【作者】 刘旭；

【导师】 周定刚；

【作者基本信息】 四川农业大学 ， 特种经济动物饲养， 2010， 硕士

【摘要】 抗菌肽是一类具有广谱抗菌活性的小分子物质,是生物天然免疫反应系统的重要成员,在生物体抵抗外界病原感染过程中起着非常重要的作用。抗菌肽分布十分广泛,并且对大多数革兰氏阴性或阳性菌、真菌、原生动物都有抑制作用,有些抗菌肽还具有抗寄生虫、病毒和肿瘤的作用。本研究根据本实验室构建的黄鳝肠道cDNA文库中获得的抗菌肽hepcidin基因ORF区(GenBank登陆号：GU997139)设计并合成特异性引物,利用RT-PCR技术从黄鳝肠道总RNA中扩增出一条约300bp的目的基因片段。将目的基因片段克隆到pMD18-T载体上,序列测定结果表明与hepcidin基因ORF区完全一致。序列分析表明：该基因ORF区长273bp,编码90个氨基酸,分子量约10236Da,包括24个氨基酸的信号肽、40个氨基酸的前域和26个氨基酸的成熟肽。结构预测显示其属于β-折叠类抗菌肽,包含有hepcidin类抗菌肽典型的8个半胱氨酸结构,形成4对二硫键。利用氨基酸序列进行同源比对,发现黄鳝抗菌肽hepcidin与多种肉食性鱼类的抗菌肽hepcidin同源性较高,其中与大黄鱼的同源性最高,为81%。分别设计带限制性内切酶BamH I和Hind III酶切位点的特异性上、下游引物,以pMD18-T-hepcidin阳性克隆质粒为模板,亚克隆黄鳝抗菌肽hepcidin基因完整的ORF区(s-hepcidin)和不含信号肽的hepcidin基因(n-hepcidin)编码序列,并插入原核表达载体pET32a(+),构建重组原核表达质粒pET32a(+)-s-hepcidin和pET32a(+)-n-hepcidin。分别转化至大肠杆菌BL21 (DE3) pLysS,在37℃,1mM IPTG诱导表达4h后,经SDS-PAGE电泳鉴定分析,pET32a(+)-s-hepcidin阳性重组菌没有表达出预期的目的蛋白；而pET32a(+)-n-hepcidin阳性重组菌明显表达出目的融合蛋白,大小约为28KDa,与预期目的融合蛋白分子量大小一致。对pET32a(+)-n-hepcidin在BL21 (DE3) pLysS中的诱导表达进行IPTG浓度和表达时间优化,发现IPTG诱导浓度在0.1mM时表达量最大,而最佳诱导时间为4h。在最佳表达条件下,pET32a (+)-n-hepcidin重组蛋白在BL21 (DE3) pLysS表达菌株中为可溶表达。更多还原

【Abstract】 Antimicrobial peptides (AMPs) are regarded as important components of the host innate immune system and play crucial roles in host defense against microbial invasion. Gene-encoded, ribosomal synthesized antimicrobial peptides are widely distributed in nature and the display strong antimicrobial activity against a broad range of microbes including Gram positive and Gram negative bacteria, protozoa, fungi and viruses. Several AMPs exhibit ant parasitic and anticancer properties.Specific primers were designed and synthesized according to Monopterus albus hepcidin gene ORF sequences(GenBank accession:GU997139)from the cDNA library, which was constructed by our lab. A treaty 300bp target gene fragment was amplified from total RNA of M.albus intestine tract by RT-PCR. The target gene cloned into pMD18-T vector and sequencing results showed that hepcidin gene ORF with exactly the same area. Further analysis of antimicrobial peptide protein sequence contains a signal peptide of 24 amino acids and pro-region part of 40 amino acids before the mature peptide of 26 amino acids, class of antimicrobial peptide hepcidin contains eight cysteine typical structure, the formation of four pairs of disulfide key; structure prediction shows that it belongs toβ-sheet type peptide. Homologous than are found with a variety of animals, M. albus hepcidin sequences are highly homologous with the similarity of Larimichthys crocea croaker up to 81%.Were designed with restriction enzyme BamH I and Hind III restriction sites of the specific upstream and downstream primers pMD18-T-hepcidin positive clone plasmid as a template, were subcloned M.albus peptide hepcidin gene complete ORF region (s-hepcidin) and without signal peptide hepcidin gene (n-hepcidin) coding sequence, and were inserted into prokaryotic expression vector pET32a (+), prokaryotic expression vector pET32a (+)-s-hepcidin and pET32a (+)-n-hepcidin. Were transformed into E. coli BL21 (DE3)pLysS, at 37℃,1mM IPTG induced for 4 hours, by SDS-PAGE electrophoresis analysis, pET32a (+)-s-hepcidin-positive recombinant strain did not express the desired protein; The pET32a (+)-n-hepcidin positive bacteria clearly express the recombinant fusion protein, the size of about 28KDa, and expected the same molecular weight fusion protein. On the pET32a (+)-n-hepcidin in the BL21 (DE3)pLysS expression in IPTG concentration and induction of expression of the time optimization, IPTG induction was found when the concentration of 0.1 mM expression level, and the best inducing time 4h. The optimal expression conditions, pET32a (+)-n-hepcidin fusion protein in BL21(DE3) pLysS strain for expression of soluble expression.更多还原