ApiIa抗菌肽基因的化学合成,克隆及其在酵母中的表达

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合成仪上的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ｐＡＦＤ１０１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。

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

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.