CN110760492A - Fucosidase and application thereof in preparation of Bombay type red blood cells - Google Patents

Abstract

The invention belongs to the fields of glycobiology technology and engineering, and particularly relates to a novel fucosidase and enzyme activity and application thereof, wherein the enzyme can effectively act on a polysaccharide structure positioned on a cell surface, namely cell surface fucosidase I (csFase I).

Description

Fucosidase and application thereof in preparation of Bombay type red blood cells

Technical Field

The invention belongs to the field of glycobiology technology and engineering, and relates to a preparation method and application of fucosidase, in particular to application in preparation of Bombay type red blood cells.

Background

The prior art discloses that glycosylation modification is a major modification on the cell surface, and plays an extremely important role in the process of intercellular recognition; fucosylation is a type of glycosylation, widely existing on various cell surfaces, and also plays an important biological function [1 ].

L-fucose is widely present in mammalian, plant and insect cells, and L-fucose-containing glycocomplexes play a crucial role in many physiological and pathological processes of these organisms, such as inflammatory reactions, bacterial and viral infections, tumor metastasis, genetic diseases, fertilization processes, immune activation effects of antibodies and allergic reactions [1,2,3] fucosylation processes are mainly mediated by fucosyltransferases (FucTs) which can link fucose on fucose donor guanosine diphosphate-fucose (GDP-Fuc) with corresponding bonds to acceptor, glycoproteins or fucosidases, and complete fucosylation processes [1] fucose is usually present at the termini of glycan structures and in part of core regions on glycoproteins, and specific terminal glycan modifications (including unique functional oligosaccharides) can confer functional properties and important functional properties on fucosylation processes of individual and cell development, and have been shown to play a very important role in the differentiation processes of fucosylation.

The prior art also discloses that fucosylation is closely related to human blood group, blood group antigen is usually determined by carbohydrate determinant [4] of glycoprotein and glycolipid, ABH antigen on erythrocyte surface determines ABO blood group system of human, O blood group is determined by immunodominant monosaccharide α -1,2 fucose, glycosyltransferase synthesis is encoded by FUT1 gene (as shown in figure 1) [5,6] with respect to ABO blood group transfusion, the principle of transfusion is currently used clinically as syngeneic blood [7,8], normally A type human transfusions A type blood, B type human transfusions B type blood of B type blood, but in some emergency situations, AB type human can accept any blood group, O type blood can transfuse to any blood group human [9], but O type individual can only accept O type red blood cells due to anti-A and anti-B antibodies contained in O type individual plasma, and if A, B, AB red cells of type are transfused into the body of A, B, AB type blood patient, immunity and hemolytic reaction will be caused.

Clinical studies have shown that there is a group of individuals called Bombay type, whose transfusions are only acceptable for Bombay donors due to the fact that fucose transferase FUT1, mutated, results in the inability to produce H antigen (as shown in FIG. 1) [10,11], the presence in Bombay blood of antibodies against the H antigen on the surface of red blood cells of type O. Surveys have shown that individuals of the global punch type are extremely rare, and Bhatia and sate detected 167404 indian punches, with frequencies around 1/7600[12 ]. The frequency of Monsanto individuals in China is much lower and only a few tens of cases are currently reported [13,14 ]. Clinical studies have also found that there are people named blood types of the Bombay type; the serum of the population can weakly react with H-antigen, research statistics shows that the probability of the Bombay blood type in Taiwan is about 1/8000 approximately [15], and the distribution is relatively high in parts of areas such as Guangxi and Fujian in China [16,17 ]; because Bombay blood is extremely rare, how to realize quick and effective matching of Bombay blood group and Bombay blood group patients is a great challenge to clinical treatment.

The prior art discloses that the H antigen on the surface of erythrocytes can be divided into three types: type 1 is obtained from plasma by erythrocytes and binds to the erythrocyte membrane; type 2 is synthesized by erythrocytes themselves and widely distributed on the surface of erythrocytes, and is the main H antigen of erythrocytes; type 3 antigens are distributed predominantly on type A erythrocytes [18]. Since most human blood types are of types A, B, AB or O, and the population distribution of each blood type is not very different, when a patient needs blood products urgently, each blood transfusion mechanism has a certain stock of four blood types to ensure the normal supply of blood, even in the case of urgent blood supply, a proper blood donor is relatively easy to find, but for the patient who buys blood types or a part of blood types, the condition is not optimistic, in the case of urgent blood products, especially the need of red blood cells, a proper donor is difficult to find, in addition, in some cases, a human body generates relatively high level of autoantibodies to induce autoimmune diseases, and the microchip has found that when the autoantibodies in the human body of 106 testers are detected by using glycan, 4 of the blood types have the autoantibodies [19] with the structure of type 2H, when a person with an autoantibody to H needs to transfuse red blood cells, transfusing Bomby red blood cells lacking H antigen is also the best solution, so the conversion of O type red blood cells into Bomby red blood cells lacking H antigen will effectively solve the above problems.

Type O blood is generally considered "pleiotropic blood" because type O red blood cells can be transfused into individuals of any one of the blood types A, B and AB, but in practice type O red blood cells are not pleiotropic and practice has shown that it cannot be transfused into individuals of Bombay or Bombay type, which is often overlooked because of the rarity of Bombay and Bombay type blood; while Bombay or Bombay-like individuals can only accept H antigen-deficient erythrocytes, clinically H-deficient Bombay-type erythrocyte donors are rare and there is currently no effective method to convert type O erythrocytes to Bombay erythrocytes.

Based on the current situation and the demand of the prior art, the inventor of the application intends to provide fucosidase and application thereof in preparing Bombay type red blood cells, and particularly relates to a novel fucosidase bFase I which can be used for efficiently and specifically carrying out enzyme digestion and hydrolysis on H antigen on O type red blood cells so as to convert the H antigen into Bombay type red blood cells.

References relevant to the present invention are:

[1].Ma B,Simala-Grant J L,Taylor D E.Fucosylation in prokaryotes andeukaryotes[J]. Glycobiology,2006,16(12):158R-184R.

[2].Becker D J,Lowe J B.Fucose:biosynthesis and biological functionin mammals[J]. Glycobiology,2003,13(7):41R-53R.

[3].Miyoshi E,Moriwaki K,Nakagawa T.Biological functionoffucosylation in cancerbiology[J]. J Biochem,2008,143(6):725-729.

[4].Watkins WM.The ABO blood group system:historicalbackground[J].Transfusion Medicine, 2001,11(4):243-265.

[5].Oriol R,Lependu J,Mollicone R.Genetics OfAbo,H,Lewis,X AndRelated Antigens[J]. Vox Sanguinis,1986,51(3):161-171.

[6].Dahiya R,Itzkowitz S H,Byrd J C,et al.Abh Blood-Group AntigenExpression,Synthesis, And Degradation In Human Colonic Adenocarcinoma Cell-Lines[J].Cancer Research,1989, 49(16):4550-4556.

[7].Milkins C,Berryman J,Cantwell C,et al.Guidelines for pre-transfusion compatibility procedures inbloodtransfusion laboratories[J].Transfusion Medicine,2013,23(1):3-35.

[8].Shanwell A,Andersson T M L,Rostgaard K,et al.Post-transfusionmortality among recipients ofABO-compatible butnon-identicalplasma[J].VoxSanguinis,2009,96(4):316-323.

[9].Nucci M L,Abuchowski A.The search for blood substitutes(vol 278,pg 72,1998)[J]. ScientificAmerican,1998,279(2):9-9.

[10].Kelly R J,Ernst L K,Larsen R D,et al.Molecular-Basis for H-Blood-Group Deficiency In Bombay(O-H)And Para-Bombay Individuals[J].Proceedings Of the National Academy Of Sciences Ofthe United StatesOfAmerica,1994,91(13):5843-5847.

[11].Kaneko M,Nishihara S,Shinya N,et al.Wide variety ofpointmutations in the H gene of Bombay andpara-Bombay individuals that inactivateH enzyme[J].Blood,1997,90(2):839-849.

[12].Bhatia H M,Sathe M S.Incidence OfBombay(Oh)Phenotype And WeakerVariants Of a AndBAntigen In Bombay(India)[J].Vox Sanguinis,1974,27(6):524-532.

[13] liu Qin, Gong Yan, Yang Shenhong, identification of Bombay blood type and autotransfusion [ J ]. college of traditional Chinese medicine of Guiyang ], 2011, 33(4):67-69.

[14] Shenlihua, Yangqing, Mengwu blood type scar uterus are delivered again in 1 case [ J ] civil military medical science 2017(11):1135 and 1136.

[15] Guo faithful, eastern, Veronica, et al, Chinese Bombay blood group FUT1 and FUT2 Gene study [ J ]. J.J.Zhonghua J.Med. Genet. 2004,21(5): 417-.

[16] Seikang, Dengshi France, once rose, etc. screening of rare blood types in the region of Panyu Guangzhou [ J ] J. China J. blood transfusion, 2007,20(4): 290-.

[17] Chenfa, Xiehai Hua, Yangxiang Jun, etc. 3 examples of the molecular genetic mechanism studies of Bombay-like blood groups [ J ] J.China J.EXPERIMENTAL HEMISTRY, 2017(6):1793-1798.

[18] Chent, li zheng jun, research progress on erythrocyte ABO blood group variation [ J ] china blood transfusion journal, 2008,21(1):66-67.

[19] Xuhua, Yu-Yu, Octopus culture, combined application of α -galactosidase and α -N-acetylgalactosamine enzyme to achieve AB → O blood group transformation [ J ] in China J.blood transfusion, 2008,21(12):917-920.

The invention content is as follows:

the invention aims to provide fucosidase and application thereof in preparing Bombay type red blood cells based on the current situation and requirements of the prior art, and particularly relates to a novel fucosidase bFase I which can efficiently and specifically carry out enzyme digestion and hydrolysis on an H antigen on O type red blood cells so as to convert the H antigen into the Bombay type red blood cells.

The present invention provides a novel fucosidase that can effectively act on the polysaccharide structure on the cell surface, named as cell surface fucosidase I (csFase I), its enzymatic activity and application, the present invention found and demonstrated that csFase I can effectively cleave α -1,2 fucose on the H-antigen on the surface of activated red blood cells, and effectively convert red blood cells of type O into rare Mongolian red blood cells.

In particular, the invention provides a preparation method of fucosidase,

the amino acid sequence of the fucosidase is optionally one of the following two types:

a) has a sequence shown as SEQ ID NO 1:

or

b) Has more than 30 percent of homology with the sequence shown in SEQ ID NO1 and has fucosidase activity.

The invention provides a recombinant vector, which comprises a nucleotide sequence for coding the fucosidase; in the embodiment of the invention, the fucosidase gene is connected to a pET28a vector.

The invention provides an engineering bacterium containing a recombinant vector;

in the embodiment of the invention, the engineering bacteria is escherichia coli BL21(DE3) for producing fucosidase.

The invention provides an expression and cloning method for encoding fucosidase gene, and the preferred mode in the embodiment of the invention is that the target gene is cloned into a prokaryotic expression vector, then is transformed into escherichia coli for expression, and the purified fucosidase is obtained by affinity chromatography and ultrafiltration methods.

More specifically, the invention provides a preparation method of the fucosidase, which comprises the following steps:

1) obtaining and amplifying the gene sequence of the fucosidase;

2) constructing a recombinant vector containing the fucosidase;

3) expressing said fucosidase;

4) separating, purifying and identifying.

The expression system can be a bacterial, yeast or insect expression system;

the production method comprises the conventional microbial fermentation production, and the expression and production in bacteria, yeast and insect expression systems by using a bioengineering technology.

In another aspect, the invention provides methods of use and use of fucosidases.

Experiments show that the fucosidase has enzyme digestion activity on various fucosylation substrates, including but not limited to artificially synthesized chromogenic substrate p-nitrobenzene- α -L-fucoside (pNP-Fuc, except for specific description, the term pNP-Fuc is equivalent to pNP- α -L-Fuc), oligosaccharide chain, glycoprotein, glycolipid and the like, the fucosylation substrates can be free and on the cell surface, and the fucosidase is efficient in enzyme digestion of the fucosylation substrates, and the enzyme digestion treatment has no obvious influence on the physiological metabolism function of cells.

The fucosidase is capable of hydrolyzing an artificially synthesized fucosyl substrate, which in some embodiments may be pNP-Fuc.

The fucosidase hydrolyzes oligosaccharide chains containing fucose residues, which in some embodiments may be milk-derived oligosaccharide chains or blood antigen oligosaccharides.

The milk-derived oligosaccharide or blood antigen oligosaccharide may be HMO, Lewis or ABH blood group antigen oligosaccharide, in some embodiments HMO, Lewis oligosaccharide may be 2' -FL, 3-FL, N2F, etc.; the blood group antigen oligosaccharides may be various types of ABH blood group antigen oligosaccharides.

The fucosylation substrate may be: free oligosaccharides, free glycoproteins or glycolipids; the free substrate can here be a single substrate distributed in the body fluid or purified.

The fucosylation substrate may be on the surface of a cell, and in still other preferred embodiments, the cell is a human red blood cell.

The enzyme digestion treatment has no obvious influence on the physiological metabolic function of the cells, and in a preferred embodiment, the physiological metabolic function is shown in that after the csFase I enzyme digests the erythrocytes, the digested supernatant hemoglobin, the hemoglobin with high iron of the erythrocytes, the intracellular ATP and the 2,3-DPG have no obvious change.

In the invention, the using method can adopt the reaction of hydrolase to carry out enzymolysis to remove fucose of various substrates. In some embodiments, the methods may use fucosidases or other enzymes having equivalent functions as described herein.

As shown in the present embodiment, the substrate on which the enzyme of the present invention acts mainly comprises 3 nonfucose modified substrates: artificial chromogenic substrate, oligose and human erythrocyte surface blood group antigen. From the simple to the complex substrate, the enzyme can effectively carry out enzyme digestion, shows the activity of hydrolyzing the fucose substrate, and has the characteristics of simple and convenient operation, high enzyme digestion efficiency and the like. Since fucosylation plays an important role in many biological processes, the enzyme of the present invention can be prepared from Montany red blood cells, and the method has important application value in clinical blood transfusion.

Provides a simple and quick new way for solving the current situation that blood supply of a Bombay blood type patient is extremely difficult; and provides a new method for the research of glycobiology and biomedicine.

Drawings

FIG. 1: ABO blood group system antigen structure diagram.

FIG. 2: csFase I is optimized for enzymatic reaction pH.

FIG. 3: csFase I is optimized for enzyme reaction temperature.

FIG. 4: the activity of csFase I on erythrocyte enzymatic cleavage was examined under a microscope.

FIG. 5: the mini gel card detects the enzymatic cleavage activity of csFase I on erythrocytes.

FIG. 6: immunofluorescence confocal detection csFase I on erythrocyte enzymatic cleavage activity.

FIG. 7: flow cytometry detects csFase I enzymatic cleavage activity on erythrocytes.

FIG. 8: enzyme activity of csFase I in erythrocyte maintenance fluids.

FIG. 9: the influence of csFase I enzyme digestion treatment on physiological metabolic functions of erythrocytes.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It is to be understood that these examples are for illustrative purposes only and are not intended to limit the present invention. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, biochemistry, and the like, which are well known to those skilled in the art. These techniques are fully described in the following documents: for example, Sambrook molecular cloning, A laboratory Manual, 2 nd edition (1989); DNA cloning, volumes I and II (D.N. Glover editor 1985); oligonucleotide synthesis (edited by m.j. gait, 1984); protein purification (richardr. burgess) or can be performed according to the instructions provided by the reagent manufacturer.

In the specific embodiment of the present invention, the enzyme digestion reaction specifically comprises the following steps:

1. preparation of reagents for enzymatic cleavage

1.1 buffers required for the reagent reactions

The buffer used in the present invention includes: 1) the phosphate solution for enzyme digestion of pNP-Fuc and oligosaccharide is 25mM phosphate solution (pH 5.0) because mass spectrum detection needs to be carried out on an enzyme digestion substrate subsequently, and the influence of salt ions in a reaction solution on the mass spectrum detection is avoided. 2) And phosphate buffer and citrate buffer for digesting the red blood cells and various red blood cell/blood maintenance solutions, wherein the phosphate buffer and the citrate buffer are isotonic solutions containing 50mM of phosphate or citrate and 100mM of NaCl (pH 6) to prevent the red blood cells from being ruptured. The aldrin solution in the red blood cell/blood maintenance solution is purchased from sigma, and other formulas are detailed in table 1 below:

TABLE 1 formulation table of red blood cell/blood nourishing liquid

1.2 preparation of the reaction substrate

Three types of substrates were used in the present invention for the demonstration of the enzyme activity.

1. The revealing substrate pNP-Fuc was purchased from carbosynth, England.

2.3-FL, Lewis X and other non-blood group antigen oligosaccharides purchased from carbosynth, UK; type 1,2, 4 oligosaccharide of blood group antigens A, B and H are purchased from Elicityl Oligotech; among them, 2' -FL is considered to be a blood type 5 antigen substance and purchased from carbosynth, UK.

3. The concentrated red blood cells are from the transfusion department of Thymelah hospital and the blood center of Shanghai city; the human ABO anti-stereogenic erythrocyte kit is from Changchun Border Biotechnology, Inc. The anti-H agent is derived from Shanghai

The substrate pNP-Fuc and each oligosaccharide were reconstituted with ultrapure water to prepare 10mmol/L of a stock solution.

2 conditions of enzyme digestion

(1) Reaction temperature: the experiment has the additional indication that the reaction temperature for the substrate pNP-Fuc and the oligosaccharide is 37 ℃ except the optimal temperature for enzyme preparation. However, when erythrocytes were digested, the reaction temperature was 26 ℃ to reduce damage to erythrocytes.

(2) Reaction time: the chromogenic substrate pNP-Fuc reacts for 30min, and the reaction time of the oligosaccharide and the human red blood cells is 1 h.

3 enzyme digestion reaction and result identification

3.1 chromogenic substrate cleavage reaction:

chromogenic substrate pNP-Fuc digestion reaction: 49 μ L of phosphate buffer, 10 μ L of chromogenic substrate pNP-Fuc mother liquor (10mM), 1 μ L of enzyme, mixing well, totaling 60 μ L, reacting at 37 ℃ for 1h, ending the reaction, adding 90 μ L of 1M Na₂CO₃The enzyme digestion reaction is terminated, and the OD measured by a microplate reader is 405 nm.

3.2 oligosaccharide cleavage reaction:

the enzyme was diluted 10-fold with an enzyme stock solution, 12.5. mu.L of phosphate buffer, 1.5. mu.L of oligosaccharide (10mM) stock solution, and 1. mu.L of diluted enzyme, totaling 15. mu.L, 37 ℃ for 1 hour. The enzyme cleaved fucose was detected by L-fucose kit, and simultaneously detected by ESI-MS.

3.3 human erythrocyte digestion reaction:

a) transferring 750 μ L volume of each erythrocyte in concentrated type (40% packed volume) to 2mLEP tube, centrifuging at 500 × g for 5min, sucking supernatant and discarding;

b) slightly washing twice with corresponding reaction solution/maintenance solution, and adding corresponding reaction solution/maintenance solution to make the whole volume be 1mL, and the packed volume of erythrocytes be 30%;

c) adding 50 mu of LcsFase I enzyme solution, placing at 26 ℃, and reacting for 1 hour by gentle oscillation;

d) after the completion of the enzyme digestion, the mixture was centrifuged at 500 Xg for 5min, and the supernatant was discarded and washed twice with physiological saline.

e) After the enzyme digestion is finished, the enzyme digestion effect is detected by a microscope, a micro gel card, immunofluorescence confocal technology and flow cytometry. The details of the method are shown in the examples.

Example 1: cFase I enzyme digestion pNP-Fuc and its enzymology property experiment

1. Fucosidase activity identification

Preparation of reaction substrate: the substrate pNP-Fuc lyophilized powder (purchased from carbosynth) was reconstituted with distilled water at a concentration of 10mM and stored as a stock solution.

Enzyme digestion reaction liquid: 25mM phosphate solution (pH 5.0)

Enzyme digestion system: the volume of the substrate pNP-Fuc stock solution was 10. mu.L and the volume of the enzyme was 1. mu.L, and the volume was adjusted to 60. mu.L by adding phosphate buffer. Table 2 below, number 1 as experimental group and number 2 as control group.

TABLE 2 arrangement of the reaction systems

Reaction conditions are as follows: at 37 ℃ for 1h, immediately adding 1M Na after the reaction is finished₂CO₃The reaction was terminated by 90. mu.L, and the OD was measured with a microplate reader at 405 nm. And judging whether the enzyme has fucosidase activity or not according to the result.

Reaction system: the pNP-Fuc stock solution was diluted with phosphate buffer and subjected to concentration gradient of 0.05, 0.1, 0.25, 0.5, 1.0, 2.0, 4.0mM, 59. mu.L of the reaction solution diluted in gradient was added to each well of a 96-well plate and 1. mu.L of the enzyme was added to each well, and each sample was repeated three times.

Reaction conditions are as follows: keeping the temperature at 37 ℃ for 15min, adding 1M Na immediately after the reaction is finished₂CO₃The reaction was terminated by 90. mu.L, and the OD was measured with a microplate reader at 405 nm.

2. Determination of optimum pH of fucosidase

The determination of the optimum pH value of fucosidase, prepare the buffer solution with pH 3.0-11.0 according to the record of Chinese pharmacopoeia, and carry out the experiment according to the above-mentioned reaction condition, each pH value is carried out three times repeatedly. The results are shown in FIG. 2: the enzyme has the maximum enzyme cutting activity on a substrate pNP-Fuc at the pH of about 6, but has higher activity at the pH of 4-7.

3. Determination of optimum reaction temperature of enzyme

According to the experimental method described in the present example, the enzyme activities of the enzymes at the total of eight temperature gradients of 4, 20, 30, 37, 45, 55, 65, and 75 ℃ were verified, and in order to ensure the reliability of the experiment, all the sample loading operations were performed at 4 ℃, and after the enzyme was finally added, the samples were immediately placed in a water bath at the corresponding temperature. All experiments were performed in triplicate. The results are shown in FIG. 3: the temperature is about 37 ℃, and the maximum enzyme activity is achieved.

Example 2: digestion experiment of oligosaccharides

Configuration of the substrate: all oligosaccharides, including blood group-related and non-blood group antigen oligosaccharides, were formulated with ultrapure water to a concentration of 10 mM.

Reaction system: mu.L of phosphate buffer solution 8. mu.L, oligosaccharide preservation solution 1. mu.L, enzyme 1. mu.L, 10. mu.L total reaction system, and reaction at 37 ℃ for 1 h.

And (3) detection of enzyme digestion reaction: in this example, two methods were used to detect the products of oligosaccharide cleavage

The method comprises the following steps: the detection is carried out according to an L-fucose detection kit (megazyme, Ireland), and the specific method refers to a kit manual, wherein fucose is oxidized by fucose dehydrogenase, NADP + is generated into NADPH, and the NADPH can be measured by an absorbance value of 340nm and quantified according to a standard curve, so that the fucose cut by the enzyme of the oligosaccharide is quantified.

The method 2 comprises the following steps: according to the mass spectrum detection, 10 mu L of reaction liquid is added into 400 mu L of ultrapure water for ESI-MS (ThermoFisher), and a cation mode is selected, and the voltage is 3500V.

After the enzyme digestion of the oligosaccharide, csFase I was detected to have the highest enzyme digestion activity on the a-1,2 glycosidic linkages, as well as on the part α -1,3 and α -1,6 oligosaccharide substrates, with the results shown in Table 3.

TABLE 3 enzyme cleavage Activity of csFase I on different glycosidic bond substrates

-: no activity was detected +: weak activity ++: medium activity ++: high activity

The enzyme digestion detection of antigen oligosaccharide related to other blood types shows that csFase I has enzyme digestion activity on all H antigens in the invention and has no enzyme digestion activity on fucose on A and B antigens, and the result is shown in Table 4, wherein 2' -Fusosyllactone is the H antigen of type 5.

TABLE 4 enzyme digestion Activity of csFase I on oligosaccharides of different blood groups antigens

-: no activity was detected +: has enzyme cutting activity.

Example 3: enzyme digestion experiment for conversion of human red blood cells to Bombay type red blood cells

csFase I digested erythrocytes

1) Transferring 750 μ L volume of each concentrated erythrocyte (40% packed volume) to 2mL EP tube, centrifuging 500g for 5min, sucking supernatant and discarding;

2) slightly washing twice with corresponding reaction solution/maintenance solution, and adding corresponding reaction solution/maintenance solution to make the whole volume be 1mL, and the packed volume of erythrocytes be 30%;

3) adding 50 mu of LcsFase I enzyme solution, placing at 26 ℃, and reacting for 1 hour by gentle oscillation;

4) after the completion of the enzyme digestion, the mixture was centrifuged at 500 Xg for 5min, and the supernatant was discarded and washed twice with physiological saline.

5) After the enzyme digestion is finished, the enzyme digestion effect is detected by a microscope, a micro gel card, immunofluorescence confocal technology and flow cytometry. The specific detection is as follows:

2. and (3) detection after enzyme digestion:

2.1 detection of erythrocytes under microscope by slide method

Washing the red blood cells subjected to the csFase I enzyme digestion treatment by using normal saline, diluting to 3% of packed volume, sucking 20 mu L of red blood cell fluid, placing the red blood cell fluid on a glass slide, sucking 20 mu L of anti-H antibody anti-H(s) and dripping the anti-H antibody anti-H(s) into the red blood cell fluid, and dripping 20 mu L of normal saline into a control; gently shaking the slide, mixing, standing for 2min, and observing erythrocyte agglutination with naked eye. And the slides were transferred to a microscope for observation and photographed for recording. The O-type erythrocytes agglutinate in a large amount under the anti-H antibody, and after enzyme digestion (O-ECR), the erythrocytes have no agglutination phenomenon with the Bombay-type erythrocytes of the negative control, and the result is shown in FIG. 4;

2.2 micro gel blood group card for testing erythrocytes

Washing erythrocytes subjected to csFase I enzyme digestion treatment with normal saline, and diluting to 0.8% of packed volume; uncovering the packaging aluminum foil of the detection card, adding 50 mu L of diluted erythrocyte liquid into the reaction chamber, then adding 50 mu L of anti-H antibody anti-H(s), dripping into the erythrocyte liquid, and dripping 50 mu L of normal saline into the contrast; placing at 37 deg.C for 15min, centrifuging at 85g for 10min, and determining according to specification standard. The O-type erythrocytes agglutinate to different degrees under the anti-H antibody and cannot be centrifuged to the bottom through the gel card; after the enzyme cutting by csFase I, the enzyme cutting by csFase I completely passes through the gel, and is centrifuged until the substrate is stuck, and the result is shown in FIG. 5;

2.3 confocal laser detection of erythrocytes

The antibody using concentration of laser confocal detection is the same as that of flow detection, and the specific steps are as follows:

1) taking about 100,000 red blood cells, washing with normal saline for three times, and adding 0.5% glutaraldehyde for fixation for 10 min;

2) washing with normal saline for three times to remove glutaraldehyde and avoid influencing subsequent experiments; adding corresponding primary antibody or lectin according to flow detection method, and standing at room temperature for 30 min;

3) washing with 150 μ L of physiological saline three times, washing the primary antibody or lectin which is not bound to the antigen;

4) 1% BSA is used for blocking erythrocytes, and the nonspecific binding is reduced after 1h at room temperature;

5) adding a second antibody Alexa Fluor 488-anti-IgM, and allowing the cells to be resuspended in a 50-mu L system; incubate for 30min and wash three times. When lectin is used, a secondary antibody does not need to be labeled since it is itself labeled with FITC. Taking care to avoid light when using agglutinin or secondary antibody labeled by fluorescein;

6) sucking 20 mu L of the red blood cells which are well resuspended and marked, dripping the red blood cells in the middle of an anti-falling glass slide which is processed by polylysine, and placing the anti-falling glass slide in a constant temperature box at 37 ℃ for 30min to ensure that the red blood cells are fixedly adsorbed on the glass slide;

7) after the cells are fixed, taking out the slide, gently sucking away the supernatant, dropwise adding 90% glycerol solution, and covering a cover slip and sealing;

8) confocal microscopy.

The detection result is shown in fig. 6, three anti-H reagents respectively mark erythrocyte surface H antigens before and after the csFase I enzyme digestion treatment, and the enzyme digestion effect is detected by an immunofluorescence confocal microscope; the anti-H(s) and the UE-lectin are two H antigen detection reagents which are widely used clinically, and the anti-H (ab) antibody is derived from scientific research reagents. The result shows that the O-type red blood cells before enzyme digestion treatment have strong fluorescent signals under a mirror, and the signals completely disappear after enzyme digestion treatment and are consistent with negative control. The csFase I has high-efficiency enzyme digestion and hydrolysis activity on O erythrocyte H antigen.

2.4 flow cytometry detection of erythrocytes

Flow cytometry is used for detecting erythrocyte surface antigens before and after enzyme digestion, three anti-H reagents are used for detecting to judge whether the enzyme digestion is complete, wherein anti-H(s) belong to a detection reagent used clinically, the detection reagent is cell culture supernatant, the concentration of antibodies is relatively low, a stock solution is directly dripped by a slide method and is used for diluting 10 times when the enzyme digestion is used as a primary antibody in a flow mode, in addition, UE-lectin-FITC belongs to lectin derived from Ulexeuropus, α -1,2 fucose can be widely recognized and is used as a reagent for detecting the H antigen clinically, the concentration of protein in flow detection is 5 mu g/mL, anti-H (ab) belongs to a reagent for scientific research, the reagent is purchased from abcam, the concentration of the antibodies used in a flow mode is 10 mu g/mL, a secondary antibody is used for goat anti-mouse IgM-Alexa Fluor 488, and the flow detection steps are as follows according to the recommended concentration:

1) approximately 500,000 red blood cells were resuspended in normal saline and fixed with 0.1% glutaraldehyde for 10 min;

2) washing with 150 mu L of normal saline for three times, and fully washing glutaraldehyde;

3) adding the prepared anti-H reagent to enable the cells to be suspended in a 50 mu L system; incubating at room temperature for 30 min;

4) washing with 150 μ L of physiological saline three times, washing the primary antibody or lectin which is not bound to the antigen;

5) 1% BSA is used for blocking erythrocytes, and the nonspecific binding is reduced after 1h at room temperature;

6) adding a second antibody Alexa Fluor 488-anti-IgM, and allowing the cells to be resuspended in a 50-mu-L system; incubate for 30min and wash three times. When lectin is used, this step is omitted since it is itself labeled with FITC; taking care to avoid light when using fluorescein labeled lectin or secondary antibody;

7) resuspend using 300 μ L of saline and analyze by flow cytometry.

In order to further explore the enzyme digestion effect of csFase I on H antigens on erythrocytes, the invention adopts flow cytometry to detect the distribution condition of the H antigens on the surfaces of erythrocytes before and after enzyme digestion, the results are shown in figure 7, the O-type erythrocyte can detect strong fluorescent signals due to the existence of a large amount of H antigens on the surface of the erythrocyte, the signals after combination of anti-H(s) and UE-lectin are most obvious, the range of the fluorescent signals of the UE-lectin is narrower, the detected H antigen has better uniformity, the detected H antigen is related to the fucose widely recognized α -1,2, the range of the fluorescent signals of anti-H (ab) is wider, the detected H antigen types are not uniformly distributed on the surface of the erythrocyte, and the O-ECR (the O-type erythrocyte after enzyme digestion conversion) has no combination effect on three anti-H antigens after the enzyme digestion treatment of the Fase I, and the signals of the O-ECR (the O-type erythrocyte after enzyme digestion conversion) basically have the same effect with negative control (without adding anti-H reagents) and the lack of the erythrocyte signals, and the effective H antigens on the O-type erythrocyte.

Example 4: clinical utility and minimal enzyme amounts of csFase I

Clinically, the erythrocyte liquid and blood maintenance liquid mainly comprise glucose, citric acid, phosphate, adenine, sodium chloride, mannitol and other components. The glucose provides an energy source, which is beneficial to maintaining the activity of the red blood cells; the buffer ion pair formed by the sodium citrate and the citric acid can stabilize the pH value of the solution; the phosphate prevents the erythrocyte from aggregating, maintains the energy metabolism phosphate balance of the erythrocyte, and slows down the descending speed of the 2, 3-DPG; adenine can improve ATP activity level during blood storage, promote synthesis of erythrocyte ATP, provide high-energy compounds for erythrocyte metabolism, and prolong preservation period of erythrocyte; sodium chloride mainly adjusts the osmotic pressure of the solution and provides sodium ions for red blood cells; mannitol dilutes and concentrates erythrocyte viscosity, increases cell membrane stability, and prevents hemolysis. According to several maintenance solutions commonly used clinically at present, a erythrocyte maintenance solution MAP commonly used clinically is selected as an enzyme digestion reaction solution of csFase I, the MAP is widely applied to preservation of erythrocytes of various blood stations/banks, and erythrocytes can be effectively preserved for 35 days at 4 ℃;

the enzyme activity of csFase I in the erythrocyte maintenance solution is detected, the enzyme cutting activity of the substrate pNP-Fuc is aimed at, the result is shown in figure 8, and the result shows that the csFase I in MAP has high enzyme cutting activity to the substrate pNP-Fuc, and the activity is improved to about 140-165% compared with the phosphate reaction solution. Determining the minimum enzyme digestion amount of csFase I for digesting erythrocytes in erythrocyte maintenance liquid through enzyme digestion experiments under different enzyme quantities, wherein the enzyme digestion method is detailed in example 3, and the detection method comprises a slide microscope and a micro gel card; the results are shown in Table 5. In summary, 1 unit (200mL) of 30% packed volume of O-type red blood cells can effectively remove H antigen on the surface of the O-type red blood cells after being digested with csFase I for 1H at 26 ℃ and less than 20 mg.

TABLE 5 minimal enzyme dose analysis of csFase I enzyme-digested H antigen in erythrocyte maintenance fluids

pRBCs 30% packed red blood cells.

37 ℃ of: after incubation for 15min at 37 ℃, centrifugal detection is carried out, and the temperature is 4 ℃: and (4) incubating for 15min at 4 ℃, and then centrifuging and detecting.

Example 5: influence of enzyme digestion on physiological metabolism function of erythrocytes

Detecting supernatant trace hemoglobin:

the specific method is slightly modified according to the instruction provided by the kit and comprises the following steps:

1) after enzyme digestion is finished, centrifuging for 3min at 500 Xg, and taking a supernatant for standby detection;

2) according to the instruction, taking a 96-well plate, and adding 250 mu L of color developing agent;

3) adding 15 mu L of supernatant into the experimental group, and adding 15 mu L of normal saline into the blank group;

4) mixing, incubating in a 37 deg.C incubator for 20min, and detecting OD₅₁₀。

And (3) detecting methemoglobin:

the specific method is slightly modified according to the instruction provided by the kit and comprises the following steps:

1) taking a 96-well plate, and adding 250 mu L of reagent second dilution application liquid;

2) after enzyme digestion is finished, adding 5 mu L of enzyme-digested erythrocytes into the application liquid, and adding 5 mu L of physiological saline into the blank group; mixing, and standing for 5 min;

3) the microplate reader detects the absorbance at 630 and 602 nm.

ATP detection:

according to the kit specification, the method comprises the following specific steps:

1) diluting an ATP standard: ATP standards provided according to the kit were diluted with physiological saline so that the final concentrations were: 10uM, 1uM, 0.1uM, 0.01uM, 0.001uM, 0.0001uM, and 0 uM; add 100 μ L of diluted ATP standard to each well of a 96-well plate;

2) after completion of the digestion, the cells were washed with physiological saline and then counted under a microscope to make 100. mu.L of the erythrocyte suspension contain 1X 10⁴One cell and transfer 100 μ L to 96-well plate;

3) add 50 μ L of lysis reagent (provided in kit) per well and shake the 96-well plate to rupture the cells;

4) adding 50 mu L of substrate reaction solution (provided by a kit), and shaking and uniformly mixing a 96-well plate;

5) placing in dark for 10min, and detecting luciferase luminescence with microplate reader.

2, 3-diphosphoglycerate detection:

binding of 2, 3-diphosphoglycerate (2,3-DPG) to deoxyhemoglobin in erythrocytes decreases hemoglobin to O₂Promoting the affinity of O₂And dissociation of hemoglobin. The content of 2,3-DPG is therefore indicative of the capacity of the erythrocytes to release oxygen; in order to detect whether the oxygen releasing capacity of erythrocytes after enzyme digestion is affected, the content of 2,3-DPG is compared before and after enzyme digestion. The detection method is as follows according to the kitThe specification is slightly modified, and the specific steps are as follows:

1) after the enzyme cutting, 1mL of about 30% packed red blood cells is added into 5mL of precooled 0.6M perchloric acid solution, and the mixture is fully and uniformly mixed by using a pipettor; centrifuging at 5,000rpm for 10 min;

2) taking 4mL of colorless supernatant, and neutralizing perchloric acid with 2.5M potassium carbonate, wherein the volume is 0.5 mL; mixing, and placing on ice for 60 min;

3) centrifuging at 5,000rpm for 10min, and sucking the supernatant for later use; the 2,3-DPG solution contained in the composition can be stably stored for one day in the environment and needs to be used as soon as possible;

4) taking a 96-well plate, respectively adding 200 mu L of solution 1, 5 mu L of solution 2 and solution 3, then adding 10 mu L of supernatant to-be-detected solution, and simultaneously adopting 10 mu L of double distilled water as a blank control;

5) mixing, reacting at 20-25 deg.C for 5min, and reading absorption value A with wavelength of 340nm with enzyme labeling instrument₁；

6) Taking out 96-well plate, adding solution 4 and solution 52 μ L each, mixing, standing for 25min, and reading absorption value A with wavelength of 340nm with microplate reader₂；

7) By A₁-A₂The ratio of the relative amount of 2,3-DPG between the samples was calculated.

In order to investigate whether the enzyme digestion treatment can influence the physiological and metabolic functions of the red blood cells, the supernatant hemoglobin, intracellular methemoglobin, intracellular ATP and intracellular 2,3-DPG after the red blood cells are digested by enzyme are detected; as shown in FIG. 9, the enzyme digestion treatment of csFase I on erythrocytes in the MAP erythrocyte maintenance solution did not cause the change of supernatant hemoglobin, indicating that the enzyme digestion treatment did not cause hemolysis of erythrocytes; the methemoglobin is not obviously changed, which shows that the enzyme digestion treatment of the csFase I can not obviously damage the oxygen carrying capacity of the erythrocytes; the ATP and 2,3-DPG contents also do not change obviously, which indicates that the activity of the red blood cells and the oxygen release capacity of the red blood cells entering tissues are not influenced; the results show that the enzyme digestion treatment of the csFase I on the erythrocytes does not influence the physiological and metabolic functions of the erythrocytes.

Example 6: production process of fucosidase

The fucosidase gene sequence is from whole genome sequencing data of a meningococcal septicemia elizakii FMS-007 strain which is completed in the early stage, gene prediction is carried out by utilizing Glimmer 3.0 software, COG, KEGG and GO are subjected to functional annotation, an Open Reading Frame (ORF) is found, the length of the sequence is 2328bp, 775 amino acids are coded, the molecular weight is about 88kDa, no signal peptide is predicted by SignalP4.1 software, but a transmembrane sequence is found at the N end of the sequence through the prediction analysis of a transmembrane region of TMHMM; amino acids 1-11 are in the cell, amino acids 12-34 form a helix structure and belong to the transmembrane region, and amino acids 35-775 are distributed outside the cell. The prediction of the conserved region through NCBI is likely to be fucosidase;

cloning a target gene into a prokaryotic expression vector by adopting a genetic engineering method, then transforming the target gene into escherichia coli for expression, and obtaining purified novel glycosidase by adopting affinity chromatography and ultrafiltration methods;

1. method and process for production

1.1 construction strategy of fucosidase gene recombinant plasmid

Obtaining a fucosidase gene sequence according to FMS-007 genome sequencing data, directionally cloning a PCR amplification product to a prokaryotic expression vector commonly used in the industry by taking genome DNA of FMS-007 as a template, such as expression vectors PET15, PET32, PET28 and the like of a PET system, and inserting a target gene into the expression vector by selecting a commonly used polyclonal enzyme cutting site to construct a recombinant plasmid;

1.2 PCR amplification of fucosidase Gene

The reaction system for PCR amplification of fucosidase gene was prepared according to Table 6, and mixed well, with amplification parameters of 98 ℃ 10s, 52 ℃ 5s, 72 ℃ 20s, 33 cycles, and 72 ℃ 5min 1 cycle. Electrophoresis at 100V for 40min, and taking pictures with a gel imaging system. The primer sequences of fucosidase are as follows:

upstream primer 2159-F:

5’-TGCCATGGTTTCGAATATCAGAGCGCAGTC-3’(SEQ ID NO 2)

downstream primer 2159-R:

5’-CGCTCGAGTCTGTAGATCTGCAAGCTAACCC-3’(SEQ ID NO 3)

TABLE 6 fucosidase PCR amplification System

1.3 directed cloning of fucosidase PCR products

(1) PCR product of fucosidase and pET28a vector double enzyme digestion

The PCR product was double-digested with two restriction enzymes NcoI and XhoI. And preparing an enzyme cutting system according to the concentration of the purified target gene csFaseI and the vector and the concentration shown in the table 7, uniformly mixing, and performing enzyme cutting for 1h at 37 ℃. And (3) after the enzyme digestion is finished, recovering the enzyme digestion product by using a DNA product purification kit, and carrying out operation steps. And (3) after double enzyme digestion of the carrier, gel tapping recovery after electrophoresis, and the operation steps are according to a gel recovery kit.

TABLE 7 double digestion System for PCR products/plasmid DNA

(2) Ligation of csFase I Gene and vector

According to the concentration of the csFase I after double enzyme digestion and purification and the concentration of the prokaryotic expression vector, the molar ratio of the csFase I to the prokaryotic expression vector is about 1:5, a connection system is prepared according to the table 8, and the csFase I and the prokaryotic expression vector are connected for 20min at 22 ℃ after being uniformly mixed.

TABLE 8 CSFase I Gene and pET-28a (+) vector ligation System

(3) Conversion of ligation products

The main principle of plasmid transformation to competent cells is that bacteria are in CaCl at 0-4 ℃₂The hypotonic solution swells into a sphere, loses part of membrane protein, becomes a state of easily absorbing exogenous DNA, and promotes the absorption of the exogenous DNA by heat shock at 42 ℃. Coli Top10 competent cells were transformed with 20 μ L of the (2) transformed ligation product, detailed procedure as follows:

1) take 50 μ L of competent cells from-80 deg.C refrigerator, melt on ice bath, add 20 μ L of ligation product, gently stir and mix, and stand in ice bath for 30 min.

2) The water bath was heat-shocked at 42 ℃ for 90s, and then the tubes were quickly transferred to ice for 2min without shaking the tubes.

3) The tube was added with 300. mu.L of sterilized LB liquid medium (containing no antibiotics), mixed well and then cultured at 37 ℃ for 1 hour with shaking at 200rpm to resuscitate the bacteria.

4) The bacterial solution was centrifuged at 5000rpm for 2min, half of the supernatant (about 150. mu.L) was discarded from the pipette tip, the precipitate was gently blown up and mixed by a pipette tip, and then the whole bacterial solution was applied to LB agar medium containing 50. mu.g/ml kanamycin antibiotic. The plates were inverted and incubated overnight at 37 deg.C (about 12 h).

5) After the plate grows out of the colonies, 8 single colonies are scattered and selected to fall on the bottom of the plate for marking, each colony is picked into a 5mLLB liquid culture medium (containing 50 mu g/mL kanamycin), and the shaking culture is carried out at the temperature of 37 ℃ and the rpm of 200 for 10-14 h.

(4) Extraction of recombinant plasmid

Plasmids were extracted using a plasmid miniprep kit (Axygen bio) with the following steps:

1) 3mL of the collected bacterial liquid is centrifuged at 12000rpm at room temperature for 1min, the supernatant is sucked off as much as possible, and the precipitate is left.

2) To the pellet from the previous step 250. mu.L of BufferA1 (ensuring RNaseA was added) was added and the bacterial cells were thoroughly suspended by pipetting or vortexing.

3) Then 250. mu.L of Buffer B1 (RNaseA is added in advance) is added into the centrifuge tube, the mixture is gently turned over 10 times to mix the thalli evenly, and then the mixture is kept stand for 5min until the solution is viscous and clear.

4) Then 350. mu.L of Buffer N1 was added to the tube and the mixture was immediately gently turned upside down and mixed several times, at which time white flocculent precipitate appeared.

5) Centrifuge at 12000rpm for 10min at room temperature, and if there is a white precipitate in the supernatant, centrifuge again.

6) Carefully sucking the centrifuged supernatant, transferring the supernatant into a DNA column with a collecting tube, taking care to avoid sucking the precipitate, centrifuging the supernatant at 12000rpm for 1min at room temperature, and pouring off the waste liquid in the collecting tube.

7) Then 500mLBuffer KB was added to the DNA column, centrifuged at 12000rpm for 1min at room temperature, and the waste liquid in the collection tube was discarded.

8) Add 500. mu.L DNAwash buffer to the spin column (ensure ethanol addition), centrifuge at 12000rpm for 1min at room temperature, and discard the waste from the collection tube.

9) Repeating the previous step.

10) The column was returned to the centrifuge and centrifuged for 5min at 12000rpm with the lid opened at room temperature to remove the residual ethanol.

11) Transferring the column to a new 1.5mL centrifuge tube, suspending and dropping 70 μ L of ElutionBuffer into the center of the adsorption membrane, standing at room temperature for 5min, centrifuging at 12000rpm for 1min, dropping the eluent again into the center of the adsorption membrane, centrifuging at 12000rpm for 1min, and collecting the eluent containing the plasmid.

12) After plasmid extraction, the concentration and purity of the product were checked by using NANODROP 2000.

(5) Double enzyme digestion identification of recombinant plasmid

And carrying out double enzyme digestion identification on the plasmid DNA extracted in the last step by using two restriction enzymes used for constructing cloning, preparing an enzyme digestion system according to the determined plasmid concentration and the table 9, and carrying out enzyme digestion for 1h at 37 ℃. The remaining plasmid was stored for future use.

TABLE 9 reaction System for double enzyme digestion identification

The cleavage products were detected by 0.8% agarose gel electrophoresis using DL 2000 and lambda-Hind III (5 min at 60 ℃ C. before use) in a DNA Marker.

(6) Sequencing of recombinant plasmids

According to the agarose gel electrophoresis result, the plasmid with positive double enzyme digestion is selected and sent to Huada Gene company for sequencing.

Prokaryotic expression of csFase I

2.1 recombinant plasmid transformation

The expression host bacterium selected in the test is E.coli BL21(DE3), and the bacterium is a protein expression host for efficiently expressing foreign genes mediated by T7RNA polymerase. The correct csFase I recombinant plasmid is transformed into BL21(DE3) competent cells by sequencing verification, and the steps are as follows:

1) take 50 μ L of competent cells from-80 deg.C refrigerator, melt on ice bath, add 1 μ L of recombinant plasmid, mix gently, stand in ice bath for 30 min.

2) The water bath was heat-shocked at 42 ℃ for 90s, then the tubes were quickly transferred to ice for 2min, and the process took care not to shake the tubes.

3) The tube was added with 300. mu.L of sterilized LB liquid medium (containing no antibiotics), mixed well and then cultured at 37 ℃ for 1 hour with shaking at 200rpm to resuscitate the bacteria.

4) 200uL of the bacterial suspension was applied to LB agar medium containing 50. mu.g/ml kanamycin antibiotic by a pipette, the plate was inverted, and the culture was carried out overnight at 37 ℃ (about 12 hours).

2.2csFase I protein expression

When the bacteria enter the optimal growth state under the condition without an inducer, the inducer IPTG is added into a culture medium, and the combination with the repressor protein is released to relieve the inhibition, so that a large amount of exogenous genes are expressed.

The induction steps are as follows:

1) BL21 containing csFase I recombinant plasmid and an empty vector are selected as controls, single colonies are picked up and respectively inoculated into 5mLLB culture medium (containing 10-200 mu g/mL kanamycin), shaking culture is carried out at the speed of 200rpm for 10-12h at the temperature of 37 ℃, 100 mu L to 1.5mL of centrifuge tubes are sampled, and the samples are preserved at the temperature of-20 ℃ and used for SDS-PAGE.

2) IPTG (final concentration is 0.1-10mM) is respectively added into the bacterial liquid, and the bacterial liquid is subjected to shaking culture at the temperature of 16-37 ℃ and the rpm of 200 for 12h to induce the expression of the target gene csFase I.

3) The bacterial liquid before and after induction is respectively 100 mu L, centrifuged at 12000rpm for 1min, and the supernatant is discarded.

4) 100. mu.L of 1 XPBS was added to the pellet to resuspend the cells, and the mixture was aspirated and mixed. mu.L of each tube was removed and placed in a new centrifuge tube, and 7.5. mu.L of 5 XSDS loading buffer was added and boiled at 95 ℃ for 10 min.

5) An Unstainedprotein MW Marker was used as a control, which was cooked for 10min at 95 ℃ before use.

6) The sample was then centrifuged at 12000rpm for 10 min.

7) And (3) sampling 15 mu L of supernatant sample, performing SDS-PAGE by using 15mA, and adjusting the current to 30mA for electrophoresis when the bromophenol blue runs to the separation gel so as to enable the bromophenol blue to run to the edge of the gel.

8) Dyeing with Coomassie brilliant blue for 1h, decolorizing with decolorizing solution for 2h, and decolorizing twice. And taking a picture and storing the result.

2.3 purification of the recombinant protein csFase I

The purification of the recombinant protein csFase I is carried out by two steps, wherein the first step is nickel column affinity chromatography and the second step is ultrafiltration. All manipulations of the purification were carried out at 4 ℃.

1) The recombinant plasmid-containing monoclonal was picked up into 5mL of LB liquid medium (containing 50. mu.g/mL kanamycin), 10 to 14 hours later, 5mL of the recombinant plasmid-containing monoclonal was aspirated and transferred to a flask containing 500mL of LB medium (containing 50. mu.g/mL kanamycin), and cultured at 37 ℃ for 6 to 8 hours with shaking at 200 rpm. After the temperature was lowered to 18 ℃ 1M IPTG was added to a final concentration of 0.5mM, and the mixture was cultured at 18 ℃ for 12 hours with shaking at 150 rpm.

2) The induced bacterial liquid is respectively added into two large centrifuge tubes, after being balanced by 1 XPBS, the mixture is centrifuged for 10min at 4 ℃ and 5000rpm, and the supernatant is sucked off as much as possible.

3) To the pellet from the previous step, 200mL of 1 XPBS was added to wash the bacterial cells, and the cells were centrifuged at 5000rpm for 10min at 4 ℃.

4) The supernatant was removed, and 7.5mL of lysine Buffer (10mM imidazole, pH 8.0) was added to each tube to resuspend the cells thoroughly.

5) Using an ultrahigh pressure homogenizer (model: FB-110X, Shanghai Shakou mechanical engineering Co., Ltd., Shanghai) for cell disruption.

6) The bacterial lysate was centrifuged at 12000rpm for 30min at 4 ℃ and the supernatant gently removed carefully to avoid aspiration of the pellet and slowly transferred along the wall into a new centrifuge tube.

7) Using Ni²⁺Purification of the target protein by means of an NTA affinity column, first washing the column with 3 column volumes of 0.5M NaOH (column regeneration); then washing the column (equilibrium column) by using lysine Buffer with the column volume of 3-5 times; after balancing, adding the supernatant of the previous step into a column, when the liquid is quickly drained, respectively eluting with Wash Buffer (25mM imidazole, pH 8.0) and Elutionbuffer (300mM imidazole, pH 8.0), sequentially collecting different eluents, and storing the collected target protein at 4 ℃.

8) All collected samples were identified by SDS-PAGE and 10. mu.L of each sample was spotted.

9) The protein of interest was purified by ultrafiltration using a 10kDa ultrafiltration tube.

10) The concentration of the target protein was measured by the BCA method, and the concentration of the target protein was measured by preparing a calibration curve from various protein standards of known concentrations using a TECAN plate reader.

3 results

3.1 csFase I/pET28a recombinant plasmid construction

Cloning the target gene csFase I fragment to pET28a vector, and verifying the success of constructing recombinant plasmid through sequencing.

3.2 IPTG inducible expression

And (3) after sequencing and identification of the recombined positive clone are correct, transforming the recombined positive clone to E.coli BL21, randomly selecting two monoclonals, respectively inoculating the monoclonals to an LB culture medium, adding 0.5mM IPTG (isopropyl thiogalactoside) for induction expression, inducing at 18 ℃ and 150rpm for 12 hours, and then sampling. SDS-PAGE was performed and expression was observed by examination.

Purification of the recombinant protein csFase I

After the recombinant protein csFase I is subjected to nickel column affinity chromatography purification, relatively pure protein can be obtained, and the purity is higher than 90%. Meets the requirements of common biochemical enzyme digestion experiments.

Sequence listing

<110> university of Compound Dan

<120> fucosidase and application thereof in preparation of Bombay type red blood cells

<160>3

<170>SIPOSequenceListing 1.0

<210>1

<211>775

<212>PRT

<213>Artificial

<400>1

Met Arg Lys Thr Pro Ser Asp Ser Gly Gly Arg Ile Leu Leu Ser Val

1 5 10 15

Thr Val Glu Lys Ser Ile Phe Leu Phe Asn Ile Leu Ile Phe Ile Phe

20 25 30

Phe Ile Val Ser Asn Ile Arg Ala Gln Ser Leu Pro Gln His Arg Leu

35 40 45

Glu Phe Thr Lys Leu Ala Pro Arg Trp Asp Glu Gly Ile Pro Leu Gly

50 55 60

Asn Gly Met Leu Gly Ser Leu Ala Trp Glu Lys Asn Gly Lys Leu Arg

65 70 75 80

Leu Ser Leu Asp Arg Ala Asp Leu Trp Asp Glu Arg Lys Ala Leu Asp

85 90 95

Leu Ser Lys Leu Asn Phe Lys Trp Val Glu Gln Gln Val Leu Lys Asp

100 105 110

Asp Tyr Lys Pro Val Gln Lys Ile Gly Asp Trp Pro Tyr Asp Asn Met

115 120 125

Pro Tyr Pro Thr Lys Leu Pro Ala Ala Ala Leu Gln Phe Asp Ile Ser

130 135 140

Gly Leu Gly Ala Val Val Ser Asn Gln Leu Glu Ile Ala Thr Ala Leu

145 150 155 160

His Thr Val Lys Phe Thr Ser Gly Val Val Phe Gln Asn Tyr Ile His

165 170 175

Ala Thr Gln Gln Ala Gly Tyr Phe Ser Phe Asp His Ile Thr Asp Glu

180 185 190

Asn Leu Met Lys Gln Leu Val Pro Lys Leu Asp Val His Asn Tyr Asn

195 200 205

Ser Gly Thr Ser Val Glu Ser Asp Asn Ser His Ala Gly Glu Gly Leu

210 215 220

Gly Lys Leu Gly Tyr Ala Lys Gly Asn Val Lys Glu Glu Glu His Thr

225 230 235 240

Ile His Tyr His Gln Pro Thr Tyr Asn Gly Arg Phe Phe Glu Val Leu

245 250 255

Val Lys Trp Lys Arg Thr Gly Lys Asp Lys Leu Thr Gly Ser Trp Thr

260 265 270

Ile Ser Gly Asn Gln Pro Ala Ala Leu Ser Ser Pro Gln Leu Ser Ile

275 280 285

Val Asp Ser Gly Glu Trp Lys Ser His Val Lys Trp Trp Lys Asp Phe

290295 300

Trp Ser Lys Ser Ser Val Lys Leu Pro Asp Glu Leu Ile Glu Lys Gln

305 310 315 320

Tyr Tyr Leu Glu Leu Tyr Lys Leu Gly Ser Val Ser Arg Lys Gly Ala

325 330 335

Pro Ala Ile Thr Leu Gln Ala Val Trp Thr Ala Asp Asn Gly Ser Leu

340 345 350

Pro Pro Trp Lys Gly Asp Phe His Asn Asp Leu Asn Thr Gln Leu Ser

355 360 365

Tyr Trp Pro Ala Tyr Thr Gly Asn His Leu Gln Glu Ala Val Ser Phe

370 375 380

Thr Asp Trp Leu Trp Lys Ile Arg Pro Val Ser Leu Gln Tyr Thr Lys

385 390 395 400

Gln Tyr Phe Gly Val Asp Gly Leu Asn Val Pro Gly Val Val Thr Leu

405 410 415

Asn Gly Asp Pro Met Gly Gly Trp Ile Gln Tyr Ser Leu Ser Pro Thr

420 425 430

Val Ser Ala Trp Cys Ala Gln Tyr Phe Tyr Trp Gln Trp Lys Tyr Ser

435 440 445

Met Asp Asp Arg Phe Leu Gln Gln Lys Ala Tyr Pro Tyr Val His Asp

450455 460

Ala Ala Val Tyr Leu Glu Asn Ile Thr Arg Leu Lys Asp Gly Val Arg

465 470 475 480

Lys Leu Pro Leu Ser Ser Ser Pro Glu Tyr Asn Asp Asn Ser Val Asn

485 490 495

Ala Trp Phe Lys Asp Trp Thr Asn Tyr Asp Leu Ser Leu Ala Arg Phe

500 505 510

Leu Phe Ser Ala Ala Ser Glu Ile Ala Lys Ala Ser Gly Lys Glu Val

515 520 525

Glu Ala Ile His Trp Lys Lys Ile Leu Gly Glu Leu Pro Asp Tyr Asn

530 535 540

Val Asn Glu Thr Gly Leu Thr Val Ala Pro Gly Gln Ser Met Glu Ser

545 550 555 560

Ser His Arg His Phe Ser Pro Tyr Met Ala Val Tyr Pro Leu Ala Leu

565 570 575

Leu Asp Val Asn Gln Pro Lys Asp Lys Glu Ile Val Asp Lys Ser Ile

580 585 590

Gln His Ile Glu Lys Leu Gly Thr Arg Ala Trp Val Gly Tyr Ser Phe

595 600 605

Thr Trp Met Ser Thr Leu Tyr Ala Arg Ala Tyr Gln Ala Glu Lys Ala

610 615620

Val Lys Gln Leu Gln Ile Phe Ala Ser Asn Phe Cys Ser Pro Asn Ser

625 630 635 640

Phe His Leu Asn Gly Asp Gln Lys Gly Gly Gln Tyr Ser Gly Phe Thr

645 650 655

Tyr Arg Pro Phe Thr Leu Glu Gly Asn Phe Ala Phe Ala Gln Gly Val

660 665 670

His Glu Leu Leu Leu Gln Ser Arg Gln Gly Tyr Ile Glu Val Phe Pro

675 680 685

Ala Ile Pro Lys Asp Trp Lys Asn Val Ser Phe Val Asn Leu Arg Ala

690 695 700

Glu Gly Ala Val Leu Val Ser Gly Lys Ile Glu Asn Glu Lys Leu Ile

705 710 715 720

Ser Val Lys Val Phe Ser Glu Lys Gly Gly Ile Val Asn Val Lys Leu

725 730 735

Pro Lys Gly Lys Ile Gln Leu Thr Asp Asn Arg Asn Val Gln Val Lys

740 745 750

Thr Leu Asn Thr Asp Lys Thr Ile Ile Asn Phe Lys Pro Gly Gly Trp

755 760 765

Val Ser Leu Gln Ile Tyr Arg

770 775

<210>2

<211>30

<212>DNA

<213>Artificial

<400>2

tgccatggtt tcgaatatca gagcgcagtc 30

<210>3

<211>31

<212>DNA

<213>Artificial

<400>3

cgctcgagtc tgtagatctg caagctaacc c 31

Claims (15)

1. A fucosidase characterized by the amino acid sequence:

has a sequence shown as SEQ ID NO 1; and various derived sequences having an enzymatic activity having a homology of 30% to the sequence.

2. A nucleic acid encoding a fucosidase having the sequence of SEQ ID NO 1.

3. A vector comprising the sequence of claim 2.

4. An engineered bacterium comprising the sequence of claim 2 or 3.

5. A fucosidase capable of hydrolyzing pNP- α -L-Fuc.

6. A fucosidase capable of hydrolyzing oligosaccharides having fucose residues.

7. A method for enzymatic removal of fucose from the surface of a cell, wherein the fucosidase of claim 1 or another fucosidase is used.

8. A fucosidase capable of hydrolyzing fucose on a cell surface.

9. The fucosidase of claim 8 wherein the cells are red blood cells.

10. The fucosidase of claim 8 wherein the cells are human red blood cells.

11. Use of the fucosidase of claim 1 for excising fucose from a cell surface.

12. The application of claim 11, wherein the application comprises: preparation and application of Montana type red blood cells and other cells are provided.

13. The method for preparing fucosidase according to claim 1, comprising the steps of:

1) obtaining and amplifying a gene sequence of the fucosidase of claim 1;

2) constructing a recombinant vector containing a gene sequence of the fucosidase of claim 1;

3) expressing the fucosidase of claim 1;

4) separating, purifying and identifying.

14. The method of claim 13, wherein the expression system is a bacterial, yeast or insect expression system.

15. The method of claim 13, wherein the method comprises conventional microbial fermentation production, expression and production in bacterial, yeast, insect expression systems using bioengineering techniques.

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