特氏杜氏藻中乙酰辅酶A合成酶的基因克

发布时间：2018-02-03 15:59

  本文关键词： 乙酰辅酶A合成酶 特氏杜氏藻 基因克隆 纯化 比酶活　出处：《现代食品科技》2017年03期 　论文类型：期刊论文

【摘要】：乙酰辅酶A合成酶(ACS)催化合成乙酰辅酶A,它是油脂代谢和醋酸盐代谢的重要节点之一。本研究利用反转录PCR(RT-PCR)技术和cDNA末端快速扩增(RACE)技术分离得到了特氏杜氏藻(Dunaliella tertiolecta)乙酰辅酶A合成酶(Dt ACS)的c DNA全长(2464 bp),预测其开放阅读框(ORF)为2184 bp,727个氨基酸由此段编码。序列比对显示Dt ACS与绿藻ACS最为相似(与衣藻Chlamydomonas reinhardtii有68%一致;与团藻Volvox carteri f.nagariensis有70%一致)。选用带有硫氧还蛋白标签(Trx-tag)的pET32a(+)作为原核表达载体,并将Dt ACS转入pET32a(+)中从而构建了pET-32a-Dt ACS质粒。将其转入BL21(DE3)感受态细胞中,同时将pET-32a空载体也转入BL21(DE3)感受态细胞中,分别得到重组菌pET-32a-Dt ACS-BL21(DE3)和对照菌种pET-32a-BL21(DE3)。将重组菌在18℃、终浓度为0.6 mmol/L的IPTG条件下诱导12 h,表达出来的带有Trx-His标签融合蛋白的Dt ACS约为8.74 ku(6.99ku+1.75 ku)。此外,将表达得到的重组蛋白经Ni2+亲和层析柱纯化,纯化后蛋白比活力为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，

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