【作者】 任耀军；

【导师】 王新华； 薄新文；

【作者基本信息】 石河子大学 ， 预防兽医学， 2008， 硕士

【摘要】 抗菌肽是广泛存在于自然界生物体中的多肽,是生物体先天免疫的重要组成部分。因其具有抗菌活性强、广谱抗菌、热稳定性及不易产生耐药性等优点而广受人们的关注。绵羊骨髓抗菌肽(SMAP-29)是1995年Bagella和Margherita Zanetti等研究小组在绵羊的骨髓mRNA中发现的,具有两亲性α-螺旋结构域,属于cathelicidins家族,含29个氨基酸残基,有很强的抗细菌、真菌、中和内毒素等生物学功能。本研究以GenBank发表的SMAP-29基因(L46854)为模板,参照毕赤巴斯德酵母(Pichia pastoris)密码子偏好性,设计合成了抗菌肽SMAP-29成熟肽目的基因片段。通过基因重组的方法将合成的基因克隆入pPIC3.5K载体中构建重组酵母胞内表达载体pPIC3.5K-SMAP-29,经酶切分析、PCR和测序鉴定证明载体构建成功。将构建成功的载体电击转化至毕赤酵母受体菌GS115中,经G418筛选高拷贝转化子,并用MM、MD板和PCR法筛选Mut+表型。对构建好的重组酵母表达基因工程菌用甲醇诱导表达,裂解酵母细胞进行Tricine-SDS-PAGE分析,结果在诱导至第2天的细胞裂解液中检测到与预测的SMAP-29分子量相当,约为3.2KD的表达带。Trizol法提取酵母总RNA,通过RT-PCR扩增SMAP-29 mRNA,发现表达期的酵母细胞中存在SMAP-29 mRNA,而对照没有检出,表明SMAP-29在毕赤酵母中存在表达。表达产物经凝胶过滤色谱处理后进行抑菌活性检测,结果表明该表达产物对金黄色葡萄球菌(Staphylococcus aureus ATCC2592)和白色念珠菌(Candida albicans ATCC2002)有明显的抑菌作用,而对大肠杆菌抑制效果不明显。更多还原

【Abstract】 Antibacterial peptides are widely existed in many creatures of nature, which are the important parts of innate immunity in organisms.They have attracted great attention for its advantages such as showed a strong antibacterial activity, has a broad antifungal spectrum, heat stability, and it is difficult to produce antimicrobial resistance and so on.Sheep myeloid antibacterial peptides-29 (SMAP-29) is a cathelicidin-derived peptide deduced from sheep myeloid mRNA by research groups of Bagella and Margherita Zanetti in 1995.It was confirmed to beα-helical, 29-amino-acid C-terminal peptide, which has the biological functions of broad spectrum anti-bacterial, anti-fungus and endotoxin-neutralizing.A gene encoding SMAP-29(sheep myeloid antibacterial peptides-29) mature peptide was synthesized according to the published SMAP-29 gene (L46854) sequence in GenBank and the biased codon usage of Pichia Pastoris. The synthesized gene was inserted into Pichia Pastoris expression vector pPIC3.5K and constructed intracellular expression plasmid pPIC3.5K-SMAP-29 for Pichia Pastoris. Then the plasmid was identified with restriction enzyme digestion, PCR and sequencing. Then the recombinated plasmid was transformed into Pichia pastoris GS115 by electroporation after lineared by SalI, and screened with G418 for high-copy recombination strain, and selected methanol utility plus phenotype with MM, MD & PCR. The recombination strain were induced in the medium containing methanol and detected by Tricine-SDS-PAGE analysis. The results showed that after induced with methanol for 2 days, the lytase gave an induced band of SMAP-29 near 3.2KD, which was identified by RT-PCR amplification of SMAP-29 mRNA extracted with Trizol in expressing yeast. The expression products purified with gel filtration chromatography were used for antibacterial assay. The recombinant SMAP-29 displayed obvious antibacterial activity against Staphylococcus aureus and Candida albicans. But it could not distinctly inhibit the growth of Escherichia coli JM109.更多还原