API2-MALT1融合蛋白質對BCL10蛋白質之影響

Effects of API2-MALT1 Fusion Protein on BCL10 Protein

蕭雅今(Ya-Jin Hsiao)

指導教授 ： 董馨蓮

國立臺灣大學/醫學院/微生物學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01600

摘要

中文摘要
胃相關黏膜組織淋巴癌的發生與幽門螺旋桿菌的感染有關，所以臨床上對於胃相關黏膜組織淋巴癌通常施以抗生素治療。在抗生素治療後，50~70﹪的患者能夠痊癒，然而仍有少部分的患者對於抗生素治療是沒有反應。研究這些對於抗生素治療沒有反應的患者基因後發現，這些患者帶有一些基因變異，包括了：t(11；18)(q21；q21)、t(1；14)(p22；q32)，而且其BCL10會異常的由細胞質轉往細胞核表現。
t(11；18)(q21；q21)染色體變異會造成11號染色體上的API2的基因位移到18號染色體上的MALT1基因之前，產生API2-MALT1融合蛋白質。在先前的研究報導認為API2-MALT1融合蛋白質的表現跟BCL10在細胞核表現有關聯性，所以在這篇論文裡我們想要探討的問題是API2-MALT1 融合蛋白質對BCL10在細胞中表現的影響。
首先，我們構築了能夠在細胞內表現不同斷點的API2-MALT1融合蛋白質的表現質體，包括了pAPI2-MALT1Δ5’UTR(0-Ig)、pAPI2-MALT1Δ5’UTR(1-Ig)、pAPI2-MALT1Δ5’UTR(2-Ig)。透過北方墨點法我們可以偵測到API2-MALT1融合基因的轉錄本表現。但是只有pAPI2-MALT1Δ5’UTR(2-Ig)表現質體能夠偵測到其蛋白質的表現。接下來，我們透過短暫轉殖技術，將其與帶有綠螢光蛋白質的BCL10送入細胞內表現，觀察API2-MALT1融合蛋白質對於BCL10蛋白質的影響。我們發現了API2-MALT1融合蛋白質能夠增加帶有綠色螢光的BCL10或是BCL10L41R的mRNA以及蛋白質表現。
另外一方面，為了避免在繁複構築的過程中造成DNA序列改變，所以我們也利用了RT-PCR技術，直接得到了胃相關黏膜組織淋巴癌患者檢體cDNA。我們意外的發現帶有t(11；18)(q21；q21)基因變異的患者之其API2-MALT1融合基因cDNA約5.5kb (預期大小約為4kb)。經過電腦程式比對後，我們發現插入的DNA片段大小約為1.3kb，屬於跳躍子 Tn10中的IS10，且其插入位置在MALT1基因的第一個Ig domain上。接著構築帶有IS10序列的API2-MALT1表現質體。透過短暫轉殖技術將此表現質體送入細胞內表現，發現患者的API2-MALT1 cDNA能夠表現mRNA卻無法有效的表達出API2-MALT1融合蛋白質。
在本篇論文中，我們建立了能夠表現API2-MALT1融合蛋白質的表現質體—pAPI2-MALT1Δ5’UTR(2-Ig)，可以做為將來研究API2-MALT1融合蛋白質與BCL10蛋白質交互關係的工具。

關鍵字

API2-MALT1融合蛋白質 ； BCL10蛋白質

並列摘要

Abstract
Gastric mucosa-associated lymphoid tissue(MALT) lymphoma is associated with the infection of Helicobacter pylori. In clinics﹐patients with MALT lymphoma were always treated with anti-H.pylori antibiotics. Approximately 50 to 70﹪of patients with MALT lymphoma can be cured. Several genetic changes were observed in patients showing no response to antibiotics treatment﹐including t(11;18)(q21;q21) ﹐t(1;14)(p22;q32) and the abnormal nuclear expression of BCL10.
Chromosome translocation t(11；18)(q21；q21) leads to the expression of a chimeric protein fusing the API2 gene on chromosome 11 to the MALT1 gene on chromosome 18. Several studies demonstrated that the presence of the API2-MALT1 fusion was associated with aberrant nuclear BCL10. In this study﹐we would like to investigate the effects of API2-MALT1 fusion proteins on the BCL10 protein﹐and possibly elucidate the mechanism(s) mediating the effects.
Firstly﹐expression vectors of API2-MALT1 fusion gene with different break points were constructed﹐including pAPI2-MALT1Δ5’UTR(2-Ig)、pAPI2-MALT1Δ5’UTR(1-Ig)、pAPI2-MALT1Δ5’UTR(0-Ig). Northern blot analysis confirmed the presence of API2-MALT1 transcripts from the three constructs. However﹐the expression of API2-MALT1 fusion protein could be detected only from cells transfected with pAPI2-MALT1Δ5’UTR(2-Ig) expression vector. Expression vector pAPI2-MALT1Δ5’UTR(2-Ig) was then cotransfected with BCL10GFP or mutant BCL10L41RGFP expression vector to examine the effect of API2-MALT1 fusion on BCL10 protein. Interstingly, API2-MALT1 fusion protein enhanced the expression of BCL10GFP or mutant BCL10L41RGFP at both protein and mRNA levels.
In addition to the complexed and multiple cutting, ligating steps from established API2, MALT1 cDNA clones, we also tried to clone API2-MALT1 fusion cDNA directly from MALT lymphoma biopsy. A 5.5 kb cDNA (larger than expected 4kb) was obtained from one MALT lymphoma biopsy. Sequence analysis indicated the insertion of a 1.3Kb IS10 DNA fragment in the first Ig domain of MALT1 gene, generating a API2-IS10-MALT1 fusion. Expression vector containing this novel API2-MALT1 fusion was constructed. Northern blot analysis demonstrated the expression of this novel API2-MALT1 fusion at mRNA level. However, we failed to detect this novel API2-MALT1 fusion protein by western blot analysis.
In this study, we were able to establish the expression system of API2-MALT1 fusion, offering a tool for further studies on the interplay between API2-MALT1 fusion and BCL10.

並列關鍵字

API2-MALT1 Fusion Protein ； BCL10 Protein

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