PHLD融合蛋白在毕赤酵母中的构建、表达及活性鉴定

发布时间：2018-06-18 10:20

本文选题：睡眠肽 + 人血清白蛋白　；参考：《兰州理工大学》2012年硕士论文

【摘要】：睡眠是人类维持生命的生理过程之一，随着现代社会节奏加快及竞争的加剧，失眠已经成为一种十分普遍的现象。因此，开发有效、安全的抗失眠药物，已成为一项迫切的医疗和社会问题。睡眠肽是一个具有促进睡眠活性的九肽，它的生理活性主要是通过诱导慢波睡眠起作用。睡眠肽在体内含量极微，但是活性却很强，且对人体无毒副作用，所以一直是国内外学者研究的焦点。但是由于其分子量较小，在体内代谢半衰期较短，所以极大地限制了其在临床上的实际应用。解决上述问题的关键是延长睡眠肽的半衰期，因而本课题设计将穿导肽（PTD）、人血清白蛋白（HSA）、连接肽（Linker）以及睡眠肽（DSIP）四个蛋白进行融合表达(简称PHLD)，以期开发一种新型、高效的抗失眠类药物。本实验挑选毕赤酵母偏爱密码子，利用PCR技术以本实验室保存的pPIC9K/HSA为模板进行扩增，将穿导肽、连接肽以及睡眠肽与人血清白蛋白基因连接获得重组基因PHLD。将PHLD基因连接到毕赤酵母分泌表达载体pPIC9k上，经过PCR鉴定、酶切鉴定以及测序后表明成功获得重组质粒pPIC9K/PHLD，然后将构建所得的重组质粒pPIC9K/PHLD经SalI线性化后电击转化入组胺酸缺陷型毕赤酵母GS115中，通过2mg/mL G418筛选出高拷贝的整合菌株，获得了高效表达PHLD融合蛋白的酵母工程菌株，并利用摇瓶对毕赤酵母发酵生产PHLD融合蛋白的条件进行了考察，初步确定目的融合蛋白的生长及制备的条件即：选用YPD作为种子培养基，摇床培养16-18h，装液量30mL/250mL三角瓶，按10%的接种量将所得菌液接种至基础盐BSM培养基中待菌体生长到48h时，开始甲醇诱导融合蛋白表达，每24h补加甲醇至终浓度1%，诱导5天后离心收集上清，经疏水层析分离、G25脱盐，得到了较纯的样品。本文探索并实现了PHLD在毕赤酵母中的表达，并对发酵产物通过疏水层析以及G25脱盐进行初步纯化并得到电泳纯样品，然后利用经典的戊巴比妥钠睡眠实验对PHLD融合蛋白的活性进行评价，，实验结果表明PHLD融合蛋白具有延长小鼠睡眠时间的作用，说明穿导肽、人血清白蛋白、连接肽以及睡眠肽四者组成的融合蛋白保留了睡眠肽的活性。
[Abstract]:Sleep is one of the physiological processes that sustain human life. With the acceleration of modern social rhythm and the intensification of competition, insomnia has become a very common phenomenon. Therefore, the development of effective and safe anti-insomnia drugs has become an urgent medical and social problem. Sleep peptide is a nine peptide which has the activity of promoting sleep. Its physiological activity is mainly by inducing slow wave sleep. Sleep peptide content in vivo is very small, but the activity is very strong, and has no side effects on human body, so it has been the focus of domestic and foreign scholars. However, because of its small molecular weight and short metabolic half-life in vivo, its clinical application is greatly limited. The key to solve the above problem is to prolong the half-life of sleep peptide. Therefore, four proteins, namely PTD, HSAA, Linker and IPDS, are designed to develop a new type of protein. Effective anti-insomnia drugs. In this experiment, Pichia pastoris codon was selected and amplified by PCR using pPIC9K / HSA stored in our laboratory as template. The recombinant gene PHLDwas obtained by linking transconductance peptide, ligand peptide and sleep peptide with human serum albumin gene. PHLD gene was cloned into Pichia pastoris secretory expression vector pPIC9k and identified by PCR. The recombinant plasmid pPIC9K / PHLDwas successfully obtained by restriction enzyme digestion and sequencing. The recombinant plasmid pPIC9K / PHLD was linearized by Sali and transformed into histamine-deficient Pichia pastoris GS115. A high-copy integrated strain was screened by 2mg / mL G418. Yeast engineering strain expressing PHLD fusion protein was obtained and the fermentation conditions of PHLD fusion protein by Pichia pastoris were investigated by shaking flask. Objective to determine the growth and preparation conditions of the fusion protein: YPD was used as seed medium, shaking bed was used for 16-18 hours, and the volume of liquid was 30 mL / 250 mL triangular flask. The bacteria solution was inoculated into the basic salt BSM medium for 48 h according to the inoculation amount. The fusion protein was induced by methanol, supplemented with methanol every 24 hours to the final concentration of 1. The supernatant was collected by centrifugation after 5 days of induction, and then desalted by hydrophobic chromatography. The pure samples were obtained. In this paper, the expression of PHLD in Pichia pastoris was studied and the fermentation product was purified by hydrophobic chromatography and desalination of G25. Then the activity of PHLD fusion protein was evaluated by classical pentobarbital sodium sleep test. The results showed that PHLD fusion protein could prolong the sleep time of mice. The fusion protein composed of ligation peptide and sleep peptide retains the activity of sleep peptide.
【学位授予单位】：兰州理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R3416

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