重组红火蚁毒素蛋白SoliⅣ致病机理的初步研究
发布时间：2019-05-15 15:18
【摘要】： 红火蚁属节肢动物门昆虫纲,是一种外来入侵害蚁,被认为是严重卫生公害。被红火蚁螫伤后,一些红火蚁毒素蛋白能引起人的过敏反应甚至死亡。本文用重组表达的红火蚁毒素蛋白Sol iⅣ注射兔子做为动物模型,来研究重组红火蚁毒素蛋白Sol iⅣ致病机理。 本实验在国内首次利用大肠杆菌表达红火蚁重组毒素蛋白Sol iⅣ。诱导表达菌经超声波破碎,表达产物存在于上清液,红火蚁重组毒素蛋白Ⅳ在表达工程菌BL-21中为可溶性表达。用镍层析树脂纯化超声波破菌后的上清液,得到的高纯度蛋白。 通过注射小鼠和兔子进行蛋白活性试验,选出合适的动物模型,发现兔子要比小鼠敏感,有40%左右的兔子出现过敏。再通过蛋白皮试试验选择敏感动物,进行蛋白免疫治疗试验,发现通过小剂量多次注射该蛋白,能起到免疫治疗效果。 通过分离兔外周血淋巴细胞进行培养,经不同浓度的重组红火蚁毒素蛋白Sol iⅣ分别和细菌脂多糖(LPS)、刀豆蛋白(ConA)共同刺激后,MTT法测定淋巴细胞的增殖情况,重组红火蚁毒素蛋白Sol iⅣ浓度为25μg/mL、50μg/mL、75μg/mL和LPS共刺激时,与单独LPS刺激的对照组比较,淋巴细胞增殖活性显著增高(P＜0.05);浓度为15μg/mL、25μg/mL、50μg/mL、75μg/mL和ConA共刺激时,与单独ConA刺激的对照组比较,淋巴细胞增殖活性显著增高(P＜0.05)。 通过兔子的致敏实验,观察受试兔子的皮肤、肝脏、肺脏、肾脏、脾脏、等组织的组织病理变化,皮肤:真皮层发生坏死,坏死灶周围出现大量的嗜酸性粒细胞和炎性细胞。肝脏:肝细胞内可见水泡变性和颗粒变性,肝窦内出现大量嗜酸性粒细胞。肺脏:肺泡壁毛充血,肿胀,肺泡壁内有多量嗜酸性粒细胞。进一步通过ELISA和荧光定量RT-PCR方法检测过敏兔子血清和组织中的IgE和细胞因子,IL-4、IFN-γ、IL-6和IL-10变化。结果显示血清中IL-4、总IgE和IL-10的水平升高,IL-4和IL-10在14h左右达到最高值,总IgE在20 h达到峰值。IFN-γ的水平降低,在20 h达到最低值,IL-6的水平没有显著的变化。重组毒素蛋白Sol iⅣ注射后32 h,实验组兔脾脏和淋巴结中除IL-6 mRNA外,IL-4和IL-10 mRNA量均极显著高于对照组兔(P＜0.01),IFN-γ则极显著低于对照组兔(P＜0.01)。实验组兔脾脏中IL-4 mRNA的表达量是对照组兔的8.3倍,IL-10 mRNA的表达量是对照组的7.2倍,而IFN-γmRNA的表达量只有对照组兔的13.4%;淋巴结中,实验组兔IL-4 mRNA的表达量是对照组兔的6倍,IL-10 mRNA实验组兔是对照组兔的16.4倍,IFN-γmRNA的表达量只有对照组兔的12.8%;而IL-6mRNA对照组和实验组均没有统计学差异 结论一定浓度的重组红火蚁毒素蛋白Sol iⅣ能引起体外培养的T、B淋巴细胞增殖,过敏体质兔在重组红火蚁毒素蛋白Sol iⅣ刺激后能引起Ⅰ型变态反应。
[Abstract]:The genus Arthropoda is a kind of alien invasive ant, which is considered to be a serious hygienic hazard. After being stung by red fire ants, some red fire ant toxin proteins can cause allergic reactions and even death. In this paper, the pathogenic mechanism of recombinant red fire ant toxin protein Sol I IV was studied by injecting recombinant red fire ant toxin protein Sol I IV into rabbits as an animal model. In this experiment, the recombinant toxin protein Sol I IV of red fire ants was expressed in E. coli for the first time in China. The induced expression strain was broken by ultrasonic wave, and the expressed product was found in the culture fluid. The recombinant toxin protein IV of red fire ant was soluble in the expression of engineering strain BL-21. The high purity protein was obtained by purifying the culture solution of ultrasonic broken bacteria with nickel chromatography resin. The protein activity test of mice and rabbits was carried out, and the suitable animal model was selected. It was found that rabbits were more sensitive than mice, and about 40% of rabbits had allergies. Then the sensitive animals were selected by protein skin test and the protein immunotherapy test was carried out. it was found that the immunotherapy effect could be achieved by injecting the protein many times in a small dose. Rabbit peripheral blood lymphocytes were isolated and cultured. The proliferation of lymphocytes was measured by MTT assay after co-stimulation with different concentrations of recombinant red fire ant toxin protein Sol I IV and bacterial lipopolysaccharide (LPS), concanavalin (ConA). When the concentration of recombinant red fire ant toxin protein Sol I IV was 25 渭 g / mL, 50 渭 g / mL, 75 渭 g / mL and LPS, the proliferation activity of lymphocytes was significantly higher than that of the control group stimulated by LPS alone (P < 0.05). When the concentration of 15 渭 g / mL, 25 渭 g / mL, 50 渭 g / mL, 75 渭 g / mL and ConA co-stimulation, the proliferation activity of lymphocytes was significantly higher than that of the control group stimulated by ConA alone (P < 0.05). Through the sensitizing experiment of rabbits, the pathological changes of skin, liver, lung, kidney, spleen and other tissues of the tested rabbits were observed. The skin: the dermis was necrotic, and a large number of eosinophils and inflammatory cells appeared around the necrotic focus. the pathological changes of the skin, liver, lung, kidney, spleen and other tissues were observed. Liver: vesicular degeneration and granule degeneration can be seen in hepatocytes, and a large number of eosinophils are found in hepatic sinuses. Lung: the alveolar wall hair is congested, swollen, and there are many eosinophils in the alveolar wall. Furthermore, the changes of IgE and cytokines, IL-4,IFN- 纬, IL-6 and IL-10 in serum and tissue of allergic rabbits were detected by ELISA and fluorescence quantitative RT-PCR. The results showed that the levels of total IgE and IL-10 in serum increased, the levels of IL-4 and IL-10 reached the highest at about 14 h, and the total IgE reached the peak at 20 h. The level of IL-4-纬 decreased and reached the lowest value at 20 h. There was no significant change in the level of IL-6. 32 hours after injection of recombinant toxin protein Sol I IV, the contents of IL-4 and IL-10 mRNA in spleen and lymph nodes of rabbits in the experimental group were significantly higher than those in the control group except IL-6 mRNA. IFN- 纬 was significantly lower than that of the control group (P < 0.01). The expression of IL-4 mRNA in spleen of experimental group was 8.3 times higher than that of control group, and the expression of IL-10 mRNA was 7.2 times of that of control group, while the expression of IFN- 纬 mRNA was only 13.4% of that of control group. In lymph nodes, the expression of IL-4 mRNA in the experimental group was 6 times higher than that in the control group, and that in the IL-10 mRNA experimental group was 16.4 times higher than that in the control group. The expression of IFN- 纬 mRNA in the experimental group was only 12.8% of that in the control group. However, there was no significant difference between the IL-6mRNA control group and the experimental group. Conclusion A certain concentration of recombinant red fire ant toxin protein Sol I IV can induce the proliferation of T, B lymphocytes in vitro. Allergic rabbits can cause type I allergy after stimulation with recombinant red fire ant toxin protein Sol I IV.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R384
本文编号：2477599
[Abstract]:The genus Arthropoda is a kind of alien invasive ant, which is considered to be a serious hygienic hazard. After being stung by red fire ants, some red fire ant toxin proteins can cause allergic reactions and even death. In this paper, the pathogenic mechanism of recombinant red fire ant toxin protein Sol I IV was studied by injecting recombinant red fire ant toxin protein Sol I IV into rabbits as an animal model. In this experiment, the recombinant toxin protein Sol I IV of red fire ants was expressed in E. coli for the first time in China. The induced expression strain was broken by ultrasonic wave, and the expressed product was found in the culture fluid. The recombinant toxin protein IV of red fire ant was soluble in the expression of engineering strain BL-21. The high purity protein was obtained by purifying the culture solution of ultrasonic broken bacteria with nickel chromatography resin. The protein activity test of mice and rabbits was carried out, and the suitable animal model was selected. It was found that rabbits were more sensitive than mice, and about 40% of rabbits had allergies. Then the sensitive animals were selected by protein skin test and the protein immunotherapy test was carried out. it was found that the immunotherapy effect could be achieved by injecting the protein many times in a small dose. Rabbit peripheral blood lymphocytes were isolated and cultured. The proliferation of lymphocytes was measured by MTT assay after co-stimulation with different concentrations of recombinant red fire ant toxin protein Sol I IV and bacterial lipopolysaccharide (LPS), concanavalin (ConA). When the concentration of recombinant red fire ant toxin protein Sol I IV was 25 渭 g / mL, 50 渭 g / mL, 75 渭 g / mL and LPS, the proliferation activity of lymphocytes was significantly higher than that of the control group stimulated by LPS alone (P < 0.05). When the concentration of 15 渭 g / mL, 25 渭 g / mL, 50 渭 g / mL, 75 渭 g / mL and ConA co-stimulation, the proliferation activity of lymphocytes was significantly higher than that of the control group stimulated by ConA alone (P < 0.05). Through the sensitizing experiment of rabbits, the pathological changes of skin, liver, lung, kidney, spleen and other tissues of the tested rabbits were observed. The skin: the dermis was necrotic, and a large number of eosinophils and inflammatory cells appeared around the necrotic focus. the pathological changes of the skin, liver, lung, kidney, spleen and other tissues were observed. Liver: vesicular degeneration and granule degeneration can be seen in hepatocytes, and a large number of eosinophils are found in hepatic sinuses. Lung: the alveolar wall hair is congested, swollen, and there are many eosinophils in the alveolar wall. Furthermore, the changes of IgE and cytokines, IL-4,IFN- 纬, IL-6 and IL-10 in serum and tissue of allergic rabbits were detected by ELISA and fluorescence quantitative RT-PCR. The results showed that the levels of total IgE and IL-10 in serum increased, the levels of IL-4 and IL-10 reached the highest at about 14 h, and the total IgE reached the peak at 20 h. The level of IL-4-纬 decreased and reached the lowest value at 20 h. There was no significant change in the level of IL-6. 32 hours after injection of recombinant toxin protein Sol I IV, the contents of IL-4 and IL-10 mRNA in spleen and lymph nodes of rabbits in the experimental group were significantly higher than those in the control group except IL-6 mRNA. IFN- 纬 was significantly lower than that of the control group (P < 0.01). The expression of IL-4 mRNA in spleen of experimental group was 8.3 times higher than that of control group, and the expression of IL-10 mRNA was 7.2 times of that of control group, while the expression of IFN- 纬 mRNA was only 13.4% of that of control group. In lymph nodes, the expression of IL-4 mRNA in the experimental group was 6 times higher than that in the control group, and that in the IL-10 mRNA experimental group was 16.4 times higher than that in the control group. The expression of IFN- 纬 mRNA in the experimental group was only 12.8% of that in the control group. However, there was no significant difference between the IL-6mRNA control group and the experimental group. Conclusion A certain concentration of recombinant red fire ant toxin protein Sol I IV can induce the proliferation of T, B lymphocytes in vitro. Allergic rabbits can cause type I allergy after stimulation with recombinant red fire ant toxin protein Sol I IV.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R384
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