本文選題：細(xì)粒棘球絳蟲 + 疫苗��； 參考：《重慶醫(yī)科大學(xué)》2010年碩士論文

【摘要】：目的 在構(gòu)建細(xì)粒棘球絳蟲轉(zhuǎn)Eg95-EgA31融合基因苜蓿疫苗的基礎(chǔ)上,提取轉(zhuǎn)基因苜蓿葉蛋白,將葉蛋白提取液采用口服灌胃和鼻腔粘膜接種免疫BALB/c小鼠,動(dòng)態(tài)觀察其誘導(dǎo)的免疫應(yīng)答的變化；將葉蛋白提取液采用口服灌胃和鼻腔粘膜接種免疫BALB/c小鼠后,再用Eg原頭節(jié)攻擊,研究該疫苗產(chǎn)生的保護(hù)力及其免疫機(jī)制。 方法 采用熱絮凝法提取轉(zhuǎn)基因苜蓿葉蛋白,用無菌雙蒸水配成20μg/μl。同時(shí)提取轉(zhuǎn)空質(zhì)粒(pBT121)苜蓿葉蛋白和正常苜蓿葉蛋白作對(duì)照。為了動(dòng)態(tài)觀察細(xì)粒棘球絳蟲轉(zhuǎn)Eg95-EgA31融合基因苜蓿疫苗免疫BALB/c小鼠后誘導(dǎo)的免疫應(yīng)答的變化,將88只雌性BALB/c小鼠隨機(jī)分為2組,每組44只�？诜辔附M：每只小鼠灌胃100μl(約含1μg融合抗原)的轉(zhuǎn)基因苜蓿葉蛋白提取液,灌胃前30 min先灌服5%的碳酸氫鈉60μl,以中和胃酸；滴鼻接種組：每鼠接種10μl(約含0.1μg融合抗原)的轉(zhuǎn)基因苜蓿葉蛋白提取液。以上各組每3d免疫1次,連續(xù)2個(gè)月。ELISA方法檢測(cè)各組鼠免疫后不同時(shí)間(免疫后0、2、4、6、8、10、12、14、16、18和20 w)血清特異性IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平以及脾細(xì)胞體外培養(yǎng)原液及在受到EgAg或ConA(LPS)刺激后分別產(chǎn)生的IFN-γ、IL-12、TNF-α和IL-10水平；用MTT比色法檢測(cè)脾細(xì)胞增殖水平；用FCM檢測(cè)各組鼠免疫后不同時(shí)間(免疫后0、2、4、6、8、10、12、14、16、18和20 w)脾細(xì)胞CD4+和CD8+亞群。 為了研究細(xì)粒棘球絳蟲轉(zhuǎn)Eg95-EgA31融合基因苜蓿疫苗免疫BALB/c小鼠后抗原頭節(jié)攻擊產(chǎn)生的保護(hù)力及其免疫機(jī)制,將32只雌性BALB/c小鼠隨機(jī)分為4組,每組8只。A組小鼠口服灌胃100μl的轉(zhuǎn)基因苜蓿葉蛋白提取液(約含1μgEg95-EgA31融合抗原),接種前30min用5%碳酸氫鈉60μl對(duì)小鼠進(jìn)行灌胃,以中和胃酸；B組小鼠滴鼻接種10μl上述提取液(約含0.1μg Eg95-EgA31融合抗原)；C組小鼠滴鼻接種10μl的轉(zhuǎn)空質(zhì)粒苜蓿葉蛋白提取液(不含Eg95-EgA31融合抗原)；D組小鼠灌胃100μl的正常苜蓿葉蛋白提取液,接種前30 min用5%碳酸氫鈉60μl對(duì)小鼠進(jìn)行灌胃,以中和胃酸。以上各組每3d免疫1次,連續(xù)2個(gè)月。4組均于末次免疫后第8w用Eg原頭節(jié)攻擊。在Eg原頭節(jié)攻擊感染后第24 w剖殺各組小鼠,分離并稱重細(xì)粒棘球蚴囊,計(jì)算囊重減少率；用ELISA方法檢測(cè)各組鼠血清特異性IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平；雙抗體夾心ELISA試劑盒檢測(cè)各組鼠脾細(xì)胞體外培養(yǎng)原液及在受到EgAg或ConA(LPS)刺激后分別產(chǎn)生的IFN-γ、IL-12、TNF-α和IL-10水平；用MTT比色法檢測(cè)脾細(xì)胞增殖水平；用FCM檢測(cè)脾細(xì)胞CD4+和CD8+亞群；用Annexin V-FITC試劑盒檢測(cè)脾細(xì)胞原液或用ConA刺激培養(yǎng)時(shí)細(xì)胞凋亡發(fā)生率。 結(jié)果 動(dòng)態(tài)觀察發(fā)現(xiàn)口服組小鼠的血清IgG、IgG2a、IgG2b、IgG1、IgG3和IgE水平分別在末次免疫后2～14周、2～14周、4～20周、6～12周、6～12周和4～16周升高,分別在末次免疫后4、4、6、8、8和10周達(dá)最高水平；脾T淋巴細(xì)胞增殖水平在末次免疫后4-10周升高,在末次免疫后6周達(dá)最高水平；脾CD4+和CD8+T細(xì)胞亞群分別在免疫后6～10周和4～12周升高,分別在免疫后6周和8周達(dá)高峰；脾細(xì)胞上清液中IL-12、IFN-γ、TNF-α和IL-10水平分別在免疫后4～6周、2～8周、2～6周和4～12周升高,分別在免疫后4、2、2和8周達(dá)最高水平。鼻腔接種組小鼠的血清IgG、IgG2a、IgG2b、IgG1、IgG3和IgE水平分別在末次免疫后2～14周、2～18周、4～10周、6～14周、8周和6～12周升高,分別在末次免疫后4、4、6、12、8和6周達(dá)最高水平；脾T淋巴細(xì)胞增殖水平在末次免疫后4-12周升高,在末次免疫后6周達(dá)最高水平；脾CD4+和CD8+T細(xì)胞亞群分別在末次免疫后4-6周和4-10周升高,分別在末次免疫后6周和8周達(dá)高峰；脾細(xì)胞上清液中IL-12、IFN-γ、TNF-α和IL-10水平分別在末次免疫后4～6周、2～10周、4～10周和6～16周升高,分別在末次免疫后6、4、6和6周達(dá)最高水平。 疫苗免疫及原頭節(jié)攻擊后發(fā)現(xiàn)與D組相比,A組小鼠檢獲棘球蚴包囊質(zhì)量降低,囊重減少率為64.10%,脾T淋巴細(xì)胞增殖水平升高,CD4+、CD8+亞群和CD4+/CD8+比值均升高,血清IgG、IgG2b和IgE水平升高,脾細(xì)胞上清液中的IFN-γ、IL-12和TNF-α水平升高,IL-10水平降低,脾細(xì)胞凋亡率降低。B組與D組相比,囊重減少率無顯著差異,脾T淋巴細(xì)胞增殖水平升高,CD4+亞群和CD4+/CD8+比值升高,血清IgG、IgG2b和IgE水平升高,脾細(xì)胞上清液中IFN-γ和TNF-α水平升高,脾細(xì)胞凋亡率降低。A組血清IgG水平、脾細(xì)胞上清液中IFN-γ和IL-12水平、CD4+亞群百分比及脾T淋巴細(xì)胞增殖水平均顯著高于B組,脾細(xì)胞凋亡率顯著低于B組,但2組囊重減少率相比無顯著差異。C組與D組相比結(jié)果無顯著差異。 結(jié)論 1.動(dòng)態(tài)觀察表明細(xì)粒棘球絳蟲轉(zhuǎn)Eg95-EgA31融合基因苜蓿葉蛋白提取液口服和滴鼻接種均可誘導(dǎo)免疫鼠產(chǎn)生有效的免疫應(yīng)答。 2.保護(hù)力實(shí)驗(yàn)提示細(xì)粒棘球絳蟲轉(zhuǎn)Eg95-EgA31融合基因苜蓿葉蛋白提取液口服接種能誘導(dǎo)免疫鼠產(chǎn)生一定的保護(hù)力,Th1型免疫應(yīng)答在其誘導(dǎo)的保護(hù)性免疫機(jī)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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

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