抗菌肽ABP3基因的克隆及其在Pichia pastoris中的表达

Cloning and Expression of Antibaterial Peptide ABP3 Gene in Pichia pastoris Project of Chinese National Programs for High Technolgy Research and Development(No.101 01 02 03).

用化学合成法合成以植物偏爱密码子编码的新抗菌肽 Ａ Ｂ Ｐ３ 基因片段，合成片段拼接后，与ｐ Ｕ Ｃ１９ 重组，经限制酶片段分析与核苷酸序列分析，获得抗菌肽 Ａ Ｂ Ｐ３ 基因。 Ａ Ｂ Ｐ３基因与表达载体ｐ Ｐ Ｉ Ｃ９ 重组，构建受乙醇氧化酶１ 基因（ Ａ Ｏ Ｘ１） 的启动子与转录终止区控制的酵母表达质粒，转化 Ｇ Ｓ１１５ 宿主菌，经表型筛选，阳性克隆用甲醇诱导表达，重组 Ａ Ｂ Ｐ３ 以分泌型表达，具抗菌活性，且符合 Ａ Ｂ Ｐ３

A 144bp DNA fragment encoding antibacterial peptide ABP3 was designed based on the amino acid sequence of ABP3 and the biased codon usage of plant.Eight oligonucleotides were chemically synthesized,linked and then cloned into pUC19 vector.After restriction enzyme analysis and DNA sequencing,the ABP3 gene was ligated with yeast expression vector pPIC9 and transfeted a special strain GS115.The positive clones screened by the phenotype were induced by methanol and were able to express rABP3 which exhibited the antibacterial activity.