融合有酸性片段的抗菌肽串连重复基因的构建

为提高抗菌肽的表达，设计在抗菌肽基因的N端融合一段编码酸性肽的片段以及减轻表达产物对宿主的毒性，通过含有酶切位点的接头将该融合肽基因以同向串连的方式连接成多拷贝基因，克隆至pUC19载体。为此，分段设计合成了编码天蚕素A-蜂毒素杂合肽和酸性肽的DNA片段。首先将其连接成融合肽全基因，然后分别与含相同粘性末端的前后接头连接。通过控制基因和接头加入的量及次序，可得到两侧有EcoRI和SalI酶切位点的同向串连的多拷贝基因。选取合适拷贝数的基因，将其克隆至pUC19载体，PCR扩增和DNA测序证明多拷贝基因构建成功且基因方向相同。结果表明，该方法能简捷高效地获得所需的多拷贝基因，为提高表达产物的量打下基因。

Construction of acidic peptide-fused multi-copy antimicrobial peptide tandem in the same direction

To improve antimicrobial peptide's yield, a novel mass-production method is proposed It is based on the neutralization of the positive charges of antimicrobial peptide by fusing to an acidic peptide to avoid the lethal effect of the expressed peptide on the host cells We designed and synthesized two DNA fragments encoding cecropin A-melittin hybrid and an acidic peptide,respectively The two fragments were ligated to a whole fusion peptide gene,next was ligated with two adapters which have the same cohesive end(ATG/TAC),respectively The whole gene was added every six hours instead of all in one time In proper time,they were mixed and continued to ligate to multi-copy in the same direction and cloned into pUC19 vector PCR amplification and DNA sequencing confirmed that 4-copy gene was correctly inserted into the vector The result showed the method can conveniently and efficiently obtain the desired muti-copy gene and be ready for next cost-effecive expression