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采用同源重组的方法,将5aG株狂犬病毒糖蛋白基因插入天坛株痘苗病毒基因组TK区段,置于痘苗病毒晚期启动子P11控制之下,通过筛选,得到一株重组病毒VVaG,实验结果表明:该株病毒可表达5aG株糖蛋白基因,表达产物位于感染细胞膜表面,分子量64×103,并可与抗狂犬病毒糖蛋白单抗特异结合,用VVaG免疫小鼠,可诱导机体产生抗狂犬病毒中和抗体,抵抗致死剂量狂犬病毒的攻击,中和抗体滴度在免疫后14d达到高峰,为1560. 相似文献
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源于大肠杆菌蛋白的表达、液相色谱复性与纯化新进展 总被引:3,自引:0,他引:3
对近两年来源于大肠杆菌(Escherichia coli,E.coli)的蛋白表达和用蛋白折叠液相色谱(protein folding liquid chromatography,PFLC)法对所形成的包涵体目标蛋白的复性并同时纯化的新近发展做了简要的介绍和评述.PFLC法用于包涵体蛋白分离、纯化很广,其特点是除了在色谱柱上将目标蛋白与其他组分分开,还同时要在色谱柱上进行包涵体蛋白折叠.可以说,现代生物技术中所用的大多数有价值蛋白产品的制备仍然有赖于不同机理的液相色谱(Lc)法.而用PFLC法对源于E.coli的蛋白的制备方法更具可塑性和容易达到规模化,其生成本可以成倍地降低.该文主要内容包括了E.coli蛋白的表达及样品前处理、PFLC的实用范围、PFLC的优化、PFLC中的新技术、新设备和新方法、PFLC的分子学机理、应用事例及对未来的展望. 相似文献
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D. A. Ledward 《高压研究》2013,33(1-6):1-10
Abstract The main targets for the pressure induced changes in proteins are those regions primarily stabilised by hydrophobic and electrostatic interactions, since hydrogen bonds are almost pressure insensitive. Thus pressure treated proteins may well have very different structures to their native or heat treated counterparts and as a consequence different functionalities. This concept is used to discuss how pressure can modify the foaming, emulsifying and gelling properties of some food proteins and can also be used to modify the activity of some enzymes of importance in dictating food quality. 相似文献
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蛋白折叠液相色谱法(PFLC)用于变性蛋白质复性并同时纯化时对流动相组成及其洗脱条件的要求远较通常的液相色谱法高。用端基为PEG-200的高效疏水作用色谱固定相对重组人干扰素-γ(rhIFN-γ)进行纯化并同时复性,详细研究了流动相组成、梯度洗脱模式和流速对rhIFN-γ质量回收率和活性的影响。分别以3.0 mol/L (NH4)2SO4 +0.05 mol/L KH2PO4(pH 7.0)和0.05 mol/L KH2PO4(pH 7.0)为流动相A和B,采用35 min非线性梯度洗脱时,所得rhIFN-γ的质量回收率最高。 相似文献
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Flt3配体(FL)是一类具有促进早期造血功能的细胞因子,在促进造血细胞生长发育及造血动员方面具有重要的临床应用价值。为了用基因工程方法获得大量用于临床和研究的重组人FL(rhFL)蛋白质,本文对在大肠杆菌(E. coli)中表达得到的Flt3配体的包涵体进行回收、洗涤,溶解于8 mol/L脲后在高效疏水相互作用色谱(HPHIC)柱上进行rhFL包涵体的复性与同时纯化,并对其保留特征和复性规律进行了研究。结果表明,在连续进样、变性蛋白质质量浓度为8.51 g/L、固定相选用端基为PEG800、流动相添加4 mol/L脲、1.8 mmol/L 还原型谷胱甘肽(GSH)和0.3 mmol/L氧化型谷胱甘肽(GSSH)、pH 7.0的优化条件下,复性与同时纯化rhFL包涵体的质量回收率为36.9%,纯度达94.5%以上。本文仅用一步HPHIC法成功地复性与同时纯化了rhFL蛋白质,为获得高活性的rhFL产品奠定了一定的工作基础。 相似文献
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Based on three-state renaturation process of denatured proteins, an equation describing the effect of denaturant concentration on renaturation yield of denatured proteins was presented. By this equation, two parameters n(m1 -m2) and Ka can be obtained. The former indicates the difference in the number of denaturant molecules between the renaturation process of n number of refolding intermediates from refolding intermediate state to native state and their aggregate process from refolding intermediate state to aggregate state, the latter denotes the apparent aggregate equilibrium constant for protein molecules aggregated from native state to aggregate state, and from them, the characteristics of the renaturation process of denatured proteins in denaturant solution can be identified. This equation was tested by the renaturation processes of denatured egg white lysozyme in guanidine hydrochloride and urea solutions, with the results to show that when guanidine hydrochloride and urea concentrations were separately higher than 1.25 and 3.00 mol/L or separately lower than 1.00 and 3.00 mol/L, the refolding intermediates of egg white lysozymes were more easily aggregated to aggregate state or more easily renatured to native state, respectively. Under different initial total egg white lysozyme concentrations in urea solution, the refolding egg white lysozyme intermediates could be deduced to have a tendency to form a bimolecular intermediate aggregate, and this inference was further confirmed by their nonreducing SDS-PAGE and size exclusion chromatography. 相似文献
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Crosslinking of gelatin membranes with ferulic acid or glutaraldehyde: Relationship between gas permeability and renaturation level of gelatin triple helices 下载免费PDF全文
Jennifer Biscarat Christophe Charmette Nathalie Masquelez José Sanchez Celine Pochat‐Bohatier 《Journal of Polymer Science.Polymer Physics》2015,53(4):280-287
This study investigated the gas separation membranes made with gelatin, crosslinked with ferulic acid (FA) and blended with polyethylene glycol (PEG) 200, by using a solvent‐free procedure. Gas permeation properties (He, N2, O2, and CO2) of these “green membranes” were studied and discussed in relation with their structure. Differential scanning calorimetric measurements were carried out to determine the gelatin triple helical renaturation level. The lowest permeability values [He and CO2 permeability (4.5 × 10?2 Barrer) with CO2/O2 selectivity of 14.5] were reached with gelatin/PEG 200 uncrosslinked membranes showing the highest renaturation level (40%). Crosslinking with FA lead to less rigid and brittle materials than GTA and to 10 times more permeable membranes compared with uncrosslinked membranes. Membranes crosslinked with glutaraldehyde broke during gas permeation measurements. Results demonstrated that higher gas permeability values were closely related to lower renaturation level of gelatin. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015 , 53, 280–287 相似文献
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Many experimental results show that aggregation is the main side reaction in the renaturation process of proteins and once the proteins aggregate, it is rather difficult to be renatured again. Therefore, the renaturation efficiency can be highly enhanced,… 相似文献
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疏水作用色谱法同时纯化及复性基因重组人干扰素-α 总被引:3,自引:0,他引:3
使用高效疏水作用色谱直接从大肠杆菌表达的基因重组人干扰素 α(rhIFN α)包涵体的裂解液中纯化了rhIFN α ,并在纯化的同时获得了高的复性效率 ,使复性和纯化一步完成 ,大大地简化了操作步骤。用凝胶排阻色谱对该法纯化的rhIFN α进行了纯度测定 ,纯度达到 95 %以上。该法的活性回收率分别比稀释法和透析法高 1 0倍和 1 6倍。 相似文献