摘要
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.
出处
《复旦学报(自然科学版)》
CAS
CSCD
北大核心
1998年第2期168-172,共5页
Journal of Fudan University:Natural Science
关键词
基因
化学合成
ApiIa抗菌肽
无性系
抗菌肽
Chemical synthesis of the gene
antibiotic peptide ApiIa
secretory expression vector
yeast