重组家蚕丝素非重复区肽段的制备及对细胞的作用

发布时间：2018-01-15 03:32

  本文关键词：重组家蚕丝素非重复区肽段的制备及对细胞的作用　出处：《苏州大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 家蚕丝素重链 非重复区肽段 大肠杆菌 表达纯化 细胞相容性

【摘要】：家蚕丝素是一种天然的动物蛋白纤维,有出色机械性能和生物相容性,越来越多地被作为组织修复用材料,不同的组织对材料的结构和性能的要求不同,结构也影响性能,尤其是蛋白质的氨基酸排列对其生物学性能有决定性影响。因此研究丝素蛋白组成与结构的关系及对细胞的作用十分重要。家蚕丝素蛋白主要是重链、轻链和P25蛋白以摩尔比6:6:1组成。其中重链是丝素蛋白的重要组成部分,由N-末端,中间核心区域和C-末端构成,其中核心区域包括重复区和非重复区(以下简称为非重复区)。为了清晰地获得各部分的肽段,基因工程是一种比较先进的技术手段。本文重点研究了核心区非重复序列的特征与性质。先前实验室对非重复区肽段的表达系统的构建已完成,在此基础上,论文主要利用大肠杆菌表达系统表达了非重复区肽段的融合蛋白,分离纯化和酶切后获得了丝素重链核心区非重复区的目的肽段,采用质谱,氨基酸组成分析和等电点测定鉴定了融合蛋白和酶切后目的肽段的正确性,初步分析了目的肽段二级结构,并研究了表达的目的肽段对内皮细胞生长的作用。(1)首先探索了非重复区基因表达载体的优化表达。将实验室已经构建好的含有非重复区序列编码的基因f(1)及其多倍体f(4),f(8)的表达载体pGEX-f(1)、pGEX-f(4)和pGEX-f(8)转化大肠杆菌,在诱导剂IPTG的诱导下获得了融合蛋白GST-F(1)、GST-F(4)和GST-F(8)。通过SDS-PAGE电泳鉴定了三种融合蛋白的初步表达。深入研究了诱导表达的起始菌密度、诱导剂IPTG浓度和诱导时间对融合蛋白表达水平的影响。SDS-PAGE电泳结果显示,融合蛋白GST-F(1)在起始菌密度OD600=1.5,IPTG诱导剂浓度0.1 mM,诱导表达时间1小时时,表达水平较高,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诱导剂浓度0.2m M,诱导表达时间4小时时,表达水平较高,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诱导剂浓度0.1 mM,诱导表达时间3小时时,表达水平较高,每升菌液可收集26.6 mg左右。(2)表达产物的纯化与目的肽段的释放。采用GST亲和层析纯化方法,获得了较高纯度的融合蛋白。经过凝血酶酶切去除GST标签,分离出目的肽段,采用SDS-PAGE电泳分析,成功获得目的蛋白F(1),F(4)和F(8)的释放且纯度较高。分离纯化得到的融合蛋白及酶切后释放的目的肽段进一步经过质谱分析,融合蛋白GST-F(1),GST-F(4)和GST-F(8)的理论分子量分别是31.0,43.0和58.9 kDa,实测值分别是31.5,43.8和59.0kDa;目的肽段F(1),F(4)和F(8)理论分子量分别是4.8,16.8和32.8kDa,实测值分别是4.8,16.8和32.8kDa,实测分子量均与理论值一致。(3)表达产物的表征。对融合蛋白和酶切后的目的肽段进行了氨基酸组成分析,结果显示实测值和理论值一致。融合蛋白GST-F(1),GST-F(4)和GST-F(8)理论等电点分别是5.35,4.56和4.22,测试结果分别是4.3,3.6和3.4;目的肽段F(1),F(4)和F(8)的实测等电点分别是3.3,3.2和3.0,这和理论等电点3.7,3.2,2.9基本一致。这些结构间接表明了表达的融合蛋白及酶切后的目的肽段是正确的。(4)目的肽段二级结构的初步分析。用红外光谱(FTIR)和圆二色光谱(CD)法初步探索了新鲜制备的目的肽段的二级结构,FTIR结果显示F(1)呈无规卷曲结构,而F(4)和F(8)均出现了无规卷曲和α螺旋结构。CD光谱显示随着分子链加长,分子构象发生改变,分子构象中部分由无规卷曲(F(1))变化到α螺旋(F(8))。(5)目的肽段对涤纶膜亲水性改性及对内皮细胞生长的研究。通过考马斯亮蓝染色、X射线光电子能谱分析(XPS)和静态水接触角分析,证明了目的肽段F(1)、F(4)和F(8)可以稳定地接枝到涤纶(PET)薄膜上,最大接枝量分别为0.020、0.008、0.006μmol/片(直径1.5cm),水接触角可以从77°分别降低到32,35和39°。探索了F(1)、F(4)和F(8)的PET膜对内皮细胞增殖的影响,结果显示接枝F(1)、F(4)和F(8)的PET膜有助于内皮细胞的生长,当接枝量分别达到0.015、0.006和0.004μmol/片可以显著提高内皮细胞的增殖。
[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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