| 抗菌肽DCD-1L基因的随机突变体在毕赤酵母中的表达 | |
| Alternative Title | The Expression of Antibacterial Peptide DCD-1L by Random Mutated in Pichia pastoris |
| 徐晓红 | |
| Subtype | 硕士 |
| Thesis Advisor | 蒲训 |
| 2007-05-10 | |
| Degree Grantor | 兰州大学 |
| Place of Conferral | 兰州 |
| Degree Name | 硕士 |
| Degree Discipline | 植物学 |
| Keyword | 抗菌肽 DCD-1L 随机突变 毕赤酵母 电穿孔 SDS-PAGE |
| Abstract | 随着抗生素的广泛应用，相应的细菌耐药性问题也随之愈发严重。在这种状况下，医药领域迫切需要寻求新的制备途径，以期获得抗菌活性更强、范围更广的新型抗菌剂。DCD是最近从人汗腺分泌物中发现的抗菌肽，DCD-1L是它的衍生物，具有广谱的抗菌活性，能够有效地抑制革兰氏阴性细菌和革兰氏阳性细菌的生长。它与抗生素不同，不会在生物体内富集，也不易诱导产生耐药菌株，是一类具有巨大发展潜力的新型抗菌药物。因此我们利用定向进化的方法在基因内部造成随机突变，选用真核表达系统来表达筛选具有更高抗菌活性的抗菌肽，有一定的经济价值和理论意义。本研究利用定向进化中寡聚核苷酸介导突变的方法，用含随机突变序列的两条引物以重叠延伸PCR制备出抗菌肽DCD-1L突变基因，长度约167bp。选用近年来发展最快和最具潜能的真核表达系统，构建含有强启动子GAP和α-factor信号肽序列的巴斯德毕赤酵母表达载体pGAPZαC-DCD-1L。重组表达载体pGAPZαC-DCD-1L酶切线性化后，用电穿孔方法整合到巴斯德毕赤酵母蛋白酶缺陷株SMDII68中，形成突变库，库容量达到1.09×104，利用抗生素Zeocin进行筛选，获得多拷贝重组菌株，并对重组菌株进行PCR鉴定和表达，用SDS-PAGE方法在培养基上清中鉴定出表达蛋白。经SDS-PAGE分析表明，重组菌株表达的培养上清中表达出分子量略大于6kD的重组蛋白，估计是重组蛋白进行了某种程度的糖基化修饰。 |
| Other Abstract | To following of the wide application of antibiotics, homologous, the bacterial resistance of conventional antibiotics also emerged more seriously. In this urgent we found a new way of preparation of antimicrobial with higher activity and broader- spectrum for drug territory. Dermcidin is a new antibacterial peptide recently found from human sweat glands, and DCD-1L is a derivant of Dermcidin which has a broad- spectrum antimicrobial peptide. It is a new class of antibacterial medicine having enormous potentiality. Therefore it has great economic value and theoretical significance to make random mutations in gene with the method of directed evolution and to express in Pichia pastorisfor screening an antibacterial peptide with even higher activityIn this research, the gene with the sequence of random mutations by overlap extention PCR and it has about 167 bp through mutation causing by oligonucleotide in orthogenesis. The eukaryotic expression system with tacho-development and most potency was choiced, the gene was inserted into the Pichia pastoris expression vector pGAPZαC Which contains GAP promotor and α-factor signal sequence. The recombinant vector was integrated in Pichia pastoris by electroporation,and which formed a mutation library with the capacity of 1.09×104. The multicopied recombinant Pichia strains were obtained by screening of Zeocin resistance. They were identified by PCR and expressed. SDS-PAGE analysis showed that the genes were expressed, and the proteins were secreted into the medium and were about 6000D which was the reason of the proteins by moderate modification of glycosylation. In this study, we have constructed a mutation library in Pichia pastoris expression vector pGAPZαC by the method of directed revolution, and achieved expression of random-mutated DCD-1L in eukaryotic system. |
| Pages | 48 |
| URL | 查看原文 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | https://ir.lzu.edu.cn/handle/262010/341818 |
| Collection | 生命科学学院 |
| Affiliation | 生命科学学院 |
| First Author Affilication | School of Life Sciences |
| Recommended Citation GB/T 7714 | 徐晓红. 抗菌肽DCD-1L基因的随机突变体在毕赤酵母中的表达[D]. 兰州. 兰州大学,2007. |
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