细粒棘球蚴重组抗原EgP-29免疫小鼠的动态研究
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摘要
目的利用已完成免疫学特性鉴定的细粒棘球蚴(Echinococcus granulosus, Eg)P-29重组蛋白免疫ICR小鼠后,用活的原头蚴攻击感染;动态观察免疫小鼠血清IgE、IgG及其亚类的变化规律;脾T细胞CD4+和CD8+亚群的变化;以及脾细胞培养上清液IFN-γ、IL-2、IL-4、TNF-α和IL-10的变化水平;观察实验动物产生的保护力;从而阐述其保护性免疫机制,为CE疫苗的开发利用提供依据。方法(1)制备抗原及定量:用已成功构建的基因工程菌株EgP-29/pET-28a/BL21(DE3)plysS进行诱导表达、鉴定、纯化、制备出重组抗原并定量;(2)实验动物分组:选取雌性ICR小鼠随机分为3组:实验组既抗原接种组、佐剂+PBS组和对照组即PBS组;(3)动物免疫:实验组注射重组抗原+佐剂+PBS100μl,共三次,每次间隔两周,后两次将弗氏完全佐剂改为弗氏不完全佐剂;佐剂+PBS组采用如上注射方法,每次注射:PBS50μl+弗氏完全佐剂50μl,(后两次亦改为弗氏不完全佐剂);PBS组则采用如上注射方法,每次注射:PBS100μl;(4)动物攻击感染:免疫后8周,各组均以2000个活的原头蚴/每只小鼠攻击感染;(5)脾细胞悬液的制备和血清的收集:小鼠接种疫苗后在0、2、4、6w及攻击感染后在10w、18w、26w分别从眼球采血收集血清并分离脾细胞,每次每组5只;(6)通过ELISA及FCM:①观察免疫后、攻击感染后不同时间IgE、IgG及其亚类的变化规律;②用ConA刺激脾细胞培养后,检测其上清液中IFN-γ、IL-2、IL-4、TNF-α和IL-10的变化水平;③分离脾细胞后,应用FCM检测T细胞CD4+和CD8+亚群的百分比变化;(7)免疫保护力观察:计算免疫保护力。结果(1)经过酶切鉴定,纯化的EgP-29重组蛋白在SDS-PAGE上分子量29KDa处见明显表达条带,抗原制备成功;(2)经“SmartSpectTM3000”微量蛋白核酸分析定量重组蛋白EgP-29的浓度为8.3μg/μl ;(3)ICR小鼠接种原头蚴26w后,切开腹腔发现数个大小不等的包囊,说明棘球蚴模型制作成功;(4)实验组(rEgP-29)与对照组(PBS)经统计学检验比较:①rEgP-29组小鼠在抗原免疫后和攻击感染后均产生高水平的IgG、IgG1、IgE、IgG3抗体(P<0.01 ),提示IgG、IgG1、IgE、IgG3可能参与保护性的免疫应答机制;②rEgP-29组小鼠在攻击感染后不仅CD4+T细胞增加(P<0.01 ),CD8+T细胞也有轻度增加(P<0.05 ),提示rEgP-29的保护机制不仅有CD4+T细胞的参与,可能也有CD8+T细胞的作用;③rEgP-29组小鼠在攻击感染后产生高水平的IL-2(P<0.05 ),低水平的IL-4和IFN-γ(P<0.05 ),提示该重组抗原以诱导Th1型免疫应答为主的反应;(5)根据Dempster公式计算:rEgP-29抗原免疫ICR小鼠可获得90%的免疫保护力,提示rEgP-29能诱导小鼠产生较强的免疫保护力。结论rEgP-29能诱导小鼠产生较强的免疫保护力,其保护性免疫机制主要通过诱导宿主产生体液免疫应答和Th1型免疫应答,表明rEgP-29是具有发展潜力的抗包虫病候选疫苗。
Objective To utilize recombinant diagnostic antigen P-29 (rEgP-29) of Echinococcus granulosus that was already identified its immunity successfully , after ICR mice immunized with rEgP-29 , all the mice were challenged with living protoscolices; then the level of change about serum IgE, IgG and its subclasses, CD4+ and CDg+ T lymphocyte subsets, IL-2、IL-4、IFN-γand TNF- a of spleen cells were dynamically observed. Test its immunological mechanism in mice immunized with rEgP-29 through molecular biological technology. Methods (1). To identify, express, purify, fixed quantity rEgP-29; (2). 132 ICR mice(female) were divided into three groups with random process;(3) All the mice of experimental group were vaccinated subcutaneously on the back with 10μl antigen dissolved in phosphate-buffered saline(PBS) and emulsified with 50ul Freund’s complete adjuvant(FCA) respectively in the first time while the mice of PBS add Adjuvant groups were vaccinated with adjuvant in PBS), two weeks after the first vaccination with the same preparation except that FCA was replaced by Freund’s incomplete adjvant(FIA), all mice were vaccinated thirdly , the same as the second time with an interval week, PBS group did merely with PBS 100ul each time, the process was as before. (4). All the mice were challenged with 2000 protoscolices after vaccinating about 8 weeks; (5) Five mice of each group were killed after each inoculation and after challenging injection (0w, 2w, 4w, 6w, 10w, 18w, 26w) in order to collect sera ; (6). Make a dynamic study on rEgP-29 through ELISA and FCM. (7) Oberve the rEgP-29 antigenicity and immunogenicity using Dempster theory; Result (1). The purified rEgP-29 could be recognized in SDS-PAGE and it had the position about 29kD. (2)Get the density of rEgP-29 by SmartSpectTM3000 ;(3) 26w after challenged with 2000 protoscolices , CE were found in the ICR mice livers, it demonstrated we made the ICR mice model successfully;(4) Compared the group of rEgP-29 with the controls:①rEgP-29 can induce mice to produce high level of IgG、IgG1、IgE、IgG3(P<0.01 ), (P<0.01 ) the low level of IgG2a and IgG2b ,it suggested that IgG、IgG1、IgE、IgG3 could participate in the mechanism of protective immune response.②After mice inoculated with rEgP-29 was challenged, CD4+ subsets obviously enhanced(P<0.01 ) meanwhile CD8+ slightly enhanced. It hinted that CD4+ and CD8+ subsets could enlist the protective immune mechanism.③After challenge, rEgP-29 induced mice to product the high level of IL-2 and IFN-γ(P<0.05 ), it indicated that rEgP-29 induced Th1 type immune reaction.(5). Using Dempster theory, its antigenicity and immunogenicity is about 90%. Conclusions We may conclude that protection against Echinococcus granulosus protoscoleces induced with rEgP-29 was associated with hunoral and Th1 cellular responses.
Objective To utilize recombinant diagnostic antigen P-29 (rEgP-29) of Echinococcus granulosus that was already identified its immunity successfully , after ICR mice immunized with rEgP-29 , all the mice were challenged with living protoscolices; then the level of change about serum IgE, IgG and its subclasses, CD4+ and CDg+ T lymphocyte subsets, IL-2、IL-4、IFN-γand TNF- a of spleen cells were dynamically observed. Test its immunological mechanism in mice immunized with rEgP-29 through molecular biological technology. Methods (1). To identify, express, purify, fixed quantity rEgP-29; (2). 132 ICR mice(female) were divided into three groups with random process;(3) All the mice of experimental group were vaccinated subcutaneously on the back with 10μl antigen dissolved in phosphate-buffered saline(PBS) and emulsified with 50ul Freund’s complete adjuvant(FCA) respectively in the first time while the mice of PBS add Adjuvant groups were vaccinated with adjuvant in PBS), two weeks after the first vaccination with the same preparation except that FCA was replaced by Freund’s incomplete adjvant(FIA), all mice were vaccinated thirdly , the same as the second time with an interval week, PBS group did merely with PBS 100ul each time, the process was as before. (4). All the mice were challenged with 2000 protoscolices after vaccinating about 8 weeks; (5) Five mice of each group were killed after each inoculation and after challenging injection (0w, 2w, 4w, 6w, 10w, 18w, 26w) in order to collect sera ; (6). Make a dynamic study on rEgP-29 through ELISA and FCM. (7) Oberve the rEgP-29 antigenicity and immunogenicity using Dempster theory; Result (1). The purified rEgP-29 could be recognized in SDS-PAGE and it had the position about 29kD. (2)Get the density of rEgP-29 by SmartSpectTM3000 ;(3) 26w after challenged with 2000 protoscolices , CE were found in the ICR mice livers, it demonstrated we made the ICR mice model successfully;(4) Compared the group of rEgP-29 with the controls:①rEgP-29 can induce mice to produce high level of IgG、IgG1、IgE、IgG3(P<0.01 ), (P<0.01 ) the low level of IgG2a and IgG2b ,it suggested that IgG、IgG1、IgE、IgG3 could participate in the mechanism of protective immune response.②After mice inoculated with rEgP-29 was challenged, CD4+ subsets obviously enhanced(P<0.01 ) meanwhile CD8+ slightly enhanced. It hinted that CD4+ and CD8+ subsets could enlist the protective immune mechanism.③After challenge, rEgP-29 induced mice to product the high level of IL-2 and IFN-γ(P<0.05 ), it indicated that rEgP-29 induced Th1 type immune reaction.(5). Using Dempster theory, its antigenicity and immunogenicity is about 90%. Conclusions We may conclude that protection against Echinococcus granulosus protoscoleces induced with rEgP-29 was associated with hunoral and Th1 cellular responses.
引文
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