【作者】 张海波；

【导师】 王秋雨；

【作者基本信息】 辽宁大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 抗菌肽(antibacterial peptides, ABP)又称抗微生物肽(antimicrobial peptides, AMP)或肽抗生素(peptide antibiotics),是生物机体为对抗外源性病原体及恶性细胞,由免疫防御系统非专一性应答产生的一类小分子活性肽类的总称。抗菌肽通常是由10-50个氨基酸组成的小分子多肽,具有抗菌谱广、热稳定性强、分子量小及免疫原性小等特点,其杀菌机制独特,不易产生耐药菌,有望开发为新一代肽类抗生素。国外对蛙类抗菌肽的研究已有数十年历史,目前已有肽类抗生素进入临床试验阶段。东北林蛙作为主要分布在我国东北地区的两栖动物,其皮肤抗菌肽分子水平的研究尚处于起步阶段,仅有零星序列的报道。天然抗菌肽尽管有很多其他抗生素无法比拟的优势,但也有其缺陷性。某些天然抗菌肽在杀菌的同时具有溶血性,还有一些抗菌肽本身具有生物毒性,无法应用于临床治疗。因此对天然抗菌肽的改造也是当前抗菌肽研究的新方向。目前对抗菌肽改造的分子设计方案主要是分析天然抗菌肽的生化性质,根据已知影响活性的因素,设计出新的抗菌肽。或者通过比较已知抗菌肽的结构,找出保守序列,筛选出具有优良抗菌活性的新的抗菌肽。Dybowskin-2CDYa(SAVGRHGRRFGLRKHRKH)是本实验室发现并命名的天然新型林蛙皮抗菌肽,富含Arg。生物信息学分析表明,其不稳定系数(Instability index)达到了52.19,而理论上认为不稳定系数低于40蛋白才能够稳定存在。根据现有的文献,改造抗菌肽dybowskin-2CDYa,将其Arg全部替换为Lys,并把第二位的Gla替换为Trp,改造后的抗菌肽命名为AWRK6。AWRK6(SWVGKHGKKFGLKKHKKH)由18个氨基酸构成,分子量为2130.5,利用HNN法预测其二级结构,其结构主要为α-螺旋,等电点为9.60,不稳定系数为-6.74,说明抗菌肽AWRK6稳定性优于Dybowskin-2CDYa。将改造后的抗菌肽AWRK6基因利用SOE-PCR方法获得AWRK6全长基因,连接酵母分泌型表达载体pPICZα-A,构建了pPICZα-AWRK6酵母表达质粒,重组质粒经Sac I酶切线性化后,电转化巴斯德毕赤酵母X33,构建真核表达系统,采用Zeocin筛选阳性菌株、PCR方法验证阳性菌株。阳性转化子经甲醇诱导表达后,利用α-factor引物、3’AOX引物对反转录cDNA进行扩增,获得300 bp左右的扩增产物,与目的基因AWRK6大小吻合,说明目的基因已插入宿主染色体中并已转录。对发酵液进行活性测定,抑菌活性显示,重组抗菌肽AWRK6对革兰氏阴性菌(大肠杆菌O157),革兰氏阳性菌(金黄色葡萄球菌)均有抑制活性。结论:将富含精氨酸抗菌肽所有的精氨酸残基替换为赖氨酸残基,可提高其抗菌活性。利用巴斯德毕赤酵母X33表达体系能够正确表达新型抗菌肽AWRK6基因。抗菌肽AWRK6的二级结构以α-螺旋结构为主的两亲性螺旋结构。AWRK6对大肠杆菌、金黄色葡萄球菌具有抗菌活性。更多还原

【Abstract】 Peptide (antibacterial peptides, ABP), also known as antimicrobial peptides (antimicrobial peptides, AMP) or peptide antibiotics (peptide antibiotics), defending exogenous pathogens and malignant cells, is a class of small molecule active peptides that is produced by the non-specific response in immune defense system in general. Antibacterial peptide is a small molecule peptide generally containing 10-50 amino acids, with the features of broad-spectrum antibacterial activity, strong thermal stability, small molecular weight and low immunogenicity, becauese of bactericidal mechanism unique and easy to produce drug-resistant, it is expected to develop a new generation of peptide antibiotics.Research on antibacterial peptide has lasted for several ten years and some peptide antibiotics are in the phase of clinical trial in abroad. It is in the initial stage of research on antibacterial peptide of Rana dybowskii in molecular level and just several sequences was reported so far. Natural antimicrobial peptides not only have many other antibiotics and incomparable advantages, but also has its inadequate. Some natural antimicrobial peptides have both haemolyticus and sterilizing, others antimicrobial peptides is biotoxicity by itself, it has not applied in clinical treatment. So the reform of natural antimicrobial peptides is a new direction at present.At present the molecular design scheme of antibacterial peptides reform is mainly to analyze the biochemical properties of the natural antimicrobial peptides, according to factors known to influence activity, to design of new antimicrobial peptides. Or by comparing the structure of the known antimicrobial peptides, to identify conserved sequences, and screen new antibacterial peptides with excellent antimicrobial activity.Dybowskin-2CDYa(SAVGRHGRRFGLRKHRKH)is a new natural antibacterial peptide which is found and named by our laboratory in frog skin, rich in Arg. Bioinformatics analysis showed that the instability coefficient (Instability index) reached 52.19, but proteins that the coefficient is less than 40 can exist stably instability in theory. According to the existing literature, reform antibacterial peptides dybowskin - 2CDYa by means of replacing all Arg by the Lys, and replacing Gla in the second place by Trp, and then antimicrobial peptides after transforming called AWRK6. AWRK6 (SWVGKHGKKFGLKKHKKH) is composed of 18 amino acids, molecular weight is 2130.5, to predict the secondary structure by the method of HNN and find out its structure, it is mainlyα-helix, isoelectric point is 9.60 and instability coefficient is -6.74, the result indicate antimicrobial peptide AWRK6 is more stable than dybowskin-2CDYa. The AWRK6 gene after transforming get full-length genes by means of SOE-PCR. Connect the yeast secretory expression vector pPICZα-A, and construct pPICZα-AWRK6 yeast expression plasmid. Recombinant plasmid is digested with SacI, and electroporatinto Pichia pastoris X33, then construct eukaryotic expression system. Use Zeocin to select positive strains, and prove positive strains by PCR. Positive transformants were induced by methanol, useα-factor primer, 3’AOX primers to amplify cDNA of reverse transcription to obtain amplification products, it is about 300 bp. It is consistent with the size of the target gene AWRK6, indicating that the gene has been inserted into the host genome and transcription. Measure the activity of fermentation liquid, and antibacterial activity showed that the recombinant antibacterial peptide AWRK6 has inhibitory activity against Gram-negative bacteria (Escherichia coli O157), and Gram-positive bacteria (Staphylococcus aureus).Conclusion: Instead all of the arginine residues of the antimicrobial peptide to lysine residues, it could improve their antimicrobial activity. New antimicrobial peptides AWRK6 genes successfully is expressed in Pasteur pichia X33 expression system. The secondary structure of antimicrobial peptide is mainlyα-helix amphiphilic spiral structure. AWRK6 has antibacterial activity to Escherichia coli and staphylococcus aureus.更多还原