串珠镰孢霉D-泛解酸内酯水解酶基因的克隆及在大肠杆菌中的表达  

Cloning and Expression of Fusarium moniliforme CGMCC 0536 D-Lactonohydrolase Gene in Escherichia coli

作　　者：柳志强[1] 孙志浩[1] 

机构地区：[1]江南大学生物工程学院生物催化研究室,无锡214036

出　　处：《生物工程学报》2005年第3期390-395,共6页Chinese Journal of Biotechnology

基　　金：国家 973项目 (No .2 0 0 3CB7160 0 8);国家自然科学基金项目 (No.2 0 4760 3 9)资助~~

摘　　要：利用D_泛解酸内酯水解酶N末端序列,并根据NCBI中公布的D_泛解酸内酯水解酶cDNA序列设计了一个特异引物,该引物结合Oligo(dT) 1 5,以串珠镰孢霉(Fusariummoniliforme)CGMCC 0 5 36mRNA反转录得到的总cDNA为模板进行扩增,获得约1 5kb左右的片段,将其克隆到T载体上进行测序,对测得的序列进行分析,重新设计了一对引物,并在引物两端分别加上限制酶EcoRⅠ和SalⅠ的识别位点序列,利用热启动PCR成功地扩增出了D_泛解酸内酯水解酶基因,基因片段长度为114 6bp ,该序列同来源于尖镰孢霉菌(F .oxysporum)AKU 370 2菌株的编码D_泛解酸内酯水解酶cDNA结构基因的同源性为90 0 6 %。将所得片段定向克隆到pTrc99a载体中,转化至JM10 9感受态细胞,筛选出了阳性克隆。经IPTG诱导阳性菌,进行SDS_PAGE电泳,检测出在约4 0kD处有一蛋白表达带。对两株重组基因工程菌的比活力进行测定,结果分别为37U和4 1U。The total cDNA obtained through reverse transcription of F. oxysporum CGMCC 0536 mRNA used as template, a fragment about 1.5kb was amplied with oligo(dT) 15 primer and a gene specific primer designed on the base of the sequence of both NH2-terminus and the cDNA sequence encoding D-lactonohydrolase of Fusarium oxysporum reported on the NCBI, then the fragment was cloned to the pMD18-T vector and sequenced. The sequence encoding D-lactonohydrolase of F. moniliforme CGMCC 0536 shows a high homology of 90.06% with that of F. oxysporum indicating that the gene encoding D-lactonohydrolase is highly conservative. Two specific primers were designed according to the sequence result, and a fragment, 1146bp, was amplied using hot start PCR with these two specific primers. Subsequently, the resulting products were digested with EcoRⅠ and SalⅠ and ligated to the pTrc99a vector digested with the same enzymes using T4 DNA ligase. the recombinant plasmid, pTrc99a-LAC, was transformed into Escherichia coli JM109. The two positive clones were induced with IPTG, and enzymes expressed in Escherichia coli JM109, the enzyme activity was about 37U and 41U respectively. The expression products were analyzed by SDS-polyacrylamide gel electrophoresis indicating that about 40kD protein was obtained.

关 键 词：串珠镰孢霉菌 D-泛解酸内酯水解酶基因 cDNA 克隆 原核表达 

分 类 号：Q785[生物学—分子生物学]