗原免疫组、 肌幼虫抗原免疫组、 佐剂组和正常对照组。成虫抗原免疫组小鼠用含蛋白100 g的成虫抗原0.1 mL与等体积的弗氏完全佐剂（FCA）乳化, 腹腔注射; 肌幼虫抗原免疫组小鼠用含蛋白100

13、 g的肌幼虫抗原0.1 mL与等体积的FCA乳化, 腹腔注射; 佐剂组小鼠用0.1 mL FCA与等体积生理盐水乳化, 腹腔注射。每隔10 d 注射1次, 共3次。正常组小鼠不做任何处理。于末次免疫注射后10 d, 用300条/只旋毛虫感染性幼虫实施攻击感染2。1.2.3  小鼠肠道旋毛虫成虫的检测 于攻击感染后7 d 每组剖杀10只小鼠, 取小肠纵向剪开, 放入盛有生理盐水的平皿中, 置37温箱4 h, 使虫体逸出, 漂洗后收集成虫, 在解剖镜下计数并计算减虫率:  减虫率（对照组检获虫体均数实验组检获虫体均数）/对照组检获虫体均数×100%。1.2.

14、4  小鼠旋毛虫肌肉幼虫的检测 于攻击感染后40 d, 将每组剩余的10只小鼠全部处死, 取全部肌肉剪碎, 加入胃蛋白酶消化液, 置于37温箱过夜。铜网过滤, 生理盐水漂洗沉淀后收集幼虫, 计数并计算减虫率。1.2.5  小鼠血清IgG抗体的检测 各组小鼠分别于抗原免疫注射当日、 攻击感染当日、 攻击感染后7 d （即剖杀小鼠检测成虫时）和40 d （即剖杀小鼠检测肌幼虫时）分4次采血, 第1、 第2次采用断尾取血, 第3、 第4次采用眶裂取血, 分离血清, -20冻存, 备用。小鼠血清IgG抗体采用酶联免疫吸附试验(ELISA)检测3, 步骤如下: 20 mg/L的羊抗鼠IgG包被液0.1 mL/孔, 置4过夜; 次日用100 mL/L灭活小牛血清0.1 mL