煙草表達(dá)粉塵螨1類變應(yīng)原及其免疫治療研究
發(fā)布時間：2018-11-25 20:28
【摘要】：目的構(gòu)建粉塵螨Ⅰ類變應(yīng)原(Der f1)瞬時表達(dá)載體TRBO-Der f1,觀察其在煙草中的表達(dá)并探討煙草表達(dá)的Der f1疫苗免疫治療過敏性哮喘的療效。 方法 1.重組質(zhì)粒TRBO-Der f1的構(gòu)建及在煙草葉片中的表達(dá) 以粉塵螨總RNA為模板,采用RT-PCR方法擴增Derf1基因并定向克隆到瞬時表達(dá)載體TRBO,轉(zhuǎn)化根癌農(nóng)桿菌(Agrobacterium tumefaciens) GV3101株后,抽提質(zhì)粒進行酶切及PCR鑒定。將含TRBO-Der f1重組質(zhì)粒的GV3101注射本氏煙葉片,SDS-PAGE電泳檢測注射3、4、5、6天后Der f1的表達(dá)。 2.煙草表達(dá)的粉塵螨Ⅰ類變應(yīng)原對哮喘小鼠氣道炎癥的免疫治療研究 100只BALB/c小鼠隨機分為5組,分別為PBS組即陰性對照組,陽性對照卵白蛋白(OVA)組,原核表達(dá)Der f1(pDer f1)組,煙草表達(dá)Der f1(tDer f1)組,煙草表達(dá)蛋白免疫治療組(tDer f1-T), PBS組用PBS,其余4組分別用OVA、pDer f1, tDer f1, tDer f1致敏激發(fā)BALB/c小鼠,于0、7、14天腹腔注射致敏,第21天霧化吸入激發(fā),連續(xù)7天,24h后,各組小鼠引頸處死；保留tDer f1-T組繼續(xù)用tDerfl免疫治療；通過肺組織病理切片觀察小鼠肺部炎癥；收集支氣管肺泡灌洗液(BALF)進行白細(xì)胞及分類計數(shù)；ELISA法檢測BALF.脾細(xì)胞培養(yǎng)上清(SCCS)的特異性細(xì)胞因子IL-2、IL-4、IL-5、IL-17和IFN-y和血清中變應(yīng)原特異性IgE. IgG1抗體濃度。 結(jié)果 1.重組質(zhì)粒TRBO-Der f1的構(gòu)建及在煙草葉片中的表達(dá) 電泳及測序表明Derfl基因克隆成功,大小為627bp。經(jīng)PCR及酶切反應(yīng)證實重組質(zhì)粒TRBO-Der f1成功轉(zhuǎn)入根癌農(nóng)桿菌。SDS-PAGE電泳檢測表明,該蛋白于第5和6天在煙草葉片中高表達(dá)。 2.煙草表達(dá)的粉塵螨Ⅰ類變應(yīng)原對哮喘小鼠氣道炎癥的免疫治療研究 煙草表達(dá)蛋白免疫治療組肺部變態(tài)反應(yīng)性炎癥病理變化較模型組明顯減輕；BALF中的細(xì)胞總數(shù)、嗜酸性粒細(xì)胞計數(shù)、IL-4、IL-5、IL-17、血清抗原特異性IgE抗體和脾細(xì)胞分泌IL-4、IL-5、IL-17均低于陽性對照組；BALF和SCCS中IL-2、IFN-y含量均高于陽性對照組。 結(jié)論構(gòu)建的瞬時表達(dá)重組載體TRBO-Der f1成功表達(dá)于煙草葉片,為進一步研究粉塵螨植物疫苗奠定了基礎(chǔ)；煙草表達(dá)的粉塵螨Ⅰ類變應(yīng)原疫苗免疫治療可抑制小鼠肺部過敏性炎癥。圖[8]表[7]參考文獻[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]
【學(xué)位授予單位】：安徽理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1
本文編號：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]
【學(xué)位授予單位】：安徽理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392.1
【參考文獻】
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