構築含人類腎上腺腦白質退化症蛋白ALDP基因的酵母菌表現質體並於酵母菌中分析其蛋白特性

Construction of a yeast expression plasmid containing the gene of human adrenoleucodystrophy protein (ALDP) and property analysis of ALDP protein in Saccharomyces cerevisiae

陳雅雯

人類腎上腺腦白質退化症；過氧化體；極長鏈脂肪酸；同源二聚體；異源二聚體。 ； Adrenoleukodystrophy；peroxisome；very long-chain fatty acids；homodimer；heterodimer.

中山醫學大學生化微生物免疫研究所學位論文

2017年

碩士

蔡榮宗

繁體中文

摘要: 人類腎上腺腦白質退化症(Adrenoleukodystrophy, ALD)是一種罕見性聯隱性遺傳疾病，85%由母親遺傳X染色體，發病率約1/20000~1/50000。與此病有關的基因是位於X染色體長臂Xq28位置的ATP-binding cassette, sub-family D, member 1基因(ABCD1)，若基因有突變會使得患者細胞中的過氧化體(peroxisome)因運輸脂肪酸功能異常，無法順利地代謝C24~C26以上之極長鏈脂肪酸(very long-chain fatty acids,VLCFA)，病患的大腦白質與腎上腺皮質會沉積大量極長鏈脂肪酸，而侵害腦神經系統的髓鞘質(myelin)，造成神經傳導出現障礙，ALD病患經診斷後通常數年內會死亡。人類ABCD1基因所表達的ALDP蛋白歸屬於ABC運輸蛋白家族，當基因正常時，過氧化體膜上的兩個半ABC運輸蛋白ALDP透過ATP水解形成同源二聚體(homodimer)，協助運輸極長鏈飽和脂肪酸。真核生物酵母菌也含有相似於人類的過氧化體半ABC運輸蛋白，稱為Pxa1p與Pxa2p，它們會形成異源二聚體(heterodimer)，在經由ATP水解輔助運輸脂肪酸進入過氧化體內進行β氧化作用方面而言是人類ALDP的同功同源蛋白，因此我們選用酵母菌作為ALDP的研究工具。我的研究是以primer-243、244擴增pCS6(BC015541)上的ALDP cDNA片段當作insert，以趙俊欽學長構築的pCC9切除PXA1-HA之後當作載體構築成pCY1，再以這個質體做為模板以primer-HS1、250擴增接上HA tag構築出pCY2，接著將pCY2送入酵母菌BJ2168中表現蛋白，採用洪偉智學長建立的快速製備酵母菌裂解物方法進行蛋白質表現分析，另外取黃傑懋學長構築的pET-15B-DH4ΔE-PXA1-CHA送入大腸桿菌BL21(DE3)，以IPTG誘導蛋白表現作為HA抗體在西方墨點法中的控制組。由西方墨點分析結果發現，與控制組BJ2168 (pRS416-Gal)相比，BJ2168 (pCY2)在預期的分子量位置並無出現任何蛋白質訊號，因此pCY2質體在酵母菌中無法表現出蛋白。將來可能需要更換成其他酵母菌的promoter (例如GAL promoter)，測試是否可以成功表達人類ALDP蛋白。能夠成功表達ALDP蛋白才能解釋突變的效應並提供ALD疾病病理的生化基礎。

Abstract Human adrenal white matter degeneration (Adrenoleukodystrophy, ALD) is a rare recessive genetic disorder. 85% of ALD patients inherit the defective gene from mother’s X chromosome and so this disease is called X-ALD. The incidence rate of X-ALD is about 1/20000 ~ 1/50000. X-ALD is genetically linked to ABCD1 gene (the ATP-binding cassette, the sub-family D). The mutation of this gene resulted in disability to transporting the very-long-chain (C24~C26 or above) saturated fatty acid (VLCFA) into peroxisome for β-oxidation. The patient's white matter and adrenal cortex will deposit a large number of VLCFA. The VLCFA accumulation will be harmful to the myelin of the brain system, resulting in nerve degenerated disorders. The ALD patients will die usually within a few years after diagnosis. ALDP protein belongs to the half-ABC transporter protein family. When the ALDP gene is normal, the two ALDP on the peroxidation membrane form a homodimer through the help of ATP hydrolysis and function in transporting VLCFA. Eukaryotic yeasts also contain human-like peroxisome half-ABC transport proteins, called Pxa1p and Pxa2p, which form heterodimers. They are the orthologous protein of human ALDP in aspects of β-oxidation and transport of long-chain fatty acids into peroxisome. Therefore, we use yeast as a research tool for ALDP. In previous studies of others, it has been known that ALDP gene can be expressed in yeast. So, I want to clone ALDP cDNA into yeast expression vector to express ALDP protein. I use a ALDP cDNA containing plasmid pCS6 (BC015541) as a PCR template and primer 243/244 to perform PCR cloning and replace PXA1 gene within pCC9 constructed by Jiun-Chin Jau, making the plasmid pCY1. In pCY1, ALDP expression was driving by the promoter of PXA1. Then, I used pCY1 as a template and primer HS1/250 to perform PCR cloning for introducing HA tag to c-terminus of ALDP, making pCY2. I transformed the plasmid pCY2 into the yeast BJ2168 to express protein. Besides, I did the transformation of pET-15B-DH4ΔE-PXA1-CHA, which was constructed by Huang Jie-Mao, into BL21 (DE3) and induced PXA1C-HA protein expression by IPTG. This overexpressed PXA1C-HA protein was used as HA antibody control in the western blot assay. I prepared the yeast lysate by the quite convenient method developed by Wei-Zhi Hong. The western blot analysis of total lysate from BJ2168 (pCY2) showed no significant protein expression as compared with BJ2168 (pRS416-Gal). This result indicated ALDP protein could not be overproduce under regulation of PXA1 promoter from pCY2. In the future, we will replace PXA1 promoter with more powerful yeast GAL promoter for successfully producing ALDP protein. The successful overproduction of ALDP in yeast will provide us a biochemical basis for pathology of ALD disease.

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