重組紅火蟻毒素蛋白SoliⅣ致病機(jī)理的初步研究
發(fā)布時(shí)間：2019-05-15 15:18
【摘要】： 紅火蟻屬節(jié)肢動(dòng)物門昆蟲(chóng)綱,是一種外來(lái)入侵害蟻,被認(rèn)為是嚴(yán)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(guò)敏反應(yīng)甚至死亡。本文用重組表達(dá)的紅火蟻毒素蛋白Sol iⅣ注射兔子做為動(dòng)物模型,來(lái)研究重組紅火蟻毒素蛋白Sol iⅣ致病機(jī)理。 本實(shí)驗(yàn)在國(guó)內(nèi)首次利用大腸桿菌表達(dá)紅火蟻重組毒素蛋白Sol iⅣ。誘導(dǎo)表達(dá)菌經(jīng)超聲波破碎,表達(dá)產(chǎn)物存在于上清液,紅火蟻重組毒素蛋白Ⅳ在表達(dá)工程菌BL-21中為可溶性表達(dá)。用鎳層析樹(shù)脂純化超聲波破菌后的上清液,得到的高純度蛋白。 通過(guò)注射小鼠和兔子進(jìn)行蛋白活性試驗(yàn),選出合適的動(dòng)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(guò)敏。再通過(guò)蛋白皮試試驗(yàn)選擇敏感動(dòng)物,進(jìn)行蛋白免疫治療試驗(yàn),發(fā)現(xiàn)通過(guò)小劑量多次注射該蛋白,能起到免疫治療效果。 通過(guò)分離兔外周血淋巴細(xì)胞進(jìn)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(xì)菌脂多糖(LPS)、刀豆蛋白(ConA)共同刺激后,MTT法測(cè)定淋巴細(xì)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(shí),與單獨(dú)LPS刺激的對(duì)照組比較,淋巴細(xì)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(shí),與單獨(dú)ConA刺激的對(duì)照組比較,淋巴細(xì)胞增殖活性顯著增高(P＜0.05)。 通過(guò)兔子的致敏實(shí)驗(yàn),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(xì)胞和炎性細(xì)胞。肝臟:肝細(xì)胞內(nèi)可見(jiàn)水泡變性和顆粒變性,肝竇內(nèi)出現(xiàn)大量嗜酸性粒細(xì)胞。肺臟:肺泡壁毛充血,腫脹,肺泡壁內(nèi)有多量嗜酸性粒細(xì)胞。進(jìn)一步通過(guò)ELISA和熒光定量RT-PCR方法檢測(cè)過(guò)敏兔子血清和組織中的IgE和細(xì)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(dá)到最高值,總IgE在20 h達(dá)到峰值。IFN-γ的水平降低,在20 h達(dá)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(shí)驗(yàn)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(duì)照組兔(P＜0.01),IFN-γ則極顯著低于對(duì)照組兔(P＜0.01)。實(shí)驗(yàn)組兔脾臟中IL-4 mRNA的表達(dá)量是對(duì)照組兔的8.3倍,IL-10 mRNA的表達(dá)量是對(duì)照組的7.2倍,而IFN-γmRNA的表達(dá)量只有對(duì)照組兔的13.4%;淋巴結(jié)中,實(shí)驗(yàn)組兔IL-4 mRNA的表達(dá)量是對(duì)照組兔的6倍,IL-10 mRNA實(shí)驗(yàn)組兔是對(duì)照組兔的16.4倍,IFN-γmRNA的表達(dá)量只有對(duì)照組兔的12.8%;而IL-6mRNA對(duì)照組和實(shí)驗(yàn)組均沒(méi)有統(tǒng)計(jì)學(xué)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(xì)胞增殖,過(guò)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(yīng)。
[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(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R384
本文編號(hào)：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(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R384
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