- 期刊
表現全長類固醇生成急性調控蛋白及其抗體之製備
Expression of Full-length StAR and Production of Its Antibodies
摘要
類固醇內泌素為維持動物體生命與延續種族繁衍所必需。在類固醇生成步驟中，所有類固醇均來自唯一的前驅物「膽固醇」，而類固醇生成急性調控蛋白(steroidogenic acute regulatory protein, StAR)負責將膽固醇由粒線體外膜運送至內膜，再經過其他酵素的修飾轉變為各式類固醇。本研究以蛋白質表現系統來大量生產高純度的重組StAR蛋白，藉以生產StAR抗血清，以利進行類固醇生成的相關研究。以人類腎上腺皮質細胞株萃取總RNA做為模版，將一對末端具有限制切位的專一性引子對以RT-PCR方式增幅StAR的全長cDNA，再將其殖入蛋白質表現載體pET-29a，以限制切割、核酸定序及胺基酸讀碼的分析將所獲得正確的StAR表現質體稱為pET-29a-StAR。將pET-29a-StAR分別轉形送入二種大腸桿菌表現宿主：BL21 (DE3)與BL21-CodonPlus-(DE3)-RIPL，再經IPTG誘導重組StAR蛋白的表現，實驗結果顯示，BL21-CodonPlus- (DE3)-RIPL可以表現較大量的重組StAR蛋白。隨後以Ni-NTA His Bind樹脂純化，獲得高純度的重組StAR蛋白。以純化的重組StAR免疫於BALB/c小鼠，再收取其血清，以西方墨點法確認此血清除了可以辨認人類來源的StAR外，也可辨認小鼠來源的StAR。本研究成功的表現出全長的StAR與產生能辨識來自人或小鼠StAR的抗血清。
並列摘要
Steroids are important for physiological homeostasis and reproduction. Steroidogenic acute regulatory protein (StAR) controls the first step in the steroidogenesis to transport cholesterol, the sole substrate initiating steroid biosynthesis, from outer to inner mitochondrial membrane. This study produced StAR antiserum by E. co/i-expressed recombinant StAR. The StAR cDNA fragment encoding full-length protein was amplified by RT-PCR using restriction enzyme site-tailed primers and human cDNA as template, and subcloned into expression vector, pET-29a, to generate a expression plasmid, pET-29a-StAR, tagged with 6xHis. After confirmation by restriction enzyme digestion and sequencing, pET-29a-StAR was transformed into E. co/i expression host, BL21 (DE3) or BL21-CodonPlus (DE3)-RIPL, and induced by IPTG to product recombinant StAR. The recombinant StAR expressed in BL21-CodonPlus-(DE3)-RIPL was much higher than that in BL21 (DE3). Subsequently, the specificity of antiserum from recombinant StAR-challenged BALB/c mice was tested by Western blotting analysis, with confirmed the StAR antiserum recognized both human- and mouse-originated StAR. This study successfully expresses full-length recombinant StAR and obtains antiserum recognizing StAR from human and mouse.
延伸閱讀
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