【作者】 王晓波；

【导师】 刘飞鹏；

【作者基本信息】 暨南大学 ， 遗传学， 2003， 硕士

【摘要】 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短肽的片段作为对抗菌肽前体的模拟，以减轻表达产物对宿主的毒性；同时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。为此，我们分段设计合成了编码天蚕素A（1-8）—蜂毒素（1-10）杂合肽和酸性短肽的DNA片段。首先将其连接成融合肽的完整基因，然后分别与含有相同粘性末端（ATG/TAC）的EcoR Ⅰ前接头和Sal Ⅰ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EcoR Ⅰ和Sal Ⅰ酶切位点的同向串连的多拷贝基因。选取合适拷贝数的串连重复基因，将其克隆至pUC19载体，双酶切、PCR扩增和DNA测序证明串连重复基因构建成功且基因方向相同。 选取构建好的5份串连基因克隆至表达载体pBV220、pGEX-4T-1和pET21a，转化E.coli DH5α。DNA测序正确后，转化不同的宿主菌中进行诱导表达，SDS-PAGE分析发现三种重组质粒在对应位置均出现特异的蛋白表达带。其中，含有5份串连基因的pET21a重组质粒在BL21（DE3）中的诱导表达量占总蛋白的25％左右。对表达产物进行可溶性分析，证明其主要以包涵体形式存在。将包涵体用8M的尿素溶解后，经Poly-His protein purification kit亲和纯化，获得纯度为99％的蛋白。纯化蛋白经溴化氢切割，再通过羧甲基纤维素离子交换层析获得最终目标CA-ME肽。以E.coli K₁₂D₃₁为指示菌进行琼脂平板空穴扩散法测定，证明CA-ME肽具有抗菌活性。更多还原

【Abstract】 Construction and Expression of tandem multi-copy gene edcodingCA(1-8)ME(1-10) hybrid fused acidic peptideTo improve antimicrobial peptide’s expression, 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(l-8)-melittin(l-10) 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. Proper multi-copy gene was selected and cloned into pUC19 vector. Restriction enzyme analysis and DNA sequencing confirmed that 5-copy gene was correctly inserted into the vector. The 5-copy gene was cloned into expression vector pBV220, pGEX-4T-l andpET21a digested with EcoR I and Sal I .After verified by sequencing, recombinant plasmids are transformed into several E.coli hosts. The 5-copy gene was expressed when induced in different hosts. SDS-PAGE analysis showed three recombinant plasimids all produced induced expression bands that accord with expected. Especially, the recombinant pET21a having 5 copy gene in E.coli BL21(DE3) got high expression and expression level was up to 25% of the total bacterial proteins. The analysis of product solubility revealed that 5-copy fusion protein mainly existed as inclusion body. The inclusion body was solulized in 8M Urea and purified by Poly-His protein purification kit. The product was further cut by CNBr and purified by CM ion-exchange chromatography and CA-ME hybrid was got. The purified CA-ME hybrid appears evident antibacterial activity with method of agar diffusion.更多还原