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標題: | 德國蟑螂主要過敏原精胺酸激酶的選殖、純化與免疫特性分析 Molecular Cloning, Purification and Immunological Characterization of Major Allergen from German Cockroach (Blattella Germanica) as Arginine Kinase |
作者: | Tang-Kuei Lin 林唐逵 |
指導教授: | 周綠蘋(Lu-Ping Chow) |
關鍵字: | 蟑螂,過敏,氣喘,選殖,純化, cockroach,allergen,asthma,cloning,purification, |
出版年 : | 2006 |
學位: | 碩士 |
摘要: | 蟑螂在1964年Bernton與Brown的研究中被認為與過敏性氣喘疾病有關。755個過敏病人中有44%在皮膚測試中對蟑螂呈現陽性反應,其中,有13%從前未被檢驗出異位性體質的病人,單獨地只對蟑螂有過敏反應。在台灣,根據國泰醫院1998年的報告,約有39.4%與36.4%的氣喘病人分別對美洲蟑螂(Periplaneta americana) 與德國蟑螂(Blattella germanica) 過敏,可見得在台灣,蟑螂過敏也是氣喘病發作重要的原因之一。
德國蟑螂是台灣地區住宅與市場中數量最高的一類,本實驗室利用德國蟑螂的總蛋白萃取液,與11個過敏病人的混和血清進行二維免疫轉漬分析,藉由蟑螂蛋白與IgE的反應情形,進一步尋找其他可能的過敏原,作為研究材料。 經本實驗室學長以LC MS/MS鑑定,推定分子量約45kDa,等電點約6.0的精胺酸激酶(Arginine kinase; EC 2.7.3.3)這個IgE結合蛋白可能是一個新的蟑螂過敏原。依據質譜分析所得的胜肽序列設計引子將cDNA選殖出來,並推演其蛋白質序列全長共有356個氨基酸,經序列比對發現其與印度穀蛾以及草蝦的精胺酸激酶(已被發表為過敏原),有80%以上的相同度(Identity)。接著利用離子交換樹酯管柱層析分離法從蟑螂蟲體中得到高度純化的精胺酸激酶,利用LC MS/MS以及抗草蝦精胺酸激酶的多株抗體進行蛋白身份的確認。經由分析質量圖譜得知成熟的精胺酸激酶之N端起始於第二個胺基酸Val上,且被乙醯化修飾成一個N端阻斷性的蛋白。 進一步進行免疫特性分析:利用德國蟑螂過敏病人血清進行ELISA,發現IgE與精胺酸激酶的陽性反應率為50.79% (n=63),可見德國蟑螂的精胺酸激酶是一個主要過敏原。另外在粗萃取物的ELISA抑制試驗中,純化的精胺酸激酶可以抑制總蛋白的IgE反應達50%以上,證明精胺酸激酶在蟑螂的過敏原中佔有相當重要的地位。比較ELISA篩檢與免疫轉漬的結果,推測德國蟑螂的精胺酸激酶主要以結構型抗原決定點和IgE結合。 為了探討精胺酸激酶在蟑螂物種中,是否為一個普遍存在的過敏原,我們也對已被報導為過敏原的美洲蟑螂精胺酸激酶進行純化,探討兩者間的免疫交叉反應,結果顯示在Dot-blot的實驗中,德蟑與美蟑的精胺酸激酶展現了抗原的交叉反應性(Antigenic cross-reactivity);在ELISA抑制反應試驗中,也可看到美蟑的精胺酸激酶會與德蟑精胺酸激酶競爭病人IgE的結合,因此我們可以推測兩者間可能具有相同的抗原決定點(Epitope)。因而認為精胺酸激酶是一種存在蟑螂物種中的共同過敏原(Pan-allergen)。 本實驗針對德國蟑螂的精胺酸激酶進行選殖與純化,分析其免疫特性並認為其不但是蟑螂物種中的一種主要過敏原(Major allergen),更是一個共通過敏原(Pan-allergen)。本研究對精胺酸激酶基因的選殖有助於進行重組過敏原的製備,希望此論文能提供學界更多資訊,有助於進一步的研究並瞭解蟑螂引發氣喘的機制,並在未來發展出更有效診斷和治療蟑螂過敏的新方法。 Cockroaches have been reported to be an important cause of asthma for over 40 years. In 1964, Bernton and Brown were the first to report positive skin test responses to cockroach allergen (44%) in a landmark study of 755 allergic patients living in New York. In that study, 13% of patients who otherwise would have been considered nonatopic were sensitized to cockroach allergen alone. There are also 39.4% and 36.4% allergy clinic patients sensitized to Periplaneta americana and Blattella germanica in the study of Taiwan in 1998. So cockroach allergy is also one of the important causes of asthma in Taiwan. The principal domestic cockroach species are Blattella germanica and Periplaneta americana and B. germanica is the most abundant one of indoor species. Previous identified allergens include Bla g 2 (Inactive aspartic protease), Bla g 4 (Calycin), Bla g 5 (Glutathione-S-transferase), Bla g 6 (Troponin), the group 1 cross-reactive allergens Bla g 1 and Per a 1, Bla g 7 and Per a 7 (Tropomyosin), and Per a 3 (Arylphorin). After observation and LC-MS/MS identification of separated proteins of cockroach crude extract on the 2D-immunoblot, we assumed that “Arginine kinase”, an IgE-binding protein, could be a putative novel allergen of German cockroach. In this study, this novel allergen was cloned and the deduced amino acid sequence was a 356 a.a. protein with an acetylated N terminus at Val2. Arginine kinase (AK) of German cockroach showed an extensive similarity with other allergic AK which are from shrimp and Indianmeal moth. Native AK from German cockroach was purified by ammonium sulfate fractionation and anion exchange chromatography. ELISA results showed a 50.79% prevalence of IgE-reactivity among German cockroach allergic patients and AK was able to inhibit 50% of the IgE-binding to total allergens from B. germanica bodies, which showed that AK is an important major allergen. In addition, 17 of 24 AK positive sera completely lost their IgE-reactivity under the SDS-denaturing condition, which indicate the major existence of conformational epitopes (70.8%). After purified P. americana AK was prepared, Dot-blot assay showed the antigenic cross-reactivity between B. germanica and P. americana. In ELISA inhibition assay, we observed that P. americana AK inhibited the IgE-binding to B. germanica AK, which indicated the same IgE epitope between AK from different cockroach species. Then, we demonstrated that AK is not only a major allergen but also a pan-allergen in cockroach species. In the future, we expect that the production of recombinant AK may be helpful to the development of improved forms of clinical diagnosis and immunotherapy for current cockroach allergy diseases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32936 |
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