细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗保护力及其免疫机制研究

发布时间：2018-07-10 16:11

本文选题：细粒棘球绦虫 + 疫苗　；参考：《重庆医科大学》2010年硕士论文

【摘要】：目的在构建细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗的基础上,提取转基因苜蓿叶蛋白,将叶蛋白提取液采用口服灌胃和鼻腔粘膜接种免疫BALB/c小鼠,动态观察其诱导的免疫应答的变化；将叶蛋白提取液采用口服灌胃和鼻腔粘膜接种免疫BALB/c小鼠后,再用Eg原头节攻击,研究该疫苗产生的保护力及其免疫机制。方法采用热絮凝法提取转基因苜蓿叶蛋白,用无菌双蒸水配成20μg/μl。同时提取转空质粒(pBT121)苜蓿叶蛋白和正常苜蓿叶蛋白作对照。为了动态观察细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗免疫BALB/c小鼠后诱导的免疫应答的变化,将88只雌性BALB/c小鼠随机分为2组,每组44只。口服灌胃组：每只小鼠灌胃100μl(约含1μg融合抗原)的转基因苜蓿叶蛋白提取液,灌胃前30 min先灌服5%的碳酸氢钠60μl,以中和胃酸；滴鼻接种组：每鼠接种10μl(约含0.1μg融合抗原)的转基因苜蓿叶蛋白提取液。以上各组每3d免疫1次,连续2个月。ELISA方法检测各组鼠免疫后不同时间(免疫后0、2、4、6、8、10、12、14、16、18和20 w)血清特异性IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平以及脾细胞体外培养原液及在受到EgAg或ConA(LPS)刺激后分别产生的IFN-γ、IL-12、TNF-α和IL-10水平；用MTT比色法检测脾细胞增殖水平；用FCM检测各组鼠免疫后不同时间(免疫后0、2、4、6、8、10、12、14、16、18和20 w)脾细胞CD4+和CD8+亚群。为了研究细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗免疫BALB/c小鼠后抗原头节攻击产生的保护力及其免疫机制,将32只雌性BALB/c小鼠随机分为4组,每组8只。A组小鼠口服灌胃100μl的转基因苜蓿叶蛋白提取液(约含1μgEg95-EgA31融合抗原),接种前30min用5%碳酸氢钠60μl对小鼠进行灌胃,以中和胃酸；B组小鼠滴鼻接种10μl上述提取液(约含0.1μg Eg95-EgA31融合抗原)；C组小鼠滴鼻接种10μl的转空质粒苜蓿叶蛋白提取液(不含Eg95-EgA31融合抗原)；D组小鼠灌胃100μl的正常苜蓿叶蛋白提取液,接种前30 min用5%碳酸氢钠60μl对小鼠进行灌胃,以中和胃酸。以上各组每3d免疫1次,连续2个月。4组均于末次免疫后第8w用Eg原头节攻击。在Eg原头节攻击感染后第24 w剖杀各组小鼠,分离并称重细粒棘球蚴囊,计算囊重减少率；用ELISA方法检测各组鼠血清特异性IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平；双抗体夹心ELISA试剂盒检测各组鼠脾细胞体外培养原液及在受到EgAg或ConA(LPS)刺激后分别产生的IFN-γ、IL-12、TNF-α和IL-10水平；用MTT比色法检测脾细胞增殖水平；用FCM检测脾细胞CD4+和CD8+亚群；用Annexin V-FITC试剂盒检测脾细胞原液或用ConA刺激培养时细胞凋亡发生率。结果动态观察发现口服组小鼠的血清IgG、IgG2a、IgG2b、IgG1、IgG3和IgE水平分别在末次免疫后2～14周、2～14周、4～20周、6～12周、6～12周和4～16周升高,分别在末次免疫后4、4、6、8、8和10周达最高水平；脾T淋巴细胞增殖水平在末次免疫后4-10周升高,在末次免疫后6周达最高水平；脾CD4+和CD8+T细胞亚群分别在免疫后6～10周和4～12周升高,分别在免疫后6周和8周达高峰；脾细胞上清液中IL-12、IFN-γ、TNF-α和IL-10水平分别在免疫后4～6周、2～8周、2～6周和4～12周升高,分别在免疫后4、2、2和8周达最高水平。鼻腔接种组小鼠的血清IgG、IgG2a、IgG2b、IgG1、IgG3和IgE水平分别在末次免疫后2～14周、2～18周、4～10周、6～14周、8周和6～12周升高,分别在末次免疫后4、4、6、12、8和6周达最高水平；脾T淋巴细胞增殖水平在末次免疫后4-12周升高,在末次免疫后6周达最高水平；脾CD4+和CD8+T细胞亚群分别在末次免疫后4-6周和4-10周升高,分别在末次免疫后6周和8周达高峰；脾细胞上清液中IL-12、IFN-γ、TNF-α和IL-10水平分别在末次免疫后4～6周、2～10周、4～10周和6～16周升高,分别在末次免疫后6、4、6和6周达最高水平。疫苗免疫及原头节攻击后发现与D组相比,A组小鼠检获棘球蚴包囊质量降低,囊重减少率为64.10%,脾T淋巴细胞增殖水平升高,CD4+、CD8+亚群和CD4+/CD8+比值均升高,血清IgG、IgG2b和IgE水平升高,脾细胞上清液中的IFN-γ、IL-12和TNF-α水平升高,IL-10水平降低,脾细胞凋亡率降低。B组与D组相比,囊重减少率无显著差异,脾T淋巴细胞增殖水平升高,CD4+亚群和CD4+/CD8+比值升高,血清IgG、IgG2b和IgE水平升高,脾细胞上清液中IFN-γ和TNF-α水平升高,脾细胞凋亡率降低。A组血清IgG水平、脾细胞上清液中IFN-γ和IL-12水平、CD4+亚群百分比及脾T淋巴细胞增殖水平均显著高于B组,脾细胞凋亡率显著低于B组,但2组囊重减少率相比无显著差异。C组与D组相比结果无显著差异。结论 1.动态观察表明细粒棘球绦虫转Eg95-EgA31融合基因苜蓿叶蛋白提取液口服和滴鼻接种均可诱导免疫鼠产生有效的免疫应答。 2.保护力实验提示细粒棘球绦虫转Eg95-EgA31融合基因苜蓿叶蛋白提取液口服接种能诱导免疫鼠产生一定的保护力,Th1型免疫应答在其诱导的保护性免疫机制中起重要作用。
[Abstract]:objective
On the basis of constructing the Eg95-EgA31 fusion gene vaccine of Echinococcus granulosus, the transgenic alfalfa leaf protein was extracted. The leaf protein extract was immunized with BALB/c mice by oral administration of stomach and nasal mucosa. The changes of immune response were observed dynamically. The extract of leaf protein was taken orally by oral administration of stomach and nasal mucosa inoculation. After vaccination with BALB/c mice, the protections and immunological mechanisms of the vaccine were studied by Eg protogenin.
Method
The transgenic alfalfa leaf protein was extracted by thermal flocculation, and the aseptic double steam water was combined with 20 g/ Mu L. to extract the empty plasmid (pBT121) alfalfa leaf protein and the normal alfalfa leaf protein as control. In order to dynamically observe the changes of immune response induced by BALB/c mice immunized with Echinococcus granulosus Eg95-EgA31 fusion gene, the immune responses of Alfalfa mice were observed. Female BALB/c mice were randomly divided into 2 groups, each group of 44. Oral gavage group: each mouse was intragastric 100 mu L (about 1 mu g fusion antigen) of transgenic alfalfa leaf protein extract, before gavage, 30 min was given to 5% sodium bicarbonate 60 mu l to neutralize gastric acid; nose drops inoculation group: transgenic alfalfa leaves were inoculated 10 mu (about 0.1 mu g fusion antigen) per mouse. Each group was immunized 1 times per 3D, and the serum specific IgG, IgG1, IgG2a, IgG2b, IgG3 and IgE levels were detected at different time (0,2,4,6,8,10,12,14,16,18 and 20 W after immunization) for 2 months after 2 months of immunization. The level of F- alpha and IL-10, the proliferation of splenocytes was detected by MTT colorimetry, and FCM was used to detect CD4+ and CD8+ subgroups of spleen cells at different times after immunization (0,2,4,6,8,10,12,14,16,18 and 20 W after immunization).
In order to study the protective force and immune mechanism of the antigen head attack after immunization of Echinococcus granulosus Eg95-EgA31 fusion gene alfalfa vaccine to BALB/c mice, 32 female BALB/c mice were randomly divided into 4 groups. Each group of 8.A mice in each group took orally 100 mu l of transgenic alfalfa white egg white extract (containing 1 Mu gEg95-EgA31 fusion antigen). The pre seed 30min was intragastric with 5% sodium bicarbonate 60 l to neutralize the gastric acid to neutralize the gastric acid, and the B group was inoculated with 10 mu L (about 0.1 mu g Eg95-EgA31 fusion antigen), and the C group was inoculated with 10 U L in the dripping nose of alfalfa leaf protein extract (without Eg95-EgA31 fusion antigenic), and the D mice were intragastric 100 mu l normal alfalfa leaf eggs. The white extract, 30 min before inoculation, was administered to mice with 5% sodium bicarbonate to neutralize gastric acid in order to neutralize gastric acid. These groups were immunized for 1 times per 3D, and in group.4 for 2 months after the last immunization, 8W was attacked by Eg original joint. After Eg original joint attack, the twenty-fourth w were killed in each group, and the Echinococcus granulosus sac was weighed and weighed, and the reduction rate of cystic weight was calculated; EL was calculated with EL. ISA method was used to detect the serum specific IgG, IgG1, IgG2a, IgG2b, IgG3 and IgE levels, and the double antibody sandwich ELISA kit was used to detect the primary liquid of spleen cells in vitro and the IFN- gamma, which was produced respectively after the stimulation of EgAg or ConA (LPS), and the proliferation of spleen cells was detected by the colorimetric method. Cell CD4+ and CD8+ subsets; Annexin V-FITC kit was used to detect the incidence of apoptosis in spleen cells or culture stimulated by ConA.
Result
The dynamic observation showed that the serum IgG, IgG2a, IgG2b, IgG1, IgG3 and IgE levels in the oral group were respectively 2~14 weeks, 2~14 weeks, 4~20 weeks, 6~12 weeks, 6~12 weeks and 4~16 Zhou Shenggao, respectively, and reached the highest level in 4,4,6,8,8 and 10 weeks respectively after the last immunization, and the proliferation level of the spleen T drenched cells increased at the last 4-10 weeks after the last immunization, at the last time. The highest level was reached at 6 weeks after immunization. The spleen CD4+ and CD8+T cell subsets increased respectively at the 6~10 and 4~12 weeks after immunization, respectively, at the peak of 6 and 8 weeks after immunization, and the levels of IL-12, IFN- gamma, TNF- A and IL-10 in the splenocytes supernatant were increased at 4~6, 2~8, 2~6 and 8 weeks after immunization, respectively, at 4,2,2 and 8 weeks after immunization, respectively. The levels of serum IgG, IgG2a, IgG2b, IgG1, IgG3 and IgE in the nasal cavity inoculation group were increased at 2~14 weeks, 2~18 weeks, 4~10 weeks, 6~14 weeks, 8 weeks and 6~12 weeks respectively, respectively, and reached the highest level in 4,4,6,12,8 and 6 weeks after the last immunization, and the level of lymph fine cell proliferation in the spleen increased at the last 4-12 weeks after the last immunization, and at the last immunization. The highest level was reached in the last 6 weeks; the spleen CD4+ and CD8+T cells subgroups were at the peak of 4-6 weeks and 4-10 Zhou Shenggao after the last immunization, respectively, at the peak of 6 and 8 weeks after the final immunization, and the levels of IL-12, IFN- gamma, TNF- A and IL-10 in the splenocytes supernatant were increased at 4~6, 2~10, 4~10 and 6~16 weeks after the last immunization, respectively, and 6,4 after the last immunization, respectively. The highest level was reached in the 6 and 6 weeks.
Compared with the D group, the mass of hydatid cyst in the A group was reduced, the weight loss rate of the cyst weight was 64.10%, the proliferation level of the spleen T lymphocyte increased, the CD4+, the CD8+ subgroup and the CD4+/CD8+ ratio increased, the serum IgG, IgG2b and IgE levels increased, and the IFN- gamma, IL-12 and TNF- alpha levels in the splenocytes supernatant were increased. Compared with group D, the reduction rate of spleen cell apoptosis was not significantly different, the proliferation level of spleen T lymphocyte increased, the ratio of CD4+ subgroup and CD4+/CD8+ increased, the level of serum IgG, IgG2b and IgE increased, the level of IFN- and TNF- alpha in the spleen cell supernatant increased, and the apoptosis rate of spleen cells decreased in the.A group and on the spleen cells and spleen cells in.B group. The level of IFN- gamma and IL-12 in the clear liquid, the percentage of CD4+ subgroup and the proliferation of spleen T lymphocyte were significantly higher than those in the B group. The apoptosis rate of spleen cells was significantly lower than that of the B group, but there was no significant difference in the reduction rate of the 2 groups compared with those in the D group.
conclusion
1. the dynamic observation showed that the oral and nasal drip inoculation of Eg95-EgA31 fusion gene of Echinococcus granulosus could induce effective immune response in immune mice.
2. the protective force experiments suggest that oral inoculation of the Eg95-EgA31 fusion gene of Echinococcus granulosus on alfalfa leaf protein extract can induce a certain protective ability of the immune mice, and the Th1 type immune response plays an important role in the induced protective immune mechanism.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【参考文献】