WO2018188672A1

WO2018188672A1 - Sialidase gene recombinant expression vector and construction method therefor, and sialidase and preparation method therefor

Info

Publication number: WO2018188672A1
Application number: PCT/CN2018/085049
Authority: WO
Inventors: 王少露; 贺丽生; 王勇
Original assignee: 中国科学院深海科学与工程研究所
Priority date: 2017-04-10
Filing date: 2018-04-28
Publication date: 2018-10-18
Also published as: CN106906236B; CN106906236A

Abstract

Provided is a preparation method for sialidase, comprising the following steps: preparing a sialidase gene fragment; cloning the sialidase gene fragment to pMD-18T to obtain a connection product, converting the connection product into E.coli JM109, and selecting a positive clone for sequencing to obtain a gene sequence as shown in SEQ ID NO: 1; connecting the positive clone and a prokaryotic expression vector to obtain a recombinant expression vector; converting the recombinant expression vector to obtain recombinant engineering bacteria; performing fermentation, induced expression and purification on the recombinant engineering bacteria to obtain the sialidase. Also provided is a preparation method for the sialidase, comprising bacterial genome DNA extraction, PCR amplification, construction of the recombinant expression vector, conversion of the recombinant expression vector into host cells for construction of recombinant expression cells, culture and induced expression of the recombinant expression cells, isolation and purification of protease from the cultured host cells, and analysis of protease enzymatic properties.

Description

Sialylase gene recombinant expression vector and construction method thereof, sialidase and preparation method thereof

Technical field

The invention relates to the technical field of an expression vector construction method, in particular to a sialidase gene recombinant expression vector and a construction method thereof, a sialidase and a preparation method thereof.

Background technique

Sialic acid is a kind of acidic amino sugar containing 9 carbon atoms and having a pyranose structure. It is widely found in animal tissues and microorganisms and is an important component of cell membrane glycoproteins and glycolipids. Sialic acid generally does not exist in a free form. It usually combines with biomacromolecules such as glycoproteins and glycolipids on the cell surface to form a conjugate, and is present at the end of the conjugate, in cell differentiation, maturation, cells and organisms. It plays an important role in physiological and biochemical processes such as intermolecular interactions. More than 50 different structures of sialic acid have been found in nature. The most important sialic acid is N-acetylneuraminic acid (Neu5Ac), which is one of the most important members of the sialic acid family. More than 99% of the sialic acid family, usually a precursor of biosynthesis of other members of the sialic acid family, has diverse biological functions. Sialic acid plays an important role in the physiological processes involved in the interaction of sugars and proteins, such as cell adhesion, signal transduction, glycoprotein stability, bacterial pathogenesis, viral infection, inflammation, and tumor metastasis. Wait. Since the content of N-acetylneuraminic acid accounts for more than 99% of the entire sialic acid family, it has been studied more deeply, and the production of N-acetylneuraminic acid (Neu5Ac) is generally obtained by the following methods: Methods from natural material extraction, chemical synthesis, enzymatic synthesis, and microbial fermentation. Enzymatic synthesis of N-acetylneuraminic acid begins with the discovery and application of sialidase or N-acetylneuraminic acid aldolase. N-acetylneuraminic acid aldolase (NAL, EC 4.1.3.3), also known as N-acetylneuraminic lyase (Neu5Ac lyase), is a terminal enzyme of sialic acid metabolism that catalyzes sialic acid (Neu5Ac) Pyrolysis to form pyruvate and N-acetylmannosamine (ManNAc), under certain conditions can also condense N-acetylmannosamine (ManNAc) and pyruvate (Pyruvate) to form N-acetylneuraminic acid (Neu5Ac) It has been reported that the enzyme synthesizes sialic acid and its analogs. Sialidase exists in animal tissues and some microorganisms. In animals, the main function of this enzyme is to control the circulation of sialic acid. In addition to regulating, microbes can not only decompose free sialic acid into available. The carbon and nitrogen sources also catalyze the synthesis of sialic acid. So far, sialidase studies on symbiotic microbial sources in deep-sea invertebrates have not been reported. Due to the special ecological environment (low temperature, high pressure and oligotrophy) in the deep sea and the lack of sampling techniques and tools, it is relatively difficult to capture organisms. Little is known about the study of sialidase from symbiotic microbial sources in deep-sea invertebrates, so little is known about the source of sialidase.

Summary of the invention

In view of this, it is necessary to provide a sialidase gene recombinant expression vector and a construction method thereof, a sialidase and a preparation method thereof.

A method for constructing a sialidase gene recombinant expression vector comprises the following steps:

Preparing a sialidase gene fragment;

The sialidase gene fragment was cloned into pMD-18T to obtain a ligation product, the ligation product was transformed into E. coli JM109, and the positive clone was selected for sequencing to obtain the gene sequence as shown in SEQ ID NO: 1.

The positive clone is ligated to a prokaryotic expression vector to obtain the sialidase gene recombinant expression vector.

In one embodiment, the method of preparing a sialidase gene fragment is as follows:

The sialidase gene fragment was obtained by polymerase chain reaction using the PowerLyzerR Powersoil DNA isolation kit as a template to extract the sialidase gene fragment; wherein the primers used in the polymerase chain reaction were as follows:

Primer F: 5'-GGATCCATGGAAAAATTAACAGGAATTTTTG-3';

Primer R: 5'-GTCGACTTATGAAAGATATTTATCAATTATGTT-3'.

In one embodiment, before the step of cloning the sialidase gene fragment into pMD-18T, the method further comprises the steps of:

The sialidase gene fragment is detected by agarose gel electrophoresis, and after detection, it is purified and recovered by a gel recovery kit.

In one embodiment, the method of joining the positive clone to the prokaryotic expression vector is as follows:

The positive clone and the prokaryotic expression vector were digested with BamHI and SalI, respectively, and the fragment was electrophoretically recovered and ligated with T4 ligase at 16 ° C for 2 h to obtain the sialidase gene recombinant expression vector.

In one embodiment, the prokaryotic expression vector is a plasmid.

A method for preparing a sialidase, comprising the steps of:

Preparing a sialidase gene fragment;

The sialidase gene fragment was cloned into pMD-18T to obtain a ligation product, and the ligation product was transformed into E. coli JM109, and the positive clone was selected for sequencing to obtain the gene sequence as shown in SEQ ID NO: 1.

The positive clone is ligated to a prokaryotic expression vector to obtain the sialidase gene recombinant expression vector;

Converting the sialidase gene recombinant expression vector to obtain a recombinant engineering strain;

The obtained recombinant engineering bacteria are subjected to fermentation-induced expression and purification to obtain the sialidase.

In one embodiment, the fermentation-induced expression in the step of fermentation-induced expression and purification of the obtained recombinant engineering bacteria is as follows:

The obtained recombinant engineering bacteria were inoculated into ampicillin-containing LB liquid medium, and cultured at 37 ° C, 150 rpm overnight, and then the whole cultured bacterial liquid was transferred to fresh LB liquid medium, followed by incubation at 37 ° C, 180 rpm. 4h until OD600=0.8-1.0, isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 1 mM, transferred to 30 ° C, induced at 180 rpm for 4 h, and the cells were collected by centrifugation.

In one embodiment, in the step of performing fermentation-induced expression and purification of the obtained engineering strain containing the sialidase gene recombinant expression vector, the purification step is as follows:

The cells collected by centrifugation were washed twice with 1×PBS, and the cells were collected by centrifugation, resuspended in 10 mL of lysis buffer containing 10 mmol of imidazole, sonicated, centrifuged to obtain a supernatant, and then the supernatant was transferred to a nickel column. Mix thoroughly to allow it to sink naturally, then rinse the protein with 40 mL of 60 mmol-80 mmol imidazole wash buffer, then elute the target protein with 4 mL of elution buffer containing 350 mmol-500 mmol of imidazole, and use a clean centrifuge tube. The protein of interest is collected to obtain the sialidase.

A sialidase gene recombinant expression vector prepared by the method for constructing the above sialidase gene recombinant expression vector.

A sialidase prepared by the preparation method of the above sialidase.

The preparation method of the above sialidase is carried out by extracting bacterial genomic DNA, PCR amplification, constructing a recombinant expression vector, transferring into a host cell, constructing a recombinant expression cell, culturing and inducing expression of the recombinant expression cell, and separating from the cultured host cell. Purification of proteases, analysis of protease enzymatic properties. The preparation method of the above sialidase has obtained the gene sequence of sialidase, and the expression of sialidase has been purified, and the related enzymatic properties of sialidase have been analyzed. The sialidase prepared by the above method for preparing sialidase is resistant to high temperature and Ph, and provides more basic materials and scientific basis for industrial and social applications.

DRAWINGS

1 is a schematic diagram showing the purification effect of SDS-PAGE according to an embodiment, wherein M is a marker, Lane 1 is an uninducer control; 2 is an unpurified induced expression protein; 3 and 4 are purified proteins;

Figure 2 is a temperature influence diagram of a sialidase catalytic reaction;

Figure 3 is a graph of the pH effect of a sialidase catalytic reaction.

detailed description

In order to make the objects, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The reagents or instruments used are not specified by the manufacturer, and are conventional products that can be obtained through purchase.

A method for preparing a sialidase according to an embodiment comprises the following steps:

S10, preparing a recombinant expression vector of a sialidase gene, that is, a recombinant engineering bacteria.

In S10, the method for constructing a sialidase gene recombinant expression vector comprises the following steps:

S110, preparing a sialidase gene fragment.

The sialidase gene fragment is prepared by cloning the nucleotide sequence of the sialidase gene (NanA) from the gastrointestinal tract of the genus. The sialidase gene fragment was obtained by polymerase chain reaction using the PowerLyzerR Powersoil DNA isolation kit to extract the genomic DNA of the gastrointestinal tract microbial bacterium.

Among them, the primers used in the polymerase chain reaction are as follows:

SEQ ID NO: 2

Primer F: 5'-GGATCCATGGAAAAATTAACAGGAATTTTTG-3';

SEQ ID NO: 3

Primer R: 5'-GTCGACTTATGAAAGATATTTATCAATTATGTT-3'.

The 5' end of primer F contains a BamHI restriction site (1st base to 6th base), and the 5' end of primer R contains a SalI restriction site (1st base to 6th base) ).

The reaction conditions of the polymerase chain reaction are as follows:

10 μL of 5×Buffer, 4 μL of dNTP, 0.5 μL of PrimeSTAR HS DNA Polymerase, 1 μL of primer F, 1 μL of primer R, 1 μL of template, 32.5 μL of H 2 O, total volume of 50 μL.

The PCR amplification conditions are as follows:

Pre-denatured 10S at 98°C, denatured at 98°C for 10s, annealed at 50°C for 15s, extended at 72°C for 1min, denatured, annealed and extended in three steps for 30 cycles, and finally finished at 72°C for 10min.

S120, the sialidase gene fragment was cloned into pMD-18T to obtain a ligation product, the ligation product was transformed into E. coli JM109, and the positive clone was selected for sequencing to obtain the gene sequence as shown in SEQ ID NO: 1.

Among them, pMD-18T was purchased from TaKaRa.

Before the step of cloning the sialidase gene fragment into pMD-18T, the following steps are also included:

The sialidase gene fragment was detected by agarose gel electrophoresis, and after detection, it was purified by a gel recovery kit. The results of agarose gel electrophoresis showed that a fragment of about 885 bp in size was obtained.

The method for sequencing the cloning product selection positive clones is:

The ligation product was transformed into Escherichia coli JM109, cultured on an LB solid plate containing ampicillin at a concentration of 100 μg/mL, and white colonies grown on the plate were picked, and positive clones were verified by colony PCR, and clones which were verified to be positive were ligated. The LB liquid medium containing ampicillin at a concentration of 100 μg/mL was cultured overnight at 37 ° C, and the plasmid was extracted by a spin column type plasmid extraction kit and sequenced (Hua Da Company). The sequencing result showed that the fragment size was 885 bp. As shown in SEQ ID NO: 1.

S130, linking the positive clone to the prokaryotic expression vector to obtain a sialidase gene recombinant expression vector.

The method of ligating a positive clone to a prokaryotic expression vector is as follows:

The positive clone and the prokaryotic expression vector were digested with BamHI and SalI, respectively, and the restriction fragment was electrophoresed and ligated with T4 ligase at 16 ° C for 2 h to obtain a sialidase gene recombinant expression vector.

Among them, the prokaryotic expression vector is a plasmid or other vector. Preferably, the prokaryotic expression vector is a pET vector series or other prokaryotic expression vector. More preferably, the prokaryotic expression vector is pET-30a.

The nucleotide sequence encoding the sialidase can be operably linked to an appropriate promoter in an expression vector to direct the synthesis of the mRNA. Representative examples of such promoters are: lac or trp promoter of E. coli, PL promoter of lambda phage, CMV early promoter of eukaryotic promoter and other known controllable genes in prokaryotic or eukaryotic cells Or a promoter expressed in the virus. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like.

The method for constructing the above sialidase gene recombinant expression vector is simple and easy to operate by extracting bacterial genomic DNA, PCR amplification, and constructing a recombinant expression vector.

S20, transforming a sialidase gene recombinant expression vector to obtain a recombinant engineering strain.

The method for transforming the sialidase gene recombinant expression vector is as follows:

The product was transformed into host cells, cultured on LB solid plate containing 100 μg/mL ampicillin, white colonies grown on the plate were picked, and positive clones were screened by colony PCR and plasmid extraction and double restriction enzyme digestion and identified by sequencing. , obtained recombinant engineering bacteria. That is, an E. coli host cell containing a sialidase gene was successfully constructed.

The host cell may be Escherichia coli, fungal cell or yeast or the like. Preferably, the host cell is Escherichia coli BL21 (DE3).

S30, performing fermentation induction-inducing expression and purification on the obtained recombinant engineering bacteria to obtain sialidase.

In S30, the method of fermentation-induced expression is as follows:

The obtained recombinant engineering bacteria were inoculated into ampicillin-containing LB liquid medium, and cultured at 37 ° C, 150 rpm overnight, and then the whole cultured bacterial liquid was transferred to fresh LB liquid medium, followed by incubation at 37 ° C, 180 rpm. 4h until OD600=0.8-1.0, add isopropyl-β-D-Thiogalactoside (IPTG) at a final concentration of 1 mM, transfer to 30 ° C, induce 3-4 h after 180 rpm The cells were collected by centrifugation.

In S30, the steps of purification are as follows:

The cells collected by centrifugation were washed twice with 1× PBS, and the cells were collected by centrifugation, resuspended in 10 mL of lysis buffer containing 10 mmol of imidazole, sonicated, centrifuged to obtain a supernatant, and then the supernatant was transferred to a nickel column and thoroughly mixed. Allow to stand naturally to sink, then rinse the protein with 40 mL of washing buffer containing 60 mmol-80 mmol of imidazole, then elute the target protein with 4 mL of elution buffer containing 350 mmol-500 mmol of imidazole, and collect it with a clean centrifuge tube. Protein, resulting in purified sialidase.

Among them, the lysis buffer includes the following components: NaH2PO4 50 mM, NaCl 300 mM, and imidazole 10 mM.

The wash buffer included the following components: NaH2PO4 50 mM, NaCl 300 mM, imidazole 60-80 mM.

The elution buffer included the following components: NaH2PO4 50 mM, NaCl 300 mM, imidazole 350-500 mM.

The protein solution is purified on a nickel column, and the nickel sulfate in the nickel column can be bound to a protein labeled with histidine (Hisidine, His) or combined with imidazole.

In order to verify whether the expressed sialidase has an enzyme activity, the purified sialidase was subjected to enzyme activity measurement and related enzymatic properties, and the results are shown in Fig. 2 and Fig. 3.

Further, a sialidase gene recombinant expression vector obtained by the method for constructing the above-described sialidase gene recombinant expression vector is also provided. And a sialidase prepared by the above-described preparation method of sialidase.

In the preparation method of the above sialidase, the sialidase gene is cloned and expressed from the symbiotic microbial mycoplasma of the deep-sea invertebrate genus, and the enzymatic properties of the enzyme are important. It helps to analyze and interpret the survival and adaptation mechanisms of deep-sea living organisms in extreme environments, facilitates the development and utilization of deep-sea biological germplasm resources and natural products, and provides a basis for expanding the industrial production of microbial-derived sialidase and sialic acid. Material and scientific basis, providing new ideas and methods for improving microbial metabolic products, has important practical significance and good application prospects.

The following further describes the embodiments in conjunction with the specific embodiments.

Example 1: The nucleotide sequence of the sialidase gene (NanA) was cloned from the gastrointestinal tract of the footworm. The PowerLyzerR Powersoil DNA isolation kit was used to extract the genomic DNA of the gastrointestinal tract of the genus Apothecary, and 1 μL was used as a template for polymerase chain reaction (PCR), and the primers (primer F and primer R) were designed in combination with the primers used in the PCR reaction. , components and amplification conditions are as follows:

Primer F: 5'-GGATCCATGGAAAAATTAACAGGAATTTTTG-3’

Primer R: 5'-GTCGACTTATGAAAGATATTTATCAATTATGTT-3’

PCR reaction system: 10 μL of 5×Buffer, 4 μL of dNTP, 0.5 μL of PrimeSTAR HS DNA Polymerase, 1 μL of primer F, 1 μL of primer R, 1 μL of template, 32.5 μL of H 2 O, and a total volume of 50 μL.

The PCR amplification conditions are as follows:

Pre-denaturation 10S at 98°C, denaturation at 98°C for 10s, annealing at 50°C for 15s, extension at 72°C for 1min, 30 cycles of variability, annealing and extension, and end of 10° at 72°C. After PCR amplification, the results of agarose gel electrophoresis showed that a fragment of about 885 bp in size was obtained and purified by a gel recovery kit (centrifugation column type), and the recovered fragment was cloned into pMD-18T (TaKaRa product). In the case, the ligation product was transformed into Escherichia coli JM109, cultured on an LB solid plate containing 100 μg/mL ampicillin, white colonies grown on the plate were picked, positive clones were verified by colony PCR, and clones verified to be positive were ligated. The medium was cultured in an LB liquid medium containing 100 μg/mL ampicillin, and cultured at 37 ° C overnight. The plasmid was extracted by a plasmid extraction kit (centrifugal column type) and sequenced (Hua Da Company). The sequencing result showed that the fragment size was 885 bp, such as SEQ. ID NO: 1 is shown.

Example 2: Construction of recombinant expression vector

The fragment amplified in Example 1 has a BamHI restriction site (1st base to 6th base) at the 5' end of the primer F, and a SalI restriction site at the 5' end of the primer R (1st base to 6th base), so the fragments were double-digested with these two enzymes, and pET-30a was digested with the same enzyme, and the digested fragments were electrophoresed and linked with T4. The enzyme was ligated at 16 ° C for 2 h to construct a recombinant expression vector. The ligation product was transformed into E. coli BL21 (DE3), cultured on LB solid plate containing ampicillin (100 μg/mL), and white colonies grown on the plate were picked and screened by colony PCR and plasmid extraction and double digestion. To the positive clone and sequence identification, E. coli host cells containing the sialidase gene were successfully constructed.

Example 3: Expression and purification of sialic acid protease

The recombinant engineering bacteria obtained in Example 2 were inoculated into 10 mL of LB liquid medium (containing 100 μg/mL ampicillin), cultured at 37 ° C, 150 rpm overnight, and the whole day was transferred to 250 mL of fresh LB liquid culture. In the medium, followed by incubation at 37 ° C, 180 rpm for 3-4 h until OD600 = 0.8-1.0, adding IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 1 mM, and transferring to 30 ° C, After induction for 4 h at 180 rpm, the cells were collected by centrifugation, and the cells were resuspended in 10 mL of a lysis buffer containing 10 mmol of imidazole, sonicated, and the supernatant was centrifuged. The protein solution is purified on a nickel column. The nickel sulfate in the nickel column can be combined with a protein having a His (histidine) tag, or can be combined with an imidazole. The protein solution is first passed through a nickel column at a certain speed to hang the protein, and then Elution was carried out with different concentrations of imidazole buffer. The above supernatant was transferred to a nickel column and thoroughly mixed to allow it to naturally sink. Then, the heteroprotein was washed with 40 mL of a washing buffer containing 60 mmol-80 mmol of imidazole, and washed with 4 mL of an elution buffer containing 350 mmol-500 mmol of imidazole. The target protein was separately collected by a clean centrifuge tube, and the eluate was detected by SDS polyacrylamide gel electrophoresis (SDS-PAGE). The purification effect is shown in FIG.

Example 4: Determination of enzyme activity

The purified protease solution was loaded into a dialysis bag, dialyzed against dialysis solution (PBS) at 4 ° C for 24 h, and the solution was changed two to three times. The dialyzed protein was placed in a concentrating tube and concentrated appropriately. A small amount of the concentrated sample was subjected to SDS-PAGE analysis, and the protein concentration was estimated based on the BSA concentration standard curve. In order to verify whether the expressed protein has enzymatic activity, the purified protein is subjected to enzyme activity assay and related enzymatic properties. The enzyme catalyzes the cleavage direction with N-acetylneuraminic acid (Neu5Ac) as a substrate, and the activity in the direction of cleavage The method of enzymatic coupling is determined by a spectrophotometer. The principle is that pyruvic acid produced by the cleavage of N-acetylneuraminic acid can be reduced to lactic acid in the presence of NADH and lactate dehydrogenase, and NADH is consumed in the process. A decrease in the absorbance of NADH at a wavelength of 340 nm is caused. One enzyme activity unit is defined as the amount of enzyme required to cleave 1 μmol of Neu5Ac to produce 1 μmol of N-acetyl-D-mannosamine and pyruvate at pH 7.0, 37 ° C for 1 min. The reaction system included 4 mmol/L Neu5Ac, 150 umol/L NADH, 0.5 U LDH, 1.5 ug enzyme. The enzyme-catalyzed synthesis direction uses N-acetylmannosamine (ManNAc) and pyruvate (Pyr) as substrates, and the synthesis direction activity is determined by HPLC. The principle is: only ManNAc, Pyr and enzyme are added, and the Neu5Ac is detected by HPLC. The amount of generation determines the amount of vitality. One enzyme activity unit is defined as the amount of enzyme required to synthesize 1 μmol of Neu5Ac in 1 min at p H 7.0, 37 °C. The reaction system included 30 mmol/L ManNAc, 100 mmol/L Pyr and 20 μg enzyme. The results of the enzyme activity test are shown in Figures 2 and 3.

The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. These improvements and retouchings should also be considered. It is the scope of protection of the present invention.

Claims

A method for constructing a sialidase gene recombinant expression vector, comprising the steps of:

Preparing a sialidase gene fragment;

The sialidase gene fragment was cloned into pMD-18T to obtain a ligation product, the ligation product was transformed into E. coli JM109, and the positive clone was selected for sequencing to obtain the gene sequence as shown in SEQ ID NO: 1.

The positive clone is ligated to a prokaryotic expression vector to obtain the sialidase gene recombinant expression vector.
The method for constructing a sialidase gene recombinant expression vector according to claim 1, wherein the method for preparing a sialidase gene fragment is as follows:

The sialidase gene fragment was obtained by polymerase chain reaction using the PowerLyzerR Powersoil DNA isolation kit as a template to extract the sialidase gene fragment; wherein the primers used in the polymerase chain reaction were as follows:

Primer F: 5'-GGATCCATGGAAAAATTAACAGGAATTTTTG-3';

Primer R: 5'-GTCGACTTATGAAAGATATTTATCAATTATGTT-3'.
The method for constructing a sialidase gene recombinant expression vector according to claim 1, wherein before the step of cloning the sialidase gene fragment into pMD-18T, the method further comprises the steps of:

The sialidase gene fragment is detected by agarose gel electrophoresis, and after detection, it is purified and recovered by a gel recovery kit.
The method for constructing a sialidase gene recombinant expression vector according to claim 1, wherein the method of ligating the positive clone and the prokaryotic expression vector is as follows:

The positive clone and the prokaryotic expression vector were digested with BamH I and SalI, respectively, and the fragment was electrophoretically recovered, and ligated with T4 ligase at 16 ° C for 2 h to obtain the sialidase gene recombinant expression vector.
The method for constructing a sialidase gene recombinant expression vector according to claim 1, wherein the prokaryotic expression vector is a plasmid.
A method for preparing a sialidase, comprising the steps of:

Preparing a sialidase gene fragment;

The sialidase gene fragment was cloned into pMD-18T to obtain a ligation product, the ligation product was transformed into E. coli JM109, and the positive clone was selected for sequencing to obtain the gene sequence as shown in SEQ ID NO: 1.

The positive clone is ligated to a prokaryotic expression vector to obtain the sialidase gene recombinant expression vector;

Converting the sialidase gene recombinant expression vector to obtain a recombinant engineering strain;

The recombinant engineered bacteria obtained are subjected to fermentation-induced expression and purification to obtain the sialidase.
The method for preparing a sialidase according to claim 6, wherein in the step of performing fermentation-induced expression and purification on the obtained recombinant engineering bacteria, the fermentation-induced expression is as follows:

The obtained recombinant engineering bacteria were inoculated into ampicillin-containing LB liquid medium, and cultured at 37 ° C, 180 rpm overnight, and then all the overnight cultured bacterial liquid was transferred to fresh LB liquid medium, followed by incubation at 37 ° C, 180 rpm - 4h until OD600=0.8-1.0, isopropyl-β-D-thiogalactopyranoside was added to a final concentration of 1 mM, transferred to 30 ° C, induced at 180 rpm for 4 h, and the cells were collected by centrifugation.
The method for producing a sialidase according to claim 7, wherein in the step of performing fermentation-induced expression and purification of the obtained engineering strain containing the sialidase gene recombinant expression vector, the purification step is as follows:

The cells collected by centrifugation were washed twice with 1×PBS, and the cells were collected by centrifugation, resuspended in 10 mL of lysis buffer containing 10 mmol of imidazole, sonicated, centrifuged to obtain a supernatant, and then the supernatant was transferred to a nickel column. Mix well to allow it to naturally sink, then rinse the protein with 40 mL of washing buffer containing 60 mmol-80 mmol of imidazole, then elute the target protein with 4 mL of elution buffer containing 350 mmol-500 mmol of imidazole, and clean with a clean centrifuge. The target protein is collected by a tube to obtain the sialidase.
A sialidase gene recombinant expression vector obtained by the method for constructing a sialidase gene recombinant expression vector according to any one of claims 1 to 5.
A sialidase prepared by the method for producing a sialidase according to any one of claims 6-8.