CN107541790A - A kind of people's agglutinin/class people lectin chip and preparation method for glycosylating detection - Google Patents
A kind of people's agglutinin/class people lectin chip and preparation method for glycosylating detection Download PDFInfo
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Abstract
People's agglutinin/class people the lectin chip and preparation method detected the invention discloses a kind of glycosylation of bio-science field, is built based on recombined human agglutinin/class people's agglutinant protein texture.The recombinant protein, which is 60 kinds, can identify and people's agglutinin/class people's agglutinant protein matter with reference to different glycosyls.Compared with traditional phytolectin chip, people's agglutinin/class people lectin chip has more special recognition capability and stronger binding ability to the complicated sugar-type of higher vertebrate sample.People's agglutinin/class people lectin chip is organized to learn to study for sugar provides new technology platform, can be widely applied to people or the glycosylation research of other mammals.
Description
Technical field
The present invention relates to bio-science field, more particularly to a kind of people's agglutinin/class people's agglutinin core for glycosylating detection
Piece and preparation method, the chip can be known by people's agglutinin/class people agglutinant protein thereon and glycosyl or sugared structural specificity
Not and combine, to analyze the change of the glycosylation pattern of biological specimen and level of glycosylation.
Background technology
Glycosylation is most important posttranslational modification in organism, and more than 50% protein exists glycosylation modified.Sugar
Base plays a crucial role in protein folding, cell growth, division, differentiation, can be used as Receptor recognition extracellular signal,
Immune response in organism etc. can be activated.Some pathological processes are also closely related with glycosylation, for example, sperm is thin
The sugar of cellular surface can influence motion and the fertility of sperm, and the sialic acid increase of cancer cell surfaces can promote cancer cell
Migration.
Compared with invertebrate, the sugar spectrum of vertebrate is more complicated.Understand human body in glycosyl composition, structure and
The change of generation can make us be better understood by glycosylated function and their roles in disease, and then instruct disease
The diagnosis and treatment of disease.Traditional glycosylation analysis method mainly has a technologies such as liquid chromatogram, mass spectrum, but these method manpowers
Material resources consumption is big, and can not carry out quick, system, in real time glycosylation analysis.
As a kind of high-throughout glycosylation analysis test platform, by its unique advantage, such as small volume, flux it is high,
Detection speed is fast, sample requirements are few etc. is analyzed, phytolectin chip has been widely used in protokaryon, eukaryotic microorganisms sugar
The analysis of base, the research of spermatoblast surface glycosylation and the glycosylated identification of cancer cell surfaces etc..Phytolectin
Chip can be very good to parse the change of the glycan structure and level of glycosylation or sugar chain structure of sample, but phytolectin core
Piece can not parse the complicated sugar-type in mammal, especially human body sometimes.
There is also an agglutinoid or agglutinoid protein in human body, most of is the cross-film egg being incorporated on cell membrane
White matter, it is soluble protein and secretory protein also to have part.People's agglutinant protein sticks regulation human inner cell's, sugar
Key effect is played in the level of albumen in the synthesis of albumen and control blood, as a kind of endogenic protein, people coagulates
Collect the complicated sugar chain in the identification human body that element or agglutinoid can be more special, and the adhesion between them is stronger.Therefore,
Compared with phytolectin chip, people's lectin chip can preferably help us to the sugar under Human Physiology or pathologic condition
Group is studied.
Because most people's agglutinin or class people agglutinin are memebrane protein, have recombination expression amount lack, isolate and purify efficiency it is low,
The shortcomings of easy in inactivation, hinder its flow of research.The report of someone's lectin chip is had no at present.
The content of the invention
For in the prior art the defects of, in order to make up deficiency of the technologies such as mass spectrum in protein glycosylation research field,
Strengthen the advantage of lectin chip, it is an object of the invention to build a kind of people's Pleurotus Ostreatus people's agglutinin core for glycosylating detection
Piece and preparation method, the chip include 60 kinds of people's agglutinins/class people's agglutinant protein matter, and chip quality inspection and people's activity of lectin are tested
Card shows that people's lectin chip can be used for the glycosylation of biological specimen to analyze.
The purpose of the present invention is achieved through the following technical solutions：
In a first aspect, the present invention provides a kind of glycosylation detection chip, the glycosylation detection albumen on the chip is behaved
Agglutinin/class people's agglutinant protein matter.
Preferably, the species number of people's agglutinin/class people's agglutinant protein matter is 60 kinds；Wherein, 60 kinds of people coagulate
The Gene Name of collection element/class people's agglutinant protein matter is respectively B3GNT3, CD207, SELE, CHODL, CLEC1A, CLEC1B,
CLEC2B, CLEC2Dv1, SELL, CLEC2Dv2, SELPLG, CLEC3A, CLEC3B, CLEC4A, CLEC4D, CLEC4E,
CLEC5A, SIGLEC12, CLECSF12, CLEC7Av6, SIGLEC6, CLEC7A, CLEC9A, COLEC10, COLEC11v1,
DAD1, FCN1, FCN2, FCN3, GALNT4, GALNT6, GALNT9vB, HSPC159, KLRC1v1, KLRC1v2, KLRF1,
SIGLEC8, KLRG1, KLRK1, OLR1, LAMP1, LAMP2, LGALS1, LGALS12, LGALS13, LGALS14,
LGALS14V2, LGALS2, MASP2v2, MASP2v1, LGALS7, LGALS8, LGALS8v2/3, LMAN1, LMAN2, LMAN2L,
MAG, MASP1v3, MASP1, REG3A.
Preferably, the preparation of people's agglutinin/class people's agglutinant protein matter is specially：The GFP will be connected with
The cloned plasmids of sequence are transferred to saccharomyces cerevisiae and are overexpressed, are i.e. available by fusion tag affinity purification.The restructuring of the purifying
Protein is that concentration is more than or equal to 10 μ g/mL.
Preferably, the chip includes some matrixes, and each matrix includes people's agglutinin/class people's agglutinant protein
Matter and control.
Preferably, in the matrix, every kind of people's agglutinin/class people's agglutinant protein matter, control set 3 repetition points.
It is further preferred that the quantity of the matrix is 12, the control includes positive control, negative control, blank
Control.Specific to the present invention, the positive control be fluorescence ConA, the negative control include glutathione s-transferase,
BSA；The blank control includes elution buffer, spotting buffer.12 detection windows can be formed, 12 samples can be detected simultaneously
Product.
From one embodiment of the present of invention, each matrix includes 60 people's agglutinins and class people's agglutinant protein matter, and 1
Individual glutathione s-transferase and 1 BSA are as negative control, and 1 ConA with fluorescence is as positive control, elution buffer
With spotting buffer as blank control.
Preferably, on the matrix, the point sample amount about 0.3- of everyone agglutinin/class people's agglutinant protein particle sampling point
0.5nL。
Preferably, the point of sample on the chip takes biochip point sample instrument point sample on chip substrate.
Second aspect, the present invention provide a kind of preparation method of the glycosylation detection chip, specifically included：By the people
Agglutinin/class people's agglutinant protein matter and control point sample are on chip substrate, you can.
Preferably, the step of preparation method also includes carrying out gained chip quality inspection, activity checking.
The third aspect, the present invention also provide a kind of active verification method of the glycosylation detection chip, specifically included：Will
The THP-1 cell pyrolysis liquids of DyLight 550NHS Ester fluorescence labelings, DyLight 550NHS Ester fluorescence labelings
Remove N- connections glycosylation THP-1 cell pyrolysis liquids and the glycosylation detection chip incubation reaction of closing, washing, it is dry after
GenePixTM4200A scanners are observed, and people's agglutinin on chip/class people agglutinin is combined with the cell pyrolysis liquid and noise
Can it be proved active combined with cell than reaching the agglutinin of the people on more than 4, or chip/class people agglutinin.
Preferably, the closing uses BSA；The time of the incubation reaction is 2h；The washing specifically includes：PBST is washed
Wash 3 times, each 5min；Ultra-pure water cleans 1 time, 1min；The mode of the drying is drying.
But on chip not combined with cell pyrolysis liquid, also people's agglutinant protein not combined with cell is also not considered as do not have
There is bioactivity, because the activity of determination each people's agglutinin one by one can not be accomplished with such a method, can only carry out complete
The checking of office's property.
It is incubated by cell pyrolysis liquid and chip, determines most people's agglutinin and class people's aggegation above people's lectin chip
Cellulose protein has biological activity.Incubated by the cell pyrolysis liquid for going N- connection glycosylase PNGase F treated with chip
Educate, determine that people's agglutinin on chip or the N- in the recognizable cell pyrolysis liquid with combination of class people agglutinin connect sugar chain.
It is incubated by different cell lines and chip, determines that different cell line has different people's lectin chip bind profiles,
Illustrate that the sugar-type of cell surface has difference.
Compared with prior art, the present invention has following beneficial effect：
People's lectin chip that the present invention is built, empirical tests people's agglutinin thereon and class people's agglutinin have good life
Thing activity, and with the characteristic for identifying and combining mammal complexity sugar chain.People's lectin chip that the present invention is built after
The advantage of phytolectin chip has been held, the glycosylation of complex samples, such as cell pyrolysis liquid can be analyzed；Can be thin to work in real time
The glycosylation of cellular surface is analyzed；It can also realize while the detection of 12 samples.
Brief description of the drawings
The detailed description made by reading with reference to the following drawings to non-limiting example, further feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 behaviours agglutinin/class people's agglutinant protein matter chip preparation flow figure；
Fig. 2 is the silver staining result of purified people's agglutinin and class people's agglutinant protein, and wherein ★ represents target protein
The position at place；
Fig. 3 is the quality inspection result of prepared people's agglutinant protein matter chip；
Wherein, A is the scanning result figure and chip point ideograph of people's lectin chip；B is the letter of people's lectin chip
Number intensity statistics result.
Fig. 4 is people's agglutinin and the active the result of class people's agglutinant protein matter on chip；
Wherein, A is the cell pyrolysis liquid with or without PNGaseF processing and the contrast knot after the incubation of people's lectin chip
Fruit；B is the quantitative result of chip signal.
Fig. 5 is the result after three kinds of cell lines are incubated with people's lectin chip.
Embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
The present embodiment provides a kind of people's agglutinin/class people agglutinant protein matter chip and active verification method, the structure of chip
Build flow and see Fig. 1.It is the detailed process and parameter of whole flow process below：
1st, the Yeast expression storehouse of people's agglutinin and class people's agglutinant protein matter is obtained
Zhu Heng of the entry clones storehouse of 60 kinds of people's agglutinins and class people's agglutinin from Johns Hopkins University is taught.
After sequencing is correct, reacted by the LR of Gateway systems, by the volume of people's agglutinin and class people's agglutinin entry clones
Code sequence is connected on purpose carrier pEGH-A, constructs expression cloning plasmid.
Finally the expression cloning plasmid is transferred in saccharomyces cerevisiae Y258 competent cell, obtains people's agglutinin and class
The Yeast expression storehouse of people's agglutinant protein matter.
2nd, the expression and purifying of people's agglutinin and class people's agglutinant protein matter
A. people's agglutinin and class people's agglutinant protein matter are expressed：Picking single bacterium colony (contains Portugal in 1mL SC-URA/Dextrose
The completely synthetic culture medium of the scarce uracil of grape sugar) in culture medium culture reach 3.0 to OD600 (light absorption value during wavelength 600nm),
By 1：2000 ratio is inoculated into 120mL SC-URA/Raffinose (the completely synthetic culture medium that uracil is lacked containing raffinose)
Culture to OD600 values are 0.6-0.8 in culture medium.The galactolipin (filtration sterilization) for adding 13mL 20% cultivates 4-6 hour
Afterwards, bacterium is received, is stored in -80 DEG C of refrigerators.
B. Purification of Human agglutinin and class people's agglutinant protein matter：Collected bacterium is taken out in -80 DEG C of refrigerators, adds oxygen
Change zirconium pearl and lysis buffer, shake 30s in 4 DEG C of environment, rearmounted 2min on ice, be repeated 4 times；The cell that 4 times are obtained cracks
Liquid is focused in 15ml centrifuge tubes, adds appropriate reduced glutathione sepharose 4B, supplement lysis buffer to 12ml, and 4
DEG C be incubated 2 hours after, centrifugation remove supernatant.After sepharose 4B is washed into 3 times respectively with cleaning buffer solution 1 and 2, eluted with 1.5ml
Buffer solution is incubated 15min with sepharose 4B, and supernatant is collected by centrifugation.Then Millipore 4 DEG C of super filter tube, 6000rpm centrifugations are used
Make volume concentration to about 20-40 μ L, take 4 μ L and 1 μ L 5 × sample-loading buffer mix after, carry out SDS-PAGE (dodecanes
Base sodium sulfonate-polyacrylamide gel electrophoresis) electrophoresis, after electrophoresis terminates, silver staining is carried out with the printing and dyeing kit in the green skies.Silver staining
Quantitative result is as shown in Fig. 2 the recombinant protein concentration being purified is all higher than being equal to 10 μ g/mL.
C. depositary's agglutinin and class people's agglutinant protein matter：Purified 60 kinds of protein presses its volume, each to add
30% glycerine, and be stored in -80 DEG C of refrigerators.
3rd, people's lectin chip is prepared
A. each protein is taken out into 10 μ L, be transferred in 384 orifice plates.Meanwhile add positive control (fluorescence labeling
ConA：Canavaline), negative control (GST：Glutathione s-transferase, BSA：Bovine serum albumin), elution buffer and point sample
Buffer solution is used as blank control (Elution buffer：Elution buffer, Printing Buffer：Spotting buffer).
In freezer, brilliant core is usedThe micro-array chip spotting systems of SmartArrayer 48, under the conditions of humidity 45-50%, according to figure
3A pattern, protein spots are formed on polymer three-dimensional substrate H surfaces, every chip there are 12 subarrays, and each array has 65
Kind of sample (wherein, including 60 kinds of people's agglutinins and class people's agglutinant protein matter, 5 kinds of controls), each sample has 3 repetition points.4
DEG C, fixing protein, that is, complete protein-chip structure overnight.It is stored in -80 DEG C, it is stand-by.
B. quality inspection
Closing：Take 0.15g bovine serum albumins to be dissolved in 5mL TBST and be used as confining liquid, chip room temperature is closed into 1h,
50rpm。
It is incubated primary antibody：According to 1:1000 volume ratio, dilute rabbit anti-GST antibody using TBST.It is incubated at room temperature 1h, 50rpm.
Washed 3 times using TBST, each 5min.
It is incubated secondary antibody：According to 1:1000 volume ratio, use the goat anti-rabbit antibody of TBST dilution Cy5 couplings.Incubation at room temperature
1h, 50rpm.Washed 3 times using TBST, each 5min.After cleaning 1min using ultra-pure water, in SliderWasherTMDried on 8
Afterwards, using GenePixTM4200A scanners, scan and record result.As a result as shown in Figure 3A, wherein people's agglutinant protein matter is believed
The statistical result of number intensity is shown in Fig. 3 B.
C. protein active is verified
THP-1 cells are handled with lysis buffer NP-40, obtained cell pyrolysis liquid is divided into two parts, it is a directly to use
DyLight 550NHS Ester carry out fluorescence labeling, after another removes N- connections glycosylation with PNGaseF, then use
DyLight 550NHS Ester carry out fluorescence labeling.After unnecessary fluorescent dye is removed with desalting column, two parts of cells are cracked
People's lectin chip incubation reaction for having closed simultaneously with BSA of liquid 2 hours, is washed 3 times, each 5min with PBST.Using ultrapure
After water cleaning 1min, in SliderWasherTMAfter being dried on 8, GenePix is usedTM4200A scanners, scan and record knot
Fruit.As shown in Figure 4 A, the statistical result of wherein signal intensity is shown in Fig. 4 B to the chip results of two parts of samples.Signal to noise ratio is taken to be more than or equal to
4 value.From Fig. 4 A, majority's agglutinin can be combined with THP-1 cell pyrolysis liquids, when with PNGaseF remove THP-1 cells
After the N- connections glycosylation of lysate, the bind profile of lysate and people's lectin chip changes；26 people are understood by Fig. 4 B
Agglutinin has combination with cell pyrolysis liquid, removes the signal that the cell pyrolysis liquid of N- glycosyls and people's agglutinin are combined and nearly all becomes
It is weak.
The cell (293T, MDA-MB-231 and Yeast) of three kinds of cultures is collected, uses CellTrackerTM Orange CMRA
The fluorescence labeling of living cells is carried out, each window that detects adds 1 × 106Individual cell, after being incubated at room temperature 1 hour, hand-held chip exists
Cleaned in PBST, after room temperature lucifuge dries chip, use GenePixTM4200A scanners, scan and record result.Three kinds
The combination situation of cell and people's lectin chip is shown in Fig. 5.The bind profile of different cells and people's lectin chip is not as shown in Figure 5
Together, three kinds of cell lines and the combination situation of lectin chip are counted, at least 30 kinds of people's agglutinant proteins are active.
In above-described embodiment, the formula of all solution：(solvent of following solution is deionized water)
(1) SC-URA/ dextrose culture-mediums：YNB 1.7g, (NH4)2SO45g, URAmix 2g, glucose 20g, addition
ddH2O to 1L, 121 DEG C of sterilizing 15min.
(2) SC-URA/Raffinose culture mediums：YNB 1.7g, (NH4)2SO45g, URAmix 2g, Raffinose20g,
Add ddH2O to 1L, 121 DEG C of sterilizing 15min.
(3) lysis buffer pH 7.5 (1L)
(4) cleaning buffer solution 1pH 7.5 (1L)
(5) cleaning buffer solution 2pH 7.5 (1L)
(6) elution buffer pH 7.5 (1L)
(7) 10 × TBS pH of buffer 7.4 (1L)
(8) 1 × TBST buffer solutions (1L)
10 × TBS buffer solutions 100mL
Polysorbas20 1mL
ddH2O constant volumes are to 1L
(9) 10 × PBSs (1L)：
(10) 1 × PBST buffer solutions (1L)
10 × PBS 100mL
Polysorbas20 1mL
ddH2O constant volumes are to 1L
(11) 5 × SDS-PAGE electrophoretic buffers (1L)：
Trishydroxymethylaminomethane 0.125M
Glycine 0.96M
SDS 0.5%
The specific embodiment of the present invention is described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various deformations or amendments within the scope of the claims, this not shadow
Ring the substantive content of the present invention.
Claims (10)
1. one kind glycosylation detection chip, it is characterised in that glycosylation detection albumen behaviour agglutinin/class people on the chip
Agglutinant protein matter.
2. glycosylation detection chip according to claim 1, it is characterised in that people's agglutinin/class people's agglutinin egg
The species number of white matter is 60 kinds；
Wherein, the Gene Name of 60 kinds of people agglutinin/class people's agglutinant protein matter is respectively B3GNT3, CD207, SELE,
CHODL, CLEC1A, CLEC1B, CLEC2B, CLEC2Dv1, SELL, CLEC2Dv2, SELPLG, CLEC3A, CLEC3B,
CLEC4A, CLEC4D, CLEC4E, CLEC5A, SIGLEC12, CLECSF12, CLEC7Av6, SIGLEC6, CLEC7A, CLEC9A,
COLEC10, COLEC11v1, DAD1, FCN1, FCN2, FCN3, GALNT4, GALNT6, GALNT9vB, HSPC159, KLRC1v1,
KLRC1v2, KLRF1, SIGLEC8, KLRG1, KLRK1, OLR1, LAMP1, LAMP2, LGALS1, LGALS12, LGALS13,
LGALS14, LGALS14V2, LGALS2, MASP2v2, MASP2v1, LGALS7, LGALS8, LGALS8v2/3, LMAN1,
LMAN2, LMAN2L, MAG, MASP1v3, MASP1, REG3A.
3. glycosylation detection chip according to claim 1 or 2, it is characterised in that the chip includes some matrixes, often
Individual matrix includes people's agglutinin/class people's agglutinant protein matter and control.
4. glycosylation detection chip according to claim 3, it is characterised in that the quantity of the matrix is 12, described
Control includes positive control, negative control, blank control.
5. glycosylation detection chip according to claim 3, it is characterised in that on the matrix, everyone agglutinin/class
The point sample amount about 0.3-0.5nL of people's agglutinant protein particle sampling point.
6. glycosylation detection chip according to claim 3, it is characterised in that the point of sample on the chip is to take life
Thing chip point sample instrument point sample is on chip substrate.
7. a kind of preparation method according to any one of claim 1 to the 6 glycosylation detection chip, it is characterised in that specific
Including：By people's agglutinin/class people's agglutinant protein matter and control point sample on chip substrate, you can.
8. the preparation method of detection chip is glycosylated according to claim 7, it is characterised in that the preparation method also includes
The step of quality inspection, activity checking are carried out to gained chip.
A kind of 9. active verification method according to any one of claim 1 to the 6 glycosylation detection chip, it is characterised in that
Specifically include：By the THP-1 cell pyrolysis liquids of DyLight 550NHS Ester fluorescence labelings, DyLight 550NHS Ester
The removal N- connections glycosylation THP-1 cell pyrolysis liquids and the glycosylation detection chip incubation reaction of closing of fluorescence labeling,
GenePix after washing, dryingTM4200A scanners are observed, and people's agglutinin/class people agglutinin on chip cracks with the cell
People's agglutinin/class people agglutinin that liquid combines and signal to noise ratio reaches on more than 4, or chip can be combined with cell, i.e.,
Prove active.
10. the active verification method of detection chip is glycosylated according to claim 9, it is characterised in that the closing uses
BSA；The time of the incubation reaction is 2h；The washing specifically includes：PBST is washed 3 times, each 5min；Ultra-pure water cleaning 1
It is secondary, 1min；The mode of the drying is drying.
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| CN110420671A (en) * | 2019-06-26 | 2019-11-08 | 南京科瑞芯生物科技有限公司 | A kind of agglutinin micro-array chip and preparation method thereof |
| CN114088669A (en) * | 2021-10-28 | 2022-02-25 | 大连理工大学 | Preparation of lectin fluorescent fusion protein and application of lectin fluorescent fusion protein in glycosylation detection |
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