家蚕抗菌肽基因Cecropin-D的克隆及其真核表达载体的构建

Cloning and Construction of the Expression Vector of Antimicrobial Peptide Cecropin D gene from Bombyx mori

从家蚕中提取总RNA,采用反转录聚合酶链式反应(RT-PCR)方法,获得了家蚕Cecropin-D基因编码区的cDNA,扩增出家蚕抗茵肽Cecropin-D成熟肽基因片段,重组克隆入pMD18-Tvector载体,经DNA测序,该基因为186bp,编码62个氨基酸。用限制性内切酶切下目的基因,插入毕赤酵母表达载体pPIC9K中,构建真核表达栽体pPlC9K-CD。本实验的研究为获得超量表达的高活性表达抗菌肽Cecropin-D以及为研制具有抗菌活性的新型基因药物奠定了基础。

The total RNA was extracted from Bombyx mori,Cecropin-D gene cDNA was obtained by reverse transcription polymerase chain reaction（RT-PCR）,and then gene of the mature peptide of Cecropin-D was amplified and cloned into pMD18-T vector.The sequence analysis showed that the cloned DNA had a length of 192bp,encoding a protein of 64 amino acids.After digested by both EcoRI and NotI. DNA fragment was linked with vector plasmid pPIC9K by T4 DNA ligase. The construction of expression vector pPIC9-CD may lead to obtaining large Cecropin D,thus providing basis for further research on developing new gene drug of antibacterial activity.