杂合抗菌肽MLH在大肠埃希菌中的融合表达及其抑菌活性研究

发布时间：2018-01-14 04:17

本文关键词：杂合抗菌肽MLH在大肠埃希菌中的融合表达及其抑菌活性研究　出处：《中国病原生物学杂志》2017年08期 　论文类型：期刊论文

【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。
[Abstract]:Objective to synthesize hybrid peptide MLH gene in Escherichia coli (Escherichia coli) expression system, fusion expression, recombinant protein with antibacterial activity. Methods the recombinant antibacterial peptide Md-Cec, human antimicrobial peptide LL-37 and Helicobacter pylori peptide Hp as parent peptide, through bioinformatic analysis identified a the antimicrobial potential novel hybrid antibacterial peptide MLH and according to the preference of the gene encoding the E.coli codon. Using PCR overlap extension (SOE-PCR) technology, the design and synthesis of 3 complementary primers required for the synthesis of the target gene, and the expression vector pET-32a (+) connection was transformed into E.coli expression strain BL21 (DE3) and the construction of recombinant strain pET-32a-MLH/BL21 (DE3), containing ampicillin (Amp) screening positive strain resistance plate, the isopropyl thiogalactoside beta -D- (IPTG) induced by SDS-PAGE electrophoresis and Western blot detection. The expression of protein. To optimize the fermentation conditions to obtain the massive expression of fusion protein by Ni method, 2+ affinity chromatography purification and renaturation of the expressed fusion protein was purified and refolded, obtain the activity of fusion protein by agar hole diffusion method determined the antibacterial activity. The successful synthesis of gene MLH and connected with the carrier to form recombinant plasmid pET-32a-MLH expression, recombinant gene engineering strain pET-32a-MLH/BL21 (DE3), the expression of relative molecular mass unit induced by IPTG (Mr) about 25 * 10~3 and the recombinant protein mainly existed in inclusion bodies. The fermentation conditions were optimized, to determine the optimal fermentation conditions were: IPTG concentration 1.0mmol/L the induction time is 4H; induction; the temperature of 37 DEG. Purification and renaturation of recombinant protein was inhibited by Staphylococcus aureus and Bacillus subtilis were passed. Conclusion Heterozygous antibacterial peptide MLH can be expressed in Escherichia coli and has certain inhibitory activity.

【作者单位】：陕西科技大学食品与生物工程学院;西安市微生物药物工程实验室;
【基金】：陕西省教育厅自然科学研究专项(No.14JK1101)
【分类号】：R3416
【正文快照】： ***sion The hybrid antimicrobial peptide MLH can be expressed in E.coli and it has a marked bacteriostatic effect on S.aureus and B.subtilis.抗菌肽(antimicrobial peptide,AMP)是一类广泛存在于自然界生物体中的小分子多肽,是生物免疫系统的主要组成部分[1-2]。由

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