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抗菌肽牛乳铁蛋白肽衍生肽在毕赤酵母中的表达及活性研究
李忠清
学位类型硕士
导师王亮
2011-05-25
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业有机化学
关键词毕赤酵母 牛乳铁蛋白肽衍生肽 抗菌肽
摘要牛乳铁蛋白肽(LfcinB)是牛乳铁蛋白在胃蛋白酶水解作用下释放出的一种含25个氨基酸残基的短肽,具有广谱抗菌、抗病毒、免疫调节等作用。经研究,不同动物的Lfcin,人源的、鼠源的、牛源的和羊源的乳铁蛋白肽,其中牛乳铁蛋白肽(LfcinB)的活性最高,这也是人们将重点移至LfcinB及其衍生物的原因,即希望能够从LfcinB及其衍生物中得到抗生素的替代品。 巴斯德毕赤酵母表达系统是一种优秀高效的外源蛋白基因表达系统,不仅具有高效表达、高稳定、高分泌、高密度生长、操作方便等特点,而且可以对表达产物进行类似真核细胞的翻译后修饰和加工等显著优点,在过去的20年中己经有超过200种不同的蛋白质在毕赤酵母中实现了成功表达。 目的:本实验拟利用基因工程技术对牛乳铁蛋白肽衍生肽建立体外表达系统,并筛选具有高效抗菌活性的衍生肽及高表达的重组菌株,为该蛋白的日后开发应用奠定基础。 方法:根据已测定的25个氨基酸残基的LfcinB的结构,并综合分析其构象关系及各个氨基酸在其抗菌活性中的作用,同时考虑到酵母偏爱密码子的特殊性,设计合成了抗菌肽牛乳铁蛋白肽衍生肽基因,该基因克隆到PMD19T载体上并对其进行酶切鉴定,挑取PMD19T-lfcinBD菌株放大培养提取质粒后用SnaBⅠ和NotⅠ进行双酶切,同时用SnaBⅠ和NotⅠ双酶切毕赤巴斯德酵母分泌表达载体pPIC9K,T4 DNA 连接酶连接,经酶切鉴定确认LfcinBD碱基序列插入多克隆位点并且开放性阅读框正确后,SacⅠ线性化重组质粒,用电击的方法转入毕赤酵母GS115中。30℃培养数天后从MD平板上挑取具有G418抗性的克隆,对比在MM和MD平板上的生长情况确定转化子的表现型。Lyticase酶裂解酵母细胞,以裂解后暴露的酵母基因组为模板,采用PCR方法确认有LfcinBD基因整合到酵母基因组中。将鉴定有LfcinBD基因整合酵母转化子接种到BMGY/BMMY培养基中发酵培养,用甲醇诱导,收集诱导不同时间的上清和细胞沉淀,用三氯醋酸(trichloroacetic acid,TCA)沉淀上清,用SDS-PAGE和抑菌实验两种方法检测目的蛋白的表达情况。 结果:LfcinBD碱基序列克隆入毕赤巴斯德酵母分泌表达载体pPIC9K;对含有LfcinBD的酵母转化子的甲醇诱导表达结果表明牛乳铁蛋白肽衍生肽LfcinBD在毕赤巴斯德酵母GS115菌株中成功表达且具有较为理想的抑菌活性。 结论:本实验设计合成了牛乳铁蛋白肽衍生肽LfcinBD,并且构建了pPIC9K-LfcinBD酵母表达质粒,SDS-PSAG电泳虽未检测到牛乳铁蛋白肽衍生肽LfcinBD的表达,但发酵液的上清检测到了抑菌活性,说明设计合成的牛乳铁蛋白肽衍生肽lfcinBD具有抗菌活性且得到了表达。 关键词:毕赤酵母,牛乳铁蛋白肽衍生肽,抗菌肽
其他摘要Bovine Lactoferricin(LfcinB) is a 25-residue peptide,which is generated by pepsin digestion of bovine lactoferin.LfcinB is a new type of antibacterial peptide, possessing broad-spectrum antibacteria,anti-virus,immunoregulation,et al.According to research,LfcinB is the best antibacterial among different animals,which is the why LfcinB has huge potential on pharmaceutical field. The Pichia pastoris expression system has being used successfully for the production of many heterologous proteins. P. pastoris is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities. Equally important, P.pastoris is capable of producing and secreting high level of various heterologous proteins,simplifying downstream purification. Additionally, P.pastoris is a eukaryote, and thereby provides the potential for producing soluble,correctly folded heterologous proteins that have undergone all the post-translational modifications required for functionality.More than 200 different proteins have been successfully expressed in the Pichia Pastoris expression system during the past 20 years. Object: To set up an effective expression system in vitro by genetic engineering in order to know bovine lactoferricin-derived peptide(LfcinBD)antimicrobial activities, and establish the foundation for later applications. Methods:This experiment designed and synthesized bovine lactoferricin-derived peptide(LfcinBD)according to the reported sequence,spatial structure,conformation,the importance of animo acids of bovine lactoferrin and the codon bias of P.pastoris.The gene encoding bovine lactoferricin-derived peptide(LfcinBD)was then cloned into PMD19T vector,after transforming into E.coli DH5α,its plasmid DNA was prepared and tested by restriction enzymes and correct results was named PMD19T-LfcinBD.Positive recombinant plasmid PMD19T-LfcinBD wasdigested with SnaBⅠand NotⅠ,at the same time,the Pichia pastoris expression vector pPIC9k was also digested with SnaBⅠand NotⅠ,then this two digested DNA fragment was ligated by T4 DNA ligase and tested by restriction enzymes. It was confirmed that the foreign gene had been inserted into the expression plasmid and the ORF was right.The expression pasmid was electroporated into PichiaPastoris GS115 after digested by Sac I.The positive transformants was selected onMD plates at 30℃ for several days,and dectected the phenotypes of methanol utilization of transformants by comparing the growth conditions on MM and MD plates.Genomic DNA of recombinants was isolated from digested yeast cells by lyticase and was used to further confirmation if the hLF gene had been integratedinto the yeast genome by PCR.According to the phenotypes of His+ transformans,the positive clones were cultured in BMGY/BMMY,inducing by methanol.We collected the supernatants and cell pellets by centrifugation,precipitated the proteinby TCA,and detected the expression of foreign protein by SDS-PAGE and antibacterial assay. Results: The gene of the LfcinBD was correctly cloned into the Pichia pastoris expression Vector pPIC9k;the remcombinant Pichia pastoris with lfcinBD gene was induced to express LfcinBD by methanol and the supernatants exhibit the antimicrobial activity against staphylococcus aureus,Escherichia coli and Ampr Escherichia coli by the measurement of the inhibition zone. Conclusion:This experiment designed and synthesized bovine lactoferricin-derived peptide (LfcinBD),and reconstruct the expression Vector pPIC9k-LfcinBD.Although SDS-PSAGE did not detect expression of the LfcinBD,the supernatants exhibit the antimicrobial activity,which is concluded that the lfcinBD possesses antimicrobial activity and was expressed successfully. Key Words:Pichia pastoris,Bovine lactoferricin-derived peptide(LfcinBD),Antibacterial peptide
文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4439
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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李忠清. 抗菌肽牛乳铁蛋白肽衍生肽在毕赤酵母中的表达及活性研究[D]. 北京. 中国科学院研究生院,2011.
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