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

【摘要】：睡眠是人類維持生命的生理過(guò)程之一，隨著現(xiàn)代社會(huì)節(jié)奏加快及競(jìng)爭(zhēng)的加劇，失眠已經(jīng)成為一種十分普遍的現(xiàn)象。因此，開(kāi)發(fā)有效、安全的抗失眠藥物，已成為一項(xiàng)迫切的醫(yī)療和社會(huì)問(wèn)題。睡眠肽是一個(gè)具有促進(jìn)睡眠活性的九肽，它的生理活性主要是通過(guò)誘導(dǎo)慢波睡眠起作用。睡眠肽在體內(nèi)含量極微，但是活性卻很強(qiáng)，且對(duì)人體無(wú)毒副作用，所以一直是國(guó)內(nèi)外學(xué)者研究的焦點(diǎn)。但是由于其分子量較小，在體內(nèi)代謝半衰期較短，所以極大地限制了其在臨床上的實(shí)際應(yīng)用。解決上述問(wèn)題的關(guān)鍵是延長(zhǎng)睡眠肽的半衰期，因而本課題設(shè)計(jì)將穿導(dǎo)肽（PTD）、人血清白蛋白（HSA）、連接肽（Linker）以及睡眠肽（DSIP）四個(gè)蛋白進(jìn)行融合表達(dá)(簡(jiǎn)稱PHLD)，以期開(kāi)發(fā)一種新型、高效的抗失眠類藥物。 本實(shí)驗(yàn)挑選畢赤酵母偏愛(ài)密碼子，利用PCR技術(shù)以本實(shí)驗(yàn)室保存的pPIC9K/HSA為模板進(jìn)行擴(kuò)增，將穿導(dǎo)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(dá)載體pPIC9k上，經(jīng)過(guò)PCR鑒定、酶切鑒定以及測(cè)序后表明成功獲得重組質(zhì)粒pPIC9K/PHLD，然后將構(gòu)建所得的重組質(zhì)粒pPIC9K/PHLD經(jīng)SalI線性化后電擊轉(zhuǎn)化入組胺酸缺陷型畢赤酵母GS115中，通過(guò)2mg/mL G418篩選出高拷貝的整合菌株，獲得了高效表達(dá)PHLD融合蛋白的酵母工程菌株，并利用搖瓶對(duì)畢赤酵母發(fā)酵生產(chǎn)PHLD融合蛋白的條件進(jìn)行了考察，初步確定目的融合蛋白的生長(zhǎng)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(chǔ)鹽BSM培養(yǎng)基中待菌體生長(zhǎng)到48h時(shí)，開(kāi)始甲醇誘導(dǎo)融合蛋白表達(dá)，每24h補(bǔ)加甲醇至終濃度1%，誘導(dǎo)5天后離心收集上清，經(jīng)疏水層析分離、G25脫鹽，得到了較純的樣品。 本文探索并實(shí)現(xiàn)了PHLD在畢赤酵母中的表達(dá)，并對(duì)發(fā)酵產(chǎn)物通過(guò)疏水層析以及G25脫鹽進(jìn)行初步純化并得到電泳純樣品，然后利用經(jīng)典的戊巴比妥鈉睡眠實(shí)驗(yàn)對(duì)PHLD融合蛋白的活性進(jìn)行評(píng)價(jià)，，實(shí)驗(yàn)結(jié)果表明PHLD融合蛋白具有延長(zhǎng)小鼠睡眠時(shí)間的作用，說(shuō)明穿導(dǎo)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(xué)位授予單位】：蘭州理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R3416

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 唱韶紅;鞏新;楊志愉;王同映;馬國(guó)昌;馬清鈞;吳軍;;人血清白蛋白和人干擾素α2b的融合蛋白在畢赤酵母中的表達(dá)[J];生物工程學(xué)報(bào);2006年02期

2 黃斯勇;伍艷蘭;李國(guó)輝;何飛;康志杰;劉利;韓驊;梁英民;;人Delta-like1~(ext)-Fc融合蛋白畢赤酵母表達(dá)載體的構(gòu)建及表達(dá)[J];細(xì)胞與分子免疫學(xué)雜志;2008年05期

3 李國(guó)輝;康志杰;黃斯勇;何飛;徐恒;張麗;伍艷蘭;牛曉麗;馬長(zhǎng)升;韓驊;梁英民;;融合蛋白hJagged1~(ext)-Fc畢赤酵母表達(dá)載體的構(gòu)建及表達(dá)[J];中國(guó)實(shí)驗(yàn)血液學(xué)雜志;2008年04期

4 丁月娣;雷楗勇;陳蘊(yùn);張蓮芬;金堅(jiān);;人腦鈉肽與人血清白蛋白融合蛋白在畢赤酵母中的分泌表達(dá)及其活性[J];中國(guó)生物制品學(xué)雜志;2009年03期

5 徐鈺;孫鈞銘;張蓮芬;竇文芳;李潔;朱威;朱書(shū)峰;金堅(jiān);;人GCSF-白蛋白融合蛋白的構(gòu)建及在畢赤酵母中的表達(dá)[J];藥物生物技術(shù);2008年02期

6 趙磊;顏煒群;;重組人uPA_(17)-KPI在畢赤酵母中的表達(dá)及鑒定[J];中國(guó)實(shí)驗(yàn)診斷學(xué);2010年11期

7 馮文華;石燕;喻光榮;沈衛(wèi)英;蔣O

本文編號(hào)：2035142