本文關(guān)鍵詞： 乙酰輔酶A合成酶 特氏杜氏藻 基因克隆 純化 比酶活　出處：《現(xiàn)代食品科技》2017年03期 　論文類(lèi)型：期刊論文

【摘要】：乙酰輔酶A合成酶(ACS)催化合成乙酰輔酶A,它是油脂代謝和醋酸鹽代謝的重要節(jié)點(diǎn)之一。本研究利用反轉(zhuǎn)錄PCR(RT-PCR)技術(shù)和cDNA末端快速擴(kuò)增(RACE)技術(shù)分離得到了特氏杜氏藻(Dunaliella tertiolecta)乙酰輔酶A合成酶(Dt ACS)的c DNA全長(zhǎng)(2464 bp),預(yù)測(cè)其開(kāi)放閱讀框(ORF)為2184 bp,727個(gè)氨基酸由此段編碼。序列比對(duì)顯示Dt ACS與綠藻ACS最為相似(與衣藻Chlamydomonas reinhardtii有68%一致;與團(tuán)藻Volvox carteri f.nagariensis有70%一致)。選用帶有硫氧還蛋白標(biāo)簽(Trx-tag)的pET32a(+)作為原核表達(dá)載體,并將Dt ACS轉(zhuǎn)入pET32a(+)中從而構(gòu)建了pET-32a-Dt ACS質(zhì)粒。將其轉(zhuǎn)入BL21(DE3)感受態(tài)細(xì)胞中,同時(shí)將pET-32a空載體也轉(zhuǎn)入BL21(DE3)感受態(tài)細(xì)胞中,分別得到重組菌pET-32a-Dt ACS-BL21(DE3)和對(duì)照菌種pET-32a-BL21(DE3)。將重組菌在18℃、終濃度為0.6 mmol/L的IPTG條件下誘導(dǎo)12 h,表達(dá)出來(lái)的帶有Trx-His標(biāo)簽融合蛋白的Dt ACS約為8.74 ku(6.99ku+1.75 ku)。此外,將表達(dá)得到的重組蛋白經(jīng)Ni2+親和層析柱純化,純化后蛋白比活力為52.87 U/mg。
[Abstract]:Acetyl coenzyme A synthase (ACSs) catalyzes the synthesis of acetyl coenzyme A. It is one of the most important nodes in lipid metabolism and acetate metabolism. In this study, reverse transcription PCR reverse transcription (RT PCR) and rapid amplification of cDNA terminal were used in this study. The c DNA of Dunaliella tertiolecta) acetyl-coenzyme A synthase (Dt ACSA) was isolated from Dunaliella tertiolecta. by this technique. The total length of c DNA was 2464 BP). The ORF was predicted to be 2184 BP. The sequence alignment shows that Dt ACS is the most similar to Chlorophyta ACS. There was 68% agreement with Chlamydomonas reinhardtii. There was 70% agreement with Volvox carteri f.nagariensis. The pET32a () with thioredoxin tag was selected. As prokaryotic expression vector. PET-32a-Dt ACS plasmid was constructed by transferring Dt ACS into pET32a (). At the same time, the empty pET-32a vector was also transferred into BL21DDE3) competent cells. The recombinant bacteria pET-32a-Dt ACS-BL21DE3) and the control strain pET-32a-BL21DE3DE3 were obtained, respectively. The recombinant bacteria were obtained at 18 鈩，

本文編號(hào)：1487781