CN111217893A

CN111217893A - Black fungus agglutinin and preparation method thereof

Info

Publication number: CN111217893A
Application number: CN202010031547.3A
Authority: CN
Inventors: 张彦龙; 赵丹丹; 曾伟民; 雷虹; 马成瑶; 黄悦; 李鹏飞
Original assignee: Heilongjiang University
Current assignee: Heilongjiang University
Priority date: 2020-01-13
Filing date: 2020-01-13
Publication date: 2020-06-02

Abstract

An auricularia auricula agglutinin and its preparing process, wherein the auricularia auricula agglutinin is prepared through preparing the coarse extract of auricularia auricula agglutinin, mixing it with the pig's gastric mucin II-cyanogen bromide activated agarose gel, centrifugal dissociation, ultrafiltering, freeze drying to obtain auricularia auricula agglutinin LAA, and features suppressing the growth of cancer cells, promoting the release of NO from macrophage, IL-1 β and TNF- α, and improving immunity.

Description

Black fungus agglutinin and preparation method thereof

Technical Field

The invention relates to a lectin and a preparation method thereof.

Background

Lectin (Lectin) refers to a glycoprotein or sugar-binding protein purified from various plants, invertebrates and higher animals, and is called Lectin because it can agglutinate red blood cells (including blood group substances). Phytoagglutinin (PNA) is commonly used, and is generally named after the plant from which it is extracted, such as concanavalin a (ConA), Wheat Germ Agglutinin (WGA), Peanut agglutinin (PNA), Soybean agglutinin (SBA), and the like, and the agglutinin is a generic name thereof.

Lectins are proteins capable of binding to sugars, which are synthesized and secreted by both animal and plant cells, and play an important role in cell recognition and adhesion reactions, mainly to promote intercellular adhesion. Lectins have more than one site for binding to sugars and are therefore involved in cell recognition and adhesion, linking different cells.

Disclosure of Invention

The invention provides a black fungus lectin and a preparation method thereof.

The black fungus agglutinin is prepared by the following steps:

firstly, preparing a black fungus lectin crude extract;

secondly, mixing the black fungus agglutinin crude extract with pig gastric mucin II type-cyanogen bromide activated agarose gel;

and thirdly, centrifuging, dissociating, ultrafiltering, and freeze-drying to obtain the auricularia auricula lectin LAA.

The auricularia auricula lectin LAA is prepared by the method.

The relative molecular mass of the auricularia auricula agglutinin LAA is determined to be 18913.22 by MALDI-TOF/TOF; the sequence of the N-terminal of the black fungus agglutinin LAA is detected to be ITAPTTTSSAATE; the black fungus agglutinin LAA is analyzed to contain the following 4 peptide segments by protein identification and comparison with a UniProt database: QIDAERK, TNHSVVVTNDK, RLNFTAGNPFPR, VRELEQVDSMTK. The existence of the same sequence is not found in the existing black fungus protein mass spectrum library, so that the black fungus lectin LAA is deduced to be a novel protein in black fungus and is determined to be a novel lectin.

The black fungus lectin can inhibit the growth of cancer cells, promote macrophages to release NO, IL-1 β and TNF- α, and has the activity of improving immunity.

The black fungus agglutinin of the invention can eliminate ABTS free radical (ABTS)⁺) Hydroxyl radical (. OH) and superoxide anion (. O)₂ ^-) Has excellent antioxidant effect.

The black fungus agglutinin of the invention has no toxicity to macrophage RAW 264.7.

Drawings

FIG. 1 is a 200-fold observation chart of Auricularia auricular lectin LAA for inhibiting proliferation of lung cancer cell A549; FIG. 1A: negative control; FIG. 1B: the LAA concentration is 50 mug/mL; FIG. 1C: the LAA concentration is 100 mug/mL; FIG. 1D: the LAA concentration is 150 mug/mL; FIG. 1E: the LAA concentration is 200 mug/mL; FIG. 1F: the LAA concentration was 250. mu.g/mL.

FIG. 2 is a graph showing the inhibition of A549 cells by auricularia auricula lectin LAA.

FIG. 3 is a 200-fold observation chart of Auricularia auricula lectin LAA for inhibiting proliferation of cancer cell MCF-7; FIG. 3A: negative control; FIG. 3B: the LAA concentration is 50 mug/mL; FIG. 3C: the LAA concentration is 100 mug/mL; FIG. 3D: the LAA concentration is 150 mug/mL; FIG. 3E: the LAA concentration is 200 mug/mL; FIG. 3F: the LAA concentration was 250. mu.g/mL.

FIG. 4 is a graph showing the inhibition of MCF-7 cells by auricularia auricula-judae lectin LAA.

FIG. 5 is a 200-fold observation chart of Auricularia auricula lectin LAA inhibiting cancer cell SGC-7901 proliferation; FIG. 5A: negative control; FIG. 5B: the LAA concentration is 50 mug/mL; FIG. 5C: the LAA concentration is 100 mug/mL; FIG. 5D: the LAA concentration is 150 mug/mL; FIG. 5E: the LAA concentration is 200 mug/mL; FIG. 5F: the LAA concentration was 250. mu.g/mL.

FIG. 6 is a graph showing the inhibition of SGC-7901 cells by auricularia auricula lectin LAA.

FIG. 7 is a 200-fold observation chart of Auricularia auricula lectin LAA for inhibiting the proliferation of cancer cell HepG-2; FIG. 7A: negative control; FIG. 7B: the LAA concentration is 50 mug/mL; FIG. 7C: the LAA concentration is 100 mug/mL; FIG. 7D: the LAA concentration is 150 mug/mL; FIG. 7E: the LAA concentration is 200 mug/mL; FIG. 7F: the LAA concentration was 250. mu.g/mL.

FIG. 8 is a graph showing the inhibition of HepG-2 cells by the auricularia auricula-judae lectin LAA.

Fig. 9 is a graph of auricularia auricula lectin LAA as a function of NO release, (n ═ 3),. p <0.05,. p <0.01,. p < 0.001.

Fig. 10 is a purity measurement chart of auricularia auricula lectin LAA, (n ═ 3),. p <0.05,. p <0.01,. p < 0.001.

Fig. 11 is a graph of inhibition of NO release by protein kinase K, (n-3), p <0.05, p <0.01, p < 0.001.

Fig. 12 shows that ATP and LPS synergistically produced IL-1 β, (n ═ 3),. p <0.05,. p <0.01,. p < 0.001.

Fig. 13 shows that auricularia auricula lectin LAA produces IL-1 β (n-3), p <0.05, p <0.01, p <0.001 in cooperation with ATP.

Fig. 14 is a trigger condition for TNF- α release, (n-3), p <0.05, p <0.01, p < 0.001.

Fig. 15 is a graph of TNF- α release versus concentrations of auricularia auricula lectin LAA, (n-3), p <0.05, p <0.01, p < 0.001.

Fig. 16 is the effect of polymyxin B on TNF- α release, (n ═ 3),. p <0.05,. p <0.01,. p < 0.001.

Fig. 17 is a graph of DPPH clearance of auricularia auricula lectin LAA.

Fig. 18 is a graph of DPPH clearance of Vc.

FIG. 19 is a graph of ABTS clearance of Auricularia auricular lectin LAA.

Fig. 20 is a graph of ABTS clearance for Vc.

FIG. 21 shows the sequence O of Auricularia auricula lectin LAA₂ ^-Clearance curves.

FIG. 22 shows the equation O of Vc₂ ^-Clearance curves.

FIG. 23 is a graph showing OH radical scavenging rate of auricularia auricula lectin LAA.

FIG. 24 is a graph showing the OH radical clearance of Vc.

FIG. 25 shows the total antioxidant capacity FeSO of auricularia auricula lectin LAA₄Standard graph of (2).

Detailed Description

The technical solution of the present invention is not limited to the following specific embodiments, but includes any combination of the specific embodiments.

The first embodiment is as follows: the black fungus lectin of the embodiment is prepared by the following steps:

firstly, preparing a black fungus lectin crude extract;

The second embodiment is as follows: the present embodiment differs from the first embodiment in that: the pig gastric mucin II type-cyanogen bromide activated agarose gel is prepared by coupling pig gastric mucin II type serving as a ligand to agarose gel (sepharose-4B). Other steps and parameters are the same as those in the first embodiment.

The third concrete implementation mode: the present embodiment is different from the first or second embodiment in that: the method comprises the following steps: adding phosphate buffer solution into Auricularia, mashing in a mashing machine to obtain viscous homogenate, freezing and centrifuging, collecting supernatant, and filtering under high pressure to obtain Auricularia lectin crude extract. The other is the same as in the first or second embodiment.

The fourth concrete implementation mode: the present embodiment is different from one of the first to third embodiments in that: the phosphate buffer concentration in step one was 10mM, pH 7.6. The others are the same as in one of the first to third embodiments.

The fifth concrete implementation mode: the present embodiment is different from one of the first to fourth embodiments in that: in the first step, the ratio of the phosphate buffer solution to the black fungus is 100mL to 5 g. The other is the same as one of the first to fourth embodiments.

The sixth specific implementation mode: the present embodiment is different from one of the first to fifth embodiments in that: in the first step, the freezing and centrifuging speed is 5000r/min, and the time is 30 min. The other is the same as one of the first to fifth embodiments.

The seventh embodiment: the present embodiment is different from one of the first to sixth embodiments in that: the supernatant collected in step one was high pressure filtered through four layers of FN15 membranes (Whatman company Filtrak FN15 chromatography cellulose membranes). The other is the same as one of the first to sixth embodiments.

The specific implementation mode is eight: the present embodiment is different from the first to seventh embodiments in that: and in the second step, the black fungus lectin crude extract is mixed with swine gastric mucin II type-cyanogen bromide activated agarose gel according to the volume ratio of 1: 1. The other is the same as one of the first to seventh embodiments.

The specific implementation method nine: the present embodiment is different from the first to eighth embodiments in that: the mixing temperature in the second step was 6 ℃. The rest is the same as the first to eighth embodiments.

The detailed implementation mode is ten: the present embodiment is different from one of the first to ninth embodiments in that: and in the second step, the mixing duration is 8-12 h. The other is the same as one of the first to ninth embodiments.

The concrete implementation mode eleven: the present embodiment is different from the first to tenth embodiments in that: centrifuging in the third step: centrifuging at 3000r/min for 5min, washing the lower-layer precipitate with PBS for three times after centrifuging, centrifuging at 3000r/min for 5min again, discarding the supernatant, adding glycine mixed liquor into the precipitate, shaking up, centrifuging at 3000r/min for 5min, taking the supernatant, adding Tris-HCl buffer solution into the supernatant, repeating the processes of adding glycine mixed liquor into the precipitate, centrifuging, taking the supernatant and adding Tris-HCl buffer solution for 2 times, and combining the supernatants added with Tris-HCl buffer solution to obtain an ultrafiltration stock solution;

wherein the glycine mixed solution is a solution with the glycine concentration of 0.1M, NaCl, the concentration of 1M and the pH value of 3.0;

wherein the concentration of the Tris-HCl buffer solution is 1M, pH 7.8.8. The rest is the same as one of the first to tenth embodiments.

The specific implementation mode twelve: this embodiment is different from the first to eleventh embodiments in that: ultrafiltration in the third step: the 5kDa ultrafiltration desalination of the ultrafiltration stock solution is carried out, and then the concentrated solution is ultrafiltered by a 10kDa ultrafiltration centrifugal tube 4400 r/min. The rest is the same as in one of the first to eleventh embodiments.

The specific implementation mode is thirteen: the present embodiment is different from the first to twelfth embodiments in that: in the ultrafiltration process, water is added for desalting for 3 times. The rest is the same as the first to twelfth embodiments.

The specific implementation mode is fourteen: the present embodiment is different from one of the first to thirteenth embodiments in that: and (4) ultrafiltering a 10kDa ultrafiltration centrifugal tube 4400r/min to obtain a concentrated solution 1/4 in volume. The rest is the same as one of the first to the thirteenth embodiments.

Example 1

The black fungus agglutinin is prepared by the following steps:

firstly, adding a phosphate buffer solution with the concentration of 10mM and the pH value of 7.6 into black fungus, stirring in a pounding machine until the black fungus is viscous and homogenate, then freezing and centrifuging, wherein the centrifugal speed is 5000r/min, the centrifugal time is 30min, collecting supernate, and filtering the supernate with four layers of FN15 membranes under high pressure, wherein the filtrate is a black fungus agglutinin crude extract; wherein the proportion of the phosphate buffer solution to the black fungus is 100mL to 5 g;

mixing the black fungus agglutinin crude extract with porcine gastric mucin II-cyanogen bromide activated agarose gel at 6 ℃ for 8-12 h, wherein the black fungus agglutinin crude extract is mixed with the porcine gastric mucin II-cyanogen bromide activated agarose gel according to the volume ratio of 1: 1;

centrifuging at 3000r/min for 5min, washing the lower-layer precipitate with PBS for three times after centrifuging, centrifuging at 3000r/min for 5min again, discarding the supernatant, adding glycine mixed liquor into the precipitate, shaking up, centrifuging at 3000r/min for 5min, taking the supernatant, adding Tris-HCl buffer solution into the precipitate, repeating the processes of adding glycine mixed liquor into the precipitate, centrifuging, taking the supernatant and adding Tris-HCl buffer solution for 2 times, and combining the supernatants added with Tris-HCl buffer solution to obtain an ultrafiltration stock solution;

wherein the concentration of the Tris-HCl buffer solution is 1M, pH 7.8.8;

the 5kDa ultrafiltration desalination of the ultrafiltration stock solution is performed, and then the concentrated solution is ultrafiltered by a 10kDa ultrafiltration centrifuge tube 4400r/min to obtain a concentrated solution 1/4. (ii) a Adding water to remove salt for 3 times in the ultrafiltration process; freeze drying to obtain Auricularia auricula lectin LAA.

Determining the relative molecular mass of the auricularia auricula agglutinin LAA to be 18913.22 by using MALDI-TOF/TOF; the sequence of the N-terminal of the black fungus agglutinin LAA is detected to be ITAPTTTSSAATE; the black fungus agglutinin LAA is analyzed to contain the following 4 peptide segments by protein identification and comparison with a UniProt database: QIDAERK, TNHSVVVTNDK, RLNFTAGNPFPR, VRELEQVDSMTK.

Anti-tumor experiments:

CCK-8 test: the Cell Counting Kit-8 Kit is used for detecting the proliferation of the auricularia auricula lectin inhibiting tumor cells.

The specific operation is as follows:

(1) ddH was added around 96-well plates₂O to prevent evaporation of the medium, and appropriate wells were selected as sample wells and control wells in a 96-well plate, and three sets of parallel wells were provided, respectively.

(2) After trypsinizing the cells in logarithmic growth phase, the cells were blown down evenly to a density of 2.5X 10 by adding the medium⁴Cell suspension of one/mL, 200. mu.l of cell suspension was inoculated into each well, and then the 96-well plate was placed at 37 ℃ in 5% CO₂The wet incubator of (1) for 24 hours.

(3) And taking out the 96-well plate the next day, sucking out the original culture solution, adding an equal volume of PBS solution, slightly shaking, sucking out, repeating for three times, adding 100 mu l of the auricularia auricula agglutinin LAA solution with different concentrations, adding an equal volume of culture medium containing 1 per thousand DMSO into the control well, and continuously culturing for 24, 48 or 72 hours.

(4) Adding 10 μ l CCK-8 solution into each well, incubating for 1h in incubator, and detecting OD with microplate reader₄₅₀。

(5) Calculating the inhibition ratio (%) of the black fungus agglutinin to the tumor cells according to the formula

Using SPSS statistical analysis software, the median inhibitory concentration of lectin on tumor cells, i.e., IC, was calculated₅₀。

The experimental results are as follows:

inhibiting proliferation of lung cancer cell a 549:

the experiment sets black fungus agglutinin LAA with different concentrations to carry out experiments on A549 cells, the experimental result is shown in figure 1, and the experimental result shows that compared with a negative control group, when the agglutinin concentration is 250 mug/mL, the A549 cells gradually lose the original irregular polygonal form, and are mostly in air bubble, the cell nucleuses gradually disappear and are in apoptosis trend, and the growth of the A549 cells is obviously inhibited.

The concentration of the auricularia auricula agglutinin LAA is 100-; the concentration of the auricularia auricula agglutinin LAA is 50 mug/mL, the A549 cell morphology is not greatly changed, only the cells are observed to begin to gather, the cell volume is increased, and the cells are judged to begin to generate air bubble.

Selecting 100 mu g/mL as the initial concentration of the auricularia auricula agglutinin LAA for inhibiting the proliferation of A549 cells; after being diluted by equal concentration gradient (100 mug/mL, 50 mug/mL, 25 mug/mL, 12.5 mug/mL and 6.25 mug/mL), the black fungus lectin LAA respectively acts on the A549 cells for 24h, 48h and 72h, and the increase inhibition condition of the black fungus lectin LAA after acting on the A549 cells at each concentration is shown in figure 2 through detection of a CCK-8 kit; the growth inhibition of the A549 cells is concentration-dependent and time-dependent, the proliferation inhibition degree of the A549 cells is gradually deepened along with the increase of the concentration of the auricularia auricula agglutinin LAA, the inhibition degree of the A549 cells is different when the auricularia auricula agglutinin LAA is treated at the same concentration for different time, and the inhibition rate of the auricularia auricula agglutinin LAA on the A549 cells reaches a maximum value of 74 percent when the concentration of the auricularia auricula agglutinin LAA is 100 mu g/mL and the action time is 72 hours.

Inhibiting proliferation of breast cancer cells MCF-7:

the primary screening result of the auricularia auricula-judae agglutinin LAA on the breast cancer cells is shown in figure 3, compared with the control group, the MCF-7 cell morphology under the treatment of 100-one 250 mu g/mL auricularia auricula-judae agglutinin LAA is obviously changed, the shuttle-shaped or multi-angle normal morphology disappears, the cell volume is increased and becomes round, and the adsorption force with the bottle bottom is reduced; MCF-7 cells treated by 50 mu g/mL auricularia auricula agglutinin LAA still keep the original cell shape, and the volume of the cells is increased and the cells become round respectively.

Selecting 100 mu g/mL auricularia auricula agglutinin LAA as the initial concentration during re-screening; the rescreening results are shown in FIG. 4, and Auricularia auricula agglutinin LAA is diluted in equal gradient and respectively mixed with MCF-7 cells at 37 deg.C with 5% CO₂The culture box is incubated for 24h, 48h and 72h, and the results show that the proliferation of MCF-7 cells is in negative correlation with the concentration of the auricularia auricula lectin LAA, namely, the higher the concentration of the auricularia auricula is, the more obvious the MCF-7 cell proliferation is inhibited, and the higher the inhibition rate of the MCF-7 cells is over time. The concentration of the auricularia auricula agglutinin LAA is 100 mug/mL, and after incubation for 72 hours, the inhibition rate of the auricularia auricula agglutinin LAA on breast cancer cells MCF-7 is up to 78%.

Inhibiting proliferation of gastric cancer cell SGC-7901:

the primary screening result of the auricularia auricula lectin LAA on SGC-7901 cells is shown in figure 5, the control cells are mostly plum blossom-shaped and are distributed in clusters, the shapes are regular and uniform, and the outlines are clear; after the auricularia auricula agglutinin LAA is added, the SGC-7901 cells begin to bubble, and the cells gradually separate from the bottom of the bottle after being deformed into a round shape.

As shown in FIG. 6, the SGC-7901 cells showed concentration and time dependence on auricularia auricula-judae lectin, and the SGC-7901 inhibition rate was only 53% after being cultured with auricularia auricula-judae lectin LAA for 72 h.

Inhibiting the proliferation of liver cancer cell HepG-2:

in the primary screening test of the auricularia auricula lectin LAA for inhibiting HepG-2, under the treatment of 50 mu g/mL of the auricularia auricula lectin LAA (shown in figure 7), the extended form of cells begins to disappear, cytoplasm shrinks, and air bubbles gradually appear; when the concentration reaches 100 mug/mL, the original form completely disappears, the volume of all cells is increased, the number is reduced and the cells begin to separate from the bottom of the bottle, and the form and the number of HepG-2 cells under the action of subsequent concentration have no great difference.

Selecting 100 mu g/mL as the initial concentration of the auricularia auricula agglutinin LAA for inhibiting HepG-2 cell proliferation; the auricularia auricula agglutinin LAA is diluted by the same volume of concentration gradient and acts on HepG-2 cells for 24h, 48h and 72h respectively (as shown in figure 8), the inhibition rate of HepG-2 is concentration-dependent with the auricularia auricula agglutinin LAA, but the inhibition rates of HepG-2 treated by 48h and 72h agglutinin LAA are not obviously different in acting time, which shows that HepG-2 has dependency on the concentration of the auricularia auricula agglutinin LAA within 48h, and the maximum inhibition rate of the auricularia auricula agglutinin LAA on HepG-2 cells reaches 80%.

Immunoregulation experiment

Cell treatment:

(3) And taking out the 96-well plate the next day, sucking out the original culture solution, adding PBS solution with the same volume, slightly shaking, sucking out, repeating for three times, adding 100 mu l of the auricularia auricula agglutinin LAA solution with different concentrations, culturing for 24h, and collecting the culture solution, namely the sample solution to be detected.

Enzyme-linked immunosorbent assay:

(1) according to the specification, 10 Xcoating Buffer is diluted to 1 Xcoating Buffer, 5 XELISA/ELISPOT is diluted to 1 XELISA/ELISPOT, and Capture Antibody and Detection Antibody are prepared respectively by using the two dilutions for standby.

(2) The standard is injected with 1mL ddH₂O, preparing a standard solution with the concentration of 15ng/mL, taking 100 mu l of the standard solution, and diluting the standard solution by 1 XELISA/ELISPOT diluent to obtain the final concentration of 1000 pg/mL.

(3) Avidin-labeled horseradish peroxidase solution was prepared for use in 1 × ELISA/ELISPOT dilutions.

(4) Mu.l of Capture Antibody was added to each well of the ELISA plate 9018, and the 96-well plate was sealed with a preservative film and cultured overnight at 2-8 ℃.

(5) Adding 250 mul of washing buffer (1 XPBS, 0.05% Tween-20) into each well of a 96-well plate, soaking for 1-2 min, then discarding, repeating the process for at least 3 times, and sucking the residual washing liquid by using a filter paper strip.

(6) 200. mu.l of 1 × ELISA/ELISPOT dilution was added to each well and incubated at room temperature for 1 h.

(7) And (5) repeating the step.

(8) Add 100. mu.l of standard solution to the first well of 96-well plate, dilute to the eighth well in a continuous 2-fold gradient, add 100. mu.l of sample solution to be tested (containing Auricularia auricular lectin LAA) to the appropriate well, set 3 biological replicates, and add 100. mu.l of 1 × ELISA/ELISPOT diluent as blank control.

(9) And (5) repeating the step.

(10) Mu.l of Detection Antibody was added to the corresponding wells and incubated for 1h at room temperature with plastic wrap sealing.

(11) And (5) repeating the step.

(12) Mu.l of Avidin-HRP was added to the corresponding well of the 96-well plate and incubated for 30min at sealed room temperature.

(13) And (5) repeating the step.

(14) Mu.l of 1 XTMB solution was added to the corresponding well of the 96-well plate and incubated for 15min at room temperature.

(15) Mu.l of stop buffer (1M H3PO4) was added to each well.

(16) The absorbance of the standard and sample was measured at 450nm using a microplate reader.

Griiss test:

cell treatment:

According to the Griess method, the content of NO released by mouse macrophages is detected. Because NO is easily oxidized into NO in vivo or in aqueous solution₂ ^-And in an acidic environment, NO₂ ^-The diazo compound is easy to react with diazo sulfanilamide to generate the diazo compound, and then the diazo compound and naphthyl ethylene diamine are subjected to coupling reaction. According to the concentration of the product formed and NO₂ ^-The NO content is calculated from the linear relationship of (1). The detailed steps are as follows:

(1) 10mM NaNO was prepared₂A standard stock solution; the stock solution was diluted 10-fold before the experiment as the initial concentration solution and continuously diluted with an equal gradient for determination of the NO standard curve.

(2) And (4) preparing a Griess reagent.

Solution A: 1.0g of anhydrous sulfanilic acid, adding 70mL of deionized water, heating to fully dissolve the sulfanilic acid, adding 6mL of 85% concentrated sulfuric acid, and fixing the volume to 100 mL.

And B, liquid B: 0.1g of N-1-naphthyl ethylenediamine hydrochloride is dissolved in deionized water, and the volume is fixed to 100 mL.

(3) 50 μ l of NaNO was added to each concentration in a 96-well plate₂Taking another blank hole, adding 50 μ l of sample solution (containing auricularia auricula agglutinin LAA with different concentrations) to be tested, and repeating the parallel test for three times.

(4) Adding 50 mu l A solution into each well, and incubating for 10min in an incubator at 37 ℃; and taking out the 96-well plate, adding 50 mu l of the solution B into each well, mixing uniformly, and then continuously incubating in an incubator at 37 ℃ for 10 min.

(5) And (4) taking out the 96-well plate, slightly oscillating for several times to avoid bubbles, and detecting the OD value of the corresponding well at the wavelength of 540nm of the microplate reader.

(6) And drawing an NO standard curve according to the measurement result, and calculating the NO content released by the mouse macrophage under different treatments according to the standard curve.

The experimental results are as follows:

the auricularia auricula lectin LAA induces NO generation, mouse mononuclear macrophage J774A.1 is selected as an immunocompetence test material, and the cell is derived from female mouse abdominal sarcoma cells, so that an immune response reaction under the stimulation of the auricularia auricula lectin LAA can be better reflected, and reliable data support is provided for test results.

The method for detecting the release amount of NO in Jew's ear agglutinin LAA acting J774A.1 cells by using a Grignard method is shown in figure 9, the release amount of NO and the concentration of Jew's ear agglutinin LAA are in a linear dependence relationship (figure 9), namely, the NO generated and released by induced macrophages is gradually increased along with the increase of the concentration of the Jew's ear agglutinin LAA, and the release of NO is verified to be LPS-independent. The elimination of errors caused by false positive results is particularly important because the materials are often subjected to false positive results caused by LPS (lipopolysaccharide) pollution in the test process, so that the test results are inaccurate. LPS is the main component of gram-negative bacteria cell wall, the heat resistance is strong, the activity is still maintained after heating in boiling water for 10min, but the black fungus lectin LAA is denatured under high temperature treatment, the biological function is lost, the release amount of NO is reduced (figure 10), the black fungus lectin is laterally verified to be free of LPS pollution, and the accuracy of the test result is ensured. Protein kinase K is a protein inhibitor, can effectively hydrolyze protein into amino acid, loses complex structure and protein function, and has the advantages that the NO release amount of the black fungus lectin LAA is remarkably reduced under the action of the protein kinase K (figure 11), and LPS (low pressure lipoprotein lipase) under the action of the protein kinase has NO phenomenon of reducing the NO release amount, so that the protein kinase K is a good lectin inhibitor and can inhibit the immunoregulation level.

Auricularia auricula agglutinin LAA induces interleukin IL-1 β production

FIG. 12 shows that the content of interleukin IL-1 β produced by macrophage of mice is tested by applying ELISA method, when ATP or LPS is added into the culture medium alone, IL-1 β is hardly released, and after ATP and LPS are added simultaneously, the result of observation shows that the release amount of IL-1 β is increased significantly, which indicates that the release of IL-1 β requires the synergistic stimulation of ATP and LPS. FIG. 13 shows that the release amount of IL-1 β is almost the same when the Auricularia auricula lectin LAA is added into the culture medium alone, and the release amount of IL-1 β is obviously increased after 24h of culture when the Auricularia auricula lectin LAA and the ATP are added into the macrophage simultaneously, and the difference is significant compared with the control group, which indicates that the Auricularia auricula lectin LAA can exist similarly to the LPS and synergistically regulate the immunological activity of organisms with the ATP.

Auricularia auricula agglutinin LAA induces tumor necrosis factor TNF- α production

ELISA detects that Jew ear agglutinin LAA stimulates J774A.1 cells to generate TNF- α as shown in figures 14-16, ATP is added into a reaction system, no TNF- α is released, after LPS is added into a culture medium, the release amount of TNF- α is obviously increased, and LPS and ATP are simultaneously added, the detected release amount is not different from the release amount of TNF- α when LPS is independently added (figure 14), the release amount of TNF- α is only related to stimulation of LPS, the result of figure 15 shows that the release amount of TNF- α is concentration-dependent to the Jew ear agglutinin LAA, polymyxin PMB is a good endotoxin inhibitor, and the result of figure 16 shows that the release amount of the Jew ear agglutinin LAA TNF- α added with PMB is not significantly different from the release amount of TNF- α not added with PMB, and is obviously reduced after the release amount of macrophage treated by LPS alone is added into the culture medium, thereby verifying the reliability of a test sample.

Oxidation resistance test

DPPH free radical scavenging assay:

(1) the DPPH powder was dissolved in methanol to prepare a 6mmol/L DPPH stock solution, which was stored at-20 ℃ for further use, diluted 100-fold with methanol, i.e., 60. mu. mol/L.

(2) Adding Auricularia auricula agglutinin LAA solution with different concentrations into 1.5mL EP tube, adding 50 μ L into cuvette, taking 50 μ L DMSO as reference solution, adding 3mL 60 μmol/L DPPH methanol solution respectively, shaking gently, and standing at room temperature in dark for 30 min.

(3) And (3) measuring the light absorption value of each solution at the wavelength of 515nm by using a spectrophotometer, zeroing by using an equal volume of methanol solution before measurement, and calculating the free radical clearance of the sample solution to be measured according to the following formula.

Wherein As is the absorbance value of the sample tube, A₀The absorbance value of the reference tube is used.

The experimental results are as follows: auricularia auricula agglutinin LAA is selected for DPPH free radical scavenging test, and IC is used₅₀The value is used for measuring the strength of the antioxidant capacity of the tea. The DPPH clearance curves of various concentrations (6.25. mu.g/mL, 12.5. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL) of auricularia auricula lectin LAA and DPPH clearance curves of various concentrations (0.00625. mu.g/mL, 0.0125. mu.g/mL, 0.025. mu.g/mL, 0.50. mu.g/mL, 0.1. mu.g/mL) of Vc are shown in FIGS. 17 and 18, and it can be seen that the clearance rate of the auricularia auricula lectin LAA on DPPH free radicals is obviously lower than that under the action of Vc, and the result shows that the clearance rate of the auricularia auricula lectin LAA on DPPH is general.

EC of Auricularia auricula lectin LAA on DPPH₅₀EC of 91.44 + -4.20 μ g/mL, and Vc vs DPPH of control group₅₀Value comparison, difference and significance (P)<0.001), indicating that the auricularia auricula lectin LAA has poor DPPH clearance rate and cannot effectively clear DPPH.

TABLE 1 elimination of DPPH

Mean±SD，n＝3，*p<0.05，versus control.

ABTS free radical scavenging assay:

ABTS is easily oxidized into green ABTS under the action of oxidant^·+ABTS in the presence of antioxidants^·+Can be inhibited, and can be measured at 734nm for ABTS^·+The antioxidant capacity of the sample is calculated from the absorbance of (b).

(1)ABTS^·+Preparing a stock solution: 5mmol/L ABTS solution is prepared by PBS, mixed with 140nmol/L potassium persulfate, diluted by PBS to the light absorption value of 0.70 +/-0.02 at 734nm, kept stand and protected from light at-20 ℃ for standby.

(2) Adding 100 μ l of Auricularia auricula agglutinin LAA solution with different concentrations into cuvette, adding 3mL of ABTS + stock solution into 100 μ l of DMSO as reference solution, shaking, and standing at room temperature in dark for 10 min.

(3) The solution was zeroed with an equal volume of PBS, and then the absorbance of each solution at 734n was measured, and the radical clearance of the sample solution to be measured (black fungus agglutinin LAA solution) was calculated according to the following formula.

The experimental results are as follows: the clearance rate of auricularia auricula lectin LAA on ABTS is shown in figure 19, and the result shows that the clearance rate of auricularia auricula lectin LAA on ABTS is gradually enhanced along with the increase of the concentration of auricularia auricula lectin, and certain concentration dependence is shown.

EC of black fungus agglutinin LAA on ABTS₅₀The value is 45.70 +/-0.11 mu g/mL, and the EC of the control group Vc on ABTS₅₀The values are not obvious in difference, so that the black fungus lectin LAA has the capability of clearing ABTS, can be used as an antioxidant to prevent organisms from being oxidized by ABTS, and accordingly related diseases and adverse symptoms are avoided.

TABLE 2 pairs of ABTS⁺EC of (1)₅₀Value of

Mean±SD，n＝3，*p<0.05，versus control.

Superoxide anion radical scavenging test

Pyrogallol, also known as pyrogallic acid, is easily autoxidized under alkaline conditions to release superoxide anions, and the occurrence of the superoxide anions accelerates the oxidation of the pyrogallol. The addition of the antioxidant can block the production of superoxide anion, thereby reducing the autoxidation rate of pyrogallol, and the ability to scavenge superoxide anion is calculated by comparing the autoxidation rates before and after the addition of the antioxidant.

Determination of the rate of auto-oxidation of pyrogallol: 4.5mL Tris-HCl solution pH8.2 with 4.2mL ddH₂Mixing O, incubating at 25 deg.C for 30min, and rapidly adding 0.3Slightly oscillating and mixing the solution of the pyrogallol mL, recording the change of absorbance at 320nm within 5min, drawing a time (t) -absorbance (A) curve, and calculating the autoxidation rate (V) of the pyrogallol₀)。

And (3) determining the autoxidation rate of pyrogallol under the action of the black fungus agglutinin LAA to be detected: 4.5mL Tris-HCl solution and 3.3mL ddH₂Mixing O, incubating at 25 deg.C for 30min, rapidly adding 0.9mL LAA gradient dilution solution of Auricularia auricula agglutinin and 0.3mL pyrogallol solution, shaking gently, mixing, recording absorbance change at 320nm within 5min, drawing time (t) -absorbance (A) curve, and calculating auto-oxidation rate (V) of pyrogallol under the action of test substance₁). The following formula:

the experimental results are as follows: auricularia auricula agglutinin LAA to superoxide anion (O)₂ ^-) The scavenging condition of (A) is shown in FIG. 21, and superoxide anion (. O) is visible₂ ^-) The clearance rate of the lectin-containing bioactive peptide is in direct linear correlation with the concentration of the lectin, has concentration dependence and has good antioxidant capacity.

Auricularia auricula agglutinin LAA to superoxide anion (O)₂ ^-) EC of the cleaning ability of₅₀The value is 35.03 +/-1.24 mu g/mL, and the statistical analysis result shows that the EC of the product is₅₀The value is not different from the control group, which indicates that the auricularia auricula agglutinin LAA has better superoxide anion (O)₂ ^-) The ability of the cell to perform.

TABLE 3 Pair.O₂ ^-EC of (1)₅₀Value of

Mean±SD，n＝3，*p<0.05，versus control.

Hydroxyl radical scavenging test:

under the condition of ferrous ions, hydrogen peroxide is easily reduced into hydroxyl radicals, and the hydroxyl radicals can be captured by adding salicylic acid to generate characteristic absorption at the wavelength of 510 nm. When an antioxidant is added into the reaction system, the ability of salicylic acid for capturing hydroxyl radicals is hindered, and further the light absorption value at a specific wavelength is reduced, so that the antioxidant ability of the substance to be tested is evaluated.

Uniformly mixing 100 mu L of to-be-detected auricularia auricula agglutinin LAA gradient diluted solution, 100 mu L of 9mmol/L ferrous sulfate solution and 100 mu L of 10mmol/L hydrogen peroxide solution, incubating in an oven at 37 ℃ for 20min, rapidly adding an isovolumetric 9mmol/L salicylic acid solution, gently mixing, continuing to incubate in the oven at 37 ℃ for 30min, and detecting the light absorption value at 510nm, ddH, by using a spectrophotometer₂And O is used as a blank control group, and the hydroxyl radical clearance rate of the sample to be detected is calculated according to the following formula:

the removal of OH free radicals by auricularia auricula lectin LAA is shown in FIG. 23, and it can be seen that the removal rate of OH free radicals is in direct proportion to the concentration of lectin, has concentration dependence, has certain antioxidant capacity, and is lower than the control group Vc in the removal capacity of OH free radicals (shown in FIG. 24).

EC of auricularia auricula agglutinin LAA on OH free radical scavenging capacity₅₀A value of 28.20. + -. 0.74. mu.g/mL, and its EC, as statistically analyzed₅₀The value is not obviously different from the control group, which shows that the auricularia auricula agglutinin LAA has better capability of eliminating OH free radicals.

TABLE 4 EC for OH₅₀Value of

Mean±SD，n＝3，*p<0.05，versus control.

Determination of total antioxidant capacity:

iron ion reducing power (FRAP) means that an antioxidant can reduce Fe in an acidic environment³⁺Reduction of TPTZ to Fe² ⁺TPTZ, whereby the change is detected at a wavelength of 593nm, in order to react with the test substanceTotal antioxidant capacity.

Preparing an FRAP working solution: 2.5mL of 300mmol/L acetate solution, 0.25mL of 10mmol/L TPTZ solution and 0.25mL of 20mmol/L ferric chloride solution.

FeSO₄Drawing a standard curve: 10 mul of FeSO with each equal concentration gradient is respectively taken₄And (3) uniformly mixing the standard solution and 100 mul of FRAP working solution, reacting at room temperature for 30min, measuring the light absorption value of 593nm by using an enzyme-labeling instrument, and drawing a standard curve according to the light absorption value.

Antioxidant activity of the sample: collecting 1mg Auricularia auricula agglutinin LAA, preparing into 10mg/mL solution with PBS, mixing 10 μ l and 100 μ l FRAP working solution at room temperature, reacting for 30min, and measuring light absorption value at 593nm to obtain FeSO with same light absorption value₄The concentration of (c) represents the total antioxidant capacity of the sample.

The experimental results are as follows: the standard curve of ferrous sulfate is shown in figure 25, and a linear regression equation is obtained: 0.5634X +0.0006 for Y, correlation coefficient squared R²The linear relation is good when the Fe content is 0.9993, and the Fe content can be used for subsequent reduction²⁺And (4) detecting the concentration. Detection of Fe in Auricularia auricula lectin³⁺In the test of reducing power, FeSO with the same light absorption value is obtained₄The iron ion reduction oxidation resistance is shown, and the result shows that the FRAP value of the black fungus agglutinin is 24.53 +/-1.60, has no obvious difference with a control group, and has good total oxidation resistance.

TABLE 5 Total antioxidant capacity

Mean±SD，n＝3，*p<0.05，versus control

The black fungus agglutinin LAA inhibits the proliferation of lung cancer cells A549, breast cancer cells MCF-7, gastric cancer cells SGC-7901 and liver cancer cells HepG2, and is time-concentration dependent, and IC detected after the agglutinin acts for 72 hours₅₀28.19 +/-1.92 mu g/mL, 30.33 +/-1.38 mu g/mL, 61.87 +/-3.76 mu g/mL and 28.79 +/-0.74 mu g/mL in sequence, the auricularia auricula agglutinin LAA promotes mouse macrophage J774A.1 to release NO, IL-1 β and TNF- α, and can improve the activity of immunity.Auricularia auricula agglutinin LAA can scavenge DPPH free radical (DPPH. cndot.) and ABTS free radical (ABTS)⁺) Hydroxyl radical (. OH) and superoxide anion (. O)₂ ^-) EC of (1)₅₀The values are 91.44 +/-4.20 mu g/mL, 45.70 +/-0.11 mu g/mL, 28.20 +/-0.74 mu g/mL and 35.03 +/-1.24 mu g/mL respectively, the auricularia auricula agglutinin LAA except DPPH has better effect of eliminating other three free radicals, the FRAP value is 24.53 +/-1.60, and the antioxidation effect is better.

Although the invention has been described above by way of general illustration and specific embodiments, it is within the scope of the invention as claimed that modifications and improvements may be made thereto without departing from the spirit of the invention.

Claims

1. A preparation method of black fungus agglutinin is characterized in that the black fungus agglutinin is prepared by the following steps:

firstly, preparing a black fungus lectin crude extract;

2. The method according to claim 1, wherein the porcine gastric mucin type II-cyanogen bromide activated Sepharose is prepared by coupling porcine gastric mucin type II as ligand to Sepharose.

3. The method for preparing black fungus lectin according to claim 1, characterized by comprising the following steps: adding phosphate buffer solution into Auricularia, mashing in a mashing machine to obtain viscous homogenate, freezing and centrifuging, collecting supernatant, and filtering under high pressure to obtain Auricularia lectin crude extract.

4. The method according to claim 1, wherein the crude extract of Auricularia auricula lectin obtained in step two is mixed with porcine gastric mucin type II-cyanogen bromide activated Sepharose at a volume ratio of 1: 1.

5. The method for preparing black fungus lectin as claimed in claim 1, wherein centrifugation is performed in step three: centrifuging at 3000r/min for 5min, washing the lower-layer precipitate with PBS for three times after centrifuging, centrifuging at 3000r/min for 5min again, discarding the supernatant, adding glycine mixed liquor into the precipitate, shaking up, centrifuging at 3000r/min for 5min, taking the supernatant, adding Tris-HCl buffer solution into the supernatant, repeating the processes of adding glycine mixed liquor into the precipitate, centrifuging, taking the supernatant and adding Tris-HCl buffer solution for 2 times, and combining the supernatants added with Tris-HCl buffer solution to obtain an ultrafiltration stock solution;

wherein the concentration of the Tris-HCl buffer solution is 1M, pH 7.8.8.

6. The method for preparing black fungus lectin as claimed in claim 5, wherein ultrafiltration is performed in step three: the 5kDa ultrafiltration desalination of the ultrafiltration stock solution is carried out, and then the concentrated solution is ultrafiltered by a 10kDa ultrafiltration centrifugal tube 4400 r/min.

7. An auricularia auricula lectin, characterized in that it is obtained by the process according to claim 1.