CN107541790B

CN107541790B - Human lectin/human-like lectin chip for glycosylation detection and preparation method

Info

Publication number: CN107541790B
Application number: CN201610472307.0A
Authority: CN
Inventors: 陶生策; 孙洋洋; 程莉; 周树敏; 郭书娟
Original assignee: Shanghai Jiao Tong University
Current assignee: Shanghai Jiao Tong University
Priority date: 2016-06-24
Filing date: 2016-06-24
Publication date: 2021-07-06
Anticipated expiration: 2036-06-24
Also published as: CN107541790A

Abstract

The invention discloses a human lectin/human-like lectin chip for glycosylation detection in the field of biological sciences and a preparation method, which are constructed based on recombinant human lectin/human-like lectin protein. The recombinant proteins were 60 human lectin/human lectin-like proteins capable of recognizing and binding different glycosyl groups. Compared with traditional plant lectin chips, human lectin/human-like lectin chips have more specific recognition ability and stronger binding ability for complex glycoforms in higher vertebrate samples. Human lectin/human-like lectin chip provides a new technical platform for glycomic research, which can be widely applied to glycosylation research in humans or other mammals.

Description

Human agglutinin/human-like agglutinin chip for glycosylation detection and preparation method thereof

Technical Field

The invention relates to the field of bioscience, in particular to a human agglutinin/human-like agglutinin chip for glycosylation detection and a preparation method thereof.

Background

Glycosylation is the most important post-translational modification in an organism, and glycosylation modification is present in more than 50% of proteins. Glycosylation plays a key role in protein folding, cell growth, division, differentiation, can recognize extracellular signals as receptors, and can also activate immune responses in the organism, and the like. Some physiopathological processes are also closely related to glycosylation, for example, sugars on the surface of sperm cells can affect sperm motility and fertilization ability, and increased sialic acid on the surface of cancer cells can promote migration of cancer cells.

The carbohydrate profile of vertebrates is more complex than invertebrates. Understanding the composition, structure and changes that occur in the human body enables us to better understand the function of glycosylation and their role in disease, and thus to guide disease diagnosis and treatment. The traditional glycosylation analysis methods mainly comprise liquid chromatography, mass spectrometry and other technologies, but the methods are high in manpower and material resource consumption and cannot perform rapid, systematic and real-time glycosylation analysis.

As a high-flux glycosylation analysis and detection platform, by virtue of its unique advantages, such as small volume, high flux, fast analysis and detection speed, less sample demand, etc., the lectin chip has been widely applied to the analysis of prokaryotic and eukaryotic microbial glycosylation, the research of sperm cell surface glycosylation and the identification of cancer cell surface glycosylation, etc. Phytohemagglutinin chips can analyze the sugar-type structure and the glycosylation level of a sample or the change in the sugar chain structure well, but phytohemagglutinin chips sometimes cannot analyze complex sugar-types in mammals, particularly in humans.

There is also a lectin-like or lectin-like protein present in humans, mostly transmembrane proteins which are integrated in the cell membrane, and also partly soluble and secreted proteins. Human lectin protein plays a key role in regulating cell adhesion, glycoprotein synthesis and controlling protein levels in blood in the human body, and as an endogenous protein, human lectin or lectin-like protein can recognize complex sugar chains in the human body more specifically and with stronger binding force therebetween. Therefore, the human lectin chip can better help us to study glycomics under physiological or pathological conditions of human body compared with the lectin chip.

Most of human agglutinin or human-like agglutinin is membrane protein, and has the disadvantages of small recombinant expression amount, low separation and purification efficiency, easy inactivation and the like, thus hindering the research process. At present, no report of human lectin chips is found.

Disclosure of Invention

Aiming at the defects in the prior art, aiming at making up the defects of technologies such as mass spectrum and the like in the field of protein glycosylation research and strengthening the advantages of lectin chips, the invention aims to construct a human lectin-like human lectin chip for glycosylation detection and a preparation method thereof, wherein the chip comprises 60 human lectin/human lectin-like proteins, and chip quality inspection and human lectin activity verification show that the human lectin chip can be used for glycosylation analysis of biological samples.

The purpose of the invention is realized by the following technical scheme:

in a first aspect, the present invention provides a glycosylation detection chip, wherein the glycosylation detection protein on the chip is human lectin/human lectin-like protein.

Preferably, the number of types of human lectin/human lectin-like proteins is 60; wherein the 60 human lectin/human lectin-like proteins are named as B3GNT, CD207, SELE, CHODL, CLEC1, CLEC1, CLEC2, CLEC2Dv, SELL, CLEC2Dv, SELPLG, CLEC3, CLEC3, CLEC4, CLEC4, CLEC4, CLEC5, SIGLEC, CLESF, CLEC7Av, SIGLEC, CLEC7, CLEC9, COLEC, COLEC11V, DAD, FCN, FCN, FCN, GALN, GALNT, GALNT, GALNT9, HSPC159, KLRC1V, KLRC1V, KLRF, SIGLEC, KLRG, KLRK, OLR, LAMP, LGALS, LGALS, LGALS, LGALS, LGALS14V, LGALS, MASP2V, MASP2V, LS8, KLLS 3, LMN, LMAN, LMAN 2V, LMAN 3, LMAN 4, CLEC4, CLEC5, CLEC9, SIGL, CLEC 9.

Preferably, the human lectin/human-like lectin protein is prepared by: and transferring the clone plasmid connected with the protein gene sequence into saccharomyces cerevisiae for overexpression, and performing affinity purification through a fusion tag to obtain the recombinant vector. The purified recombinant protein is at a concentration of greater than or equal to 10 μ g/mL.

Preferably, the chip comprises a number of matrices, each matrix comprising the human lectin/human-like lectin protein and a control.

Preferably, 3 replicate spots are set for each human lectin/human lectin-like protein, control in the matrix.

Further preferably, the number of the matrix is 12, and the control comprises a positive control, a negative control and a blank control. Specifically, the positive control is fluorescent ConA, and the negative control comprises glutathione S transferase and BSA; the blank control comprises elution buffer and spotting buffer. 12 detection windows can be formed, and 12 samples can be detected simultaneously.

As can be seen from one embodiment of the present invention, each matrix contains 60 human lectins and human lectin-like proteins, 1 glutathione S transferase and 1 BSA as negative controls, 1 fluorescent ConA as positive control, elution buffer and spotting buffer as blank controls.

Preferably, the amount of each lectin/lectin-like protein spot deposited on the matrix is about 0.3-0.5 nL.

Preferably, the spots on the chip are spotted on the chip substrate by using a biochip spotting instrument.

In a second aspect, the present invention provides a method for preparing the glycosylation detection chip, which specifically comprises: and (3) spotting the human agglutinin/human-like agglutinin protein and the control on a chip substrate.

Preferably, the preparation method further comprises the steps of performing quality inspection and activity verification on the obtained chip.

In a third aspect, the present invention further provides an activity verification method for the glycosylation detection chip, specifically comprising: carrying out incubation reaction on Dylight 550NHS Ester fluorescence labeled THP-1 cell lysate, Dylight 550NHS Ester fluorescence labeled N-linked glycosylation-removed THP-1 cell lysate and closed glycosylation detection chip, washing and drying^TM4200A scanner, the human agglutinin/human-like agglutinin on the chip is combined with the cell lysate and the signal to noise ratio is more than 4, or the human agglutinin/human-like agglutinin on the chip can be combined with the cell, which proves to be active.

Preferably, the blocking is with BSA; the incubation reaction time is 2 h; the washing specifically comprises: PBST washing 3 times, each time for 5 min; cleaning with ultrapure water for 1 time and 1 min; the drying mode is spin drying.

However, human lectin proteins that do not bind to cell lysates or to cells on the chip are not considered biologically inactive, since it is not possible to determine the activity of each individual human lectin individually by this method, but only globally.

The cell lysate is incubated with the chip to determine that most of the human agglutinin and human-like agglutinin protein on the human agglutinin chip have biological activity. The cell lysate treated with the N-linked glycosylase PNGase F was incubated with the chip to confirm that the human lectin or human-like lectin on the chip recognizes and binds to the N-linked sugar chains in the cell lysate.

Different cell lines are incubated with the chip to determine that different cell lines have different human lectin chip binding spectra, which indicates that the glycoforms on the cell surface are different.

Compared with the prior art, the invention has the following beneficial effects:

the human agglutinin and human-like agglutinin on the human agglutinin chip constructed by the invention have good biological activity and have the characteristic of recognizing and combining complex carbohydrate chains of mammals. The human lectin chip constructed by the invention inherits the advantages of the plant lectin chip and can analyze glycosylation of complex samples, such as cell lysate; the glycosylation on the surface of the living cell can be analyzed in real time; and the detection of 12 samples at the same time can be realized.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1 is a flow chart of the preparation of a human lectin/human-like lectin protein chip;

FIG. 2 shows the silver staining results of purified human lectin and human lectin-like protein, wherein::representsthe position of the target protein;

FIG. 3 shows the results of quality control of the prepared human lectin protein chips;

wherein A is a scanning result diagram and a chip dot system mode diagram of the human lectin chip; b is the signal intensity statistics of the human lectin chip.

FIG. 4 shows the results of activity assays for human lectins and human lectin-like proteins on a chip;

wherein A is the comparison result of the cell lysate treated with or without PNGaseF and the human lectin chip after incubation; b is the quantitative result of the chip signal.

FIG. 5 shows the results of the incubation of three cell lines with the human lectin chip.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

This example provides a human lectin/human-like lectin protein chip and an activity verification method, the chip construction process is shown in FIG. 1. The detailed process and parameters of the whole process are as follows:

1. obtaining yeast expression library of human lectin and human-like lectin protein

The entry clone pool of 60 human lectins and human-like lectins was derived from professor hippophae of john hopkins university.

After the sequencing is correct, the coding sequences of the human lectin and the human lectin-like entry clone are connected to a target vector pEGH-A through LR reaction of a Gateway system, and an expression clone plasmid is constructed.

And finally, transferring the expression cloning plasmid into a competent cell of saccharomyces cerevisiae Y258 to obtain a yeast expression library of the human lectin and the human-like lectin protein.

2. Expression and purification of human lectins and human-like lectin proteins

a. Expression of human lectins and human-like lectin proteins: single colonies were picked and cultured in 1mL SC-URA/Dextrose (complete synthetic medium with glucose-deficient uracil) medium until OD600 (absorbance at wavelength 600 nm) reached 3.0, as 1: 2000 in a ratio of 0.6 to 0.8 in 120mL SC-URA/Raffinose (complete synthetic medium containing Raffinose and uracil-lacking) medium. Adding 13mL of 20% galactose (filter sterilized) and culturing for 4-6 hr, collecting bacteria, and storing at-80 deg.C in refrigerator.

b. Purification of human lectins and human-like lectin proteins: taking out the collected bacteria in a refrigerator at-80 deg.C, adding zirconia beads and lysis buffer, shaking at 4 deg.C for 30s, placing on ice for 2min, and repeating for 4 times; the cell lysate obtained 4 times is collected in a 15ml centrifuge tube, an appropriate amount of reduced glutathione agarose beads is added, lysis buffer is supplemented to 12ml, after incubation for 2 hours at 4 ℃, the supernatant is removed by centrifugation. After washing the agarose beads 3 times with washing buffer 1 and 2, respectively, the agarose beads were incubated with 1.5ml of elution buffer for 15min, and the supernatant was collected by centrifugation. Then, the mixture was centrifuged at 6000rpm at 4 ℃ in an ultrafiltration tube of Millipore to concentrate the volume to about 20 to 40. mu.L, 4. mu.L of the mixture was mixed with 1. mu.L of 5 Xloading buffer solution, and subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis), followed by silver staining using a dye-printing kit on Byunyan day after the electrophoresis. As shown in FIG. 2, the concentration of the purified recombinant protein was 10. mu.g/mL or more.

c. Preservation of human lectin and human-like lectin proteins: the purified 60 proteins were added to 30% glycerol by volume, respectively, and stored in a refrigerator at-80 ℃.

3. Preparation of human lectin chip

a. 10 μ L of each protein was removed and transferred to 384-well plates. Meanwhile, positive control (fluorescent-labeled ConA: canavalin), negative control (GST: glutathione S transferase, BSA: bovine serum albumin), Elution Buffer and spotting Buffer were added as blank controls (Elution Buffer, Printing Buffer). In the cold storageIn (1), use a crystal core

SmartArrayer 48 microarray spotting System spots proteins onto the surface of a polymeric three-dimensional substrate H in the pattern of FIG. 3A under 45-50% humidity, with 12 sub-arrays per chip, 65 samples per array (including 60 human lectin and human lectin-like proteins, 5 controls), and 3 replicate spots per sample. And (4) fixing the protein at 4 ℃ overnight to finish the construction of the protein chip. Storing at-80 deg.C for use.

b. Quality inspection

And (3) sealing: 0.15g of bovine serum albumin was dissolved in 5mL of TBST as a blocking solution, and the chip was blocked at room temperature for 1h at 50 rpm.

Incubating the primary antibody: the rabbit anti-GST antibody was diluted with TBST at a volume ratio of 1: 1000. Incubate at room temperature for 1h, 50 rpm. Wash 3 times 5min each using TBST.

Incubation of secondary antibody: cy 5-coupled goat anti-rabbit antibodies were diluted with TBST at a volume ratio of 1: 1000. Incubate at room temperature for 1h, 50 rpm. Wash 3 times 5min each using TBST. After rinsing with ultrapure water for 1min, the plates were washed in a SlideWasher ^TM8 after spin-drying, GenePix was used^TM4200A scanner, scan and record the results. The results are shown in FIG. 3A, in which the statistical results of the signal intensity of human lectin protein are shown in FIG. 3B.

c. Verification of protein Activity

THP-1 cells were treated with lysis buffer NP-40, and the resulting cell lysate was divided into two portions, one portion was directly fluorescently labeled with Dylight 550NHS Ester, and the other portion was fluorescently labeled with Dylight 550NHS Ester after N-linked glycosylation was removed with PNGaseF. After removing the excess fluorescent dye with desalting column, two cell lysates were incubated with BSA-blocked human lectin chip for 2 hr, washed with PBST for 5min 3 times. After rinsing with ultrapure water for 1min, the plates were washed in a SlideWasher ^TM8 after spin-drying, GenePix was used^TM4200A scanner, scan and record the results. The results of the two samples are shown in FIG. 4A, where the statistical results of the signal intensities are shown in FIG. 4B. The signal-to-noise ratio is 4 or more. As can be seen from FIG. 4A, most human lectins can bind to THP-1 cell lysates, and the binding spectra of the lysates and human lectin chips changed after the PNGaseF was used to remove N-linked glycosylation from THP-1 cell lysates; as is clear from FIG. 4B, the binding of 26 human lectins to the cell lysate was observed, and almost all of the signals from the cell lysate with the N-glycosyl groups removed and the human lectins were weak.

Three cultured cells (293T, MDA-MB-231 and Yeast) were collected and used with CellTracker^TMOrange CMRA fluorescence labeling of live cells, 1X 10 addition per detection window⁶After incubating the cells for 1 hour at room temperature, the chip is washed in PBST by hand, and after drying the chip in the dark at room temperature, GenePix is used^TM4200A scanner, scan and record the results. The binding of the three cells to the human lectin chip is shown in FIG. 5. As can be seen from FIG. 5, the binding spectra of different cells and the human lectin chip are different, and at least 30 human lectin proteins were active when counting the binding of the three cell lines to the lectin chip.

In the above examples, the formulations of all solutions were: (the solvents of the following solutions are all deionized water)

(1) SC-URA/glucose medium: YNB 1.7g, (NH)₄)₂SO₄5g, URAmix 2g, glucose 20g, add ddH₂O to 1L, and sterilizing at 121 deg.C for 15 min.

(2) SC-URA/Raffinose medium: YNB 1.7g, (NH)₄)₂SO₄5g, URAmix 2g, Raffinose20g, add ddH₂O to 1L, and sterilizing at 121 deg.C for 15 min.

(3) Lysis buffer pH 7.5(1L)

(4) Wash buffer 1pH 7.5(1L)

(5) Wash buffer 2pH 7.5(1L)

(6) Elution buffer pH 7.5(1L)

(7)10 × TBS buffer pH 7.4(1L)

(8)1 XTSST buffer (1L)

10 × TBS buffer 100mL

Tween 201 mL

ddH₂O constant volume is 1L

(9)10 XPBS buffer (1L):

(10)1 XPBST buffer (1L)

10 XPBS buffer 100mL

Tween 201 mL

ddH₂O constant volume is 1L

(11)5 XSDS-PAGE electrophoresis buffer (1L):

tris 0.125M

Glycine 0.96M

SDS 0.5％

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. A glycosylation detection chip, wherein the glycosylation detection protein on the chip is a human lectin/human-like lectin protein; the number of types of the human lectin/human-like lectin protein is 60 kind;

Wherein, the gene names of the 60 human lectins/human lectin-like proteins are 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 LAMP1, LAMP2, LGALS1, LGALS12, LGALS13, LGALS14, LGALS14v2, LGALS2, MASP2v2, MASP2v1, LGALS7, LGALS8, LGALS8v2/3, LMAN1, LMAN2, LMAN2L, MAG, MASP1v3, MASP1, REG3A;

The preparation of the human lectin/human lectin-like protein is as follows:

S1. Obtain the yeast expression library of human lectin and human lectin-like proteins:

a. After the sequencing is correct, through the LR reaction of the Gateway system, the coding sequences of human lectin and human-like lectin entry clones are connected to the destination vector pEGH-A to construct an expression cloning plasmid;

b. After the sequencing is correct, through the LR reaction of the Gateway system, the coding sequences of the human lectin and human-like lectin entry clones are connected to the target vector pEGH-A to construct an expression cloning plasmid;

S2. Expression and purification of human lectins and human lectin-like proteins:

a. Expression of human lectin and human lectin-like protein: Pick a single colony and culture it in 1mL SC-URA/Dextrose medium until the OD600 reaches 3.0, then inoculate it into 120mL SC-URA/Raffinose medium at a ratio of 1:2000 Cultivate to OD600 value of 0.6-0.8; add 13mL of 20% galactose and cultivate for 4-6 hours, collect the bacteria, and store in -80℃ refrigerator;

b. Purification of human lectin and human lectin-like protein: Take out the collected bacteria from the -80°C refrigerator, add zirconia beads and lysis buffer, shake at 4°C for 30 s, then place on ice for 2 minutes, repeat 4 times; Collect the cell lysate obtained four times into a 15ml centrifuge tube, add reduced glutathione agarose beads, supplement the lysis buffer to 12ml, incubate at 4°C for 2 hours, and remove the supernatant by centrifugation; After washing three times with washing buffer 1 and 2 respectively, incubate with agarose beads with 1.5 ml of elution buffer for 15 min, and collect the supernatant by centrifugation; the concentration of purified recombinant protein is greater than or equal to 10 μg/mL;

The purification is by fusion tag affinity purification.

2 . The glycosylation detection chip according to claim 1 , wherein the chip comprises several matrices, and each matrix comprises the human lectin/human lectin-like protein and a control. 3 .

3 . The glycosylation detection chip according to claim 2 , wherein the number of the matrix is 12, and the control includes a positive control, a negative control, and a blank control. 4 .

4 . The glycosylation detection chip according to claim 2 , wherein, on the matrix, the amount of each human lectin/human lectin-like protein spotting spot is 0.3-0.5nL. 5 .

5. A method for preparing a glycosylation detection chip according to any one of claims 1 to 4, characterized in that it specifically comprises: spotting the human lectin/human lectin-like protein and a control on a chip base On the chip, you can.

6 . The preparation method of the glycosylation detection chip according to claim 5 , wherein the preparation method further comprises the steps of performing quality inspection and activity verification on the obtained chip. 7 .

7 . The method for preparing a glycosylation detection chip according to claim 5 , wherein the spotting spots on the chip are spotted on the chip substrate by using a biochip spotter. 8 .

8. according to the activity verification method of the described glycosylation detection chip of any one of claim 1 to 4, it is characterized in that, specifically comprises: the THP-1 cell lysate of DyLight 550NHS Ester fluorescence labeling, DyLight 550NHS Ester fluorescence The labeled de-N-linked glycosylated THP-1 cell lysate was incubated with the blocked glycosylation detection chip, washed and dried, and observed on a GenePix ^™ 4200A scanner. The human lectin/human-like lectin on the chip was observed. If it is combined with the cell lysate and the signal-to-noise ratio reaches 4 or more, or the human lectin/human-like lectin on the chip can bind to the cell, it is proved to be active.

9. The method for verifying the activity of the glycosylation detection chip according to claim 8, wherein the blocking adopts BSA; the incubation reaction time is 2h; the washing specifically comprises: washing with PBST 3 times, each time 5min; washed with ultrapure water once, 1min; the drying method is spin drying.