旋毛蟲成蟲抗原和肌幼蟲抗原保護(hù)性免疫的比較研究

1、旋毛蟲成蟲抗原和肌幼蟲抗原保護(hù)性免疫的比較研究         11-01-28 14:46:00     編輯：studa20            作者：毛立群, 唐潔, 楊靜, 李覃, 牛秀瓏, 葉路, 孫奕 【摘要】  目的: 比較旋毛蟲成蟲抗原和肌幼蟲抗原誘導(dǎo)小鼠產(chǎn)生保護(hù)性免疫效果的異同。方法: 制備旋毛蟲成蟲抗原和肌幼蟲抗原, 分組免疫

2、小鼠, 并于末次免疫后10 d用旋毛蟲感染性幼蟲對各組小鼠實施攻擊感染, 檢測攻擊感染后各組小鼠腸道成蟲和肌肉幼蟲數(shù)并計算減蟲率, 同時采用ELISA法檢測各組小鼠血清IgG抗體水平。結(jié)果: 成蟲抗原免疫組小鼠和肌幼蟲抗原免疫組小鼠檢獲的腸道成蟲數(shù)和肌幼蟲數(shù)均顯著少于對照組（P0.05), 其中以成蟲抗原免疫組小鼠減蟲率最高（P0.01）。成蟲抗原免疫組和肌幼蟲抗原免疫組小鼠于實施旋毛蟲感染性幼蟲進(jìn)行攻擊感染的當(dāng)日所檢測的血清IgG抗體水平與其免疫前相比均升高（P0.01, P0.05), 與對照組相比亦升高（P0.01, P0.05), 其中也以成蟲抗原免疫組小鼠血清IgG抗體升高最為顯著（

3、P0.01）。結(jié)論: 旋毛蟲成蟲抗原和肌幼蟲抗原均能激發(fā)小鼠產(chǎn)生保護(hù)性免疫, 其中成蟲抗原免疫組小鼠顯示出更好的抗感染保護(hù)性。 【關(guān)鍵詞】  旋毛蟲; 成蟲抗原; 肌幼蟲抗原; 保護(hù)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旋毛蟲病是一種人畜共患病, 對人類及畜牧業(yè)危害嚴(yán)重。旋毛蟲抗原成分復(fù)雜, 各種抗原誘導(dǎo)產(chǎn)生的保護(hù)性免疫效果也各有不同1, 給旋毛蟲病的預(yù)防工作帶來很大困難。我們應(yīng)用旋毛

10、蟲成蟲抗原和肌幼蟲抗原分別免疫小鼠, 再采用相同數(shù)量的感染性幼蟲對其進(jìn)行攻擊感染, 通過檢測減蟲率及小鼠血清IgG水平來評價旋毛蟲成蟲抗原和肌幼蟲抗原免疫后小鼠所獲得的抗感染能力, 以研究旋毛蟲不同發(fā)育時期的抗原所誘導(dǎo)產(chǎn)生的保護(hù)性免疫效果, 為進(jìn)一步研究旋毛蟲的人工預(yù)防提供實驗數(shù)據(jù)。1  材料和方法1.1  材料 昆明種小鼠購自武警醫(yī)學(xué)院動物飼養(yǎng)場。旋毛蟲蟲種由武警醫(yī)學(xué)院寄生蟲學(xué)教研室提供, 為小鼠保種傳代的豬源旋毛蟲。1.2  方法1.2.1  旋毛蟲抗原的制備旋毛蟲成蟲可溶性抗原的制備: 小鼠灌胃感染300條/只旋毛蟲感染期幼蟲, 7 d

11、后處死, 取小腸縱行剪開并剪成23 cm長小段, 無菌生理鹽水漂洗, 加入37預(yù)溫的無菌生理鹽水, 置37恒溫培養(yǎng)箱孵育45 h, 使成蟲從腸壁鉆出, 反復(fù)無菌生理鹽水沉淀、 漂洗后收集純凈的成蟲。將成蟲研磨成勻漿, 超聲粉碎儀間歇粉碎, 4冰箱過夜, 以10 000 r/min 4離心30 min, 取上清即為成蟲可溶性抗原。紫外分光光度計測定蛋白含量, -20凍存, 備用。旋毛蟲肌幼蟲抗原的制備: 小鼠罐胃感染300條/只旋毛蟲感染期幼蟲, 40 d 后處死, 取全部肌肉剪碎, 加入胃蛋白酶消化液, 置37溫箱812 h, 待肌肉被完全消化后, 銅網(wǎng)過濾, 自然沉淀, 再用生理鹽水漂洗3次

12、, 收集脫囊肌幼蟲。將肌幼蟲中加入無菌生理鹽水, 用人工玻璃勻漿器研磨成勻漿, 超聲波粉碎儀粉碎3次, 每次10 min, 置4冰箱過夜, 以10 000 r/min 4離心30 min, 收集上清即為肌幼蟲可溶性抗原。紫外分光光度計測定蛋白含量, -20 凍存, 備用。1.2.2 小鼠的免疫接種和攻擊感染 小鼠80只, 1月齡, 體質(zhì)量1822 g, 每組20只, 隨機(jī)分為4組, 即成蟲抗原免疫組、 肌幼蟲抗原免疫組、 佐劑組和正常對照組。成蟲抗原免疫組小鼠用含蛋白100 g的成蟲抗原0.1 mL與等體積的弗氏完全佐劑（FCA）乳化, 腹腔注射; 肌幼蟲抗原免疫組小鼠用含蛋白100

13、 g的肌幼蟲抗原0.1 mL與等體積的FCA乳化, 腹腔注射; 佐劑組小鼠用0.1 mL FCA與等體積生理鹽水乳化, 腹腔注射。每隔10 d 注射1次, 共3次。正常組小鼠不做任何處理。于末次免疫注射后10 d, 用300條/只旋毛蟲感染性幼蟲實施攻擊感染2。1.2.3  小鼠腸道旋毛蟲成蟲的檢測 于攻擊感染后7 d 每組剖殺10只小鼠, 取小腸縱向剪開, 放入盛有生理鹽水的平皿中, 置37溫箱4 h, 使蟲體逸出, 漂洗后收集成蟲, 在解剖鏡下計數(shù)并計算減蟲率:  減蟲率（對照組檢獲蟲體均數(shù)實驗組檢獲蟲體均數(shù)）/對照組檢獲蟲體均數(shù)×100%。1.2.

14、4  小鼠旋毛蟲肌肉幼蟲的檢測 于攻擊感染后40 d, 將每組剩余的10只小鼠全部處死, 取全部肌肉剪碎, 加入胃蛋白酶消化液, 置于37溫箱過夜。銅網(wǎng)過濾, 生理鹽水漂洗沉淀后收集幼蟲, 計數(shù)并計算減蟲率。1.2.5  小鼠血清IgG抗體的檢測 各組小鼠分別于抗原免疫注射當(dāng)日、 攻擊感染當(dāng)日、 攻擊感染后7 d （即剖殺小鼠檢測成蟲時）和40 d （即剖殺小鼠檢測肌幼蟲時）分4次采血, 第1、 第2次采用斷尾取血, 第3、 第4次采用眶裂取血, 分離血清, -20凍存, 備用。小鼠血清IgG抗體采用酶聯(lián)免疫吸附試驗(ELISA)檢測3, 步驟如下: 20 mg/L的羊抗鼠IgG包被液0.1 mL/孔, 置4過夜; 次日用100 mL/L滅活小牛血清0.1 mL