WO2016119536A1 - 高正电荷荧光蛋白在糖胺聚糖及其类似物分析检测中的应用 - Google Patents

高正电荷荧光蛋白在糖胺聚糖及其类似物分析检测中的应用 Download PDF

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WO2016119536A1
WO2016119536A1 PCT/CN2015/097648 CN2015097648W WO2016119536A1 WO 2016119536 A1 WO2016119536 A1 WO 2016119536A1 CN 2015097648 W CN2015097648 W CN 2015097648W WO 2016119536 A1 WO2016119536 A1 WO 2016119536A1
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glycosaminoglycan
sample
fluorescent protein
fluorescence
gags
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李福川
王文爽
张晓茹
韩乃寒
韩文君
李瑞娟
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Shandong University
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence

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  • the invention relates to the application of a high positive green fluorescent protein in the study of glycosaminoglycans, and belongs to the field of biochemical technology.
  • Glycosaminoglycans also known as mucopolysaccharides, are linear polyanionic polysaccharides composed of repeating disaccharide units. Mainly including Hyaluronic Acid (HA), Heparin/Heparan Sulfate (Hep/HS), Chondroitin Sulfate/Dermatan Sulfate (CS/DS), Keratin Sulfate (Keratan Sulfate, KS).
  • HA Hyaluronic Acid
  • Hep/HS Heparin/Heparan Sulfate
  • CS/DS Chondroitin Sulfate/Dermatan Sulfate
  • Keratin Sulfate Keratan Sulfate
  • the disaccharide units of other GAGs are derived from hexuronic acid (D-glucuronic acid/L-Edu Uronic acid) is composed of hexosamine (N-acetylglucosamine/N-acetylgalactosamine).
  • the HA structure is relatively simple, and a disaccharide unit composed of D-glucuronic acid and N-acetylglucosamine is linked by a ⁇ -1,4-glycosidic bond.
  • GAGs make the sugar chain extremely complicated due to the action of various modified enzymes, mainly in the sulfation of hydroxyl (-OH) and amino (-NH 2 ) in different parts of the sugar chain, and D-glucuronic acid in C5- Under the action of epimerase, it is converted into L-iduronic acid, acetylation of the di-amino group of hexosamine, and the like.
  • the high complexity of the GAGs sugar chain structure is not random, but has a high temporal and spatial specificity, which is caused by the expression level of various sugar chain synthesis related enzymes in different developmental stages of different cell tissues and organs. Structures have different functions, and the complexity of the structure gives them a diversity of functions.
  • GAGs are widely distributed on the cell surface and cell matrix of animal cells. They participate in various physiological and pathological processes such as cell proliferation and differentiation, cell-to-cell recognition, cell transfer, tissue morphogenesis, and carcinogenesis through specific interactions with various proteins. [1-4], a series of important biological functions of GAGs make it an important bioactive molecule, widely used in medicine and functional foods, such as Hep, CS, HA, etc. [5,6].
  • GAGs The structure of GAGs is more complex than nucleic acids and proteins, and its structural complexity and diversity pose great challenges for studying the relationship between structure and function.
  • Various cationic dyes eg, alixin blue, toluidine blue, and amide black
  • alixin blue, toluidine blue, and amide black are often used for the staining and detection of GAGs in samples.
  • dyes have many disadvantages, such as poor biocompatibility, and are not suitable for staining living cells and tissues; low selectivity, resulting in high background values; low sensitivity.
  • various novel cationic chromophores have been synthesized and used for high sensitivity detection of Hep in buffer or serum, but their biocompatibility and application in the detection of other glycosaminoglycans need further study. Therefore, it is urgent to develop a biocompatible, highly sensitive multi-purpose GAG probe for the observation and detection of GAGs in various biological samples.
  • Green fluorescent protein was first discovered in a jellyfish named Aequorea victoria. Because GFP and biofilm reporter genes for living organisms are used in cell biology and molecular biology research. And GFP can be used as a molecular probe for real-time monitoring of living organisms. Recently, by mutating some non-conservative amino acids on GFP to basic amino acids such as lysine and arginine, a large amount of positive charge is carried out without affecting the fluorescence characteristics of GFP. Charge green fluorescent protein has been successfully used as a transport vector for proteins and nucleic acids to enter cells, and as a highly sensitive probe for nucleic acid detection [7,8].
  • the invention aims at the deficiencies of the prior art, and provides a novel means for detecting glycosaminoglycans, that is, qualitative and quantitative analysis and detection of glycosaminoglycans by using high positive green fluorescent protein.
  • the glycosaminoglycan analog is selected from a natural or artificial semi-synthetic and fully synthetic polyanionic polysaccharide; further preferably, the glycosaminoglycan analog is selected from the group consisting of: a sulfated polysaccharide, and more Polysialic acid, alginate, carrageenan, fucoidan or lipopolysaccharide.
  • the sample to be tested is subjected to fluorescence measurement, and the content of glycosaminoglycan and/or glycosaminoglycan analogue in the sample is qualitatively and/or quantitatively analyzed according to the fluorescence intensity.
  • the sample in step (1) is a liquid, a solid phase carrier, a cell, a tissue, an organ.
  • the contact concentration of the high positively charged fluorescent protein is 0.01 to 5.0 ⁇ g/ml.
  • the fluorescence quencher is selected from the group consisting of graphene, nano gold, and/or an organic small molecule fluorescence quencher.
  • the quantitative analysis is to draw a standard curve by detecting the fluorescence intensity of the standard sample of different dilutions, and obtain a linear equation according to the linear relationship between the concentration of the standard curve and the fluorescence intensity, The fluorescence intensity value of the sample to be tested is substituted into the obtained linear equation, and the content of glycosaminoglycan and/or glycosaminoglycan analog in the sample is calculated.
  • the fluorescence is determined by qualitative analysis using a fluorescence microscope, a flow cytometer or a living imaging device.
  • the present invention is the first to use high-positive GFP (ScGFP) as a biocompatible, highly sensitive and highly selective fluorescent probe for visualization of GAGs for living cell surface, high sensitivity quantitative analysis of GAGs in serum, heparin Chondroitin sulfate contamination analysis of persulfation. Therefore, it overcomes the shortcomings of traditional cationic dyes in the detection of GAGs, such as poor biocompatibility, low selectivity and low sensitivity. It has important application value and can be widely used in basic and applied research, clinical diagnosis, medicine and food related to GAGs. Detection, etc.
  • FIG. 1 Polyacrylamide gel electrophoresis pattern (SDS-PAGE) of recombinant high positive green fluorescent protein (ScGFP) expression and purification.
  • the samples added in each lane were: M: protein molecular weight standard, the strip size was 116kD, 66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD from top to bottom; lane 1: pre-broken cells of the control strain, The sample volume was 10 ⁇ L, lane 2: the pre-debrided cell was pre-broken, the sample volume was 10 ⁇ L, lane 3: the supernatant of the recombinant strain was broken, and the sample volume was 10 ⁇ L. Lane 4: ScGFP purified by nickel column, loading amount 10 ⁇ L.
  • Figure 2 Effect of different concentrations of glycosaminoglycans on inhibition of graphene oxide quenching ScGFP fluorescence.
  • A hyaluronic acid (HA);
  • B chondroitin sulfate A (CS-A);
  • C dermatan sulfate (DS);
  • D heparin (Hep).
  • the glycosaminoglycan concentrations were 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, respectively. 0.95, 1 ⁇ g/ml.
  • Figure 3 Different concentrations of heparin in serum inhibit the effect of graphene oxide on quenching ScGFP fluorescence. From bottom to top, the concentrations of Hep are 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1 ⁇ g, respectively. /ml.
  • FIG. 4 Effect of heparin containing different persulfated chondroitin sulfate (OSCS) on inhibition of graphene oxide quenching of ScGFP fluorescence after heparinase degradation.
  • OSCS persulfated chondroitin sulfate
  • Figure 5 Confocal microscopy assisted detection of glycosaminoglycans on the cell surface.
  • Figure 6 Flow cytometry assisted detection of glycosaminoglycans on the cell surface.
  • A not treated with enzyme;
  • B treated with chondroitin sulfate degrading enzyme ABC;
  • C treated with heparinase I, II and III;
  • D co-sulfated chondroitin-degrading enzyme ABC and heparinase I, II and III deal with.
  • the amino acid sequence of ScGFP (+36) was obtained by literature reports [7], and the obtained amino acid sequence was converted into the corresponding base sequence, and the corresponding base was codon optimized to make it easier in Escherichia coli. expression.
  • the corresponding DNA sequence was fully synthesized by Biotech Engineering.
  • the ScGFP gene obtained in Example 1 was used as a template to carry out PCR amplification.
  • the primers are as follows:
  • the forward primer is underlined with the restriction endonuclease Nde I site
  • the reverse primer is underlined with the restriction endonuclease Xho I site.
  • the PCR product was double-digested, and the double-digested PCR product was also subjected to double digestion.
  • the pET-22b vector was ligated and the positive recombinant plasmid (pET22b-ScGFP) was screened.
  • the gene sequencing results showed that the ScGFP gene was inserted between the Nde I and Xho I restriction sites of pET-22b, and the insertion direction was correct.
  • pET22b-ScGFP was transformed into E. coli strain BL21 (DE3) (purchased from Novagen, USA), and then subjected to recombinant high-positive green fluorescent protein pET22b-ScGFP-induced expression according to the procedure provided by the company.
  • the purification of recombinant ScGFP was detected by polyacrylamide gel electrophoresis. The results showed that the purified recombinant ScGFP showed a single band on the electrophoresis gel and its position was consistent with the predicted molecular weight.
  • the amino acid sequence was as follows. SEQ ID NO. 1 is shown.
  • Example 3 Different concentrations of glycosaminoglycan inhibit the effect of graphene oxide on quenching scGFP fluorescence
  • a suitable amount of A549 cells were cultured on a 35 mm diameter cover glass culture dish, and a total of 5 plates were cultured, one of which was treated with heparinase I, II, and III, and one plate was treated with chondroitin sulfate degrading enzyme ABC.
  • the plates were co-treated with two enzymes, and two plates were treated without enzymes and treated at 30 ° C for 1-5 h. All 5 cells were stained with DAPI, except that one cell without enzyme treatment was not added with ScGFP as a negative control, and the rest was added with 0.1-10 ⁇ g ScGFP (200 ⁇ l PBS) for 5-30 min, then 100-1,000 ⁇ l PBS. Wash 2-5 times.
  • Two plates of 293T cells were cultured in a 10 cm diameter cell culture dish, and then the cells were suspended and divided into 4 portions on average.

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Abstract

高正电荷绿色荧光蛋白在糖胺聚糖研究中的应用，所述高正电荷荧光蛋白的氨基酸序列如SEQ ID NO.1所示。将高正电GFP作为生物相容性、高灵敏度和高选择性荧光探针，应用于活细胞表面的GAGs的可视化，血清中GAGs的高灵敏性定量分析，肝素中过硫酸化的硫酸软骨素污染分析等。从而克服了传统阳离子染料在GAGs检测中的生物相容性差、选择性低、灵敏度低等缺点，具有重要应用价值，可被广泛应用于GAGs相关的基础和应用研究、临床诊断、医药和食品的检测等。

Description

高正电荷荧光蛋白在糖胺聚糖及其类似物分析检测中的应用 技术领域
本发明涉及高正电荷绿色荧光蛋白在糖胺聚糖研究中的应用，属于生物化学技术领域。
背景技术
糖胺聚糖(Glycosaminoglycans，GAGs)又称为粘多糖，是重复二糖单位组成的直链聚阴离子多糖。主要包括透明质酸(Hyaluronic Acid，HA)，肝素/硫酸乙酰肝素(Heparin/Heparan Sulfate，Hep/HS)，硫酸软骨素/硫酸皮肤素(Chondroitin Sulfate/Dermatan Sulfate，CS/DS)，硫酸角质素(Keratan Sulfate，KS)。除硫酸角质素的二糖单位是由中性的D-半乳糖和N-乙酰葡糖胺组成以外，其它GAGs的二糖单位均是由己糖醛酸(D-葡萄糖醛酸/L-艾杜糖醛酸)与己糖胺(N-乙酰葡萄糖胺/N-乙酰半乳糖胺)组成。其中HA结构相对简单，由D-葡萄糖醛酸和N-乙酰葡糖胺组成的二糖单位经β-1,4-糖苷键连接而成。其它GAGs则由于各种修饰酶的作用使糖链变得异常复杂，主要表现在糖链中不同部位羟基(-OH)和氨基(-NH2)的硫酸化，D-葡萄糖醛酸在C5-差向异构酶的作用下转变为L-艾杜糖醛酸，己糖胺二位氨基的乙酰化等。GAGs糖链结构的这种高度复杂性不是随机发生的，而是具有高度的时空特异性，是各种糖链合成相关酶在不同细胞组织和器官的不同发育阶段表达调控水平所致，不同的结构使其具有不同的功能，结构的复杂性赋予其功能的多样性。
GAGs广泛存在于动物细胞细胞表面和细胞基质中，通过与各种蛋白的特异相互作用参与了细胞的增值和分化、细胞间的识别、细胞转移、组织形态发生、癌变等各种生理和病理过程[1-4]，GAGs所具有的一系列重要的生物学功能使其成为重要的生物活性分子，在医药及功能食品中得到广泛应用，如Hep、CS、HA等[5,6]。
GAGs的结构相对于核酸和蛋白质来说更为复杂，它的结构复杂性和多样性为研究其结构与功能的关系带来了很大的挑战。各种阳离子染料(如：阿利新蓝,甲苯胺蓝和酰胺黑等)常被用于样品中GAGs的染色和检测。然而这类染料具有很多缺点，如生物相容性较差，不适用于对活细胞和组织染色；选择性较低，致使出现很高的背景值；灵敏度较低等。近年来，各种新型的阳离子发色团被合成并用于缓冲液或血清中的Hep进行高灵敏度检测，但其生物相容性及在其它糖胺聚糖检测方面的应用有待进一步研究。因此，亟待发展一种生物相容性好、灵敏度高的多用途GAG探针用于各种生物样品中GAGs的观察和检测。
绿色荧光蛋白(GFP)是最早在一种学名为Aequorea victoria的水母中发现的。因为GFP与活体生物的生物相报道基因被用于细胞生物学和分子生物学研究。并且GFP可以作为分子探针用于活体的实时监测。最近，通过将GFP上的一些非保守氨基酸突变为赖氨酸、精氨酸等碱性氨基酸，从而在不影响GFP荧光特性的情况下使其带大量的正电荷，这种高正 电荷绿色荧光蛋白已被成功用于蛋白与核酸进入细胞的运输载体，并作为核酸检测的高灵敏度探针[7,8]。
发明内容
本发明针对现有技术的不足，提供一种新的检测糖胺聚糖的手段，即运用高正电绿色荧光蛋白对糖胺聚糖进行定性定量分析检测。
本发明的技术方案如下：
高正电荷荧光蛋白作为荧光标记物在糖胺聚糖和/或糖胺聚糖类似物分析检测中的应用，所述高正电荷荧光蛋白的氨基酸序列如SEQ ID NO.1所示。
根据本发明优选的，所述的糖胺聚糖类似物选自天然或人工半合成及全合成的聚阴离子多糖；进一步优选，所述的糖胺聚糖类似物选自：硫酸化多糖、多聚唾液酸、褐藻胶、卡拉胶，岩藻聚糖或脂多糖。
根据本发明优选的，具体步骤如下：
(1)使样品与高正电荷荧光蛋白接触，进行孵育结合，孵育时间5～30min，孵育后待测样品；
(2)去除未与待检测样品中糖胺聚糖和/或糖胺聚糖类似物结合的高正电荷荧光蛋白，或者采用荧光淬灭剂淬灭与待检测样品中糖胺聚糖和/或糖胺聚糖类似物结合的高正电荷荧光蛋白，制得待检测样品；
(3)对待检测样品进行荧光测定，根据荧光强度对样品中糖胺聚糖和/或糖胺聚糖类似物含量进行定性和/或定量分析。
根据本发明优选的，所述步骤(1)中的样品为液体、固相载体、细胞、组织、器官。
根据本发明优选的，所述步骤(1)中，高正电荷荧光蛋白的接触浓度为0.01-5.0μg/ml。
根据本发明优选的，所述步骤(2)中，荧光淬灭剂选自：石墨烯、纳米金和/或有机小分子荧光淬灭剂。
根据本发明优选的，所述步骤(3)中，定量分析是通过检测不同稀释度的标准样品的荧光强度绘制标准曲线，根据标准曲线的浓度与荧光强度之间的线性关系获得线性方程，将待测样品的荧光强度数值代入所得线性方程，计算获得样品中糖胺聚糖和/或糖胺聚糖类似物的含量。
根据本发明优选的，所述步骤(3)中荧光测定为采用荧光显微镜、流式细胞仪或活体成像设备进行定性分析。
有益效果
本发明首次将高正电GFP(ScGFP)作为生物相容性、高灵敏度和高选择性荧光探针，应用于用于活细胞表面的GAGs的可视化，血清中GAGs的高灵敏性定量分析，肝素中过硫酸化的硫酸软骨素污染分析等。从而克服了传统阳离子染料在GAGs检测中的生物相容性差、选择性低、灵敏度低等缺点，具有重要应用价值，可被广泛应用于GAGs相关的基础和应用研究、临床诊断、医药和食品的检测等。
附图说明
图1：重组高正电绿色荧光蛋白(ScGFP)表达及纯化情况的聚丙烯酰胺凝胶电泳图(SDS-PAGE)。各泳道加入的样品分别是：M：蛋白质分子量标准，条带自上至下大小为116kD，66.2kD，45kD，35kD，25kD，18.4kD，14.4kD；泳道1：对照菌株破壁前菌体，上样量10μL，泳道2：重组菌破壁前菌体，上样量10μL，泳道3：重组菌破壁后上清，上样量10μL，泳道4：经镍柱纯化的ScGFP，上样量10μL。
图2：不同浓度的糖胺聚糖对抑制氧化石墨烯淬灭ScGFP荧光的作用。A：透明质酸(HA)；B：硫酸软骨素A(CS-A)；C：硫酸皮肤素(DS)；D：肝素(Hep)。由下向上，糖胺聚糖的浓度分别为0,0.05,0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.55,0.6,0.65,0.7,0.75,0.8,0.85,0.9,0.95,1μg/ml.
图3：血清中不同浓度的肝素抑制氧化石墨烯淬灭ScGFP荧光的作用。由下向上，Hep的浓度分别为0,0.05,0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5,0.55,0.6,0.65,0.7,0.75,0.8,0.85,0.9,0.95,1μg/ml.
图4：含不同过硫酸化硫酸软骨素(OSCS)的肝素经肝素酶降解后对抑制氧化石墨烯淬灭ScGFP荧光的作用。
图5：共聚焦显微镜辅助检测细胞表面的糖胺聚糖。
图6：流式细胞仪辅助检测细胞表面的糖胺聚糖。A：未用酶处理；B：经硫酸软骨素降解酶ABC处理；C：经肝素酶I、II和III处理；D：经硫酸软骨素降解酶ABC和肝素酶I、II和III共处理。
具体实施方式
以下实施例的阐述，是为了全面公开本发明如何实施的一些常用技术，而不是为了限制本发明的应用范围。发明人已经尽最大努力确保实施例中各参数的准确性(例如量，温度，等等)，但是一些实验误差和偏差也应该予以考虑。
实施例1、ScGFP基因的获得
通过文献报道获得ScGFP(+36)的氨基酸序列[7]，并将所得的氨基酸序列转变为相应的碱基序列，并对所对应的碱基进行密码子优化，使其更容易在大肠杆菌中表达。所对应的DNA序列由生工生物工程公司全合成。
实施例2、ScGFP(+36)蛋白的表达与纯化
以实施例1得到的ScGFP基因为模板，进行PCR扩增。引物如下：
正向引物ScGFP-F：
(gCATATGATGGGTCACCATCATCATCATCACG)
反向引物ScGFP-R：
(gCTCGAGTTTGTAGCGTTCGTCACGACCGTG)
正向引物下划线标注的是限制性内切酶Nde I位点，反向引物下划线标注的是限制性内切酶Xho I位点。对PCR产物进行双酶切，并将经过双酶切的PCR产物与同样经过双酶切 pET-22b载体连接，并且筛选阳性的重组质粒(pET22b-ScGFP)。基因测序结果表明，在pET-22b的Nde I和Xho I酶切位点之间插入ScGFP基因，且插入方向正确。
将pET22b-ScGFP转化大肠杆菌菌株BL21(DE3)(购自美国Novagen公司)，然后按照该公司提供的操作步骤进行重组高正电绿色荧光蛋白pET22b-ScGFP诱导表达。并用Ni SepharoseTM 6Fast Flow(GE)凝胶对scGFP进行纯化，纯化条件按照GE公司的产品手册操作。用聚丙烯酰胺凝胶电泳检测重组ScGFP的纯化情况，结果如图1所示，纯化后的重组ScGFP在电泳胶上呈单一条带，且位置与预测的分子量相吻合；经测序，氨基酸序列如SEQ ID NO.1所示。
实施例3、不同浓度的糖胺聚糖抑制氧化石墨烯淬灭scGFP荧光的作用
在10mM Tris-HCl 100mM NaCl(pH7.0)溶液中加入0.01-0.5μg的ScGFP，然后分别加入肝素(Hep)、硫酸软骨素A(CS-A)、硫酸皮肤素(DS)、透明质酸(HA)使其终浓度为0-1μg/ml，总体积为190μl，室温放置10min后，加入10μl的氧化石墨烯，混匀、室温放置10min。用荧光分光光度计(F-4600，日立)进行荧光强度测定。结果显示，在糖胺聚糖浓度为0-1μg/ml时，反应体系的荧光强度与糖胺聚糖的浓度存在线性关系(如图2)。
在用10mM Tris-HCl 100mM NaCl(pH7.0)稀释10倍的血清中加入0.01-0.5μg的ScGFP，然后分别加入不同量的肝素(Hep)使其终浓度为0-1μg/ml，总体积为190μl，室温放置10min后，加入10μl的氧化石墨烯，混匀、室温放置10min。用荧光分光光度计(F-4600，日立)进行荧光强度测定。结果显示，在血清中，荧光强度与Hep的浓度也存在线性关系(如图3)。该方法可以用于检测病人血清中肝素的含量。
实施例4、对血清样品中的肝素进行定量分析
标准曲线的绘制，将肝素标准品溶解在血清中配制成100μl浓度为0-10μg/ml的标准溶液。取20μl标准溶液，加入0.1μg的ScGFP，并用PBS定容至190μl，室温放置10min后，加入10μl的氧化石墨烯，混匀、室温放置10min。用荧光分光光度计(F-4600，日立)进行荧光强度测定，并依据标准溶液浓度和相对应的荧光强度进行标准曲线的绘制。所得的标准曲线为：F＝2.98C+126.09，其中F代表的为荧光强度，C代表的是肝素的浓度。
取20μl待测样品，然后加入0.1μg的ScGFP，并用PBS定容至190μl，室温放置10min后，加入10μl的氧化石墨烯，混匀、室温放置10min。用荧光分光光度计(F-4600，日立)进行荧光强度测定。测得的荧光强度为140.2，带入标准曲线可得血清样品中肝素的浓度为4.73μg/ml。
实施例5、用ScGFP对被过硫酸化硫酸软骨素(OSCS)污染的肝素进行检测
含有不同含量OSCS的肝素10μg，用肝素酶I、II、和III适度降解。向10mM Tris-Cl100mM NaCl溶液中加入0.01-0.5μg的ScGFP，然后向溶液中加入污染的肝素使其终浓度为0-50μg/ml，室温放置10min后，加入10μl的氧化石墨烯，补加10mM Tris-Cl 100mM NaCl缓冲液使其总体积为200μl，混匀、室温放置10min.最后用荧光分光光度计(F-4600， 日立)进行测定。结果显示，OSCS的含量大于10-6％时，用本发明所述的方法检测，结果如图4所示，比现在存在的大部分检测肝素污染的方法都要简单和灵敏。
实施例6、用ScGFP对细胞表面的糖胺聚糖进行可视化检测
在直径为35mm的盖玻片底培养皿上培养适量的A549细胞，共培养5盘，其中一盘用肝素酶I、II、和III处理，一盘用硫酸软骨素降解酶ABC处理，一盘用两种酶共处理，还有两盘不用酶处理,30℃处理1-5h。将所有的5盘细胞都用DAPI进行染色，除了一盘不用酶处理的细胞不加入ScGFP作为阴性对照，其余的加入0.1-10μg ScGFP(200μl PBS)孵育5-30min，然后用100-1,000μl PBS洗涤2-5次。进行共聚焦显微镜观察(LSM 700，Zeiss)。激发波长分别为405nm和488nm。结果显示，与对照相比，ScGFP对细胞表面强烈染色，用酶肝素酶或硫酸软骨素酶分别处理，细胞的ScGFP荧光染色显著减弱；加入两种酶时，细胞表面荧光染色基本消除(如图5)。说明ScGFP可以特异性的与细胞表面的糖胺聚糖结合，同时验证ScGFP有较好的组织相容性，可以用于后细胞和组织的糖胺聚糖的检测。
用直径为10cm细胞培养皿培养满2板293T细胞，然后将细胞吹悬，平均分成4份。一份用肝素酶I、II、和III处理，一份用硫酸软骨素降解酶ABC处理，一盘用两种酶共处理，还有两份不用酶处理,用PBS补齐500μl，30℃处理1-5h。处理后用PBS洗涤2-5次，每次1-5ml。然后每份加入0.5-3μg ScGFP(100μl)，室温放置10min。用PBS洗涤2-5次，每次1-5ml。最后过滤进行流式细胞仪(FACS Aria III，BD)分析。结果显示，与对照相比，用酶处理的细胞的ScGFP荧光强度变弱；加入两种酶时，荧光强度最弱(如图6)。这也说明ScGFP可以特异性的与细胞表面的糖胺聚糖结合，并且利用该方法可以对细胞表面的糖胺聚糖进行定量分析。
说明书中涉及的参考文献
1.Takagaki K,Nakamura T,Takeda Y,Daidouji K,Endo M.A new endo-beta-galactosidase acting on the Gal beta 1-3Gal linkage of the proteoglycan linkage region.J Biol Chem 1992,267:18558-18563.
2.Silbert JE,Sugumaran G.Biosynthesis of chondroitin/dermatan sulfate.Iubmb Life 2002,54:177-186.
3.Sugahara K,Mikami T,Uyama T,Mizuguchi S,Nomura K,Kitagawa H.Recent advances in the structural biology of chondroitin sulfate and dermatan sulfate.Curr Opin Struct Biol 2003,13:612-620.
4.Izumikawa T,Kitagawa H,Mizuguchi S,Nomura KH,Nomura K,Tamura J,et al.Nematode chondroitin polymerizing factor showing cell-/organ-specific expression is indispensable for chondroitin synthesis and embryonic cell division.J Biol Chem 2004,279:53755-53761.
5.Jiang D,Liang J,Noble PW.Hyaluronan in tissue injury and repair.In:Annu Rev Cell Dev Biol 2007.pp.435-461.
6.Noble PW.Hyaluronan and its catabolic products in tissue injury and repair.Matrix Biol 2002,21:25-29.
7.Lawrence MS, Phillips KJ, Liu DR. Supercharging proteins can impart unusual resilience. J Am Chem Soc 2007, 129: 10110-10112.
8.Lei C, Huang Y, Nie Z, Hu J, Li L, Lu G, Han Y, Yao S. A supercharged fluorescent protein as a versatile probe for homogeneous DNA detection and methylation analysis. Angew Chem Int Ed Engl. 2014, 53: 8358-8362.

Claims (9)

  1. 高正电荷荧光蛋白作为荧光标记物在糖胺聚糖和/或糖胺聚糖类似物分析检测中的应用，所述高正电荷荧光蛋白的氨基酸序列如SEQ ID NO.1所示。
  2. 如权利要求1所述的应用，其特征在于，所述的糖胺聚糖类似物选自天然或人工半合成及全合成的聚阴离子多糖。
  3. 如权利要求2所述的应用，其特征在于，所述的糖胺聚糖类似物选自：硫酸化多糖、多聚唾液酸、褐藻胶、卡拉胶，岩藻聚糖或脂多糖。
  4. 如权利要求1所述的应用，其特征在于，具体步骤如下：
    (1)使样品与高正电荷荧光蛋白接触，进行孵育结合，孵育时间5～30min，孵育后待测样品；
    (2)去除未与待检测样品中糖胺聚糖和/或糖胺聚糖类似物结合的高正电荷荧光蛋白，或者采用荧光淬灭剂淬灭与待检测样品中糖胺聚糖和/或糖胺聚糖类似物结合的高正电荷荧光蛋白，制得待检测样品；
    (3)对待检测样品进行荧光测定，根据荧光强度对样品中糖胺聚糖和/或糖胺聚糖类似物含量进行定性和/或定量分析。
  5. 如权利要求4所述的应用，其特征在于，所述步骤(1)中的样品为液体、固相载体、细胞、组织、器官。
  6. 如权利要求4所述的应用，其特征在于，所述步骤(1)中，高正电荷荧光蛋白的接触浓度为0.01-5.0μg/ml。
  7. 如权利要求4所述的应用，其特征在于，所述步骤(2)中，荧光淬灭剂选自：石墨烯、纳米金和/或有机小分子荧光淬灭剂。
  8. 如权利要求4所述的应用，其特征在于，所述步骤(3)中，定量分析是通过检测不同稀释度的标准样品的荧光强度绘制标准曲线，根据标准曲线的浓度与荧光强度之间的线性关系获得线性方程，将待测样品的荧光强度数值代入所得线性方程，计算获得样品中糖胺聚糖和/或糖胺聚糖类似物的含量。
  9. 如权利要求4所述的应用，其特征在于，所述步骤(3)中荧光测定为采用荧光显微镜、流式细胞仪或活体成像设备进行定性分析。
PCT/CN2015/097648 2015-01-28 2015-12-17 高正电荷荧光蛋白在糖胺聚糖及其类似物分析检测中的应用 Ceased WO2016119536A1 (zh)

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