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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。
Objective To design a synthetic hybrid peptide MLH gene and express it in Escherichia coli expression system with high efficiency and obtain the recombinant protein with antibacterial activity. Methods A new hybrid antimicrobial peptide MLH with antibacterial potential was screened out by bioinformatics analysis using the Musca domestica antimicrobial peptide Md-Cec, human antimicrobial peptide LL-37 and Helicobacter pylori Hp as the parent peptide. Preference codons for codons. The desired gene was synthesized by designing and synthesizing three complementary primers by overlap extension gene amplification (SOE-PCR) and ligated with the expression vector pET-32a (+) and transformed into E. coli expression strain BL21 (DE3) Construction of genetically engineered bacteria pET-32a-MLH / BL21 (DE3), positive strains were screened with ampicillin (Amp) resistant plates and induced with isopropyl-β-D-thiogalactoside SDS-PAGE electrophoresis and Western blot detection of the target protein expression. The fermentation conditions were optimized to obtain the large amount of fusion protein. Purified and refolded fusion protein was purified by Ni 2+ affinity chromatography and dialysis renaturation. The fusion protein was obtained by using agarose hole The antibacterial activity was measured by diffusion method. Results The gene MLH was successfully synthesized and ligated with the expression vector to form the recombinant plasmid pET-32a-MLH. The recombinant gene engineering strain pET-32a-MLH / BL21 (DE3) was constructed and induced with IPTG for about 25 × 10 ~ 3 recombinant protein and mainly in the form of inclusion bodies. The fermentation conditions were optimized to determine the optimal fermentation conditions: final concentration of IPTG 1.0mmol / L; induction time 4h; induction temperature 37 ℃. Through purification and renaturation, the recombinant protein can inhibit Staphylococcus aureus and Bacillus subtilis. Conclusion The hybrid antimicrobial peptide MLH can be fused and expressed in Escherichia coli and has certain antibacterial activity.