烟草表达粉尘螨1类变应原及其免疫治疗研究
发布时间：2018-11-25 20:28
【摘要】：目的构建粉尘螨Ⅰ类变应原(Der f1)瞬时表达载体TRBO-Der f1,观察其在烟草中的表达并探讨烟草表达的Der f1疫苗免疫治疗过敏性哮喘的疗效。 方法 1.重组质粒TRBO-Der f1的构建及在烟草叶片中的表达 以粉尘螨总RNA为模板,采用RT-PCR方法扩增Derf1基因并定向克隆到瞬时表达载体TRBO,转化根癌农杆菌(Agrobacterium tumefaciens) GV3101株后,抽提质粒进行酶切及PCR鉴定。将含TRBO-Der f1重组质粒的GV3101注射本氏烟叶片,SDS-PAGE电泳检测注射3、4、5、6天后Der f1的表达。 2.烟草表达的粉尘螨Ⅰ类变应原对哮喘小鼠气道炎症的免疫治疗研究 100只BALB/c小鼠随机分为5组,分别为PBS组即阴性对照组,阳性对照卵白蛋白(OVA)组,原核表达Der f1(pDer f1)组,烟草表达Der f1(tDer f1)组,烟草表达蛋白免疫治疗组(tDer f1-T), PBS组用PBS,其余4组分别用OVA、pDer f1, tDer f1, tDer f1致敏激发BALB/c小鼠,于0、7、14天腹腔注射致敏,第21天雾化吸入激发,连续7天,24h后,各组小鼠引颈处死；保留tDer f1-T组继续用tDerfl免疫治疗；通过肺组织病理切片观察小鼠肺部炎症；收集支气管肺泡灌洗液(BALF)进行白细胞及分类计数；ELISA法检测BALF.脾细胞培养上清(SCCS)的特异性细胞因子IL-2、IL-4、IL-5、IL-17和IFN-y和血清中变应原特异性IgE. IgG1抗体浓度。 结果 1.重组质粒TRBO-Der f1的构建及在烟草叶片中的表达 电泳及测序表明Derfl基因克隆成功,大小为627bp。经PCR及酶切反应证实重组质粒TRBO-Der f1成功转入根癌农杆菌。SDS-PAGE电泳检测表明,该蛋白于第5和6天在烟草叶片中高表达。 2.烟草表达的粉尘螨Ⅰ类变应原对哮喘小鼠气道炎症的免疫治疗研究 烟草表达蛋白免疫治疗组肺部变态反应性炎症病理变化较模型组明显减轻；BALF中的细胞总数、嗜酸性粒细胞计数、IL-4、IL-5、IL-17、血清抗原特异性IgE抗体和脾细胞分泌IL-4、IL-5、IL-17均低于阳性对照组；BALF和SCCS中IL-2、IFN-y含量均高于阳性对照组。 结论构建的瞬时表达重组载体TRBO-Der f1成功表达于烟草叶片,为进一步研究粉尘螨植物疫苗奠定了基础；烟草表达的粉尘螨Ⅰ类变应原疫苗免疫治疗可抑制小鼠肺部过敏性炎症。图[8]表[7]参考文献[75]
[Abstract]:Objective to construct the transient expression vector TRBO-Der F1 of Der F1 and to observe its expression in tobacco and to investigate the efficacy of Der F1 vaccine in the treatment of allergic asthma. Method 1. Construction of recombinant plasmid TRBO-Der F1 and its expression in tobacco leaves using total RNA of Dermatophagoides farinae as template, Derf1 gene was amplified by RT-PCR method and cloned into TRBO, which was transformed into (Agrobacterium tumefaciens) GV3101 strain of Agrobacterium tumefaciens. The plasmids were digested by enzyme and identified by PCR. The expression of Der F1 was detected by SDS-PAGE electrophoresis after the GV3101 containing the recombinant plasmid of TRBO-Der F1 was injected into the leaf slices of Bensch tobacco, and the expression of Der F1 was detected by SDS-PAGE electrophoresis. 2. Immunotherapy of Airway inflammation in Asthmatic mice with Dermatophagoides farinae Type I allergen expressed in Tobacco 100 BALB/c mice were randomly divided into 5 groups: PBS group (negative control group) and positive control group (ovalbumin (OVA) group). Der F1 (pDer F1) group, Der F1 (tDer F1) group and tDer f1-T), PBS group (tDer f1-T), PBS group) were sensitized to BALB/c mice with OVA,pDer f1, tDer F1 and tDer F1, respectively. The mice were sensitized by intraperitoneal injection for 14 days, and stimulated by atomization inhalation on the 21st day. After 7 days and 24 hours, the mice in each group were killed. TDer f1-T group continued to be treated with tDerfl immunotherapy; lung inflammation was observed by pathological sections of lung tissue; (BALF) was collected from bronchoalveolar lavage fluid for leukocyte count and classified count; BALF. was detected by ELISA method. Specific cytokines IL-2,IL-4,IL-5,IL-17 and IFN-y and allergen-specific IgE. in serum of spleen cell culture supernatant (SCCS) IgG1 antibody concentration. Result 1. The construction of recombinant plasmid TRBO-Der F1 and its expression in tobacco leaves by electrophoresis and sequencing showed that the Derfl gene was cloned successfully with a size of 627bp. The recombinant plasmid TRBO-Der F1 was successfully transformed into Agrobacterium tumefaciens by PCR and enzyme digestion. SDS-PAGE electrophoresis showed that the protein was highly expressed in tobacco leaves on the 5th and 6th day. 2. Immunotherapy of bronchial inflammation in asthmatic mice with tobacco-expressed dermatophagoides farinae; the pathological changes of pulmonary allergic inflammation in the tobacco expression protein immunotherapy group were significantly less than those in the model group. The total number of cells in BALF, eosinophil count, IL-4,IL-5,IL-17, serum antigen-specific IgE antibody and splenocyte IL-4,IL-5,IL-17 secretion were lower than those in positive control group. The content of IL-2,IFN-y in BALF and SCCS was higher than that in positive control group. Conclusion the transient expression vector TRBO-Der F1 was successfully expressed in tobacco leaves, which laid a foundation for further research on the plant vaccine of Dermatophagoides farinae. Immunotherapy with tobacco-expressed class I allergen vaccine can inhibit allergic inflammation of lung in mice. Figure [8] Table [7] refs [75]
【学位授予单位】：安徽理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1
本文编号：2357290
[Abstract]:Objective to construct the transient expression vector TRBO-Der F1 of Der F1 and to observe its expression in tobacco and to investigate the efficacy of Der F1 vaccine in the treatment of allergic asthma. Method 1. Construction of recombinant plasmid TRBO-Der F1 and its expression in tobacco leaves using total RNA of Dermatophagoides farinae as template, Derf1 gene was amplified by RT-PCR method and cloned into TRBO, which was transformed into (Agrobacterium tumefaciens) GV3101 strain of Agrobacterium tumefaciens. The plasmids were digested by enzyme and identified by PCR. The expression of Der F1 was detected by SDS-PAGE electrophoresis after the GV3101 containing the recombinant plasmid of TRBO-Der F1 was injected into the leaf slices of Bensch tobacco, and the expression of Der F1 was detected by SDS-PAGE electrophoresis. 2. Immunotherapy of Airway inflammation in Asthmatic mice with Dermatophagoides farinae Type I allergen expressed in Tobacco 100 BALB/c mice were randomly divided into 5 groups: PBS group (negative control group) and positive control group (ovalbumin (OVA) group). Der F1 (pDer F1) group, Der F1 (tDer F1) group and tDer f1-T), PBS group (tDer f1-T), PBS group) were sensitized to BALB/c mice with OVA,pDer f1, tDer F1 and tDer F1, respectively. The mice were sensitized by intraperitoneal injection for 14 days, and stimulated by atomization inhalation on the 21st day. After 7 days and 24 hours, the mice in each group were killed. TDer f1-T group continued to be treated with tDerfl immunotherapy; lung inflammation was observed by pathological sections of lung tissue; (BALF) was collected from bronchoalveolar lavage fluid for leukocyte count and classified count; BALF. was detected by ELISA method. Specific cytokines IL-2,IL-4,IL-5,IL-17 and IFN-y and allergen-specific IgE. in serum of spleen cell culture supernatant (SCCS) IgG1 antibody concentration. Result 1. The construction of recombinant plasmid TRBO-Der F1 and its expression in tobacco leaves by electrophoresis and sequencing showed that the Derfl gene was cloned successfully with a size of 627bp. The recombinant plasmid TRBO-Der F1 was successfully transformed into Agrobacterium tumefaciens by PCR and enzyme digestion. SDS-PAGE electrophoresis showed that the protein was highly expressed in tobacco leaves on the 5th and 6th day. 2. Immunotherapy of bronchial inflammation in asthmatic mice with tobacco-expressed dermatophagoides farinae; the pathological changes of pulmonary allergic inflammation in the tobacco expression protein immunotherapy group were significantly less than those in the model group. The total number of cells in BALF, eosinophil count, IL-4,IL-5,IL-17, serum antigen-specific IgE antibody and splenocyte IL-4,IL-5,IL-17 secretion were lower than those in positive control group. The content of IL-2,IFN-y in BALF and SCCS was higher than that in positive control group. Conclusion the transient expression vector TRBO-Der F1 was successfully expressed in tobacco leaves, which laid a foundation for further research on the plant vaccine of Dermatophagoides farinae. Immunotherapy with tobacco-expressed class I allergen vaccine can inhibit allergic inflammation of lung in mice. Figure [8] Table [7] refs [75]
【学位授予单位】：安徽理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1
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