摘要
目的构建牛乳铁多肽(Lactoferricin B)的真核表达质粒pYES2/Lactoferricin B,实现其在酿酒酵母S.cerevisiae中的表达,并初步检测其不同变异的体外抗菌活性。方法通过分别合成Lactoferricin B基因两条单链的部分序列,让其互为模板、引物进行PCR扩增,得到Lactoferricin B与3种变异的基因序列。将它们克隆到穿梭质粒pYES2中,构建pYES2/Lactoferricin B及3种变异基因重组质粒,转化到大肠杆菌Top10中,让其大量增殖。提取重组质粒经纯化后将其转化到S.cerevisiae中,通过营养缺陷型筛选获得重组酵母菌并通过半乳糖诱导使其表达目的蛋白。经离子交换柱纯化收集目的蛋白后,通过体外抑菌实验比较Lactoferricin B及3种变异重组蛋白的抗菌活性。结果经PCR扩增检验、DNA测序表明成功构建pYES2/Lactoferricin B与3种变异基因重组质粒。提取诱导后的蛋白进行SDS-PAGE电泳及质谱检测,证实目的蛋白的存在,相对分子质量约为3.4×103。在大肠埃希菌及金黄色葡萄球菌的抑菌实验中观测到Lactoferricin B和A17-Lactoferricin B产生了抑菌圈。结论成功构建pYES2/Lactoferricin B及变异基因重组质粒,该重组质粒能在S.cerevisiae中诱导表达目的蛋白,为进一步研究其生物功能及抗菌活性奠定了基础。
Objective To construct the eucaryotic recombinant plasmld of pYES2/LactoferricinB expressing in yeast of S. cerevisiae, of which the expressed protein antibacterial activity was verified in preliminary. Methods By self-template PCR method, the gene of Lactoferrlcin B and its several sequence mutations were amplified with the parts of the pre-synthesized single chains. And then Lactoferricin B gene and its mutants were cloned into the vector of pYES2 to construct the recombined expression plasmid pYES2/Lactoferricin B etc. extracted and used to transform the yeast S. cerevisiae. The expressions of proteins were determined after induced by galactose. The expression proteins were collected and purified by hydronium-exchange column, and the bacterial inhibited test was applied to identify the protein antibacterial activities, Results The PCR amplifying and DNA sequencing tests indicated that the purpose plasmld contained the Lactoferricln B gene and several mutations. The induced target proteins were confirmed by SDS-PAGE electrophoresis and mass spectrum test. The protein antibacterial activities of mutations were verified in preliminary. Conclusion The recombined plasmid pYES2/Lactoferricin B etc. are successfully constructed and induced to express in yeast cell of S. cerevisiae;the obtained recombined protein of Lactoferricin B provides a basis for further research work on the biological function and antibacterial activity.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2007年第4期578-582,共5页
Journal of Sichuan University(Medical Sciences)
关键词
抗菌肽
乳铁蛋白B
基因序列变异
酵母表达
Antibacterial peptide Lactoferricin B Gene sequence mutating Yeast expression
