ApiIa抗菌肽基因的化学合成、克隆及其在酵母中的表达

Chemical Synthesis, Cloning and Expression of Antibiotic Peptide Apila Gene in Yeast Pichia pastoris

用固相亚磷酰胺法合成了ＡｐｉＩａ抗菌肽基因，全长为８０个核苷酸．它被分成４个寡 核苷酸片段，分别在ＤＮＡ合成仪上合成．经分离纯化后的寡核苷酸片段经酶促连接，然后被 克隆到分泌型载体质粒ｐＦＤ１０１上．经限制酶酶切、ＰＣＲ模板检测以及双链 ＤＮＡ序列分析 检测，证明合成的ａｐｉＩａ基因和设计的完全一致．将含有ａｐｉＩａ基因的质粒ｐＦＤ１００１经ＢｇｌⅡ 酶切后，转化毕氏酵母．所得转化子经摇瓶发酵，上清液经２５％ＳＤＳ－ＰＡＧＥ检测，有 ＡｐｉＩａ抗 菌肽表达．用抑菌圈试验作定性实验表明，表达的ＡｐｉＩａ有明显的生物活性．

A gene coding for antibiotic peptide Apila has been chemically synthesized by solid-phase phosphoramidite method. The 80hp of synthetic DNA duplex consists of 18 codons and a leading peptide coding sequence encoding for six consecutive histidines (a Ni-binding domain). The synthesis of the gene Involved enzymatic joining of 4 oligonucleotides to form DNA duplex which was cloned into the expression vector pFD101. The synthetic gene was identified by restriction enzyme digestion and PCR amplifiction. Finally, DNA sequence analysis confirmed the seqence of synthetic gene to be identical with that of designed one. After being digested with a proper restriction enzyme (such as Bgl II),the plasmid pFD1001 carrying api Ia gene was transformed into yeast PICHIA pastoris. The inhibition zone experiments and 25% SDS-PAGE showed that the yeast trans formats could express and seers Apila.