一种快速简便的多肽基因合成方法

A Rapid and Simple Method for the Synthesis of Peptide Gene

在目前的基因合成中,不管是大的蛋白质基因的合成,还是一小段基因的合成都是采用Khorana等所建立的双链的多片段连接方法,不过因合成仪的使用更多地采用了较长的合成片段(如50聚左右),从而可以减少合成片段的数目和一次能连接成较长的双链,节约了合成的时间。另一个改进的方面是以合成的单链为模板,再用DNA聚合酶来复制得到双链的方法,这不仅大大节省了用于化学合成的消耗,而且也节省了纯化学合成DNA片段的时间。Ita-

A rapid and simple method for the synthesis of peptide gene wasdescribed through the synthesis of Lys3-trichosanthes trypsin inhibitor-Ⅰ(Lys3-TTI-Ⅰ)gene as an example. The TTI-Ⅰis a new trypsin inhibitorpurified from Trichosanthes and composed of 27 amino acid residues withthree pairs of disulfide bonds. A108 nucleotide fragment was synthesizedby a DNA synthesizer.This synthetic 108 mer is negative strand of Lys3-TTI-Ⅰgene including the coding region, the initiation codon ATG andthe double stop codon TAG TAA. It also has a BamHI recognition sequ-ence on the 5′ end and a 10-nucleotide palindromic sequence encompassinga HindⅢ cleavage sile on the 3′ end,so that it can be directly convertedto double-stranded homoduplex form by mutually primed extention withKlenow fragment.The double-stranded homoduplex was successivly cleavedby BamHⅠ and HindⅢ and then cloned into plasmid pUC19. Finaly,through screering bacterial colonies the cloned gene was obtained and theDNA sequence was proved to be correct by dideoxy DNA sequencingwith 5′-labeled oligonucleotide primers.