本文關(guān)鍵詞：重組家蠶絲素非重復(fù)區(qū)肽段的制備及對(duì)細(xì)胞的作用　出處：《蘇州大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 家蠶絲素重鏈 非重復(fù)區(qū)肽段 大腸桿菌 表達(dá)純化 細(xì)胞相容性

【摘要】：家蠶絲素是一種天然的動(dòng)物蛋白纖維,有出色機(jī)械性能和生物相容性,越來越多地被作為組織修復(fù)用材料,不同的組織對(duì)材料的結(jié)構(gòu)和性能的要求不同,結(jié)構(gòu)也影響性能,尤其是蛋白質(zhì)的氨基酸排列對(duì)其生物學(xué)性能有決定性影響。因此研究絲素蛋白組成與結(jié)構(gòu)的關(guān)系及對(duì)細(xì)胞的作用十分重要。家蠶絲素蛋白主要是重鏈、輕鏈和P25蛋白以摩爾比6:6:1組成。其中重鏈?zhǔn)墙z素蛋白的重要組成部分,由N-末端,中間核心區(qū)域和C-末端構(gòu)成,其中核心區(qū)域包括重復(fù)區(qū)和非重復(fù)區(qū)(以下簡稱為非重復(fù)區(qū))。為了清晰地獲得各部分的肽段,基因工程是一種比較先進(jìn)的技術(shù)手段。本文重點(diǎn)研究了核心區(qū)非重復(fù)序列的特征與性質(zhì)。先前實(shí)驗(yàn)室對(duì)非重復(fù)區(qū)肽段的表達(dá)系統(tǒng)的構(gòu)建已完成,在此基礎(chǔ)上,論文主要利用大腸桿菌表達(dá)系統(tǒng)表達(dá)了非重復(fù)區(qū)肽段的融合蛋白,分離純化和酶切后獲得了絲素重鏈核心區(qū)非重復(fù)區(qū)的目的肽段,采用質(zhì)譜,氨基酸組成分析和等電點(diǎn)測(cè)定鑒定了融合蛋白和酶切后目的肽段的正確性,初步分析了目的肽段二級(jí)結(jié)構(gòu),并研究了表達(dá)的目的肽段對(duì)內(nèi)皮細(xì)胞生長的作用。(1)首先探索了非重復(fù)區(qū)基因表達(dá)載體的優(yōu)化表達(dá)。將實(shí)驗(yàn)室已經(jīng)構(gòu)建好的含有非重復(fù)區(qū)序列編碼的基因f(1)及其多倍體f(4),f(8)的表達(dá)載體pGEX-f(1)、pGEX-f(4)和pGEX-f(8)轉(zhuǎn)化大腸桿菌,在誘導(dǎo)劑IPTG的誘導(dǎo)下獲得了融合蛋白GST-F(1)、GST-F(4)和GST-F(8)。通過SDS-PAGE電泳鑒定了三種融合蛋白的初步表達(dá)。深入研究了誘導(dǎo)表達(dá)的起始菌密度、誘導(dǎo)劑IPTG濃度和誘導(dǎo)時(shí)間對(duì)融合蛋白表達(dá)水平的影響。SDS-PAGE電泳結(jié)果顯示,融合蛋白GST-F(1)在起始菌密度OD600=1.5,IPTG誘導(dǎo)劑濃度0.1 mM,誘導(dǎo)表達(dá)時(shí)間1小時(shí)時(shí),表達(dá)水平較高,每升菌液可收集58.8 mg左右。融合蛋白GST-F(4)在起始菌密度OD600=1.2,IPTG誘導(dǎo)劑濃度0.2m M,誘導(dǎo)表達(dá)時(shí)間4小時(shí)時(shí),表達(dá)水平較高,每升菌液可收集39.2 mg左右。融合蛋白GST-F(8)在起始菌密度OD600=1.5,IPTG誘導(dǎo)劑濃度0.1 mM,誘導(dǎo)表達(dá)時(shí)間3小時(shí)時(shí),表達(dá)水平較高,每升菌液可收集26.6 mg左右。(2)表達(dá)產(chǎn)物的純化與目的肽段的釋放。采用GST親和層析純化方法,獲得了較高純度的融合蛋白。經(jīng)過凝血酶酶切去除GST標(biāo)簽,分離出目的肽段,采用SDS-PAGE電泳分析,成功獲得目的蛋白F(1),F(4)和F(8)的釋放且純度較高。分離純化得到的融合蛋白及酶切后釋放的目的肽段進(jìn)一步經(jīng)過質(zhì)譜分析,融合蛋白GST-F(1),GST-F(4)和GST-F(8)的理論分子量分別是31.0,43.0和58.9 kDa,實(shí)測(cè)值分別是31.5,43.8和59.0kDa;目的肽段F(1),F(4)和F(8)理論分子量分別是4.8,16.8和32.8kDa,實(shí)測(cè)值分別是4.8,16.8和32.8kDa,實(shí)測(cè)分子量均與理論值一致。(3)表達(dá)產(chǎn)物的表征。對(duì)融合蛋白和酶切后的目的肽段進(jìn)行了氨基酸組成分析,結(jié)果顯示實(shí)測(cè)值和理論值一致。融合蛋白GST-F(1),GST-F(4)和GST-F(8)理論等電點(diǎn)分別是5.35,4.56和4.22,測(cè)試結(jié)果分別是4.3,3.6和3.4;目的肽段F(1),F(4)和F(8)的實(shí)測(cè)等電點(diǎn)分別是3.3,3.2和3.0,這和理論等電點(diǎn)3.7,3.2,2.9基本一致。這些結(jié)構(gòu)間接表明了表達(dá)的融合蛋白及酶切后的目的肽段是正確的。(4)目的肽段二級(jí)結(jié)構(gòu)的初步分析。用紅外光譜(FTIR)和圓二色光譜(CD)法初步探索了新鮮制備的目的肽段的二級(jí)結(jié)構(gòu),FTIR結(jié)果顯示F(1)呈無規(guī)卷曲結(jié)構(gòu),而F(4)和F(8)均出現(xiàn)了無規(guī)卷曲和α螺旋結(jié)構(gòu)。CD光譜顯示隨著分子鏈加長,分子構(gòu)象發(fā)生改變,分子構(gòu)象中部分由無規(guī)卷曲(F(1))變化到α螺旋(F(8))。(5)目的肽段對(duì)滌綸膜親水性改性及對(duì)內(nèi)皮細(xì)胞生長的研究。通過考馬斯亮藍(lán)染色、X射線光電子能譜分析(XPS)和靜態(tài)水接觸角分析,證明了目的肽段F(1)、F(4)和F(8)可以穩(wěn)定地接枝到滌綸(PET)薄膜上,最大接枝量分別為0.020、0.008、0.006μmol/片(直徑1.5cm),水接觸角可以從77°分別降低到32,35和39°。探索了F(1)、F(4)和F(8)的PET膜對(duì)內(nèi)皮細(xì)胞增殖的影響,結(jié)果顯示接枝F(1)、F(4)和F(8)的PET膜有助于內(nèi)皮細(xì)胞的生長,當(dāng)接枝量分別達(dá)到0.015、0.006和0.004μmol/片可以顯著提高內(nèi)皮細(xì)胞的增殖。
[Abstract]:Silk is a natural animal protein fiber, has excellent mechanical properties and biological compatibility, are increasingly being used as tissue repair materials, different structures and properties of materials have different requirements, the structure also affects the performance, especially the protein amino acid sequences have decisive influence on its biological properties. Therefore study on the relationship between the composition and structure of silk fibroin and effect on cells is very important. Silk fibroin is mainly heavy chain and light chain and P25 protein in a molar ratio of 6:6:1. The heavy chain is an important part of silk protein, by the end of N-, the middle of the core area and C- terminal, which includes the core area repeat region and non repeat region (hereinafter referred to as non repeat regions). In order to clearly get peptides and each part of the genetic engineering is an advanced technology. This paper focuses on the core area of non heavy The characteristics and properties of complex sequences. The construction has been completed the previous laboratory on the expression system of non repeat regions of peptides, on this basis, the main use of Escherichia coli expression system expressed non repeat peptide fusion protein purification and enzyme digestion were obtained after fib-h core non target peptide and repeat region by mass spectrometry, amino acid analysis and isoelectric point determination accuracy of the fusion protein and enzyme digestion to peptides, preliminary analysis of the two level structure of objective peptide and study objective peptide expression on the growth of endothelial cells. (1) first explored by gene expression optimization non repeats expression vector. The constructed containing non sequence encoding gene f repeat region (1) and f (4), f polyploid (8) expression vector pGEX-f (1), pGEX-f (4) and pGEX-f (8) were transformed into E.coli, the inducer IPTG under the The fusion protein GST-F (1), GST-F (4) and GST-F (8). Through SDS-PAGE electrophoresis preliminary expression of three fusion protein was studied. The initial bacterial density induced the expression of inducer IPTG concentration and induction time on the expression of fusion protein.SDS-PAGE electrophoresis effect level showed that the fusion protein GST-F (1) in the initial bacterial density of OD600=1.5, IPTG induced a concentration of 0.1 mM, the time expression induced by 1 hours, the expression level is higher, per liter of bacterial liquid can be collected about 58.8 mg. The fusion protein of GST-F (4) in the initial cell density OD600=1.2 IPTG 0.2m M, inducer concentration, induction time of 4 hours, the expression of high levels of bacteria per liter of liquid can be collected about 39.2 mg. The fusion protein GST-F (8) in the initial bacterial density of OD600=1.5, IPTG induced a concentration of 0.1 mM, the time expression induced by 3 hours, the expression level is higher, per liter of bacterial liquid can be collected about 26.6 mg. (2) the expression product of pure With the purpose of peptide release by GST affinity chromatography method, obtained high purity fusion protein. After digested with thrombin to remove the GST label, isolated objective peptides were analyzed by SDS-PAGE electrophoresis, successfully obtained F protein (1), F (4) and F (8) release and high purity. To release peptide fusion protein and enzyme digestion of purified after further by mass spectrometry analysis, fusion protein GST-F (1), GST-F (4) and GST-F (8) the theoretical molecular weight were 31.0,43.0 and 58.9 kDa, the measured values are 31.5,43.8 and 59.0kDa; the purpose of peptide F (1), F (4) and F (8) theory of molecular weight were 4.8,16.8 and 32.8kDa respectively, the measured value is 4.8,16.8 and 32.8kDa, the measured molecular weight was consistent with the theoretical value. (3) the expression products. For the purpose of characterization of peptide fusion protein and enzyme digestion of the amino acid composition analysis, the results show the measured and theoretical values 鑷，

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