CN109402090B

CN109402090B - Beta-1,3 endoglucanase with immune enhancing activity and derived from scapharca broughtonii and encoding polynucleotide thereof

Info

Publication number: CN109402090B
Application number: CN201811349746.8A
Authority: CN
Inventors: 于荣敏; 李春磊; 朱建华
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-11-14
Filing date: 2018-11-14
Publication date: 2022-10-21
Anticipated expiration: 2038-11-14
Also published as: CN109402090A

Abstract

The invention relates to endoglucanase with immune enhancing activity and a gene nucleotide sequence for coding the endoglucanase, belonging to the technical field of biology. The invention provides a beta-1,3 endoglucanase protein sequence and a gene nucleotide sequence thereof, which are one of key enzymes in a glucanase hydrolase system. The enzyme is derived from Arca inflata Reeve and contains 332 amino acids, the total length of the corresponding coding gene nucleotide is 999bp, the content of G + C is 39.34 percent, and the enzyme is a new glucan hydrolase resource. The engineering strain constructed by the enzyme gene nucleotide can efficiently express beta-1,3 endoglucanase, the Kd value of the enzyme is 13.09 mu M when laminarin is used as a substrate, and the produced enzyme preparation can be used for industries of food, medicine, feed, washing and the like, not only can solve the problem of recycling of biomass waste rich in glucan, but also can obtain rich glucan oligosaccharide to generate considerable economic benefit.

Description

Beta-1,3 endoglucanase with immune enhancing activity and derived from scapharca broughtonii and encoding polynucleotide thereof

Technical Field

The invention relates to a dextran hydrolase beta-1,3 endoglucanase with immune enhancing activity from Arca inflata Reeve and coding gene nucleotide thereof, belonging to the field of biotechnology.

Background

Glucan is the main component of plant and microorganism cell walls, accounts for 30% -50% of the total dry weight of plants, is a renewable carbon source compound which is widely distributed on the earth and most abundant in content, and accounts for 40% of the total biomass of the earth. According to the report, the glucan-rich biomass such as the photo crop straws, the rice stalks, the kelp and the like in China per year is more than 5 hundred million tons, and the plant biomass produced by the photosynthesis in the world per year is as high as 1.55 multiplied by 10 ⁹ 89% of the total amount of the straws and the rice are not utilized by peopleThe utilization rate of glucan-rich biomass such as stalks and the like is low, and most glucan-rich biomass is treated by a combustion method, so that environmental pollution is caused, the physicochemical properties of soil are damaged, and organic matter components are lost. Therefore, the full utilization and effective conversion of glucan biomass have great significance for solving the current energy crisis, food shortage, environmental pollution and the like.

The glucanase is an enzyme for decomposing glucan, can decompose the glucan into monomolecular glucose or low-molecular-weight oligosaccharide, and fully utilizes glucan biological resources. Since the discovery of beta-1,4 glucanase in snail digestive juice by Serlieres in 1906, the research and application of endoglucanase have received great attention of scholars at home and abroad, and great progress has been made. At present, scholars at home and abroad find the glucanase by screening enzyme-producing strains and separating and purifying, and then apply the glucanase to industries such as food, medicine, feed, textile, washing, energy and the like, thereby not only solving the problem of recycling the glucan, but also obtaining considerable economic benefit.

Glucanase is a complex enzyme system of multiple enzyme components, mainly from mollusks, plants, fungi and bacteria. According to different enzyme functions, the enzyme is divided into three main categories: endoglucanase, exoglucanase and beta-glucosidase. The construction of high-efficiency expression strains by adopting genetic engineering means can realize the mass expression of the glucanase, and at present, more than 100 glucanase genes can be cloned and expressed in escherichia coli, mainly endoglucanase and beta-glucosidase. The acquisition of the endoglucanase gene has great application potential in the field of efficient utilization of glucan biomass, and simultaneously, the biological activity of the endoglucanase can be explored to better develop and utilize endoglucanase resources, so that the conversion and utilization of biomass and the multi-functionalization of enzyme preparations are realized, and the endoglucanase gene has very important significance on the sustainable development of human beings.

Arca inflata Reeve belongs to the phylum Amanita, class of lamellibranchia, order of Arca, family Arca, and is one of marine organisms with abundant resources in China. The scapharca broughtonii is large and fat, the meat quality is delicious, the scapharca broughtonii is recorded in ancient books to have the effects of being eaten by people, benefiting blood color, eliminating blood clots and reducing phlegm accumulation, the nutritional value and the economic value are high, and the scapharca broughtonii is one of important economic shellfish in China and coastal areas of Asia. According to the related literature data, foreign scholars express and express a polypeptide with antibacterial activity and immunoregulatory activity from arca inflata reeve, and the research on the endoglucanase of the arca inflata reeve is not reported. Therefore, the intraglucosylase from Arca inflata reeve can utilize marine biological resources of Arca inflata reeve with high value, has great economic significance while expanding the resources of the intraglucosylase, and provides theoretical basis for reasonable and efficient utilization of the marine resources.

Disclosure of Invention

Technical problem

The invention aims to provide beta-1,3 endoglucanase with immune enhancement activity and coding gene nucleotide thereof, which belong to the field of biotechnology, and the produced enzyme preparation is used in the industries of food, medicine, feed, washing and the like, can not only solve the problem of recycling of glucan-rich biomass waste, but also can obtain rich glucan oligosaccharide to generate considerable economic benefit.

Technical scheme

The beta-1,3 endoglucanase of the Arca inflata Reeve provided by the invention has the molecular weight of 51.03kDa, and the amino acid sequence of the endoglucanase is SEQ ID NO.1.

The Arca inflata Reeve beta-1,3 endoglucanase related by the invention is prepared according to the following method:

peeling fresh Arca inflata Reeve, taking out the content, washing, adding phosphate buffer solution, homogenizing, extracting, centrifuging, taking the supernatant, carrying out fractional precipitation by ammonium sulfate, taking 70-100% salting-out precipitation, redissolving, dialyzing, desalting, separating by adopting ion exchange column chromatography, collecting the enzyme activity peak, separating by using a hydrophobic chromatographic column after dialysis desalting, collecting the enzyme activity peak, and separating by using a space exclusion gel column after dialysis desalting to obtain the beta-1,3 endoglucanase in the Arca inflata Reeve.

The invention also discloses a coding gene of beta-1,3 endoglucanase separated from Arca inflata Reeve, wherein the full length of the gene (from an initial codon to a stop codon) is 999bp, the content of G + C is 43.77 percent, and the nucleotide sequence is as follows: SEQ ID NO.2.

The invention also discloses a recombinant expression plasmid containing the coding gene of the Arca inflata Reeve beta-1,3 endoglucanase and a recombinant microorganism E.coli BL21 (DE 3) containing the recombinant plasmid.

The recombinant expression plasmid of Arca inflata Reeve beta-1,3 endoglucanase and the recombinant microorganism E.coli BL21 (DE 3) containing the recombinant plasmid are prepared according to the following method:

amplifying a beta-1,3 endoglucanase gene segment of the scapharca broughtonii from the positive clone by using forward and reverse PCR primers respectively containing NdeI and HindIII; carrying out enzyme linkage on NdeI and HindIII double enzyme digestion and pET-28a (+) which is cut by the two enzymes, converting enzyme-linked pET-28a (+) recombinant plasmids containing arca inflata beta-1,3 staphyloinase genes into expression host bacteria E.coli BL21 (DE 3) to obtain recombinant microorganisms E.coli BL21 (DE 3), transferring the recombinant microorganisms E.coli BL21 (DE 3) to a plate containing 50mg/L kanamycin, culturing for 16h at 37 ℃, picking positive transformants, and storing after sequencing verification that the gene sequence is error-free.

The Arca inflata Reeve beta-1,3 endoglucanase and the coding gene thereof can be applied to the industries of food, medicine, feed, washing and the like.

Advantageous effects

1. The marine economic scapharca broughtonii is taken as a material, and a beta-1,3 endoglucanase (one of key enzymes in the glucan degradation process) and an encoding gene thereof are separated and purified by applying modern biochemical separation technologies, such as phosphate buffer solution extraction, ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography and steric exclusion chromatography.

2. The enzyme contains 332 amino acids, the total length of the coding gene is 999bp, and the content of G + C is 39.34%.

3. The complete beta-1,3 endoglucanase gene fragment containing NdeI and HindIII cleavage sites at the end was amplified by PCR technique, ligated to NdeI and HindIII cleavage sites of E.coli high expression vector pET-28a (+) (available from Novegen) and transformed into expression host strain E.coli BL21 (DE 3) (available from Invitrogen) for IPTG inducible expression.

4. The invention carries out enzyme activity determination on the separated Scapharca broughtonii beta-1,3 endoglucanase and the product expressed by the coding gene thereof, can efficiently hydrolyze laminarin (laminarin), has the specific activity of 90.01U/mg, has the Kd value of 13.09 mu M when the laminarin (laminarin) is taken as a substrate, and can hydrolyze the laminarin into glucan-oligosaccharide molecules by Thin Layer Chromatography (TLC).

5. The test results of the optimum pH and the pH stability of the beta-1,3 endoglucanase of the scapharca broughtonii show that the optimum pH of the beta-1,3 endoglucanase of the scapharca broughtonii is 6.0, the beta-1,3 endoglucanase is stable in the pH range of 5.5-7.5, and the residual enzyme activity is more than 80% after the beta-1,3 endoglucanase is treated in the pH range for 60 min. The test results of the optimum temperature and the heat stability of the beta-1,3 endoglucanase of the scapharca broughtonii show that the optimum temperature of the beta-1,3 endoglucanase of the scapharca broughtonii is 40 ℃, the temperature is raised for 60min at the temperature of 45 ℃, and the enzyme activity is still kept over 60 percent. The test results of the influence of different metal ion chemical reagents on the beta-1,3 endoglucanase show that most of the metal ions and the chemical reagents have no obvious influence on the activity of the beta-1,3 endoglucanase, and Mn has no obvious influence on the activity of the beta-1,3 endoglucanase ²⁺ And Mg ²⁺ The activity of beta-1,3 endoglucanase of the scapharca broughtonii is promoted at 2mM, and Cu ²⁺ 、Zn ²⁺ And Ba ²⁺ The activity of beta-1,3 endoglucanase of scapharca broughtonii is obviously inhibited when the concentration is 2mM.

6. The arca inflata reeve beta-1,3 endoglucanase can act on laminarin, and has a certain degradation effect on pachyman (pachyman) and barley glucan (barley glucan). Its degradation ability to pachyman was 78.72% relative to laminarin, and its degradation ability to barley glucan was 40.18% relative to laminarin.

7. Beta-1,3 endoglucanase of Arca inflata Reeve can remarkably enhance the immunocompetence of RAW264.7 cells and induce the release of nitric oxide, cell factors TNF-alpha and interleukin IL-6.

8. The recombinant engineering strain constructed by the gene can efficiently express the beta-1,3 endoglucanase (the content of the active beta-1,3 endoglucanase protein accounts for about 30 percent of the total protein content of cells), and the produced enzyme preparation can be used for industries such as food, medicine, feed, washing, energy and the like.

4. Description of the drawings

FIG. 1SDS-PAGE detects separated scapharca broughtonii beta-1,3 endoglucanase, wherein M is protein marker, and J2C1G1 is scapharca broughtonii beta-1,3 endoglucanase;

FIG. 2 binding constant K of beta-1,3 endoglucanase to laminarin _d Measuring results;

FIG. 3 shows the results of the determination of the optimum pH of beta-1,3 endoglucanase in Arca inflata Reeve;

FIG. 4 shows the results of pH stability measurement of beta-1,3 endoglucanase of Arca inflata Reeve;

FIG. 5 results of measurement of optimum temperature for beta-1,3 endoglucanase of Arca inflata Reeve

FIG. 6 results of measurement of thermostability of beta-1,3 endoglucanase in Arca inflata Reeve;

FIG. 7 determination result of influence of Anadara inflata beta-1,3 endoglucanase on RAW264.7 cell activity

FIG. 8 shows the results of measurement of the effect of beta-1,3 endoglucanase on the phagocytic activity of RAW264.7 cells

FIG. 9 shows the measurement results of the effect of beta-1,3 endoglucanase on the release of nitric oxide from RAW264.7 cells

FIG. 10 shows the results of measurement of the effect of beta-1,3 endoglucanase on the release of tumor necrosis factor (TNF-alpha) from RAW264.7 cells

FIG. 11 shows the measurement results of the effect of Anadara inflata beta-1,3 endoglucanase on the release of RAW264.7 cell interleukin 6 (IL-6)

5. Detailed description of the preferred embodiments

1. Separation and purification of arca inflata reeve beta-1,3 endoglucanase

Peeling the Arca inflata Reeve, taking the content of the Arca inflata Reeve, washing the content of the Arca inflata Reeve with low-temperature distilled water for three times, weighing the Arca inflata Reeve after drying, and weighing the Arca inflata Reeve according to the weight ratio of 1:3 (pH =8.0,0.03mol/L, sodium phosphate buffer solution), homogenizing at 8000 Xg intervals for 3min by a high-speed tissue triturator until the homogenate is fine and blockless, placing the homogenate in a low-frequency ultrasonic instrument for ultrasonic extraction for 40min, using a low-temperature high-speed centrifuge, 10000 Xg, 4 ℃, centrifuging for 30min to remove residues, measuring the volume of a supernatant, placing the supernatant in an ice bath environment, adding 70% of saturated ammonium sulfate, continuing stirring and salting out for 1h, then centrifuging at high speed to remove precipitates, taking the supernatant, measuring the volume, adding 100% of saturated ammonium sulfate, continuing stirring and salting out for 1h, 10000 Xg, 4 ℃, centrifuging for 30min, and collecting the precipitates. Dissolving the precipitate with a small amount of extractive solution, dialyzing in 3000D dialysis bag for 24 hr to remove salt, and replacing peripheral distilled water every 6 hr. Separating the salting-out component with enzyme activity by using DEAE-Sepharose Fast Flow anion exchange chromatography, using 0.03mol/L Tris-HCl buffer solution with pH of 8.0 as initial eluent, performing stage elution by increasing the concentration of sodium chloride, detecting an absorption peak at 280nm, collecting eluent eluted by 0.1mol/L sodium chloride with beta-1,3 endoglucanase activity, dialyzing and desalting, and separating by using Phenyl Sepharose CL-4B hydrophobic chromatography column. Eluting with 1.0mol/L ammonium sulfate-phosphate buffer solution with pH of 8.0, sequentially eluting with 0.5mol/L and 0mol/L ammonium sulfate-sodium phosphate buffer solution (pH of 8.0), detecting absorption peak at 280nm, collecting eluate eluted with 1.0mol/L ammonium sulfate-containing sodium phosphate buffer solution, dialyzing for desalting, and separating with molecular exclusion gel Sephadex G50 chromatographic column. And (3) performing isocratic elution by using a sodium phosphate buffer solution containing 0.1mol/L sodium chloride, collecting a first elution peak, concentrating, freezing and drying to obtain the beta-1,3 endoglucanase in the scapharca broughtonii. The purity of the separated enzyme was determined to be electrophoretically pure by SDS-PAGE using polyacrylamide gel electrophoresis (FIG. 1). The molecular weight of the enzyme is 51027.83Da determined by matrix-assisted laser desorption ionization time of flight mass spectrometry MALDI-TOF-MS, and the secondary structure composition of the enzyme is analyzed by circular dichroism spectrum and Jasco protein secondary structure evaluation software. Beta-1,3 endoglucanase of the scapharca broughtonii is subjected to enzymolysis by pancreatin and electrospray ionization tandem mass spectrometry ESI-MS/MS, and is compared with a scapharca broughtonii transcriptome database to analyze the amino acid sequence and the coding gene nucleotide sequence of the endoglucanase.

2. High-efficiency expression of scapharca broughtonii beta-1,3 endoglucanase gene in E.coli BL21 (pET-28 a (+))

2.1 Arca inflata Reeve RNA extraction and cDNA reverse transcription

RNA from Arca inflata Reeve was extracted using E.Z.N.A.Total RNA Kit I (R6834-01, manufactured by omega Biotech). And (3) carrying out liquid nitrogen quick-freezing and grinding on fresh arca inflata reeve tissues, adding 30mg of ground tissue powder into 500ul of TRK lysis buffer solution to lyse cells, and centrifuging for 5min at 15000 g. Transfer supernatant to a localization Spin Column, centrifuge for 3min at 13000g, transfer the centrifuged effluent to a new EP tube. Adding 50% volume of anhydrous ethanol into the cracking product, mixing, and vortexing for 20s. The sample was transferred to a Hibind RNA Mini column and centrifuged at 10000g for 30s at room temperature, and the effluent (liquid that flowed out to the centrifuge tube through the column) was discarded. The column was placed in a fresh 2mL collection tube, the column was washed sequentially with 500ul RNA wash buffer I and 500ul RNA wash buffer II, and the effluent was discarded by centrifugation at 13000 g. DNase I digestion, removing possible residual DNA. The column was transferred to a new 1.5mL Ep tube, 50. Mu.L of DEPC water was added to elute the column, 13000g was centrifuged for 1min to recover the RNA in the EP tube.

cDNA synthesis was performed using a Timestaver cDNA Synthesis kit (produced by pharmacia Biotech). First 5. Mu.g of mRNA was dissolved in 20. Mu.l of sample buffer. After heat denaturation at 65 ℃ for 10 minutes, the mixture was added to the first strand synthesis mixture together with a dithiothreitol solution and an oligomer (dT) primer, and reacted at 37 ℃ for 1 hour. Then adding the two to the second strand synthesis mixture, reacting for 30 minutes at 12 ℃, and reacting for 1 hour at 22 ℃ to obtain arca inflata reeve cDNA.

2.2 PCR amplification of the beta-1,3 endoglucanase Gene

With the forward primer:

5'-GGAATTCCATATGGAATTCCGATCACACTAAGCCGTTAACACCTCA-3' and reverse primer:

5'-CCCAAGCTTGGGTCAATTTTGGTTCATTACCTCTAAAATGACTTCCA-3' as primer, beta-1,3 endoglucanase gene fragment was amplified from Arca inflata Reeve cDNA by PCR.

An amplification system:

PCR amplification procedure:

denaturation at 98 ℃ for 10sec, annealing at 55 ℃ for 10sec, and elongation at 72 ℃ for 1.5min, for 30 cycles;

b.72 ℃ extension for 10min;

c.15 ℃ for 10min.

2.3PCR product was digested with NdeI and HindIII.

Enzyme digestion system:

reacting for more than 3 hours in a water bath at 37 ℃.

The digested product was recovered by electrophoresis on 0.75% agarose gel.

2.4pET-28a (+) was digested with NdeI and HindIII (see 2.2).

2.5 transformation and expression

The recovered fragment of 2.3 was enzymatically ligated with digested pET-28a (+) of 2.4. The enzyme-linked pET-28a (+) recombinant plasmid containing beta-1,3 endoglucanase gene is transformed into expression host bacterium E.coli BL21 (DE 3) to obtain recombinant microorganism E.coli BL21 (DE 3). Plates containing 50mg/L kanamycin were plated, positive transformants were picked and the gene sequence was verified to be correct by sequencing. Positive clones were cultured in LB medium at 37 ℃ to OD600 between 0.5 and 0.6, IPTG was added to a concentration of 0.5mM and culture was continued at 18 ℃ for 24h. After the collected bacteria are resuspended by citric acid-sodium citrate buffer solution (pH 6.0), the bacteria cells are broken by ultrasonic treatment, 20000g is centrifuged for 15min, the obtained supernatant is crude enzyme liquid, and the purified recombinant arca inflata beta-1,3 endoglucanase is obtained by nickel column separation and 500mM imidazole elution.

2.6 hydrolysis function of Arca inflata Reeve beta-1,3 endoglucanase on different polysaccharide substrates

200 mu g of Arca inflata Reeve beta-1,3 endoglucanase is added into 1ml of 50mM sodium citrate buffer solution containing 1% of various polysaccharide substrates (laminarin, zymosan, pachyman, sodium carboxymethylcellulose, agarose and barley glucan) to react for 30min at 40 ℃, and then the generation amount of the product glucose is measured by a DNS method. The enzyme activity unit U is defined as the hydrolysis yield of 1. Mu. Mol glucose per minute at 40 ℃ reaction conditions. The results show that (Table 1) Arca inflata Reeve beta-1,3 glucanThe best hydrolysis effect of laminarin is achieved by the endoprotease, the enzyme activity is 90.01U/mg, namely, 90.01 mu mol glucose can be obtained by 1mg of the enzyme which takes 1% laminarin as a substrate and reacts every minute under the optimal condition. The Scapharca broughtonii beta-1,3 endoglucanase can rapidly hydrolyze laminarin to generate oligosaccharide molecules such as monosaccharide, disaccharide, trisaccharide, tetrasaccharide and the like. The binding constant K of the enzyme to laminarin was determined using a micro thermophoresis apparatus, and the results showed (FIG. 2) that the binding constant K was determined when laminarin was used as a substrate _d The value was 13.09. Mu.M. The arca inflata reeve beta-1,3 endoglucanase can be used in the industries of food, medicine, feed, textile, washing, energy and the like.

TABLE 1 enzymatic Activities of the Anadara inflata beta-1,3 endoglucanase on different polysaccharide substrates

2.7 Arca inflata Reeve beta-1,3 endoglucanase optimum temperature, optimum pH, pH and temperature stability, influence of metal ion chemical reagent on enzyme activity

The optimum temperature of the arca inflata reeve beta-1,3 endoglucanase is measured, and enzymatic reaction is carried out by taking laminarin as a substrate in a citric acid-sodium citrate buffer solution (pH6.0) buffer solution system and at different temperatures. The temperature tolerance is determined by treating dextranase at different temperatures (20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C and 55 deg.C) for 60min, and determining enzyme activity at 40 deg.C with laminarin as substrate. The optimal temperature result of the enzyme reaction shows that the optimal temperature of the scapharca broughtonii beta-1,3 endoglucanase (figure 3) is 40 ℃. The results of enzyme thermal stability determination show that (figure 4) scapharca broughtonii beta-1,3 endoglucanase has complete enzyme activity below 40 ℃, and the enzyme activity is still maintained by more than 60 percent after incubation for 60min at 45 ℃.

The optimum pH value of the arca inflata reeve beta-1,3 endoglucanase is measured by using different buffer solution systems (citric acid buffer solution, glycine buffer solution, tris buffer solution, MOPS buffer solution and MES buffer solution), and performing enzymatic reaction by taking laminarin as the fifth in different pH reaction systems at 40 ℃. pH stability experiments dextranase activity assays were performed at 40 ℃ in different buffers at different pH. The optimal pH result of the enzyme reaction shows that (figure 5) the optimal pH of the scapharca broughtonii beta-1,3 endoglucanase is 6.0. The results of the pH stability measurement of the enzyme show that (figure 6) the beta-1,3 endoglucanase of the scapharca broughtonii is stable in the pH range of 5.5-7.5, and the residual enzyme activity is more than 80% after the endoglucanase is treated in the pH range for 60 min.

Different metal ions and chemical reagents with different concentrations are added into an enzymatic reaction system, the influence of the enzymatic reaction system on the activity of the beta-1,3 endoglucanase of the scapharca broughtonii is studied, and the final concentration of each substance is 2mM. The enzyme activity was measured at 40 ℃ and pH 6.0. The results show that (Table 2) most of metal ions and chemical reagents have no obvious influence on the activity of the beta-1,3 endoglucanase of scapharca broughtonii, and Mn ²⁺ And Mg ²⁺ The activity of beta-1,3 endoglucanase of the scapharca broughtonii is promoted at 2mM, and Cu ²⁺ 、Zn ²⁺ And Ba ²⁺ Has obvious inhibiting effect on the activity of beta-1,3 endoglucanase of the scapharca broughtonii at 2mM.

TABLE 2 Effect of different metal ions on the endoglucanase activity of B-1,3 in Arca inflata Reeve

2.8 Anadara inflata beta-1,3 endoglucanase immune activity test

2.8.1 Effect of Arca inflata Reeve beta-1,3 endoglucanase on RAW264.7 cell proliferation

Mixing RAW264.7 cells with a DMEM solution containing 10% Fetal Bovine Serum (FBS), 100U/mL penicillin and 100. Mu.g/mL streptomycin at 5% CO ₂ Are incubated together at 37 ℃. Cells were seeded into 96-well plates (2X 10) ⁴ Individual cells/well) and incubated for 24 hours. Cells were treated with different concentrations of arca inflata β -1,3 endoglucanase for 48 hours. Cell viability was determined by MTT assay. All experiments were repeated 3 times and data are expressed as mean ± Standard Deviation (SD).

RAW264.7 cells were treated with a series of Arca inflata Reeve β -1,3 endoglucanases at a concentration of 31.25-500 μ g/mL and Lipopolysaccharide (LPS) at 0.1 μ g/mL, respectively. LPS has a significant ability to activate macrophages. The results show (figure 7) that the beta-1,3 endoglucanase enzyme of arca inflata reeve does not significantly affect the cell viability even at a high concentration of 500 μ g/mL. Therefore, the arca inflata β -1,3 endoglucanase is not cytotoxic to RAW264.7 cells.

2.8.2 Effect of Arca inflata Reeve beta-1,3 endoglucanase on phagocytosis

RAW264.7 cells were seeded into 96-well plates (1X 10) ⁴ Cells/well) and incubated for 24 hours. Cells were treated with different concentrations of arca inflata β -1,3 endoglucanase for 24 hours. Medium and 1. Mu.g/mL Lipopolysaccharide (LPS) were used as blank and positive controls, respectively. Phagocytic activity of RAW264.7 cells was determined by a neutral red uptake assay. The enhancement of phagocytic activity is one of the most significant features of macrophage activation. The results show (fig. 8) that all concentrations of arca inflata β -1,3 endoglucanase tested significantly enhanced phagocytes compared to LPS control, and presented a dose-dependent profile. Therefore, the beta-1,3 endoglucanase of the scapharca broughtonii has obvious immunity enhancing activity.

2.8.3 Effect of Arca inflata Reeve beta-1,3 endoglucanase on Nitric Oxide (NO) and cytokine release

RAW264.7 cells were seeded into 96-well plates (1X 10) ⁵ Individual cells/well) and incubated for 24 hours. Cells were treated with different concentrations of arca inflata β -1,3 endoglucanase for an additional 24 hours. Medium and LPS (1. Mu.g/mL) were used as blank and positive controls, respectively. Then, the NO content in the medium in each well was detected using an NO detection kit. Quantification of the cytokines tumor necrosis factor TNF-alpha and interleukin IL-6 in the culture medium of each well was performed by ELISA. Medium and LPS (1. Mu.g/mL) were used as blank and positive controls, respectively. Activated macrophages are important effectors of innate immunity. When macrophages are activated, they can secrete an increasing number of pro-inflammatory molecules, including NO and cytokines such as TNF- α and IL-6. The results show (fig. 9) that the arca inflata beta-1,3 endoglucanase can greatly induce RAW264.7 macrophages to release NO compared with the control group. In addition, the results show (fig. 10 and 11) that after the RAW264.7 cells are treated by endoglucanase beta-1,3 of arca inflata reeve with different concentrations, the production of TNF-alpha and IL-6 is effectively improved. Therefore, the beta-1,3 endoglucanase of the scapharca broughtonii has obvious immunityEnhancing the activity.

Sequence listing

<110> Yu Rongmin

<120> beta-1,3 endoglucanase with immune enhancing activity and derived from scapharca broughtonii and encoding polynucleotide thereof

<130> 2018.11.10

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 332

<212> PRT

<213> Arca inflata Reeve

<400> 1

Asp His Thr Lys Pro Leu Thr Pro Gln Gln Phe Val Asp Tyr Ile Gly

1 5 10 15

Tyr Gly Phe Thr Thr His Tyr Phe Lys Thr Leu Asp Phe Thr Leu Tyr

20 25 30

Asn Lys Gln Asn Ile Ala Asp Ile Lys Ala Lys Asn Phe Thr Asn Leu

35 40 45

Arg Val Arg Cys Arg Gln Asp Leu Pro Gly Leu Asn Met Thr Val Phe

50 55 60

Leu Asn Asn Leu Glu Ile Val Ile Asp Asp Cys Leu Glu Tyr Asn Val

65 70 75 80

Thr Pro Ile Ile Ser Trp Ile His His Glu Gly Glu Ala Val Ala Asn

85 90 95

Glu Thr His Arg Gln Met Phe Leu Gln Trp Trp Arg Asp Val Ala Thr

100 105 110

Lys Leu Lys Asp Lys Asp Tyr Arg Val Ala Phe Asn Leu Phe Thr Glu

115 120 125

Leu Gly Thr Asp Glu Cys Thr Lys Arg Gly Tyr Ser Cys Ala Glu Ser

130 135 140

Leu Arg Lys Asn Pro Leu Lys Thr Asn Asn Trp Thr Lys Glu Val Val

145 150 155 160

Asp Ile Ile Arg Asn Ser Gly Gly Asn Asn Ala Gln Arg Ile Ile Ile

165 170 175

Leu Gly Ser Pro Glu Lys Thr Ala Lys Gly Leu Asp Lys Ile Asp Pro

180 185 190

Asn Ile Tyr Ile Asn Glu Thr Tyr Met Met Val Glu Trp His Ile Tyr

195 200 205

Ala Ser Gly Pro Ser Lys Lys Val Gly Ser Glu Lys Phe Trp Glu Gly

210 215 220

Asp Gly Thr Gly Leu Gly Lys Gln Asn Val Asp Thr Ala Ile Asn Met

225 230 235 240

Gly Lys Asp Phe Met Asn Arg Thr Gly Leu Leu Ser Tyr Phe Ala Glu

245 250 255

Trp Met Pro Gln Asp Asn Asn Asn Gly Arg Ile Asn Gln Thr Glu Ala

260 265 270

Ile Tyr Phe Thr Arg Tyr Phe Leu Gln Gln Leu Lys Pro Ile Pro Trp

275 280 285

Ser Leu Asn Thr Ile Lys Arg Tyr Tyr Asn Thr Arg Lys Asn Arg Trp

290 295 300

Val Thr Gly Thr Glu Val Ile Ala Asn Gln Thr Leu Asn Phe Pro Glu

305 310 315 320

Leu Leu Glu Val Ile Leu Glu Val Met Asn Gln Asn

325 330

<210> 2

<211> 999

<212> DNA

<213> Arca inflata Reeve

<400> 2

gatcacacta agccgttaac acctcaacaa tttgtggatt acattggata cggattcact 60

acacattatt ttaaaaccct cgactttacg ctatacaaca agcagaatat tgcagacatc 120

aaagccaaaa atttcaccaa tttgagagtg cgatgcagac aagatctacc cggtttgaat 180

atgacagtct ttctaaataa cttagaaatt gttattgatg actgtctgga gtacaacgta 240

acgcctatta tatcttggat ccatcatgaa ggggaggctg tggccaacga gacacacaga 300

cagatgttct tgcaatggtg gagagatgtg gcaactaagt tgaaggacaa ggactataga 360

gtagcattca acttgtttac ggaattgggc actgatgaat gtaccaaaag agggtattca 420

tgcgctgaga gtttaagaaa aaacccactc aaaactaata actggactaa ggaggtcgtg 480

gatatcatac ggaactctgg tggaaacaat gcacaacgca tcattatttt aggatcaccg 540

gagaaaactg caaaaggctt agataaaata gacccaaaca tctacataaa tgaaacgtac 600

atgatggtag agtggcatat atatgcttca ggaccatcta aaaaagtggg gtcagaaaaa 660

ttttgggagg gtgatggtac tggcttaggt aaacaaaacg tcgacacggc aatcaacatg 720

ggcaaagatt tcatgaacag aacaggtctg ctgtcgtatt ttgctgaatg gatgccacag 780

gataacaaca atggccgtat caaccaaaca gaagccatat actttactag gtacttcctt 840

caacaactga agccgatacc ttggtcttta aatacaatca aacgatatta taataccaga 900

aagaacagat gggtcacggg tacggaggta attgcaaatc aaactttaaa ttttcctgaa 960

ttattggaag tcattttaga ggtaatgaac caaaattga 999

<210> 3

<211> 46

<212> DNA

<213> Artificial sequence

<400> 3

ggaattccat atggaattcc gatcacacta agccgttaac acctca 46

<210> 4

<211> 47

<212> DNA

<213> Artificial sequence

<400> 4

cccaagcttg ggtcaatttt ggttcattac ctctaaaatg acttcca 47

Claims

1. The beta-1,3 endoglucanase with the immune enhancing activity has the amino acid sequence as follows: SEQ ID NO.1.

2. The method of claim 1 for the isolation and preparation of an immunopotentiating activity of β -1,3 endoglucanase comprising the steps of:

peeling Arca inflata Reeve, taking the content of the Arca inflata Reeve, washing the Arca inflata Reeve with low-temperature distilled water for three times, weighing the Arca inflata Reeve after drying, adding an extraction buffer solution of sodium phosphate with the pH =8.0 and 0.03mol/L according to the weight ratio of 1:3, homogenizing the mixture at intervals of 8000 Xg for 3min by adopting a high-speed tissue triturator until the mixture is fine and blockless, placing the homogenized mixture in a low-frequency ultrasonic instrument for ultrasonic extraction for 40min, using a low-temperature high-speed centrifuge, 10000 Xg and 4 ℃, centrifuging for 30min to remove residues, measuring the volume of a supernatant, placing the supernatant in an ice bath environment, adding 70% of ammonium sulfate, continuously stirring and salting out for 1h, removing the precipitate through high-speed centrifugation, taking the supernatant, adding 100% of ammonium sulfate after the volume, further stirring and salting out for 1h, 10000 Xg, 4 ℃, centrifuging for 30min, and collecting the precipitate; dissolving the precipitate with a small amount of extractive solution, dialyzing in 3000D dialysis bag for 24 hr to remove salt, and replacing peripheral distilled water every 6 hr; separating the salting-out component with enzyme activity by using DEAE-Sepharose Fast Flow anion exchange chromatography, using 0.03mol/L Tris-HCl buffer solution with the pH of 8.0 as initial eluent, performing stage elution by increasing the concentration of sodium chloride, detecting an absorption peak at 280nm, collecting eluent eluted by 0.1mol/L sodium chloride with beta-1,3 endoglucanase activity, dialyzing and desalting, and separating by using Phenyl Sepharose CL-4B hydrophobic chromatographic column; eluting with 1.0mol/L ammonium sulfate-phosphate buffer solution with pH of 8.0, sequentially eluting with 0.5mol/L ammonium sulfate-sodium phosphate buffer solution with pH of 8.0, detecting absorption peak at 280nm, collecting eluate eluted with sodium phosphate buffer solution containing 1.0mol/L ammonium sulfate, dialyzing for desalting, and separating with molecular exclusion gel Sephadex G50 chromatographic column; and (3) performing isocratic elution by using a sodium phosphate buffer solution containing 0.1mol/L sodium chloride, collecting a first elution peak, concentrating, freezing and drying to obtain the beta-1,3 endoglucanase in the scapharca broughtonii.

3. A gene encoding the β -1,3 endoglucanase of claim 1, having the sequence: SEQ ID NO.2.

4. A pET-28a (+) recombinant plasmid containing the gene for β -1,3 endoglucanase of claim 3.

5. Coli BL21 (DE 3) which comprises the recombinant plasmid of claim 4.

6. The recombinant microorganism E.coli BL21 (DE 3) according to claim 5, which is constructed as follows:

the beta-1,3 endoglucanase gene of claim 3 amplified from positive clones using forward and reverse PCR primers NdeI and HindIII, respectively; carrying out enzyme linkage on the NdeI and HindIII double enzyme digestion and pET-28a (+) which is cut by the two enzymes, converting the enzyme-linked pET-28a (+) recombinant plasmid containing a gene of beta-1,3 endoglucanase into an expression host bacterium E.coli BL21 (DE 3) to obtain a recombinant microorganism E.coli BL21 (DE 3), transferring the recombinant microorganism E.coli BL21 (DE 3) to a kanamycin plate with a mass ratio of 50mg/L, culturing for 16h at 37 ℃, picking out a positive transformant, and storing after sequencing to verify that a gene sequence has no error.

7. The use of the gene for beta-1,3 endoglucanase of scapharca broughtonii of claim 1 or beta-1,3 endoglucanase of claim 3 for hydrolysis of beta-1,3 glucan.

8. The application of the gene of beta-1,3 endoglucanase of scapharca broughtonii of claim 1 or the gene of beta-1,3 endoglucanase of claim 3 in the preparation of an immunopotentiator.