CN112048495A - Alpha galactosidase and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of glycobiology, and relates to a novel alpha galactosidase, and a preparation method and application thereof. The invention particularly relates to alpha galactosidase polypeptide (Galase) obtained from elizasa meningitidis (Elizabethkingia meningoseptica, FMS-007), polynucleotide for coding the alpha galactosidase polypeptide, and a preparation method and application thereof.

Description

Alpha galactosidase and preparation method and application thereof

Technical Field

The invention belongs to the technical field of glycobiology, relates to molecular biology, biochemistry and pathogenic biology, and particularly relates to a novel alpha galactosidase and a preparation method and application thereof.

Background

The prior art discloses that glycosylation is a post-translational modification of carbohydrates to proteins or lipids by the action of glycosyltransferases. Studies have shown that glycosylated proteins or lipids have a significantly increased diversity due to almost all aspects of glycosylation, such as: the sites of attachment of glycosidic linkages to glycans, glycan structures that are straight or branched, the different types of sugars that make up the glycosylation, and the length of the glycans, among others. The types of glycosylation at present are reported to be mainly N-glycosylation, O-glycosylation, phosphoserine glycosylation, C-mannosylation, etc.

The literature states that α galactosylation modifications are widespread in various proteins and oligosaccharides, the common glycosidic linkages being α 1,3, 1,4 and 1,6 linked, with terminal α -Gal modifications having important tissue and race specificities in organisms, and not only that the terminal α -Gal structures play an important role in the infection process of certain microorganisms.

At present, the clinical blood transfusion still has the problem of shortage of blood sources, and along with the exploration of xenotransplantation, xenotransfusion becomes a new research field. Research shows that the shape, size, structure and function of pig red blood cell are the same as those of human red blood cell, and the living environment of pig red blood cell is similar to that of human. Due to the species difference between swine and human, the presence of xenogeneic antigens on the surface of swine erythrocytes can be divided into α -Gal antigens and non- α -Gal antigens, wherein α -Gal antigens are terminal Gal- α 1,3-Gal (as shown in FIG. 9), while human IgG 1% is Anti- α 1,3-Gal, which is the major xenogeneic antigen causing hyperacute immune rejection in xenotransplantation^[1]Therefore, the alpha-Gal antigen of pig red blood cells becomes an urgent problem to be solved in xenotransfusion^[2]。

It has been discovered that alpha 1,3 and alpha 1,4 linked galactose provide an important receptor signal during microbial infection, and the outer layer of the cell wall of gram-negative bacteria has the structure of LPS^[3]LPS consists of lipid A, core polysaccharide and O antigen (FIG. 10)^[4]In which some bacterial O antigens terminate with an alpha 1,3-Gal structure, e.g. pneumoniaKlebsiella sp^[5]It is not known what effect the deletion of this structure will have on the bacterial LPS action; infections caused by shigella dysenteriae 1 are often associated with complications such as hemolytic uremic syndrome or leukemia, and cytotoxins similar to shiga toxins produced by entero-hemorrhagic escherichia coli are prone to hemorrhagic diarrhea and often cause a severe form of gastroenteritis known as Hemorrhagic Colitis (HC); these biological infections may cause serious sequelae such as hemolytic uremic syndrome or thrombotic thrombocytopenic purpura; it has been reported that shiga toxin recognition of target cells is dependent on α 1,4 linked galactose on the target cells, thus showing that α galactosylation plays an important role in the infection process^[6]。

Alpha galactosidase is a glycoside hydrolase that is capable of hydrolyzing alpha galactosyl moieties from glycolipids and glycoproteins, which are widely found in animals, plants, and microorganisms^[7]. Carbohydrate-Active enZYmes Database (CAZy, http:// www.cazy.org) is a Database dedicated to displaying and analyzing genomic, structural and biochemical information of Carbohydrate-Active enZYmes, the Glycoside Hydrolases family being Glycoside Hydrolases (GHs) which produce different classes based on their amino acid sequences. The catalytic mechanisms and the like of the same family are relatively conserved and can therefore be used to predict the characteristics of any novel glycosidase enzyme that has not yet been characterized. The currently known alpha galactosidases mainly belong to the GH4,27,36,57 and 110 families^[8]. Alpha-galactosidases are reported to have a wide range of applications, for example in the medical field, food industry and other industries such as feed^[9]. The removal of the terminal α -Gal is of great significance: for example, the removal of terminal alpha 1,3-Gal on porcine red blood cells and organs can obviously reduce the hyperacute immune rejection reaction in xenotransfusion and xenotransplantation, the alpha galactosidase is used as a tool to help research the role of O antigen as part of LPS in the infection and bacterial survival process, and the property of alpha galactosidase for cutting terminal alpha 1,4-Gal can be used for cutting terminal alpha 1,4-GalThe process of blocking the shigella from infecting receptor cells through shiga toxin is achieved, and the like, so that the method has great potential application value. Based on the current situation and the foundation of the prior art, the inventor intends to provide a novel alpha galactosidase, and a preparation method and a use thereof.

Prior art references relevant to the present invention are:

[1]Milland J,Christiansen D,Lazarus B D,et al.The molecular basis for gal alpha(1,3)gal expression in animals with a deletion of the alpha1,3galactosyltransferase gene.[J].Journal of Immunology,2006,176(4):2448-2454.

[2]Liu Q P,Yuan H,Bennett E P,et al.Identification of a GH110Subfamily of alpha 1,3-Galactosidases:NOVEL ENZYMES FOR REMOVAL OF THEα1,3GAL XENOTRANSPLANTATION ANTIGEN[J].Journal of Biological Chemistry,2008,283(13):8545-8554.

[3]Gangloff S C,Hijiya N,Goyert H S M.Lipopolysaccharide Structure Influences the Macrophage Response via CD14-Independent and CD14-Dependent Pathways[J]. Clinical Infectious Diseases,1999,28(3):491-496.

[4] study on a technique for analyzing bacterial lipopolysaccharide and an oligosaccharide chain structure thereof by Zhouyugu bright, Li Qian, Huang Chun Cui, et al, progress on biochemistry and biophysics m2017(01):50-58.

[5]Vinogradov,E.Structures of Lipopolysaccharides from Klebsiella pneumoniae. ELUCIDATION OF THE STRUCTURE OF THE LINKAGE REGION BETWEEN CORE AND POLYSACCHARIDE O CHAIN AND IDENTIFICATION OF THE RESIDUES AT THE NON-REDUCING TERMINI OF THE O CHAINS[J].Journal of Biological Chemistry,2002,277(28):25070-25081.

[6]Cooling L L,Walker K E,Gille T,et al.Shiga toxin binds human platelets via globotriaosylceramide(P^k antigen)and a novel platelet glycosphingolipid.[J].Infection&Immunity,1998,66(9):4355.

[7] The research progress of Haiguanjuan, Zhang Kai, Wang Zhi, et al, alpha-galactosidase [ J ] animal doctor in China, 2013,40(3):149-154.

[8]Folmer F.Crystal Structure ofα-galactosidase from Lactobacillus acidophilus NCFM:Insight into Tetramer Formation and Substrate Binding[J].J Mol Biol,2011,412:466-480.

[9] Catalytic mechanism and substrate specificity of photinia, prune suhong, xujie, et al.

Disclosure of Invention

The invention aims to provide a novel alpha galactosidase, a preparation method and application thereof based on the current situation and the foundation of the prior art.

The invention provides a novel alpha galactosidase polypeptide (Galase) obtained from Elizabethigia meninosis (FMS-007), a polynucleotide for coding the alpha galactosidase polypeptide, a preparation method and an application thereof. The invention also provides a method for preparing the alpha galactosidase, the invention can simultaneously enzyme-cut the terminal alpha 1,3, 1,4, 1,6Gal, provides a new tool for glycobiology and other applications, and has wide application prospect.

Specifically, the amino acid sequence of the alpha galactosidase of the invention is any one of the following two optional types:

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

or

b) Has more than 25 percent of homology with the sequence shown in SEQ ID NO1 and has alpha galactosidase activity.

The invention provides a recombinant vector, which comprises a nucleotide sequence for coding alpha galactosidase.

As a preferred mode of embodiment, the α -galactosidase gene is ligated to pET28a vector.

The invention provides an engineering bacterium containing a recombinant vector.

As a preferred mode of embodiment, the engineering bacterium is Escherichia coli BL21(DE3) for producing alpha galactosidase. The invention provides an expression and cloning method for coding the alpha galactosidase gene, which is an optimal method of an embodiment.

The invention provides a preparation method of the alpha galactosidase, which comprises the following steps:

1) obtaining and amplifying the gene sequence of the alpha galactosidase of claim 1;

2) constructing a recombinant vector containing the alpha galactosidase;

3) expressing the alpha galactosidase of claim 1;

4) separating, purifying and identifying.

The expression system may 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 the use of an alpha galactosidase enzyme.

Experiments show that the alpha galactosidase not only has hydrolase activity on an artificially synthesized chromogenic substrate pNP-alpha-D-Gal (except for special specification, the term pNP-Gal is equivalent to pNP-alpha-D-Gal in the invention), but also has hydrolytic activity on oligosaccharide chain terminals alpha 1,3, 1,4 and 1,6-Gal, and can catalyze and hydrolyze alpha 1,3-Gal at the sugar chain terminals on cell surface glycoprotein.

The alpha galactosidase enzyme hydrolyzes a synthetic galactose substrate, which in some embodiments may be p-pNP-alpha-D-Gal.

The alpha galactosidase hydrolyzes oligosaccharide chains that contain alpha 1,3, 1,4, 1,6-Gal at their termini, which in some embodiments can be blood antigen oligosaccharides or other oligosaccharides.

The blood antigen oligosaccharide or other oligosaccharide may be a p blood group determining antigen or other oligosaccharide, and in some embodiments the p blood group determining antigen or other oligosaccharide may be p^kEither antisense or Isoglobotriose, Melibiose.

The sugar chain of the cell surface glycoprotein may be a sugar chain of an animal cell surface glycoprotein, and in some embodiments, the animal cell may be a hog erythrocyte.

As shown in the present embodiment, the substrate on which the enzyme of the present invention acts mainly comprises 3 substrates modified with α -Gal, artificially synthesized chromogenic substrates, oligosaccharide chains and cell surface glycoprotein sugar chains. From the simple to the complex substrate, the enzyme can effectively carry out enzyme digestion, shows the activity of hydrolyzing the terminal alpha-Gal substrate, and has the characteristics of simple and convenient operation, high enzyme digestion efficiency and the like. Because the terminal alpha-Gal plays an important role in the race specificity and the infection of the host cell by the pathogen, the enzyme can be used for enzyme digestion of the sugar chain structure of the terminal alpha 1,3, 1,4, 1,6-Gal, the pig red blood cell with the race specificity removed from the terminal alpha 1,3-Gal is prepared, a tool enzyme is provided for the implementation of xenotransfusion, the enzyme can also be used for the research on the functions of the alpha-Gal in the identification process of the pathogen and the host cell surface, and the like, and has application value in the research of the process of interrupting the infection of the host by the pathogen.

The invention provides alpha-galactosidase, namely glycosidase with the function of removing end alpha 1,3, 1,4 and 1,6-Gal in Iressa meningitidis, provides a novel tool enzyme for glycobiology research, helps to develop erythrocytic blood transfusion from different sources, and provides a potential therapeutic tool for treating infectious diseases caused by pathogenic factors of pathogenic bacteria through recognizing end alpha-Gal signals on receptors, such as a drug product for treating the infectious diseases.

Drawings

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

FIG. 1: the enzyme kinetic parameters of Galase on the substrate pNP-Gal.

FIG. 2: the reaction pH range of Galase on the substrate pNP-Gal.

FIG. 3: reaction temperature range of Galase to pNP-Gal as a substrate.

FIG. 4: the influence of the ions on the enzyme activity of the Galase.

FIG. 5: the enzyme cutting result of Galase on the oligosaccharide (detected by a D-Galactose kit).

FIG. 6: and (4) carrying out enzyme cutting on the oligosaccharide by Galase (mass spectrometry detection).

FIG. 7: and (4) carrying out enzyme digestion on the surface antigen of the pig red blood cell by Galase (microscopic detection).

FIG. 8: and (3) carrying out enzyme digestion on the surface antigen of the pig red blood cell by Galase (flow detection).

FIG. 9: the alpha-Gal antigen is the terminal Gal-alpha 1, 3-Gal.

FIG. 10: LPS consists of lipid a, core polysaccharide and O antigen.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the 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 (Richard r. burgess) or can be performed according to the instructions provided by the reagent manufacturer.

Example 1: alpha galactosidase activity identification and enzymology property experiment

1. Alpha galactosidase Activity identification

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

Enzyme digestion reaction buffer solution: PBS (pH7.4)

Enzyme digestion system: the substrate pNP-alpha-D-Gal stock solution (5 ul), the enzyme (2 ul) and PBS were added to make up to a volume of 50 ul. In Table 1, the reference numeral 1 was used as an experimental group, and the reference numeral 2 was used as a control group.

TABLE 1 configuration of the reaction System

Reaction conditions are as follows: 30min at 37 ℃, and immediately adding 1M Na after the reaction is finished₂CO₃The reaction was terminated at 75ul and measured at OD 405nm using an enzyme standard. And judging whether the enzyme has alpha galactosidase activity according to the result.

(2) For the determination of the kinetic parameters of the enzyme:

standard curve: eight concentrations of pNPs were prepared as shown in Table 2, diluted with PBS in a 60ul reaction system, 59ul of the reaction solution diluted in a gradient was added to each well of a 96-well plate, 1ul of Galase diluted five times was added to each well, and each sample was repeated three times as a standard curve.

TABLE 2 setting of concentration gradient

Reaction conditions are as follows: keeping the temperature at 37 ℃ for 15min, adding 1M Na immediately after the reaction is finished₂CO₃The reaction was terminated at 75ul, and the OD 405nm was measured by a microplate reader.

And (4) processing a result: enzyme kinetic parameter K_m，K_cat，V_maxThe isoconstants were obtained by nonlinear regression statistics using GraphPad Prism (La Jolla, Calif.) software.

As a result, as shown in FIG. 1, the maximum reaction rate of the cleavage of pNP-Gal as a substrate was 0.19uM/s, and Km was 0.22 mM.

2. Determination of the optimum pH value of alpha galactosidase

53ul of pH7.4PBS, 5ul of 10mM pNP-Gal, 2ul of Galase per well, pH 1.1,2,3,4,5,6,7,8,9,10,11,12, and reaction at 37 ℃ for 30min according to the record of the Chinese pharmacopoeia, three replicates for each pH, 75ul of 1M Na₂CO₃The reaction was terminated, and OD 405nm was measured by a microplate reader. The results are shown in FIG. 2: the pH value is between 4 and 8, and the substrate pNP-Gal has large enzyme cutting activity.

3. Determination of optimum reaction temperature

54ul pH7.4PBS per well, 5ul 10mM pNP-Ga per welll, 1ul enzyme reaction for 15min, verifying enzyme activity under nine temperature gradients of 4 ℃,20 ℃, 30 ℃, 37 ℃, 45 ℃, 55 ℃, 65 ℃, 75 ℃ and 85 ℃, all sample adding operations are carried out at 4 ℃ to ensure the reliability of the experiment, and after the enzyme is added, the sample is immediately put into a water bath kettle at the corresponding temperature, all experiments are carried out for three times, and 75ul 1M Na is used for repeating₂CO₃The reaction was terminated, and OD 405nm was measured by a microplate reader. The results are shown in FIG. 3: the temperature is between 4 and 45 ℃, the activity is the largest at 20 ℃.

4. Determination of the Effect of Metal ions on glycosidase Activity

K with a concentration of 1M⁺，Mg²⁺，Mn²⁺，Ca²⁺，Zn²⁺，Ni²⁺，Fe²⁺，Cu²⁺EDTA and SDS as a storage solution, and 50ul of pH6.8CH as a storage solution according to the above-mentioned experimental method₃COONH₄In the system, each corresponding ion or EDTA, SDS was added to give a final concentration of 10mM, and all experiments were performed in triplicate. The results are shown in FIG. 4: at a concentration of 10mM, Zn²⁺、Mn²⁺、Ni²⁺、Mg²⁺、K⁺、Ca²⁺Has no obvious influence on the galactosidase activity of Galase, and Cu²⁺And Fe²⁺Has different degrees of inhibition on the enzyme activity of the Galase.

Example 2: digestion experiment of oligosaccharides

Configuration of the substrate: oligosaccharide Isoglotriose, p^Kanti, Melibiose, was formulated with ultrapure water to a concentration of 10 mM.

Method 1 (kit method):

reaction system: 2ul Galase, 3.11ul 10mM oligosaccharide substrate and 8.89ul pH7.4PBS, reacted at 37 ℃ for 2 h.

The detection is carried out according to D-Galactose kit (megazyme, Ireland), and the specific method refers to a kit handbook, the kit generates NADPH by NADP +, and the NADPH can be measured by absorbance value of 340nm and quantified according to standard, thereby quantifying Galactose cut by enzyme digestion oligosaccharide.

Method 2 (mass spectrometric detection):

reaction system: 10mM oligosaccharide substrate 1ul each, 5-fold diluted Galase 1ul, 9ul pH4CH₃COOH, reacting for 3h and 37 ℃, and adding 200ul of ultrapure water into each tube of reaction product for dilution.

And (3) carrying out high-resolution tandem multistage mass spectrometry on the LTQ Orbitrap XL.

The results are shown in FIGS. 5 and 6: galase detects the isologotriose, p after the oligosaccharide is cut by Galase^KThe three oligosaccharides of antisense and Melibiose have obvious enzyme cutting activity, which shows that the oligosaccharide has terminal alpha 1,3, 1,4 and 1,6 galactosidase activity.

Example 3: cell surface glycoprotein sugar chain cleavage experiment

The collected pig blood and the CPDA-1 blood preservation solution are mixed according to the proportion of 10: 1.5. Centrifuging at 4000r for 7min, removing supernatant and leukocyte layer, and resuspending red blood cells at ratio of remaining red blood cells to CPDA-1 of 10: 1.5. 1ml of 10% erythrocyte suspension is centrifuged at 1500r 2min and PCBS (60mmol/l NaH) is used₂PO₄25mmol/L NaCitrate, 75mmol/L NaCl) were washed 2 times and brought to 40% volume by pressure for use.

Method 1 (microscopic examination):

control and experimental reaction systems were prepared according to table 3.

TABLE 3 control and Experimental groups of reaction systems

After one hour reaction at room temperature, the erythrocytes were washed 2 times with 1500r, 2min, pH7.4PBS, and the erythrocytes were brought to 10% volume, 10ul of erythrocytes were mixed with 2ul of Bsi-B4 on a slide and observed by a microscope (10X 40).

Method 2 (flow assay):

TABLE 4 reaction systems for control and experimental groups

Reacting at room temperature for 1h, and keeping the mixture uniformly mixed without sedimentation. After reaction, each tube is added with 200ul of PBS (pH7.4PBS), washed for 2-3 times at 1500r for 2min, 200ul of PBS with pH value of 7.4 is used for resuspension, 30ul of resuspension liquid is taken, 500ul of 0.1% glutaraldehyde is added for fixation for no more than 15min, washed for 2-3 times at pH value of 7.2PBS, and 200ul of PBS with pH value of 7.2 is used for resuspension. Bsi-B4 was added as per Table 5.

TABLE 5 reaction systems for control and experimental groups

The reaction is carried out for 1h in a dark place, and the mixture is uniformly mixed without sedimentation. After the reaction, the cells were washed 2 to 3 times with PBS (pH 7.2), diluted to a certain number of cells, and then examined by FACS Calibur (BD Co., U.S.A.).

The results are shown in FIGS. 7 and 8: the microscopic examination and the flow detection prove that the Galase can cut the alpha 1,3-Gal antigen on the surface of the pig red blood cell.

Example 4: process for producing alpha galactosidase

The alpha galactosidase gene sequence is from whole genome sequencing data of a meningitis 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 on genes, an Open Reading Frame (ORF) is found, the length of the sequence is 1103bp, 368 amino acids are coded, the molecular weight is about 41.6kDa, and no signal peptide is possibly predicted by SignalP 4.1 software. The alpha galactosidase is likely to be predicted by NCBI for its conserved regions.

The invention adopts a genetic engineering method to clone a target gene into a prokaryotic expression vector, then the target gene is transformed into escherichia coli for expression, and the purified novel glycosidase is obtained through affinity chromatography.

1.1 methods and Processes of production

1.1.1 construction strategy of recombinant plasmid of alpha galactosidase gene

Obtaining an alpha galactosidase 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.

1.1.2 PCR amplification of the alpha galactosidase Gene

The reaction system for PCR amplification of the alpha galactosidase gene was prepared according to Table 6, mixed well, with amplification parameters of 98 ℃ 10s, 55 ℃ 15s, 72 ℃ 20s, 34 cycles, 72 ℃ 5 min. Electrophoresis was performed at 100V for 30min and photographed using a gel imaging system. The primer sequences for alpha galactosidase were as follows:

an upstream primer: 5'-TCCATATGATGTTGTTTTCTCAAAAAGCCAAAC-3' (SEQ ID NO 2)

A downstream primer: 5'-CGCTCGAGCTAATAAGTCTTCAGAACAATGATA-3' (SEQ ID NO 3)

TABLE 6 alpha galactosidase PCR amplification System

1.1.3 Targeted cloning of the alpha galactosidase PCR product

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

The DNA product purification kit recovers the PCR product and double-cleaves the PCR product and the vector with two restriction enzymes Nde I and Xho I. Preparing an enzyme cutting system according to the concentration of the purified target gene Galase and the vector and the concentration shown in the table 7, uniformly mixing, and performing enzyme cutting for 1h at 37 ℃. After the enzyme digestion is finished, recovering the enzyme digestion product by using a DNA product purification kit, and operating steps are as per the instruction.

TABLE 7 double digestion System for PCR products/plasmid DNA

(2) Ligation of Galase Gene and vector

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

TABLE 8 Galase 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 ℃. The transformation ligation products of (2) were all transformed into E.coli DH 5. alpha. competent cells, 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, mix gently, 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 1-2min without shaking the tubes.

3) The tube was added with 800. mu.l of sterilized LB liquid medium, mixed well and cultured at 37 ℃ for 4 hours with shaking at 200rpm to resuscitate the bacteria.

4) The bacterial liquid is centrifuged for 2min at 5000rpm, about half of the supernatant is discarded, the sediment is slightly blown up and uniformly mixed by a gun head, and then 100ul of the bacterial liquid is taken and coated on LB agar culture medium containing 50 ug/ml kanamycin antibiotic. The plate was inverted and incubated overnight at 37 deg.C (about 12 h).

5) After the plate grows out of the colonies, 8 large and full single colonies are selected in a dispersing way and are streaked respectively, and the single colonies are cultured overnight at 37 ℃.

6) Each streaked single colony was picked up and cultured in 5mL of LB liquid medium (containing 50. mu.g/mL kanamycin) at 37 ℃ for 10-14 hours with shaking at 200 rpm.

(4) PCR of bacterial liquid

1) Taking 100ul of cultured bacterial liquid, carrying out water bath at 95 ℃ for 10min, and centrifuging at 10000r for 1 min;

2) 1ul of the supernatant was used as a PCR template, and the other systems were prepared as in Table 4, with the same reaction conditions.

(5) Sequencing of bacterial solutions

According to the electrophoresis result of the PCR product of the bacterial liquid, 200ul of bacterial liquid with a single target band corresponding source is selected and sent to Jinzhi Gene company for sequencing.

(6) Extraction of recombinant plasmid

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

1) centrifuging the residual bacteria solution at 12000rpm for 1min at room temperature, sucking the supernatant as much as possible, and taking the precipitate.

2) To the pellet from the previous step 250. mu.l Buffer A1 (ensuring RNase A was added) was added and the bacterial cells were thoroughly suspended by pipetting or vortexing.

3) Then 250 μ LBuffer B1 (RNase A was added in advance) was added to the centrifuge tube, gently turned up and down 10 times to mix the bacteria evenly, and then left to stand for 5min until the solution was 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) 500mL Buffer KB was added to the DNA column, centrifuged at 12000rpm for 1min at room temperature, and the waste solution in the collection tube was decanted.

8) Add 500. mu.L of DNA wash buffer to the spin column (ensure ethanol addition), centrifuge at 12000rpm for 1min at room temperature, and discard the 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 centrifugal column into a new 1.5mL centrifugal tube, suspending and dropping 70 μ L of precipitation Buffer into the center of the adsorption membrane, standing at room temperature for 5min, centrifuging at 12000rpm for 1min, dropping the eluent into the center of the adsorption membrane again, 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.

1.2.1 prokaryotic expression of Galase

1.2.1.1 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 Galase recombinant plasmid was transformed into BL21(DE3) competent cells by sequencing verification, 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 with correct sequencing, mix gently, and stand on 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 600. mu.l of sterilized LB liquid medium, mixed well and cultured at 37 ℃ for 4 hours with shaking at 200rpm to resuscitate the bacteria.

4) 100ul of the resulting bacterial suspension was applied to LB agar medium containing 50. mu.g/ml kanamycin antibiotic by a pipette, and the plate was inverted and incubated overnight at 37 ℃ (about 12 hours).

1.2.1.2Galase protein expression

Firstly, under the condition of no inducer, when bacteria enter an optimal growth state, adding an inducer IPTG into a culture medium, and combining with repressor protein to remove inhibition, so that a large amount of exogenous genes are expressed;

the induction steps are as follows:

1) BL21 containing the Galase recombinant plasmid and an empty vector are selected as controls, single colonies are picked up and respectively inoculated into 5ml of LB culture medium (containing 50 mu g/ml kanamycin), the culture is carried out at 37 ℃ and 200rpm under shaking until OD600 is 0.6-0.8, 100ul to 1.5ml of centrifuge tubes are sampled, and the samples are stored at 4 ℃ and used for SDS-PAGE.

2) IPTG (final concentration of 0.1-10mM) is added into the bacterial liquid respectively, and shaking culture is carried out for 12h at 28 ℃ and 160rpm, so as to induce the expression of the target gene Galase.

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, 7.5. mu.L of 5 XSDS loading buffer was added, the mixture was boiled at 95 ℃ for 10min, and the sample was centrifuged at 12000rpm for 10 min.

6) And (3) sampling 8 mu L of supernatant sample, sampling 6ul of marker, performing SDS-PAGE by using 80V, and adjusting the voltage to 120V 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.

7) Dyeing with Coomassie brilliant blue for 1h, decolorizing with decolorizing solution overnight, and photographing to store the result.

1.2.2 purification of the recombinant protein Galase

And purifying the recombinant protein Galase through nickel column affinity chromatography. All manipulations of the purification were carried out at 4 ℃.

A. Purification by nickel column

1) The expression strain Galase-pet28a-BL21(DE3) was expanded to 1L at 200r 37 ℃.

2) The induced bacteria liquid is respectively added into two large centrifuge tubes, centrifuged for 10min at the temperature of 4 ℃ and the rpm of 5000, and the supernatant is discarded.

3) To the pellet of the previous step, 30mL of 1 XPBS was added, respectively, to thoroughly resuspend the cells.

4) Using an ultrahigh pressure homogenizer (model: FB-110X, Shanghai Shakou mechanical engineering Co., Ltd., Shanghai) for disrupting the mycelia.

5) 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.

6) The supernatant was fully combined with a nickel column prepared in advance at 4 ℃ for 2h and centrifuged at 400r for 2 min. The packing was loaded on an empty column during which washing with 25mM imidazole was performed 2-3 times, and then the packing was washed with 1ml of different concentrations (50mM, 100mM, 200mM, 500mM) of imidazole buffer, and the eluates were collected separately. Selecting proper eluent by SDS-PAGE, dialyzing by using PBS (pH7.4PBS) as a dialysate, changing the dialysate 1 time every 6h for 4 times in total, and collecting the Galase after dialysis. The Pierce BCA protein quantitative assay kit determines the enzyme concentration.

2 the results show that:

2.1Galase/pET28a recombinant plasmid construction

The target gene is cloned to pET28a vector, and the sequencing is carried out to verify that the recombinant plasmid is successfully constructed.

2.2IPTG inducible expression

And (3) after sequencing and identification of the recombinant positive clone are correct, transforming the recombinant positive clone to E.coli BL21, randomly selecting two monoclonals, respectively inoculating the monoclonals to an LB culture medium, adding 0.5mMIPTG to induce 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 Galase

The recombinant protein Galase can obtain relatively pure protein with purity higher than 90% after nickel column affinity chromatography purification. Satisfies the common biochemical enzyme digestion experiment.

Experiments prove that the alpha-galactosidase provides a new tool enzyme for glycobiology research, helps to develop erythrocytic transfusion from different sources, and provides a potential therapeutic tool for treating infectious diseases caused by pathogenic factors through recognizing terminal alpha-Gal signals on receptors.

SEQUENCE LISTING

<110> university of Compound Dan

<120> alpha galactosidase and preparation method and application thereof

<130> 20190607

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<170> PatentIn version 3.3

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Met Leu Phe Ser Gln Lys Ala Lys Pro Pro Ile Met Gly Trp Ser Ser Trp Asn Asn Phe

Arg Ile Asn Ile Asn Glu Gln Leu Ile Lys Glu Gln Ala Asp Ala Leu Val Ser Ser Gly

Leu Tyr Ala Ala Gly Tyr Arg Tyr Ile Asn Val Asp Asp Gly Tyr Phe Gly Gly Arg Asp

Glu Lys Gly Asn Leu Ile Thr Asp Asn Lys Lys Phe Pro Ser Gly Met Lys Asn Leu Ala

Ala Tyr Ile His Ser Lys Gly Leu Lys Ala Gly Ile Tyr Ser Asp Ala Gly Lys Asn Thr

Cys Gly Ser Ile Trp Asp Asn Asp Lys Gln Gly Phe Gly Val Gly Leu Tyr Gly His Leu

Asp Gln Asp Ala Asp Leu Phe Phe Lys Asp Trp Lys Tyr Asp Phe Leu Lys Val Asp Trp

Cys Gly Gly Glu Gln Met Lys Leu Asn Glu Gln Glu Glu Tyr Thr Lys Ile Ile Asn Lys

Val Lys Ser Ile Asp Pro Asn Ile Val Phe Asn Val Cys Arg Trp Gln Phe Pro Gly Glu

Trp Ala Ile Lys Ile Ala Asp Ser Trp Arg Val Ser Gly Asp Ile Ser Ala Lys Phe Ser

Ser Ile Leu His Ile Ile Asp Leu Asn Lys Asn Leu Tyr Ser Tyr Ala Ser Ala Gly His

Tyr Asn Asp Met Asp Met Leu Gln Val Gly Arg Gly Met Ser Tyr Asp Glu Asp Lys Thr

His Phe Ser Met Trp Ala Leu Leu Asn Ser Pro Leu Leu Ala Gly Asn Asp Leu Arg Ser

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Claims (10)

1. An alpha galactosidase enzyme having the amino acid sequence of either:

a. has a sequence shown as SEQ ID NO 1;

or

b. Has more than 25 percent of homology with the sequence shown in SEQ ID NO1 and has alpha galactosidase activity.

2. A nucleic acid encoding an alpha galactosidase enzyme, wherein said nucleic acid encodes the alpha galactosidase enzyme of claim 1.

3. A vector comprising the nucleic acid of claim 2.

4. The method for preparing alpha galactosidase according to claim 1, comprising the steps of:

1) obtaining and amplifying the gene sequence of the alpha galactosidase of claim 1;

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

3) expressing the alpha galactosidase of claim 1, said system of expression being a bacterial expression system;

4) separating, purifying and identifying.

5. The method of claim 4, wherein the method comprises conventional microbial fermentation production, expression and production in a bacterial expression system using bioengineering techniques.

6. The use of an alpha galactosidase of claim 1 for hydrolyzing enzymatic proteins, wherein said alpha galactosidase is capable of hydrolyzing pNP- α -D-Gal, oligosaccharides comprising terminal galactose residues and galactose capable of hydrolyzing terminal sugar chains of cell surface glycoproteins.

7. Use of the α -galactosidase according to claim 1 for cleaving a cell surface glycoprotein sugar chain terminal galactosyl group.

8. The use according to claim 6, wherein the junction of the glycoprotein carbohydrate chain is a terminal galactose connected to α 1,3, 1,4, 1, 6.

9. The use of claim 6, wherein said glycoprotein substrate is a porcine red blood cell surface antigen.

10. Use of an alpha galactosidase according to claim 1 for the manufacture of a pharmaceutical product for the treatment of an infectious disease, wherein said infectious disease is an infectious disease caused by pathogenic agents of a pathogen by recognition of a receptor terminal alpha-Gal signal.

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