CN112795583A - Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method - Google Patents

Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method Download PDF

Info

Publication number
CN112795583A
CN112795583A CN202011275437.8A CN202011275437A CN112795583A CN 112795583 A CN112795583 A CN 112795583A CN 202011275437 A CN202011275437 A CN 202011275437A CN 112795583 A CN112795583 A CN 112795583A
Authority
CN
China
Prior art keywords
amuc
recombinant
sialic acid
expression vector
exonuclease
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202011275437.8A
Other languages
Chinese (zh)
Inventor
孙正龙
顾依然
关淼
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Kunming University of Science and Technology
University of Shanghai for Science and Technology
Original Assignee
Kunming University of Science and Technology
University of Shanghai for Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Kunming University of Science and Technology, University of Shanghai for Science and Technology filed Critical Kunming University of Science and Technology
Priority to CN202011275437.8A priority Critical patent/CN112795583A/en
Publication of CN112795583A publication Critical patent/CN112795583A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag

Abstract

The invention discloses a preparation method of recombinant sialic acid exonuclease, an expression gene, a recombinant expression vector and a construction method, wherein the expression gene comprises a nucleotide sequence shown as SEQ ID No.1, a restriction enzyme site NdeI, a restriction enzyme site PstI and 6 xHisTag positioned at the carboxyl end of a recombinant protein. The recombinant sialic acid exonuclease AMUC _0625 gene can realize a large amount of soluble expression in escherichia coli, and the expressed sialic acid exonuclease AMUC _0625 has the biological activity specific to sialic acid, can be used for degrading alpha-sialic acid at the tail end of a glycoprotein sugar chain, and has important significance for biological pharmacy and clinical treatment; the recombinant protein AMUC _0625 provided by the invention is a novel sialic acid exonuclease with application prospect.

Description

Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of recombinant sialic acid exonuclease, an expression gene, a recombinant expression vector and a construction method.
Background
Sialic acid is a generic name for a group of acidic amino sugars having 9 carbon atoms and a pyranose structure, and is a natural carbohydrate, and more than 50 derivatives thereof, mainly including three types: -acetylneuraminic acid (Neu5Ac), -hydroxyneuraminic acid (Neu5Gc) and-keto-deoxynonaketonic acid (KDN). The so-called sialidon refers to N-acetylneuraminic acid, usually in the form of an α -glycoside at the non-reducing end of a glycoprotein or glycolipid. Sialic acids are involved directly or indirectly in a variety of cellular activities, including 1) themselves as recognized receptors, 2) intercellular information transfer, and molecules such as hormones, lectins, antibodies, etc. are all ligands for sialic acids, 3) modulation of immune responses and cellular adhesion processes, and masking by preventing or attenuating exposure of cellular molecules to specific recognition sites. For example, sialic acid at the end of a sugar chain can mask recognition sites and prevent recognition of some important antigen sites on the cell surface; meanwhile, the protective cap is also a protective cap of the sugar chain, so that the viscosity of the mucin is increased, the sugar chain in the mucin can be effectively prevented from being degraded by specific glycosidase, and the protein can be protected from being degraded by protease.
Sialidases include 4 species: (1) sialic acid endonuclease (2) sialic acid exonuclease (3) transfer sialidase (4) sialic acid glycosyltransferase, wherein sialic acid exonuclease is a glycoside hydrolase that can hydrolyze sialic acid residues that cleave α - (2 → 3), α - (2 → 6), and α - (2 → 8) bonds, and remove sialic acid from the non-reducing end of a sugar chain. The sialidase has application potential in various fields such as biological pharmacy, clinical medicine, food and the like.
Prokaryotic expression is a biological technology that foreign target genes are constructed into expression plasmid vectors through experimental means such as molecular cloning, fragment connection and the like, the expression plasmids are poured into proper expression strains, a specific induction means is utilized to induce a large amount of expression of target proteins, and then separation and purification are carried out to obtain the target proteins. Prokaryotic expression combines various molecular biological means including gene engineering, cell engineering, protein engineering and the like, has the advantages of short period, simple and convenient operation and low cost, and is provided with various optional expression elements, expression vector plasmids and host bacteria. The target protein obtained by prokaryotic expression can play a role in the research and application of biomedicine, structural biology, cell biology and other fields.
However, no scheme for stably expressing the sialic acid exonuclease by using a prokaryotic expression system exists at present.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of recombinant sialic acid exonuclease, an expression gene, a recombinant expression vector and a construction method aiming at the defects in the prior art.
In order to solve the technical problems, the invention adopts the technical scheme that: provides an expression gene of a recombinant sialic acid exonuclease AMUC _0625, which comprises a nucleotide sequence shown as SEQ ID No.1, a restriction enzyme cutting site NdeI, a restriction enzyme cutting site PstI and 6 XHis Tag positioned at the carboxyl terminal of a recombinant protein.
The invention also provides a prokaryotic expression vector of the recombinant sialic acid exonuclease AMUC _0625, which comprises the expression gene of AMUC _ 0625.
Preferably, the prokaryotic expression vector selected is pMAL-c 5X.
The invention also provides a construction method of the prokaryotic expression vector of the recombinant sialic acid exonuclease AMUC _0625, which comprises the following steps:
1) acquiring a nucleotide sequence of sialic acid exonuclease AMUC _0625, and designing an amplification primer;
2) obtaining the whole cDNA of Achromobacter incarnatum;
3) cloning the AMUC _0625 gene from the cDNA by using the amplification primer;
4) and connecting the cloned AMUC _0625 gene with the prokaryotic expression vector pMAL-c5X to obtain a recombinant prokaryotic expression vector pMAL-c5X-Amuc _ 0625.
Preferably, the step 1) specifically includes:
obtaining a nucleotide sequence of the sialic acid exonuclease AMUC _0625 according to transcriptome sequencing data and proteome comparison of Achromobacter incarnatum recorded in a GenBank database, and removing a C-terminal signal peptide through bioinformatics analysis;
designing an amplification primer: a primer for amplifying the AMUC _0625 gene was obtained by adding a restriction site NdeI to the 5 'end of the primer, and adding an amino acid sequence of 6 XHis Tag and a restriction site PstI to the 3' end of the primer.
Preferably, the amplification primers comprise an upstream primer shown as SEQ ID No.3 and a downstream primer shown as SEQ ID No. 4.
The invention also provides a recombinant fusion protein AMUC _0625 which is obtained by encoding the nucleotide sequence shown in SEQ ID No.1, and the amino acid sequence of the recombinant fusion protein AMUC _0625 is shown in SEQ ID No. 2.
The invention also provides a preparation method of the recombinant fusion protein AMUC _0625, which comprises the following steps:
a) transforming a recombinant prokaryotic expression vector pMAL-c5X-Amuc _0625 into Escherichia coli Rossata (DE 3);
b) inducing pMAL-c5X-Amuc _0625 to express in Escherichia coli Rossata (DE3) to obtain a fusion protein MBP-AMUC _ 0625;
c) and carrying out enzyme digestion and purification on the obtained fusion protein MBP-AMUC _0625 to obtain a recombinant protein AMUC _ 0625.
The invention has the beneficial effects that:
(1) the recombinant sialic acid exonuclease AMUC _0625 gene can realize a large amount of soluble expression in escherichia coli, and the expressed sialic acid exonuclease AMUC _0625 has the biological activity specific to sialic acid, can be used for degrading alpha-sialic acid at the tail end of a glycoprotein sugar chain, and has important significance for biological pharmacy and clinical treatment;
(2) the recombinant protein AMUC _0625 provided by the invention is a novel sialic acid exonuclease with application prospect;
(3) the prokaryotic expression vector of the invention utilizes the technical means of enzyme digestion and enzyme linkage, the construction is simple and convenient, the expression system adopts the fusion expression of MBP and the target protein, the soluble expression of the target protein is enhanced, the purification system is mature, and the recombinant protein AMUC _0625 with biological activity can be stably obtained;
(4) compared with the NCBI published sequence, the amino acid sequence of the recombinant protein AMUC _0625 obtained by the invention cuts off a C-terminal signal peptide to prevent the secretion expression of the recombinant protein from being influenced, and the recombinant protein has biological activity, thereby proving the operability of the whole scheme provided by the invention.
Drawings
FIG. 1 shows the results of cloning and screening of the prokaryotic expression vector pMAL-c5X-Amuc _0625 for recombinant proteins of the sialic acid exonuclease AMUC _0625 in an example of the present invention;
FIG. 2 is a plasmid map of a prokaryotic expression vector pMAL-c5X-Amuc _0625 of a recombinant protein of the sialic acid exonuclease AMUC _0625 in an embodiment of the present invention;
FIG. 3 is a SDS-PAGE electrophoresis of a fusion protein MBP-AMUC _0625 of maltose binding protein and sialyl exonuclease purified by Ni column in an example of the present invention;
FIG. 4 is a graph showing the reaction rate of the sialic acid exonuclease AMUC _0625 of the present invention at different temperatures;
FIG. 5 is a graph showing the reaction rate of the sialic acid exonuclease AMUC _0625 of the present invention under different pH conditions.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The invention provides an expression gene of recombinant sialic acid exonuclease AMUC _0625, which comprises a nucleotide sequence shown as SEQ ID No.1, a restriction enzyme cutting site NdeI, a restriction enzyme cutting site PstI and 6 × His Tag positioned at the carboxyl terminal of a recombinant protein.
The invention also provides a prokaryotic expression vector of the recombinant sialic acid exonuclease AMUC _0625, which comprises the expression gene. In a preferred embodiment, the prokaryotic expression vector selected is pMAL-c 5X. The invention further provides a construction method of the prokaryotic expression vector of the recombinant sialic acid exonuclease AMUC _ 0625.
The invention also provides a recombinant fusion protein AMUC _0625, wherein the amino acid sequence of the recombinant fusion protein AMUC _0625 is shown in SEQ ID No.2, and the nucleotide sequence coding amino acid sequence shown in SEQ ID No.1 is shown in SEQ ID No.2, so that the recombinant fusion protein AMUC _0625 is provided. The invention further provides a preparation method of the recombinant fusion protein AMUC _ 0625.
The present invention will be further described with reference to the following examples.
Example 1 preparation method of recombinant sialyl exonuclease AMUC _0625, expression gene, recombinant expression vector, and vector construction method thereof
1. Primer design
A Genbank database Amuc _0625 gene sequence (NM _053615) in the American National Center for Biotechnology Information was queried, and after bioinformatics analysis to remove the C-terminal signal peptide, PCR primers were designed to amplify the Amuc _0625 gene (SEQ ID NO: 1) by adding an NdeI cleavage site at the 5 ' end, a 6 XHis Tag sequence at the 3 ' end, and a PstI cleavage site at the 5 ' end of Amuc _ 0625.
Upstream primer 5'-CATATGAGCGCCGGGGAGGGTAATCCC-3' (SEQ ID NO: 3)
Downstream primer 5'-CTGCAGCTACTTGAGAACAGGAGC-3' (SEQ ID NO: 4)
2. Culture of Achiminella anserinum and RNA acquisition
Inoculating Achiminella anserinum into a special culture medium, wherein the components of the culture medium are shown in tables 1 and 2, culturing under the culture conditions of anaerobic property, 37 ℃ and 100rpm until 0D600 is 1, centrifuging at the room temperature of 12000rpm for 10min, removing supernatant, collecting bottom thalli, adding 1.0ml of Trilzol reagent into every 1g of wet weight thalli, and uniformly mixing by shaking; standing at room temperature for 5 minutes, adding 0.2ml of chloroform into 1ml of the mixture, standing at room temperature for 5 minutes, and centrifuging at 4 ℃ and 12000rpm for 15 minutes to separate layers; transferring the upper aqueous phase into a clean EP tube, adding equal volume of isopropanol, standing at room temperature for 10 minutes, and centrifuging at 4 ℃ and 12000rpm for 10min to precipitate RNA; washing the precipitate with 75% ethanol for 2 times; the pellet was dissolved with rnase-free DEPC water. Centrifuging (12000g, 10 min), collecting precipitate, adding RNaseH, and heating at 37 deg.C for 20 min; storing at-20 deg.C.
TABLE 1 basic Medium ingredient Table
Figure BDA0002778877490000051
TABLE 2 ingredient tables for acid trace solution, alkali trace solution and vitamin solution
Figure BDA0002778877490000052
3. Obtaining cDNA
Reverse transcription of RNA to cDNA was performed by RT-PCR using a reverse transcription kit from TOYOBO, comprising the steps of:
3-1, RNA denaturation
Incubating RNA at 65 deg.C for 5min, immediately cooling on ice;
3-2, removing genome DNA, wherein the total system is as follows:
4×DN MASTER MIX 10μl
gDNA Remover 0.2μl
RNA 2μg
addition of Nuclear-free water to 40. mu.l
Mixing the reaction solution uniformly, and incubating for 5min at 37 ℃;
3-3, reverse transcription reaction
Mu.l of 5 XTT MASTER MIXII was added to the above reaction solution, and the reaction was carried out according to the following procedure:
1)37℃15min；
2)50℃5min；
3)98℃5min；
4)4℃HOLD；
after the reaction, the obtained cDNA was stored at-20 ℃.
4. Cloning and obtaining Amuc _0625 gene
Cloning Amuc _0625 gene from cDNA using specific primers for Amuc _0625 gene, and performing PCR using high fidelity enzyme from Takara corporation, the total system is 50 μ L, and comprises:
2×PrimeSTAR Max Premix 25μl
primer-F (10. mu.M) 1. mu.l
primer-R (10. mu.M) 1. mu.l
cDNA 200μg
ddH2O to 50. mu.l;
the amplification procedure was:
1)95℃5min；
2) 10s at 95 ℃; 15s at 59 ℃; 30s at 72 ℃; 34 cycles of treatment;
3)72℃10min。
5. construction of recombinant plasmid pMAL-c5X-Amuc _0625
By utilizing the technical means of enzyme digestion and enzyme linkage, the Amuc _0625 gene obtained by PCR cloning and the pMAL-c5X plasmid are integrated to obtain a recombinant expression plasmid pMAL-c5X-Amuc _0625, and the specific steps are as follows:
5-1, restriction enzyme
The double-restriction reaction system of the original plasmid pMAL-c5X is as follows:
10×BUFFER 5μl
pMAL-c5X plasmid 2. mu.l
NdeI 2μl
PstI 2μl
ddH2O to 50. mu.l;
the digestion conditions were 37 ℃ for 2 h.
The double enzyme digestion reaction system of Amuc _0625 is as follows:
10×BUFFER 5μl
Amuc_0625 2μl
NdeI 2μl
PstI 2μl
ddH2O to 50. mu.l;
the digestion conditions were 37 ℃ for 2 h.
After the digestion reaction, 5. mu.l of 10 XLOADING BUFFER was added to terminate the reaction, and after electrophoresis in 1% agarose gel, the target band was cut and purified and recovered using a TIANGEN kit
5-2, enzymatically linked to
Amuc _0625 and pMAL-c5X were enzymatically ligated using complementary cohesive ends formed after double digestion
The enzyme connecting system is as follows:
10×T4 Ligase buffer 5μl
amuc _ 062550 ng after double enzyme digestion
After double digestion, pMAL-c5X 50ng
T4 DNA Ligase 2μl
ddH2O to 50. mu.l;
reacting at 4 ℃ overnight, cutting the product after enzyme connection and recovering the product to obtain the recombinant plasmid pMAL-c5X-Amuc _ 0625.
5-3, recombinant plasmid transformation of escherichia coli and positive clone screening
Competent cells, Rossata (DE3) (100. mu.l), were removed from a-80 ℃ freezer and lysed on ice; mu.l of recombinant plasmid pMAL-c5X-Amuc _0625 was added to Rossata (DE3) and incubated on ice for 30 min; the plasmid and competent cell mixture was heat-shocked in water bath at 42 ℃ for 30s, immediately transferred to ice and incubated for 2 min; adding 900 μ l LB culture medium without antibiotic, culturing at 37 deg.C and 200rpm in shaker for 1 h; uniformly spreading 200 μ l of the transformant on LB medium plate containing 100 μ g/ml ampicillin with a coating rod, placing the plate in a 37 deg.C incubator for 20min, inverting, and culturing overnight; selecting a single clone for culturing, extracting plasmids for identification, wherein the identification result is shown in figure 1, which indicates that the expression plasmids are successfully constructed.
Positive clones were selected and sequenced, which indicated that the gene sequence was consistent with that published by Genebank, and the recombinant expression plasmid pMAL-c5X-Amuc _0625 is shown in FIG. 2.
6. Expression of the fusion protein MBP-AMUC _0625
6-1, according to 1: 1000, adding 50 μ l of the monoclonal culture into 50ml LB liquid medium containing 100 μ g/ml ampicillin, culturing at 37 ℃ and 200rpm for about 4h, wherein OD600 is 0.6-0.8, adding isopropyl- β -D-thiogalactoside (IPTG), and culturing in a constant temperature shaking table. Different groups are set according to different induction conditions: firstly, inducing for 6 hours at 37 ℃ and 100rpm with IPTG concentration of 0.1 mM; ② inducing for 6h at 37 ℃ and 100rpm with IPTG concentration of 0.5 mM; ③ inducing for 6 hours at 37 ℃, 100rpm and IPTG concentration of 1.0 mM; fourthly, inducing for 6 hours at 20 ℃, 100rpm and 0.1mM IPTG concentration; inducing at 20 deg.c and 100rpm for 6 hr with IPTG concentration of 0.5 mM; sixthly, inducing for 6h at 20 ℃, 100rpm and 1.0mM IPTG. Each group of cultures was collected in 50ml centrifuge tubes, centrifuged at 8000rpm for 10min at 4 ℃ to precipitate the cells, washed 2 times with PBS, resuspended in 15ml PBS containing 1mM PMSF and 1mM lysozyme, and placed in a-20 ℃ freezer overnight.
6-2, taking the bacterial suspension out of the temperature of minus 20 ℃, dissolving on ice, resuspending the bacteria by using a vortex oscillator, carrying out ultrasonic disruption in ice bath for 15min at the power of 300W for 5s at intervals of 5s, carrying out centrifugation for 10min at 12000rpm for cell disruption suspension at the temperature of 4 ℃, collecting the supernatant, and adding 15ml of PBS into the precipitate for resuspension.
6-3, respectively taking the supernatant and the precipitate 40 μ l, adding 5 × Loading buffer 10 μ l, carrying out metal bath at 100 ℃ for 10min to completely denature the protein, and carrying out 10% SDS-PAGE electrophoresis detection, wherein as can be seen in a figure X, the MBP-AMUC _0625 fusion protein has higher expression level in host bacteria Rossette (DE3), most of the MBP-AMUC _0625 fusion protein is located in the supernatant, and a certain amount of the MBP-AMUC _0625 fusion protein also exists in inclusion bodies.
6-4, purifying the supernatant containing the target fusion protein MBP-AMUC _0625 by a Ni column, eluting by 20, 50, 100, 250 and 500mM imidazole (pH 7.4), collecting the eluate, quantifying by using a BCA protein quantification kit, and carrying out SDS-PAGE electrophoresis detection, wherein the result is shown in FIG. 3, and the 250mM imidazole can elute high-concentration target protein.
6-5, the eluted target protein was concentrated using an ultrafiltration tube, and finally dissolved in Column buffer (20mM Tris-HCl, 0.2M NaCl, 1mM EDTA, 1mM DTT). Factor Xa enzymes recognize specific Ile- (Glu/Asp) -Gly-Arg between the tag proteins MBP and AMUC _0625 and hydrolyze arginine residues. Adding the purified fusion protein MBP-AMUC _0625 into the protein according to the ratio of 1: adding Xa factor enzyme according to the weight ratio of 100, and reacting for 24h at room temperature to obtain MBP and AMUC _0625 after enzymolysis separation. And (3) passing the enzyme-digested protein through an Amylose purification Column, collecting flow-through liquid to obtain separated AMUC _0625, and eluting with Column buffer containing 10mM maltose to obtain MBP.
Example 2 Activity and Biochemical index detection of recombinant protein AMUC _0625
The activity of 4MU-NANA (4-methylumbelliferone-alpha-acetylneuraminic acid) is detected as a substrate of an sialic acid exonuclease AMUC _0625, and the AMUC _0625 can hydrolyze 4MU-NANA to generate 4MU and acetylneuraminic acid, wherein the 4MU is a fluorescent dye, the maximum absorption light wavelength is 365nm, and the maximum emission light wavelength is 450 nm. After drawing the 4MU standard curve, 4MU fluorescence values were detected, which can reflect the rate of AMUC _0625 hydrolysis of 4 MU-NANA.
The reaction system is as follows:
1mM 4MU-NANA 20μl
48μg/ml AMUC_0625 1μl
79 μ l of reaction buffer;
after 20min at room temperature, 200. mu.l of a reaction terminator (100mM glycine/sodium hydroxide, pH10.7) was added to terminate the reaction, and the fluorescence value was measured. Setting different reaction temperatures (25-65 ℃) in the same reaction system, and detecting fluorescence values; under the same reaction system, after changing the pH value of the reaction buffer solution, the reaction is carried out, the fluorescence value is detected, and the optimal reaction temperature and the optimal pH value of AMUC _0625 are obtained by analysis, as shown in figure 4 and figure 5, the optimal reaction temperature of AMUC _0625 is 35 ℃, and the optimal pH value is 5.6.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
The applicant: university of Shanghai Kunming science university
The invention name is as follows: preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method
SEQ NO.1
AGCGCCGGGGAGGGTAATCCCTATGCGTCCATCCGTATTCCCGCCCTGCTCAGTATCGGCAAGGGCCAGCTTCTGGCATTCGCCGAAGGACGGTACAAAAATACCGACCAGGGGGAGAACGATATTATCATGAGCGTCAGCAAGAATGGCGGGAAGACCTGGTCCCGTCCCCGGGCGATAGCCAAGGCCCATGGCGCCACGTTCAATAATCCGTGCCCCGTTTATGATGCCAAAACCAGGACCGTGACTGTCGTATTCCAGCGTTACCCTGCCGGGGTCAAGGAGCGGCAGCCCAATATCCCGGACGGATGGGATGATGAAAAGTGCATCCGCAATTTCATGATTCAGAGCAGGAACGGAGGTTCTTCCTGGACGAAGCCGCAGGAGATCACGAAGACGACCAAGCGTCCTTCCGGAGTGGATATTATGGCGTCCGGCCCGAATGCGGGAACCCAGCTGAAGAGCGGCGCCCACAAGGGCCGCCTGGTGATTCCGATGAATGAAGGGCCGTTCGGCAAATGGGTGATTTCCTGCATTTACAGCGATGACGGCGGCAAGAGCTGGAAGCTGGGCCAGCCGACTGCCAATATGAAGGGCATGGTGAACGAGACGTCCATTGCGGAAACGGATAACGGCGGCGTTGTGATGGTTGCGCGCCATTGGGGCGCAGGCAATTGCCGCCGTATTGCGTGGTCCCAGGATGGCGGGGAGACCTGGGGACAGGTGGAGGACGCTCCGGAGCTGTTTTGCGACAGTACCCAGAATTCCCTGATGACGTATTCCCTGAGCGACCAGCCTGCCTATGGCGGCAAAAGCCGCATTCTGTTTTCCGGGCCCAGTGCGGGCCGGCGCATTAAGGGACAGGTGGCCATGAGCTATGACAACGGCAAGACCTGGCCGGTGAAGAAATTGCTGGGCGAGGGCGGTTTTGCCTATTCCAGCCTTGCCATGGTGGAACCCGGCATCGTTGGGGTGCTTTATGAGGAGAACCAGGAGCATATTAAAAAGCTGAAGTTTGTTCCCATTACCATGGAATGGCTGACGGACGGAGAAGACACAGGGCTGGCTCCCGGCAAAAAAGCTCCTGTTCTCAAGCATCATCACCATCACCATTAA
SEQ NO.2
SAGEGNPYASIRIPALLSIGKGQLLAFAEGRYKNTDQGENDIIMSVSKNGGKTWSRPRAIAKAHGATFNNPCPVYDAKTRTVTVVFQRYPAGVKERQPNIPDGWDDEKCIRNFMIQSRNGGSSWTKPQEITKTTKRPSGVDIMASGPNAGTQLKSGAHKGRLVIPMNEGPFGKWVISCIYSDDGGKSWKLGQPTANMKGMVNETSIAETDNGGVVMVARHWGAGNCRRIAWSQDGGETWGQVEDAPELFCDSTQNSLMTYSLSDQPAYGGKSRILFSGPSAGRRIKGQVAMSYDNGKTWPVKKLLGEGGFAYSSLAMVEPGIVGVLYEENQEHIKKLKFVPITMEWLTDGEDTGLAPGKKAPVLKHHHHHH
SEQ NO.3
5′-CATATGAGCGCCGGGGAGGGTAATCCC-3′
SEQ NO.4
5′-CTGCAGCTACTTGAGAACAGGAGC-3′

Claims (8)

1. An expression gene of a recombinant sialic acid exonuclease AMUC _0625, which is characterized by comprising a nucleotide sequence shown as SEQ ID No.1, a restriction enzyme site NdeI, a restriction enzyme site PstI and 6 XHis Tag positioned at the carboxyl terminal of a recombinant protein.
2. A prokaryotic expression vector of recombinant sialyl exonuclease AMUC _0625, comprising the expressed gene of claim 1.
3. The recombinant exosialidase AMUC _0625 prokaryotic expression vector of claim 2, wherein the prokaryotic expression vector selected is pMAL-c 5X.
4. A method for constructing a prokaryotic expression vector of the recombinant sialyl exonuclease AMUC _0625 as claimed in claim 2 or 3, comprising the steps of:
1) acquiring a nucleotide sequence of sialic acid exonuclease AMUC _0625, and designing an amplification primer;
2) obtaining the whole cDNA of Achromobacter incarnatum;
3) cloning the AMUC _0625 gene from the cDNA by using the amplification primer;
4) and connecting the cloned AMUC _0625 gene with the prokaryotic expression vector pMAL-c5X to obtain a recombinant prokaryotic expression vector pMAL-c5X-Amuc _ 0625.
5. The method for constructing the prokaryotic expression vector of the recombinant sialyl exonuclease AMUC _0625 as claimed in claim 4, wherein the step 1) specifically comprises:
obtaining a nucleotide sequence of the sialic acid exonuclease AMUC _0625 according to transcriptome sequencing data and proteome comparison of Achromobacter incarnatum recorded in a GenBank database, and removing a C-terminal signal peptide through bioinformatics analysis;
designing an amplification primer: a primer for amplifying the AMUC _0625 gene was obtained by adding a restriction site NdeI to the 5 'end of the primer, and adding an amino acid sequence of 6 XHis Tag and a restriction site PstI to the 3' end of the primer.
6. The method for constructing the prokaryotic expression vector of the recombinant sialyl exonuclease AMUC _0625 as claimed in claim 5, wherein the amplification primer comprises an upstream primer shown in SEQ ID No.3 and a downstream primer shown in SEQ ID No. 4.
7. A recombinant fusion protein AMUC _0625 encoded by the nucleotide sequence of SEQ ID No.1 as defined in claim 1, wherein the amino acid sequence of said recombinant fusion protein AMUC _0625 is as set forth in SEQ ID No. 2.
8. A method of preparing the recombinant fusion protein AMUC _0625 according to claim 7, comprising the steps of:
a) transforming the recombinant prokaryotic expression vector pMAL-c5X-Amuc _0625 obtained in claim 4 into E.coli Rossata (DE 3);
b) inducing pMAL-c5X-Amuc _0625 to express in Escherichia coli Rossata (DE3) to obtain a fusion protein MBP-AMUC _ 0625;
c) and carrying out enzyme digestion and purification on the obtained fusion protein MBP-AMUC _0625 to obtain a recombinant protein AMUC _ 0625.
CN202011275437.8A 2020-11-16 2020-11-16 Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method Pending CN112795583A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011275437.8A CN112795583A (en) 2020-11-16 2020-11-16 Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011275437.8A CN112795583A (en) 2020-11-16 2020-11-16 Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method

Publications (1)

Publication Number Publication Date
CN112795583A true CN112795583A (en) 2021-05-14

Family

ID=75806138

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011275437.8A Pending CN112795583A (en) 2020-11-16 2020-11-16 Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method

Country Status (1)

Country Link
CN (1) CN112795583A (en)

Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994026908A1 (en) * 1993-05-17 1994-11-24 Genentech, Inc. Cho cell sialidase by recombinant dna technology
US5401650A (en) * 1990-10-24 1995-03-28 The Mount Sinai School Of Medicine Of The City University Of New York Cloning and expression of biologically active α-galactosidase A
WO1998038289A1 (en) * 1997-02-28 1998-09-03 Japan Tobacco Inc. β-GALACTOSIDE $G(a)-2,6-SIALYLTRANSFERASE
JPH10327873A (en) * 1996-09-20 1998-12-15 Ngk Insulators Ltd Plasmid, its preparation, microorganism containing the same plasmid, production of endosialidase using the same microorganism and production of sialic acid trimer using the same endosialidase
US5858751A (en) * 1992-03-09 1999-01-12 The Regents Of The University Of California Compositions and methods for producing sialyltransferases
WO2006002402A2 (en) * 2004-06-23 2006-01-05 Children's Hospital & Research Center At Oakland Polysaccharide derivatives and uses in induction of an immune response
US7094530B1 (en) * 1993-03-29 2006-08-22 Kyowa Hakko Kogyo Co., Ltd. α-1,3-fucosyltransferase
WO2007075921A2 (en) * 2005-12-23 2007-07-05 Children's Hospital & Research Center At Oakland De-n-acetyl sialic acid antigens, antibodies thereto, and methods of use in cancer therapy
KR20080109333A (en) * 2007-06-12 2008-12-17 한국생명공학연구원 Novel bacterial trans-sialidase and its use for the production of sialoconjugates
US20090203055A1 (en) * 2005-04-18 2009-08-13 Massachusetts Institute Of Technology Compositions and methods for RNA interference with sialidase expression and uses thereof
US20100167344A1 (en) * 2007-02-20 2010-07-01 Albertus Alard Van Dijk Novel sialidases
CN102911955A (en) * 2012-10-24 2013-02-06 山西大学 Synthetic human enterokinase gene and expression and purification method thereof
CN103562214A (en) * 2011-05-13 2014-02-05 格力康公司 Diversification of human milk oligosaccharides (HMOS) or precursors thereof
CN103562401A (en) * 2011-05-13 2014-02-05 格力康公司 Method for generating human milk oligosaccharides (HMOs) or precursors thereof
CN104046603A (en) * 2014-05-21 2014-09-17 江南大学 Sialidase with metal ion tolerance, heat stability and acid stability and application thereof
CN106906236A (en) * 2017-04-10 2017-06-30 中国科学院深海科学与工程研究所 Sialidase gene recombinant expression carrier and its construction method, sialidase and preparation method thereof
CN107365780A (en) * 2017-08-10 2017-11-21 广东省生物资源应用研究所 A kind of clone of eastern subterranean termite salivary gland β glycosidase genes and prokaryotic expression method
CN108118066A (en) * 2016-11-28 2018-06-05 南方医科大学珠江医院 The prokaryotic expression carrier and its construction method of the recombinant protein of casein kinase i α
CN109627298A (en) * 2018-11-23 2019-04-16 武汉大学 It is a kind of with the recombination flagellin and its encoding gene of anti-HIV-1 effect and application
CN111183221A (en) * 2017-05-26 2020-05-19 杰诺维斯公司 Proteases and binding polypeptides for O-glycoproteins
CN111936517A (en) * 2018-01-26 2020-11-13 瑞泽恩制药公司 anti-TMPRSS 2 antibodies and antigen binding fragments
CN112739374A (en) * 2018-07-20 2021-04-30 安迅生物制药公司 Delivery of sialidases to cancer cells, immune cells and tumor microenvironment
CN114875013A (en) * 2022-06-21 2022-08-09 南京林业大学 Method for secreting natural intracellular beta-galactosidase by using recombinant bacillus subtilis
CN115247173A (en) * 2021-11-11 2022-10-28 南京启真基因工程有限公司 Gene editing system for constructing TMPRSS6 gene mutant iron deficiency anemia pig nuclear transplantation donor cells and application thereof

Patent Citations (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5401650A (en) * 1990-10-24 1995-03-28 The Mount Sinai School Of Medicine Of The City University Of New York Cloning and expression of biologically active α-galactosidase A
US5858751A (en) * 1992-03-09 1999-01-12 The Regents Of The University Of California Compositions and methods for producing sialyltransferases
US7094530B1 (en) * 1993-03-29 2006-08-22 Kyowa Hakko Kogyo Co., Ltd. α-1,3-fucosyltransferase
WO1994026908A1 (en) * 1993-05-17 1994-11-24 Genentech, Inc. Cho cell sialidase by recombinant dna technology
JPH10327873A (en) * 1996-09-20 1998-12-15 Ngk Insulators Ltd Plasmid, its preparation, microorganism containing the same plasmid, production of endosialidase using the same microorganism and production of sialic acid trimer using the same endosialidase
WO1998038289A1 (en) * 1997-02-28 1998-09-03 Japan Tobacco Inc. β-GALACTOSIDE $G(a)-2,6-SIALYLTRANSFERASE
WO2006002402A2 (en) * 2004-06-23 2006-01-05 Children's Hospital & Research Center At Oakland Polysaccharide derivatives and uses in induction of an immune response
US20090203055A1 (en) * 2005-04-18 2009-08-13 Massachusetts Institute Of Technology Compositions and methods for RNA interference with sialidase expression and uses thereof
WO2007075921A2 (en) * 2005-12-23 2007-07-05 Children's Hospital & Research Center At Oakland De-n-acetyl sialic acid antigens, antibodies thereto, and methods of use in cancer therapy
US20100167344A1 (en) * 2007-02-20 2010-07-01 Albertus Alard Van Dijk Novel sialidases
KR20080109333A (en) * 2007-06-12 2008-12-17 한국생명공학연구원 Novel bacterial trans-sialidase and its use for the production of sialoconjugates
CN103562214A (en) * 2011-05-13 2014-02-05 格力康公司 Diversification of human milk oligosaccharides (HMOS) or precursors thereof
CN103562401A (en) * 2011-05-13 2014-02-05 格力康公司 Method for generating human milk oligosaccharides (HMOs) or precursors thereof
CN102911955A (en) * 2012-10-24 2013-02-06 山西大学 Synthetic human enterokinase gene and expression and purification method thereof
CN104046603A (en) * 2014-05-21 2014-09-17 江南大学 Sialidase with metal ion tolerance, heat stability and acid stability and application thereof
CN108118066A (en) * 2016-11-28 2018-06-05 南方医科大学珠江医院 The prokaryotic expression carrier and its construction method of the recombinant protein of casein kinase i α
CN106906236A (en) * 2017-04-10 2017-06-30 中国科学院深海科学与工程研究所 Sialidase gene recombinant expression carrier and its construction method, sialidase and preparation method thereof
CN111183221A (en) * 2017-05-26 2020-05-19 杰诺维斯公司 Proteases and binding polypeptides for O-glycoproteins
CN107365780A (en) * 2017-08-10 2017-11-21 广东省生物资源应用研究所 A kind of clone of eastern subterranean termite salivary gland β glycosidase genes and prokaryotic expression method
CN111936517A (en) * 2018-01-26 2020-11-13 瑞泽恩制药公司 anti-TMPRSS 2 antibodies and antigen binding fragments
CN112739374A (en) * 2018-07-20 2021-04-30 安迅生物制药公司 Delivery of sialidases to cancer cells, immune cells and tumor microenvironment
CN109627298A (en) * 2018-11-23 2019-04-16 武汉大学 It is a kind of with the recombination flagellin and its encoding gene of anti-HIV-1 effect and application
CN115247173A (en) * 2021-11-11 2022-10-28 南京启真基因工程有限公司 Gene editing system for constructing TMPRSS6 gene mutant iron deficiency anemia pig nuclear transplantation donor cells and application thereof
CN114875013A (en) * 2022-06-21 2022-08-09 南京林业大学 Method for secreting natural intracellular beta-galactosidase by using recombinant bacillus subtilis

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
LÉA CHUZEL等: "Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156)", 《J. BIOL. CHEM.》 *
ZHAO,F.等: "Akkermansia muciniphila strain DSM 22959 chromosome, complete genome", 《GENBANK DATABASE》 *
李蕾;仇萌;邹先彪;: "白念珠菌天冬氨酸蛋白酶基因SAP2在大肠杆菌中的表达及产物纯化" *
杨安钢等主编: "《生物化学与分子生物学实验技术》", 30 June 2008, 高等教育出版社 *
沈丹虹: "重组唾液酸酶在转化多唾液酸神经节苷脂中的应用", 《中国优秀博硕士学位论文全文数据库(硕士) 基础科学辑》 *
王亚娟等: "唾液酸酶和唾液酸糖基转移酶的结构、功能与催化反应研究进展", 《有机化学》 *
王寅竹等: "重组蛋白融合信号肽在大肠杆菌周质空间表达的研究 进展" *
郑振宇等主编: "《基因工程》", 31 March 2015, 华中科技大学出版社 *

Similar Documents

Publication Publication Date Title
CN111423486A (en) Renaturation method of new type coronavirus recombinant protein inclusion body
CN106906236B (en) Sialidase gene recombinant expression vector and construction method thereof, sialidase and preparation method thereof
CN110468116A (en) A kind of expression and purification method of all-round endonuclease
CN113801240B (en) D-psicose-3-epimerase activity aggregate and preparation method and application thereof
CN109486804B (en) Novel chitosanase CsnM with heat recovery characteristic and application thereof
CN111088183A (en) Marine vibrio and application thereof in preparation of iota-carrageenase with thermal stability
JP6236512B2 (en) Agarase, compositions comprising said, and applications thereof
US6407208B1 (en) Chimeric proteins with a cellulose binding domain
CN109022405A (en) A kind of Cold tolerance algin catenase AlgA5 and its application
CN103468661B (en) Neoagarobiose hydrolase and application thereof
CN101280001B (en) Preparation of human SDF-1 alpha, human SDF-1 alpha obtained therefrom and use thereof
CN112795583A (en) Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method
CN104419688A (en) Fructosyl transferase as well as gene, secreted expression vector and application thereof
CN114958893B (en) Construction method of lactase required by preparation of suckling pig high-temperature creep feed
CN101605807A (en) The expression system for recombinant human arginase i of improvement
CN109022404A (en) A kind of novel Cold tolerance algin catenase AlgA7 and its application
CN114164224B (en) Preparation method of low-temperature debittering enzyme
CN105132400A (en) Enzyme with function of catalyzing formaldehyde for synthesis of 1,3-dihydroxyacetone and preparation method of enzyme
CN113234704B (en) Method for preparing recombinant serratia marcescens nuclease
CN109022406A (en) It is a kind of with the algin catenase AlgA1 of acclimatization to cold characteristic and its application
CN102140444B (en) Low temperature alkaline phosphatase and preparation method thereof
CN112430610B (en) Low-temperature esterase functional gene DcaE and application thereof
CN114214302A (en) Agarase, coding gene, recombinant vector, host cell and application thereof, novel agaro-oligosaccharide and preparation method thereof
WO2022213477A1 (en) Enzymatic method for preparing glucosamine from glucose, and enzymatic use
CN116144632B (en) Tea tree neutral/alkaline invertase CsINV protein and preparation method and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20210514