【作者】 蒋瑞瑞；

【导师】 康相涛；

【作者基本信息】 河南农业大学 ， 动物遗传育种与繁殖， 2007， 硕士

【摘要】 B-防御素是鸡体内最重要的抗菌肽,除了具有直接的杀菌、抗病毒作用外,它还参与机体免疫反应,有重大的开发利用潜力。本文构建了表达鸡B-防御素-11和β-防御素-12(Gal-11和Gal-12)的大肠杆菌表达系统,研究了重组鸡β-防御素在大肠杆菌系统中高水平表达规律和蛋白纯化技术,为其进一步研究开发奠定了基础。本研究共分四部分:①鸡Gal-11基因cDNA的克隆根据genebank中AY621326序列设计了一对引物,运用RT-PCR技术,从固始鸡L系-崇仁麻鸡肾脏总RNA中扩增出片断大小为315bp的鸡Gal-11编码cDNA序列全长。测序结果与AY621326比对,同源性达99%。②鸡Gal-12成熟肽DNA的克隆根据genebank中AY621327设计上下游引物,从基因组DNA中扩增出大小为159bp的目的片断测序结果EF044309与序列AY621327,AY534898和DQ858309比对,序列完全一致;③鸡Gal-11和Gal-12基因的原核表达以pET32a为表达质粒构建了重组质粒pET32a-Gal-11和pET32a-Gal-12,并在BL21(DE3)pLySs中进行融合表达。SDS-PAGE电泳结果表明在目标位置约27.175KD和22.896KD出现条带。诱导剂IPTG在0.2mM-1.2mM的对重组蛋白的产量无明显影响,加入诱导剂1h开始有可检出量重组蛋白质的表达,在2h时其表达水平已接近最大量。④鸡Gal-11和Gal-12基因表达产物的活性检测融合表达蛋白经过初步分离,变性,组氨酸亲和层析柱纯化,复性,对纯化的表达蛋白进行琼脂糖弥散试验检测生物学活性。结果表明:蛋白进行抑菌试验对大肠杆菌、白色念球菌、金黄色葡萄球菌没有产生抗菌活性。鸡Gal-11和Gal-12在pET32a质粒表达体系中成功表达为鸡防御素多肽下一步的研究奠定基础。更多还原

【Abstract】 β-defensin Gallinacin is the most important member of a large family of antimicrobial peptides in chicken.β-defensin Gallinacin displays a strong antimicrobial activity against bacteria and fungi.Furthermore,it also plays indispensable function in triggering adaptive immune responses.So it could act as a substitute for antibiotic.In this paper,systems for high-level expression of theβ-defensin fusions in Escherichia toll were constructed,and expression regulation and the technology of protein purification were investigated.There are four parts in this paper:①Cloning of chicken Gal-ll gene:A pair of primers were designed and synthesized based on the publised gene sequence of Gal-11(AY621326).The target DNA,which contains 315bps,were amplified by RT-PCR on the template of tatal RNA isolated from kidney.The sequencing result displaied the target DNA was homologous with AY621326 by 99%.②Cloning of chicken Gal-12 mature peptides gene:A pair of primers were designed and synthesized based on the publised gene sequence of Gal-12(AY621327). The target DNA were amplified by PCR on the template of genome DNA isolated from blood.The sequencing result was completely consistent with AY621327,AY534898 and DQ858309.③Expression of chicken Gal-11 and Gal-12 gene:Gal-11 and Gal-12 gene was expressed in the form of fusion protein useing pET32a as expression vectors and BL21 (DE3)pLySs as expression host.Expression products with molecular weight of approximately 27.175KD and 22.896KD were examined by SDS-PAGE.Top yield of recombinant Gal-11 and Gal-12 could be achieved with as less as 0.2 mmol/L IPTG induction after 2 hours.④Biological activity detection of the purified protein:Recombinant fusion protein was extracted roughly from dissolved BL21 and purified with Ni-NTA resin affinity column.The biological activity was measured by the minimum inhibitory concentrations (MICs)the radial diffusion plate assay method.The result demonstrated that the purified protein has no antimicrobial activity against Escherichia.coli O78,Candida.albicans ATCC10231 and Staphylococcus.aureus 1056MRSA.Further study will be continued based on the results of this research.更多还原