斑馬魚脂肪酸去飽和酶 2之選殖

利用生物資訊資料庫 NCBI （National Center for Biotechnology Information） 中的搜尋比對工具 BLAST （Basic Local Alignment Search Tool），搜尋到一組為 Delta-6 Desaturase 的人類（編號NP_004256/NM_00426）與斑馬魚（編號NP_571720.2/NM_131645）脂肪酸去飽和酶 2 (fatty acid desaturase-2； FADS2)。此斑馬魚脂肪酸去飽和酶 2 cDNA 之開放閱讀框架（Open Reading Frame）含有1335個鹼基對( base pair； bp)，可轉譯成444個胺基酸，估計分子量52.14 kDa，已完全成功地被放大出來，作為表達質體。人類脂肪酸去飽和酶 2 cDNA之開放閱讀框架含有1335個鹼基對，可轉譯成444個胺基酸，估計分子量52.27 kDa。但人類脂肪酸去飽和酶 2 未成功放大其開放閱讀框架之序列。藉由 pQE 表達質體系統，將斑馬魚脂肪酸去飽和酶 2表達與純化出來，再以 SDS-PAGE 分析此重組蛋白質，結果顯示斑馬魚蛋白質分子量如預期所估計為52.14 kDa。另外發現大腸桿菌表現系統可能不是穩定大量表現這重組蛋白質的系統。另外此酵素經北方墨點分析(Northern Blotting)，發現在 Sprague-Dawley 大鼠的腦、心、胃、肝、睪丸皆有表現。

Based on the data mining against NCBI（National Center for Biotechnology Information） data band. The analysis found a fatty acid desaturase-2 (FADS2) of human（NP_004256/NM_00426）and zebrafish（NP_571720.2/NM_131645）. The FADS2 cDNA of zebrafish were successfully amplified with a continuous open reading frame of 1335 bp encoding a protein of 444 amino acid. But the FADS2 cDNA of human were not successfully amplified with a continuous open reading frame of 1335 bp .The FADS2 of human and zebrafish were with a calculated molecular mass of 52.27 and 52.14 kDa .Zebrafish FADS2 were cloned and expressed with a 6-histidine tag for specific purification. The purified recombinant protein have mobility of 52.14 kDa on sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. We discovered that E. coli expression system may not be an appropriate system to express stable abundance of the recombinant protein。In addition, Northern blotting analysis showed that fatty acid desaturase-2 is expressed in the Sprague-Dawley rat tissues, such as brain, heart, liver, stomach, and testis.