Pettersson R F, Lundström K, Chattopadhyaya J B, Josephson S, Philipson L, Kääriäinen L, Palva I
Gene. 1983 Sep;24(1):15-27. doi: 10.1016/0378-1119(83)90127-0.
We have chemically synthesized an oligonucleotide 5'd(TGATTGATTGA)3' 3'd(ACTAACTAACT)5' that encodes the translation termination codon TGA in all three reading frames. After ligation of appropriate restriction endonuclease linkers to the ends, the double-stranded oligonucleotide (STOP-oligonucleotide) was joined to the plasmid pBR322 between the EcoRI and BamHI, or HindIII and BamHI sites, and the hybrid plasmids were transformed into Escherichia coli HB101. Four different constructions were obtained: (i) EcoRI-STOP-BamHI (STOP-oligonucleotide flanked by EcoRI and BamHI linkers; pKTH606), (ii) HindIII-STOP-BamHI (pKTH601), (iii) BamHI-STOP-HindIII (pKTH604), and (iv) HindIII-STOP-POTS-BamHI (two STOP-oligonucleotides in opposite orientation; pKTH605). The inserts in pKTH606 and pKTH601 were excised and transferred to a modified plasmid constructed previously for the expression and secretion of foreign gene products from Bacillus subtilis. The resulting secretion plasmids now contain the promoter/signal sequence region of the alpha-amylase gene from Bacillus amyloliquefaciens joined to the STOP-oligonucleotide by EcoRI or HindIII linkers. Foreign genes can be cloned into these sites. The plasmids can be used to express foreign genes truncated at their C-terminal end and therefore lacking their own translation termination codon. One such plasmid has been successfully used to express the Semliki Forest virus (SFV) membrane protein E1 truncated at its C-terminus.
我们化学合成了一种寡核苷酸5'd(TGATTGATTGA)3' 3'd(ACTAACTAACT)5'，它在所有三个阅读框中都编码翻译终止密码子TGA。在将适当的限制性内切酶接头连接到末端后，将双链寡核苷酸（终止寡核苷酸）连接到质粒pBR322的EcoRI和BamHI之间，或HindIII和BamHI位点之间，然后将杂种质粒转化到大肠杆菌HB101中。获得了四种不同的构建体：(i) EcoRI-终止-BamHI（终止寡核苷酸两侧为EcoRI和BamHI接头；pKTH606），(ii) HindIII-终止-BamHI（pKTH601），(iii) BamHI-终止-HindIII（pKTH604），以及(iv) HindIII-终止-POTS-BamHI（两个终止寡核苷酸方向相反；pKTH605）。将pKTH606和pKTH601中的插入片段切除，并转移到先前构建的用于从枯草芽孢杆菌表达和分泌外源基因产物的修饰质粒中。所得的分泌质粒现在含有来自解淀粉芽孢杆菌的α-淀粉酶基因的启动子/信号序列区域，通过EcoRI或HindIII接头与终止寡核苷酸相连。外源基因可以克隆到这些位点。这些质粒可用于表达在其C末端被截短且因此缺乏自身翻译终止密码子的外源基因。一种这样 的质粒已成功用于表达在其C末端被截短的Semliki森林病毒（SFV）膜蛋白E1。
