利用分离型内含肽DnaE表达抗菌肽

摘要

In order to investigate the feasibility of producing antimicrobial peptide by the two natural fracture type inteins Ssp DnaE and Npu DnaE which can circumvent toxic effects to the host caused by the expression of antimicrobial peptides directly.The DNA sequence of antimicrobial peptide ADP-1 was split into two parts at special sites.The N-terminal of ADP-1 peptide was fused to the N-terminal of Npu DnaE, and the C-terminal peptide was fused to the C-terminal of Ssp DnaE.Then they were cloned into the prokaryotic expression vector pET-23a(Amp+) and pET-30a(Kan+) respectively, resulting the recombinant expression plasminds pET-23a-ADP-1-N-Npu and pET-30a-Ssp-ADP-1-C.Then they were transformed into BL21(DE3) alone or together, induced by IPTG, and detected by SDS-PAGE.We found they were transformed into BL21(DE3) strain, and both were expressed after inducing.But the expression products did not have antibacterial activity in strain which was transformed by only one recombinant plasmid pET-23a-ADP-1-N-Npu or pET-30a-Ssp-ADP-1-C.However, the antibacterial activity was detected in the concentration and purification supernatant of the strain which was co-transformed with pET-23a-ADP-1-N-Npu and pET-30a-Ssp-ADP-1-C plasmid.The two natural fracture type inteins Ssp DnaE and Npu DnaE are used to express the defensin ADP-1 C terminal and N terminal respectively, and the two fused protein can form the trans-splicing reaction in BL21(DE) and promote the spliced ADP-1 to obtain the antibacterial activity.%拟验证天然断裂内含肽Npu DnaE、Ssp DnaE的反式剪接反应对嵌合蛋白形成的影响,探讨依据此方法表达抗菌肽的可行性,规避抗菌生物表达中对宿主的毒害作用.将花蝉属抗菌肽ADP-1(Amblyomma defensin peptide 1)拆分为N端和C端两部分,其N端与断裂型内含肽Npu DnaE的N端融合,C端与Ssp DnaE的C端融合,并分别构建到原核表达载体pET-23a(Amp+)和pET-30a(Kan+)上,获得重组表达质粒pET-23a-ADP-1-N-Npu和pET-30a-Ssp-ADP-1-C,然后单独或共转化大肠杆菌感受态细胞BL21.实验结果表明,两个重组质粒分别单独或组合转化至Bl21(DE3),经IPTG诱导后,SDS-PAGE检测转化的菌株中都表达了目的蛋白,共转化的菌株诱导表达破菌后其上清液有抑菌活性.该实验验证以两种天然断裂型内含肽Ssp DnaE和Npu DnaE及搭载的ADP-1 N端和C端可在大肠杆菌中获得表达,且共转化的两段内含肽可在离体上清中发生反式剪接,促进融合在内含肽上的ADP-1两部分形成完整具有抑菌活性产物.