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  本文選題：睡眠肽 + 人血清白蛋白�。� 參考：《蘭州理工大學》2012年碩士論文

【摘要】：睡眠是人類維持生命的生理過程之一，隨著現(xiàn)代社會節(jié)奏加快及競爭的加劇，失眠已經成為一種十分普遍的現(xiàn)象。因此，開發(fā)有效、安全的抗失眠藥物，已成為一項迫切的醫(yī)療和社會問題。睡眠肽是一個具有促進睡眠活性的九肽，它的生理活性主要是通過誘導慢波睡眠起作用。睡眠肽在體內含量極微，但是活性卻很強，且對人體無毒副作用，所以一直是國內外學者研究的焦點。但是由于其分子量較小，在體內代謝半衰期較短，所以極大地限制了其在臨床上的實際應用。解決上述問題的關鍵是延長睡眠肽的半衰期，因而本課題設計將穿導肽（PTD）、人血清白蛋白（HSA）、連接肽（Linker）以及睡眠肽（DSIP）四個蛋白進行融合表達(簡稱PHLD)，以期開發(fā)一種新型、高效的抗失眠類藥物。 本實驗挑選畢赤酵母偏愛密碼子，利用PCR技術以本實驗室保存的pPIC9K/HSA為模板進行擴增，將穿導肽、連接肽以及睡眠肽與人血清白蛋白基因連接獲得重組基因PHLD。將PHLD基因連接到畢赤酵母分泌表達載體pPIC9k上，經過PCR鑒定、酶切鑒定以及測序后表明成功獲得重組質粒pPIC9K/PHLD，然后將構建所得的重組質粒pPIC9K/PHLD經SalI線性化后電擊轉化入組胺酸缺陷型畢赤酵母GS115中，通過2mg/mL G418篩選出高拷貝的整合菌株，獲得了高效表達PHLD融合蛋白的酵母工程菌株，并利用搖瓶對畢赤酵母發(fā)酵生產PHLD融合蛋白的條件進行了考察，初步確定目的融合蛋白的生長及制備的條件即：選用YPD作為種子培養(yǎng)基，搖床培養(yǎng)16-18h，裝液量30mL/250mL三角瓶，按10%的接種量將所得菌液接種至基礎鹽BSM培養(yǎng)基中待菌體生長到48h時，開始甲醇誘導融合蛋白表達，每24h補加甲醇至終濃度1%，誘導5天后離心收集上清，經疏水層析分離、G25脫鹽，得到了較純的樣品。 本文探索并實現(xiàn)了PHLD在畢赤酵母中的表達，并對發(fā)酵產物通過疏水層析以及G25脫鹽進行初步純化并得到電泳純樣品，然后利用經典的戊巴比妥鈉睡眠實驗對PHLD融合蛋白的活性進行評價，，實驗結果表明PHLD融合蛋白具有延長小鼠睡眠時間的作用，說明穿導肽、人血清白蛋白、連接肽以及睡眠肽四者組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.
【學位授予單位】：蘭州理工大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R3416

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本文編號：2035142