重組紅火蟻毒素蛋白SoliⅣ致病機理的初步研究
發(fā)布時間：2019-05-15 15:18
【摘要】： 紅火蟻屬節(jié)肢動物門昆蟲綱,是一種外來入侵害蟻,被認為是嚴重衛(wèi)生公害。被紅火蟻螫傷后,一些紅火蟻毒素蛋白能引起人的過敏反應甚至死亡。本文用重組表達的紅火蟻毒素蛋白Sol iⅣ注射兔子做為動物模型,來研究重組紅火蟻毒素蛋白Sol iⅣ致病機理。 本實驗在國內(nèi)首次利用大腸桿菌表達紅火蟻重組毒素蛋白Sol iⅣ。誘導表達菌經(jīng)超聲波破碎,表達產(chǎn)物存在于上清液,紅火蟻重組毒素蛋白Ⅳ在表達工程菌BL-21中為可溶性表達。用鎳層析樹脂純化超聲波破菌后的上清液,得到的高純度蛋白。 通過注射小鼠和兔子進行蛋白活性試驗,選出合適的動物模型,發(fā)現(xiàn)兔子要比小鼠敏感,有40%左右的兔子出現(xiàn)過敏。再通過蛋白皮試試驗選擇敏感動物,進行蛋白免疫治療試驗,發(fā)現(xiàn)通過小劑量多次注射該蛋白,能起到免疫治療效果。 通過分離兔外周血淋巴細胞進行培養(yǎng),經(jīng)不同濃度的重組紅火蟻毒素蛋白Sol iⅣ分別和細菌脂多糖(LPS)、刀豆蛋白(ConA)共同刺激后,MTT法測定淋巴細胞的增殖情況,重組紅火蟻毒素蛋白Sol iⅣ濃度為25μg/mL、50μg/mL、75μg/mL和LPS共刺激時,與單獨LPS刺激的對照組比較,淋巴細胞增殖活性顯著增高(P＜0.05);濃度為15μg/mL、25μg/mL、50μg/mL、75μg/mL和ConA共刺激時,與單獨ConA刺激的對照組比較,淋巴細胞增殖活性顯著增高(P＜0.05)。 通過兔子的致敏實驗,觀察受試兔子的皮膚、肝臟、肺臟、腎臟、脾臟、等組織的組織病理變化,皮膚:真皮層發(fā)生壞死,壞死灶周圍出現(xiàn)大量的嗜酸性粒細胞和炎性細胞。肝臟:肝細胞內(nèi)可見水泡變性和顆粒變性,肝竇內(nèi)出現(xiàn)大量嗜酸性粒細胞。肺臟:肺泡壁毛充血,腫脹,肺泡壁內(nèi)有多量嗜酸性粒細胞。進一步通過ELISA和熒光定量RT-PCR方法檢測過敏兔子血清和組織中的IgE和細胞因子,IL-4、IFN-γ、IL-6和IL-10變化。結(jié)果顯示血清中IL-4、總IgE和IL-10的水平升高,IL-4和IL-10在14h左右達到最高值,總IgE在20 h達到峰值。IFN-γ的水平降低,在20 h達到最低值,IL-6的水平?jīng)]有顯著的變化。重組毒素蛋白Sol iⅣ注射后32 h,實驗組兔脾臟和淋巴結(jié)中除IL-6 mRNA外,IL-4和IL-10 mRNA量均極顯著高于對照組兔(P＜0.01),IFN-γ則極顯著低于對照組兔(P＜0.01)。實驗組兔脾臟中IL-4 mRNA的表達量是對照組兔的8.3倍,IL-10 mRNA的表達量是對照組的7.2倍,而IFN-γmRNA的表達量只有對照組兔的13.4%;淋巴結(jié)中,實驗組兔IL-4 mRNA的表達量是對照組兔的6倍,IL-10 mRNA實驗組兔是對照組兔的16.4倍,IFN-γmRNA的表達量只有對照組兔的12.8%;而IL-6mRNA對照組和實驗組均沒有統(tǒng)計學差異 結(jié)論一定濃度的重組紅火蟻毒素蛋白Sol iⅣ能引起體外培養(yǎng)的T、B淋巴細胞增殖,過敏體質(zhì)兔在重組紅火蟻毒素蛋白Sol iⅣ刺激后能引起Ⅰ型變態(tài)反應。
[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- 緯, 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-緯 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- 緯 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- 緯 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- 緯 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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R384
本文編號：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- 緯, 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-緯 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- 緯 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- 緯 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- 緯 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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R384
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