蛇毒纤溶酶ALF基因在大肠杆菌中的表达

Expression of snake venom fihrino(geno) lytic enzyme alfimeprase in E.coli

根据蛇毒纤溶酶(A lfim eprase,ALF)氨基酸序列和大肠杆菌密码子偏爱性,设计了14条单链DNA,采用重叠延伸PCR方法人工合成了ALF基因编码区。通过克隆使其分别连接在融合标签N usA,T h ioredox-in,Skp,H is T ag和信号肽pe lB的C端,构建成了多种ALF表达载体p43-A lf,p25-A lf,p32-A lf,p15-A lf和pSkp-A lf,转化大肠杆菌后进行了诱导表达,并对表达产物进行了SDD-PAGE分析。实验结果表明,合成的ALF基因长度为603 bp;构建的表达载体在大肠杆菌中均获得了很好的表达,其中pSkp-A lf表达量最高,目的蛋白含量高达菌体总蛋白的45%。

According to the amino acid sequence of fihrino （geno）lytic emzyme Alfimeprase （ALF） and optimal codon usage orE. coli,the cDNA of alfimeprase was amplified by recursive PCR,and cloned into pET43, la, p25b （＋）, pET32a, pET15b （＋） and pETSkp vectors to generate fusions with NusA, Thioredoxin,Skp, 6 × His and signal peptide pelB respectively. Expression was induced after transformation into the E. coli strain. The results showed that the length of ALF coding sequence was about 603 bp,and all of the fusion vectors had high level expression in E. coli,the highest being the expression level of pSkp-Alf, reaching as high as 45 percent in proportion to the total protein.