作　　者： (魏媛）; (龚国利）;

机构地区： 陕西科技大学食品与生物工程学院,陕西西安712000

出　　处： 《中国病原生物学杂志》 2017年第8期741-746,共6页

摘　　要： 目的设计合成杂合肽MLH基因并在大肠埃希菌(Escherichia coli)表达系统中高效融合表达,获得具有抑菌活性的重组蛋白。方法以家蝇抗菌肽Md-Cec、人源抗菌肽LL-37和幽门螺杆菌肽Hp为母体肽,通过生物信息学分析筛选出一条具有抗菌潜力的新型杂合抗菌肽MLH并根据E.coli密码子的偏好性编码基因。采用重叠延伸基因扩增(SOE-PCR)技术,通过设计合成3条互补引物合成所需的目的基因,与表达载体pET-32a(+)连接后转化至E.coli表达菌株BL21(DE3),构建基因工程菌pET-32a-MLH/BL21(DE3),用含氨苄青霉素(Amp)的抗性平板筛选阳性菌株,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导后采用SDS-PAGE电泳和Western blot检测目的蛋白的表达。对发酵条件进行优化以获得融合蛋白的大量表达,采用Ni 2+亲和层析柱纯化和透析复性的方法将表达的融合蛋白进行纯化和复性,获取具有活性的融合蛋白,采用琼脂孔穴扩散法测定其抑菌活性。结果成功合成基因MLH并与表达载体连接形成重组质粒pET-32a-MLH,构建重组基因工程菌株pET-32a-MLH/BL21(DE3),经IPTG诱导表达相对分子质量单位(Mr)约25×10~3的重组蛋白且主要以包涵体形式存在。对发酵条件进行优化,确定最优发酵条件为:IPTG终浓度为1.0mmol/L;诱导时间为4h;诱导温度为37℃。通过纯化以及复性处理得到重组蛋白对金黄色葡萄球菌和枯草芽孢杆菌均有抑制作用。结论杂合抗菌肽MLH可在大肠埃希菌中融合表达且有一定抑菌活性。 Objectives To design and synthesize a gene encoding a hybrid peptide,MLH,and to efficiently express that peptide in an Escherichia coli expression system in order to obtain a recombinant protein with antibacterial activity.Methods Md-Cec,LL-37,and a Helicobacter pylori peptide were used as parent peptides.Bioinformatic analysis was used to screen for a new hybrid MLH peptide with antibacterial potential and a gene with preferred codons for E.coli.The target gene was synthesized using SOE-PCR with three complementary primers and ligated into the expression vector pET-32a（＋）.The vector was transformed into the E.coli expression strain BL21（DE3）to construct pET-32a-MLH/BL21.Strains were screened for resistance to Amp.Expression was induced with IPTG under optimized conditions.SDSPAGE protein electrophoresis and Western blotting were used to detect the target protein,and fermentation conditions were optimized to obtain substantial expression of the fusion protein.Ni^（2＋）affinity chromatography and renaturation were used to obtain an active hybrid peptide,and the bacteriostatic activity of the hybrid peptide was preliminary determined using inhibition zones. Results The gene MLH was successfully synthesized in two rounds of PCR using three overlapping primers and the gene was ligated into the recombinant plasmid pET-32a-MLH to construct the recombinant pET-32aMLH/BL21（DE3）.After expression of the recombinant protein was induced,the protein was mainly found in the form of inclusions.The fermentation conditions were optimized as follows：an IPTG concentration of 1.0mmol/L;an induction time of 4h;and an induction temperature of 37 ℃.The recombinant protein was purified using an Ni^（2＋）affinity chromatography column.The fusion protein was obtained,accounting for 90% of the total protein.After renaturation by dialysis,the recombinant protein had a marked bacteriostatic effect on Staphylococcus aureus and Bacillus subtilis. Conclusion The hybrid antimicrobial peptide MLH can be expressed in E.co

关 键 词： 家蝇抗菌肽 人源抗菌肽 幽门螺杆菌抗菌肽 杂合抗菌肽 融合表达 抑菌活性