CN104678092B - Application of the positive charge fluorescin high in glycosaminoglycan analysis detection - Google Patents
Application of the positive charge fluorescin high in glycosaminoglycan analysis detection Download PDFInfo
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- G—PHYSICS
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- 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
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- 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
Abstract
The present invention relates to application of the positive charge green fluorescent protein high in glycosaminoglycan research, the amino acid sequence of the positive charge fluorescin high is as shown in SEQ ID NO.1.The present invention is first using positive electricity GFP high as biocompatibility, high sensitivity and high selectivity fluorescence probe, it is applied to the visualization of the GAGs for liver cell surface, the high sensitivity quantitative analysis of GAGs, chondroitin sulfate contamination analysis oversulfated in heparin etc. in serum.So as to overcome poor biocompatibility, selectivity low, sensitivity low shortcoming of the traditional cation dyestuff in GAGs detections, with significant application value, detection of the related basic and applied research of GAGs, clinical diagnosis, medicine and food etc. can be widely used in.
Description
Technical field
The present invention relates to application of the positive charge green fluorescent protein high in glycosaminoglycan research, belong to Measurement for Biochemistry
Field.
Background technology
Glycosaminoglycan (Glycosaminoglycans, GAGs) is also called mucopolysaccharide, is the straight of repetition disaccharide unit composition
Chain polyanionic polysaccharide.Mainly include hyaluronic acid (Hyaluronic Acid, HA), heparin/Heparan sulfate
(Heparin/Heparan Sulfate, Hep/HS), chondroitin sulfate/dermatan sulfate (Chondroitin Sulfate/
Dermatan Sulfate, CS/DS), keratan sulfate (Keratan Sulfate, KS).The disaccharide unit of sulfuric acid keratan
Beyond being made up of neutral D- galactolipins and N-acetyl-glucosamine, the disaccharide unit of other GAGs is by hexuronic acid (D-
Glucuronic acid/L- iduronic acids) constituted with hexosamine (N-Acetyl-D-glucosamine/N- acetylgalactosamines).Wherein HA is tied
Structure is relatively easy, and the disaccharide unit being made up of D-Glucose aldehydic acid and N-acetyl-glucosamine is formed by connecting through β-Isosorbide-5-Nitrae-glycosidic bond.
Other GAGs then make sugar chain become complex due to the effect of various modification enzymes, are mainly manifested in different parts hydroxyl in sugar chain
(- OH) and amino (- NH2) sulphation, D-Glucose aldehydic acid is changed into L- idose aldehyde in the presence of C5- epimerases
Acid, acetylation of the bit amino of hexosamine two etc..This high complexity of GAGs sugar chain structures is not random generation, but tool
There is the Space-time speciality of height, be that various sugar chain synthesis relevant enzymes are expressed in the different developmental phases of different cell tissues and organ
Caused by regulation and control level, different structures makes it have different functions, and the complexity of structure assigns the diversity of its function.
GAGs is widely present in zooblast cell surface and cellular matrix, by the special phase interaction with various albumen
With various physiology such as increment and differentiation, intercellular identification, cell transfer, tissue morphology generation, the cancerations that take part in cell and
Pathologic process [1-4], a series of important biological function that GAGs has becomes important bioactive molecule,
It is used widely in medicine and functional food, such as Hep, CS, HA [5,6].
The structure of GAGs is increasingly complex for nucleic acid and protein, and its structural complexity and diversity is research
Its Structure and Function brings very big challenge.The various dyes of positive ion are (such as：A Li Xinlan, toluidine blue and acid amides
It is black etc.) it is commonly used for the dyeing and detection of GAGs in sample.But this kind of dyestuff tool has disadvantages that, such as biocompatibility compared with
Difference, is not suitable for living cells and tissue staining；Selectivity is relatively low, causes and background value very high occurs；Sensitivity is more low.Closely
Nian Lai, various new cationic chromophores are synthesized and carry out high-sensitivity detection for the Hep in buffer solution or serum, but
Its biocompatibility and other glycosaminoglycan context of detection application need further research.It would therefore be highly desirable to develop a kind of life
The multipurpose GAG probes that thing compatibility is good, sensitivity is high are used for the observation and detection of GAGs in various biological samples.
Green fluorescent protein (GFP) is most to be found in a kind of scientific name is for the jellyfish of Aequorea victoria.Cause
For the biofacies reporter of GFP and living body biological is used for Celluar and Molecular Biology research.And GFP can make
It is used for the real-time monitoring of live body for molecular probe.Recently, by by some nonconserved amino acids on GFP sport lysine,
The basic amino acids such as arginine are this high just so as to make it in the case where GFP fluorescent characteristics are not influenceed with substantial amounts of positive charge
Electric charge green fluorescent protein has been employed successfully in the transport agent that albumen enters cell with nucleic acid, and as the Gao Ling of detection of nucleic acids
Sensitivity probe [7,8].
The content of the invention
The present invention is in view of the shortcomings of the prior art, there is provided a kind of means of new detection glycosaminoglycan, i.e., with positive electricity high
Green fluorescent protein carries out qualitative and quantitative analysis detection to glycosaminoglycan.
Technical scheme is as follows：
Positive charge fluorescin high is as fluorescent marker in glycosaminoglycan and/or the similar thing analysis detection of glycosaminoglycan
Application, the amino acid sequence of the positive charge fluorescin high is as shown in SEQ ID NO.1.
Natural or artificial semi-synthetic and fully synthetic gathering is selected from according to the similar thing of currently preferred, described glycosaminoglycan
Anion polysaccharide；Further preferably, the similar thing of described glycosaminoglycan is selected from：Sulfated polysaccharides, polysialic acids, algin,
Carragheen, fucosan or lipopolysaccharides.
According to currently preferred, comprise the following steps that：
(1) sample is contacted with positive charge fluorescin high, carry out hatching combination, 5~30min of incubation time, after incubation
Testing sample；
(2) the positive charge fluorescence high that removal thing not similar with glycosaminoglycan in detected sample and/or glycosaminoglycan is combined
Albumen, or the height that thing similar with glycosaminoglycan in detected sample and/or glycosaminoglycan is combined is quenched using fluorescence quenching
Positive charge fluorescin, is obtained detected sample；
(3) fluoremetry is carried out to detected sample, according to fluorescence intensity to glycosaminoglycan in sample and/or glycosaminoglycan
Analog content carries out qualitative and/or quantitative analysis.
According to currently preferred, the sample in the step (1) is liquid, solid phase carrier, cell, tissue, organ.
According to currently preferred, in the step (1), the exposure concentration of positive charge fluorescin high is 0.01-5.0 μ
g/ml。
According to currently preferred, in the step (2), fluorescence quenching is selected from：Graphene, nm of gold and/or organic
Small molecule fluorescent quencher.
According to currently preferred, in the step (3), quantitative analysis is the standard sample by detecting different dilution factors
Fluorescence intensity draw standard curve, linear side is obtained according to the linear relationship between the concentration and fluorescence intensity of standard curve
Journey, gained linear equation is substituted into by the florescent intensity value of testing sample, and glycosaminoglycan and/or osamine are poly- in calculating acquisition sample
The content of sugar analogue.
According to currently preferred, fluoremetry is using fluorescence microscope, flow cytometer or work in the step (3)
Body imaging device carries out qualitative analysis.
Beneficial effect
The present invention is visited positive electricity GFP (ScGFP) high as biocompatibility, high sensitivity and high selectivity fluorescence first
Pin, is applied to the visualization of the GAGs for liver cell surface, the high sensitivity quantitative analysis of GAGs, over cure in heparin in serum
Chondroitin sulfate contamination analysis of acidifying etc..So as to overcome biocompatibility of the traditional cation dyestuff in GAGs detections
Poor, the selectivity low shortcoming of low, sensitivity, with significant application value, can be widely used in the related bases of GAGs and answer
Detection with research, clinical diagnosis, medicine and food etc..
Brief description of the drawings
Fig. 1：Recombinate the polyacrylamide gel electrophoresis figure of positive electricity green fluorescent protein (ScGFP) expression high and purifying situation
(SDS-PAGE).The sample that each swimming lane is added is respectively：M：Protein molecular weight standard, size is 116kD to band from top to bottom,
66.2kD, 45kD, 35kD, 25kD, 18.4kD, 14.4kD；Swimming lane 1：Thalline before control strain broken wall, the μ L of applied sample amount 10, swimming lane
2：Thalline before recombinant bacterium broken wall, the μ L of applied sample amount 10, swimming lane 3：Supernatant after recombinant bacterium broken wall, the μ L of applied sample amount 10, swimming lane 4：Through nickel post
The ScGFP of purifying, the μ L of applied sample amount 10.
Fig. 2：The effect of ScGFP fluorescence is quenched to suppressing graphene oxide for the glycosaminoglycan of various concentrations.A：Hyaluronic acid
(HA)；B：Chondroitin sulfate A (CSA) (CS-A)；C：Dermatan sulfate (DS)；D：Heparin (Hep).From bottom to top, the concentration of glycosaminoglycan
Respectively 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.
Fig. 3：The effects of heparin graphene oxide of various concentrations is quenched the effect of ScGFP fluorescence in serum.From bottom to top,
The concentration of Hep is respectively 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, and 0.65,
0.7,0.75,0.8,0.85,0.9,0.95,1μg/ml.
Fig. 4：Heparin containing different oversulfated chondroitin sulfates (OSCS) is after heparinase degraded to suppressing graphite oxide
Alkene is quenched the effect of ScGFP fluorescence.
Fig. 5：The glycosaminoglycan of Laser Scanning Confocal Microscope auxiliary detection cell surface.
Fig. 6：The glycosaminoglycan of flow cytometer auxiliary detection cell surface.A：Unused ferment treatment；B：Through chondroitin sulfate
Digestive enzyme ABC treatment；C：Through Heparinase I, II and III treatment；D：Through chondroitin sulfate digestive enzyme ABC and Heparinase I, II and
III coprocessing.
Specific embodiment
The elaboration of following examples, be for comprehensive disclosure some common technologies how to implement of the present invention, rather than for
Limitation range of application of the invention.Inventor tried one's best ensure the accuracy of each parameter in embodiment (for example measure,
Temperature, etc.), but some experimental errors and deviation should also pay attention to.
The acquisition of embodiment 1, ScGFP genes
The amino acid sequence [7] of ScGFP (+36) is obtained by document report, and the amino acid sequence of gained is changed into
Corresponding base sequence, and codon optimization is carried out to corresponding base, make it easier in expression in escherichia coli.Institute is right
The DNA sequence dna answered is fully synthetic by Sheng Gong bio-engineering corporations.
The expression and purification of embodiment 2, ScGFP (+36) albumen
The ScGFP genes obtained with embodiment 1 enter performing PCR amplification as template.Primer is as follows：
Forward primer ScGFP-F：
(gCATATGATGGGTCACCATCATCATCATCACG)
Reverse primer ScGFP-R：
(gCTCGAGTTTGTAGCGTTCGTCACGACCGTG)
Forward primer underscore mark is restriction enzyme Nde I sites, and reverse primer underscore mark is limit
Property restriction endonuclease Xho I sites processed.Carry out double digestion to PCR primer, and by by the PCR primer of double digestion with also pass through double enzymes
The connection of pET-22b carriers is cut, and screens the recombinant plasmid (pET22b-ScGFP) of the positive.Gene sequencing result shows,
ScGFP genes are inserted between Nde I and Xho the I restriction enzyme sites of pET-22b, and direction of insertion is correct.
PET22b-ScGFP is converted into coli strain BL21 (DE3) (be purchased from Novagen companies of the U.S.), then according to
The operating procedure that the said firm provides carries out recombinating positive electricity green fluorescent protein pET22b-ScGFP induced expressions high.And use Ni
SepharoseTM6Fast Flow (GE) gel is purified to scGFP, and purification condition is grasped according to the product manual of GE companies
Make.The purifying situation of restructuring ScGFP is detected with polyacrylamide gel electrophoresis, as a result as shown in figure 1, restructuring after purification
ScGFP matches in single band, and position on running gel with the molecular weight of prediction；Through sequencing, amino acid sequence such as SEQ
Shown in ID NO.1.
The glycosaminoglycan of embodiment 3, various concentrations suppresses the effect that graphene oxide is quenched scGFP fluorescence
The ScGFP of 0.01-0.5 μ g is added in 10mM Tris-HCl 100mM NaCl (pH7.0) solution, is then distinguished
Adding heparin (Hep), chondroitin sulfate A (CSA) (CS-A), dermatan sulfate (DS), hyaluronic acid (HA) makes its final concentration of 0-1 μ g/
Ml, cumulative volume is 190 μ l, after room temperature places 10min, adds the graphene oxide of 10 μ l, and mixing, room temperature place 10min.With glimmering
Light spectrophotometer (F-4600, Hitachi) carries out fluorescent strength determining.Result shows, is 0-1 μ g/ml in GAG concentration
When, there is linear relationship (such as Fig. 2) with the concentration of glycosaminoglycan in the fluorescence intensity of reaction system.
Add 0.01-0.5 μ g's in 10 times of serum is diluted with 10mM Tris-HCl 100mM NaCl (pH7.0)
ScGFP, being then respectively adding different amounts of heparin (Hep) makes its final concentration of 0-1 μ g/ml, and cumulative volume is 190 μ l, and room temperature is put
After putting 10min, the graphene oxide of 10 μ l is added, mixing, room temperature place 10min.With sepectrophotofluorometer (F-4600, day
It is vertical) carry out fluorescent strength determining.Result shows, in serum, fluorescence intensity there is also linear relationship (as schemed with the concentration of Hep
3).The method can be used for detecting the content of heparin in patients serum.
Embodiment 4, quantitative analysis is carried out to the heparin in blood serum sample
The drafting of standard curve, Heparin Standard product are dissolved in serum the mark for being configured to that 100 μ l concentration are 0-10 μ g/ml
Quasi- solution.20 μ l standard liquids are taken, the ScGFP of 0.1 μ g is added, and 190 μ l are settled to PBS, after room temperature places 10min, plus
Enter the graphene oxide of 10 μ l, mixing, room temperature place 10min.It is strong fluorescence to be carried out with sepectrophotofluorometer (F-4600, Hitachi)
Degree is determined, and establishing criteria solution concentration and corresponding fluorescence intensity carry out the drafting of standard curve.The standard curve of gained
For：What F=2.98C+126.09, wherein F were represented is fluorescence intensity, and what C was represented is the concentration of heparin.
20 μ l testing samples are taken, the ScGFP of 0.1 μ g is subsequently adding, and 190 μ l are settled to PBS, room temperature places 10min
Afterwards, the graphene oxide of 10 μ l is added, mixing, room temperature place 10min.Carried out with sepectrophotofluorometer (F-4600, Hitachi)
Fluorescent strength determining.The fluorescence intensity for measuring be 140.2, bring into standard curve can obtain heparin in blood serum sample concentration be 4.73
μg/ml。
Embodiment 5, with ScGFP to being detected by the heparin that oversulfated chondroitin sulfate (OSCS) pollutes
The μ g of heparin 10 containing different content OSCS, are moderately degraded with Heparinase I, II and III.To 10mM Tris-
The ScGFP of 0.01-0.5 μ g is added in Cl100mM NaCl solutions, then to adding the heparin of pollution to make its final concentration in solution
It is 0-50 μ g/ml, after room temperature places 10min, adds the graphene oxide of 10 μ l, adds 10mM Tris-Cl 100mMNaCl and delay
Fliud flushing makes its cumulative volume for 200 μ l, and mixing, room temperature are placed 10min. and finally entered with sepectrophotofluorometer (F-4600, Hitachi)
Row is determined.Result shows that the content of OSCS is more than 10-6During %, with method of the present invention detect, as a result as shown in figure 4, than
The method of the most of detection heparin pollution for existing now will be simply and sensitive.
Embodiment 6, Visual retrieval is carried out to the glycosaminoglycan of cell surface with ScGFP
Appropriate A549 cells are cultivated on the cover glass bottom culture dish of a diameter of 35mm, 5 disks are co-cultured, wherein a disk is used
Heparinase I, II and III treatment, disk chondroitin sulfate digestive enzyme ABC treatment, a disk two kinds of enzyme coprocessing, also two
Without ferment treatment, 30 DEG C process 1-5h to disk.All of 5 disk cell is all dyeed with DAPI, except a disk is without ferment treatment
Cell be added without ScGFP as negative control, remaining add 0.1-10 μ g ScGFP (200 μ l PBS) be incubated 5-30min,
Then washed 2-5 times with 100-1,000 μ l PBS.Carry out confocal microscopy (LSM 700, Zeiss).Excitation wavelength point
Wei not 405nm and 488nm.Result shows, compared with the control, ScGFP to the strong dyeing of cell surface, with enzyme heparinase or sulfuric acid
Chondroitinase is processed respectively, and the ScGFP fluorescent stainings of cell are obviously reduced；When adding two kinds of enzymes, cell surface fluorescence dyeing base
This elimination (such as Fig. 5).Illustrate that ScGFP can be combined with specific with the glycosaminoglycan of cell surface, at the same verify ScGFP have compared with
Good histocompatbility, can be used for the detection of the glycosaminoglycan of rear cell and tissue.
With the full 2 plate 293T cells of a diameter of 10cm Tissue Culture Dish culture, cell is then blown outstanding, be divided into 4 parts.One
Part is gone back with Heparinase I, II and III treatment, portion chondroitin sulfate digestive enzyme ABC treatment a, disk with two kinds of enzyme coprocessing
There are two parts without ferment treatment, with PBS polishings 500 μ l, 30 DEG C for the treatment of 1-5h.Washed 2-5 times with PBS after treatment, each 1-5ml.
Then every part adds 0.5-3 μ g ScGFP (100 μ l), and room temperature places 10min.Washed 2-5 times with PBS, each 1-5ml.Finally
Filtering carries out flow cytometer (FACS Aria III, BD) analysis.Result shows, compared with the control, with the cell of ferment treatment
ScGFP fluorescence intensities die down；When adding two kinds of enzymes, fluorescence intensity is most weak (such as Fig. 6).This also illustrates that ScGFP can be with specific
Combined with the glycosaminoglycan of cell surface, and quantitative analysis can be carried out to the glycosaminoglycan of cell surface using the method.
Bibliography
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galactosidase acting on the Gal beta 1-3Gal linkage of the proteoglycan
linkage region.J Biol Chem1992,267:18558-18563.
2.Silbert JE,Sugumaran G.Biosynthesis of chondroitin/dermatan
sulfate.Iubmb Life2002,54:177-186.
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 Biol2003,13:612-620.
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
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Annu Rev Cell Dev Biol 2007.pp.435-461.
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Claims (6)
1. application of the positive charge fluorescin high as fluorescent marker in glycosaminoglycan analysis detection, comprises the following steps that：
（1）Sample is contacted with positive charge fluorescin high, carry out hatching combination, incubation time 5~30 min is to be measured after incubation
Sample；
（2）The positive charge fluorescin high not combined with glycosaminoglycan in testing sample after removal incubation, or quenched using fluorescence
Agent of going out is quenched the positive charge fluorescin high not combined with glycosaminoglycan in testing sample after being incubated, and detected sample is obtained；
（3）Fluoremetry is carried out to detected sample, according to fluorescence intensity GAG content in sample is carried out it is qualitative and/or
Quantitative analysis；
The amino acid sequence of the positive charge fluorescin high is as shown in SEQ ID NO.1.
2. application as claimed in claim 1, it is characterised in that the step（1）In sample be liquid, it is solid phase carrier, thin
Born of the same parents, tissue, organ.
3. application as claimed in claim 1, it is characterised in that the step（1）In, the contact of positive charge fluorescin high is dense
It is 0.01-5.0 μ g/ml to spend.
4. application as claimed in claim 1, it is characterised in that the step（2）In, fluorescence quenching is selected from：Graphene, receive
Rice gold and/or organic molecule fluorescence quenching.
5. application as claimed in claim 1, it is characterised in that the step（3）In, quantitative analysis is different dilute by detecting
The fluorescence intensity of the standard sample of degree of releasing draws standard curve, according to the linear pass between the concentration and fluorescence intensity of standard curve
System obtains linear equation, and the florescent intensity value of testing sample is substituted into gained linear equation, calculates osamine in obtaining sample and gathers
The content of sugar.
6. application as claimed in claim 1, it is characterised in that the step（3）Middle fluoremetry be using fluorescence microscope,
Flow cytometer or living imaging equipment carry out qualitative analysis.
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| PCT/CN2015/097648 WO2016119536A1 (en) | 2015-01-28 | 2015-12-17 | Application of high-positive-charge fluorescent protein in analysis and detection of glycosaminoglycans and analogues thereof |
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| CN105606827B (en) * | 2016-03-14 | 2017-12-12 | 山东大学 | A kind of kit for proteoglycans high-sensitivity detection and preparation method thereof |
| CN107085109A (en) * | 2017-05-15 | 2017-08-22 | 山东大学深圳研究院 | Application of the high positive charge green fluorescent protein in hepatocarcinoma early diagnosis kit is prepared |
| CN107460179B (en) * | 2017-09-22 | 2021-06-29 | 青岛农业大学 | Polysaccharide degrading enzyme and coding gene and application thereof |
| CN107991294B (en) * | 2017-11-28 | 2020-11-06 | 陕西慧康生物科技有限责任公司 | Method for detecting polysialic acid |
| CN108195808B (en) * | 2017-12-26 | 2020-11-17 | 中国石油大学(华东) | Method for detecting oversulfated chondroitin sulfate impurities in heparin sodium |
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| A Supercharged Fluorescent Protein as a Versatile Probe for Homogeneous DNA Detection and Methylation Analysis;Chunyang Lei 等;《Angewandte Chemie International Edition》;20140624;第53卷(第24期);摘要，第8358页右栏第2段-第8359页左栏第1段，第8359页右栏第2段-第8360页左栏第1段，Scheme 1，图2 * |
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