杜仲蛋白纯化、序列分析及抗菌活性研究

【作者】 刘世会；

【导师】 赵德刚；

【作者基本信息】 贵州大学 ， 农药学， 2008， 博士

【摘要】 论文对杜仲皮和杜仲胶颗粒中的蛋白进行了分离纯化、序列分析和体外抗菌活性研究,获得以下研究结果:用凝胶层析、反相高效色谱层析等分离纯化方法,从杜仲树皮中分离到分子量为4.16的一个新杜仲抗真菌蛋白;用SDS-PAGE方法从杜仲胶颗粒中分离到分子量约为14、16、18、22、24、28、30、32和55 kDa的9个杜仲胶颗粒结合蛋白。用液相色谱质谱联用（LC-MS/MS）技术,鉴定了4.16、4.2、8.3、16和18 kDa蛋白序列,获得14个肽片段,在抗菌肽数据库（APD）中对这14个肽片段进行结构分析、抗菌活性预测和序列比对,结果显示有11个肽片段具有抗菌肽典型结构。这11个肽片段是:-.LYQQLIAGITLNK.-、-.PCNAGLCCSIYGYCGSGNAYCGAGNCR.-、-.LRLDGAMPPSYPSSWSVLPR.-、-.QIITHPSMDDYVRK.-、-.LPLGAGVNLSGGPVVK.-、-.LPVAVKTGAEVVYSK.-、-.ISTLNSHNLPILR.-、-.ISLTEGIRR.-、-.RHGNDHDVHPK.-、-.TNGPVVNLKAFLKK.-和-.GRSAPVLAATVAGDTR.-。用纸片法对EAFP3进行体外抗菌活性检测,抑菌活性检测结果表明:EAFP3可抑制酵母、酵母类真菌、霉菌和植物病原真菌的生长,EAFP3对白色念珠球菌、黄曲霉、番茄灰霉病菌的IC₅₀值分别为31.25,31.25和62.5μg/mL。EAFP3对高温有一定耐受性,在100℃加热30 min仍有显著抑制活性;可适应较宽pH范围,在pH从5.8-8.0范围内都有显著抑制活性。以上研究结果可能为创制新型生物农药和生物药物提供了先导化合物信息,为克隆编码杜仲抗菌蛋白的基因提供了引物和探针设计序列。更多还原

【Abstract】 In this thesis,proteins of Eucommia bark and Eucommia rubber particle were isolated,and their amino acid sequences and antimicrobial activity were determined.The research results are mentioned below.A novel Eucommia antifungal peptide,named EAFP3,was isolated from the bark of E. ulmoides by the method of gel filtration and reversed-phase high-performance liquid chromatography.On the other hand,nine proteins with molecular mass of 14,16,18,22,24,28, 30,32 and 55 kDa,respectively,were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） from Eucommia rubber particle.Among the eleven proteins,only five proteins with molecular mass 4.16,4.2,8.3,16 and 18 kDa were identified and 14 peptide fragments were obtained through liquid chromatography mass/mass spectrometry（LC-MS/MS）.The structure,sequence and function of these 14 peptide fragments were confirmed and accumulated in antimicrobial peptide database（APD）.The results show that eleven peptides:-.LYQQLIAGITLNK.-,-.PCNAGLCCSIYGYCGSGNAYCGGNCR.-, -.LRLDGAMPPSYPSSWSVLPR.-,-.QIITHPSMDDYVRK.-,-.LPLGAGVNLSGGPVVK.-,-.L PVAVKTGAEVVYSK.-,-.ISTLNSHNLPILR.-,-.ISLTEGIRR.-,-.RHGNDHDVHPK.-,-.TNGP VVNLKAFLKK.-and-.GRSAPVLAATVAGDTR.-have the typical structural feature of antimicrobial peptide.Besides,the result of antifungal activity indicates that EAFP3 exhibits broad spectrum antifungal activity and can inhibit the growth of yeast,yeast-like,molds and phytopathogenic fungi. The IC₅₀ values of EAFP3 for Candida albicans,Aspergillus flavus and Botrytis cinere are 31.25, 31.25 and 62.5μg/mL.After heating at 100℃for 30 min,EAFP3 can keep its antifungal activity and thermal stability,and display a broad pH-activity,profile in the range from 5.8 to 8.0.The above results indicate that lead compounds for creating new biological pesticides and drugs as well as primer and probe sequences for cloning gene of antifungal protein could be obtained.更多还原