CN114395545B

CN114395545B - Sialidase for resisting helicobacter pylori colonization in stomach

Info

Publication number: CN114395545B
Application number: CN202110436490.XA
Authority: CN
Inventors: 杜方川; 童文德; 朱白梅; 童武学; 蒲凯
Original assignee: Chengdu Olymvax Biopharmaceuticals Inc
Current assignee: Chengdu Olymvax Biopharmaceuticals Inc
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2023-05-12
Anticipated expiration: 2041-04-22
Also published as: CN114395545A

Abstract

The invention discloses sialidase for resisting helicobacter pylori colonization in stomach. Belongs to the technical field of genetic engineering vaccines. The amino acid sequence of sialidase is shown as SEQ ID NO. 1; the nucleotide sequence of the coding gene is shown as SEQ ID NO.2, and a construction method and application are provided. The invention adopts genetic engineering technology to clone and express helicobacter pylori sialidase recombinant protein, has high expression level of supernatant, simple and convenient separation and purification steps and high immune titer, and has protection. The sialidase recombinant protein can be directly combined with mucosal adjuvant LT (B) ₅ Is matched with the medicine for oral immunization.

Description

Sialidase for resisting helicobacter pylori colonization in stomach

Technical Field

The invention relates to the technical field of genetic engineering vaccines, in particular to sialidase for resisting the colonization of helicobacter pylori in the stomach.

Background

Helicobacter pylori (Hp) is a gram-negative bacterium that, after infection of humans, can often cause digestive system diseases such as chronic gastritis, peptic ulcer, gastric lymphoproliferative lymphoma, gastric cancer, etc. in patients. Helicobacter pylori has been increasingly becoming lower from standard triple therapy in the past to quadruple therapy. The development of an effective helicobacter pylori vaccine can avoid the problem of reduced eradication rate in the current eradication methods.

In the last 30 years of helicobacter pylori vaccine research, development of helicobacter pylori vaccine has been very slow, but preclinical studies, clinical studies and a phase 3 clinical study in the chinese range have proved successful in prevention and treatment strategies. This lays a solid foundation for the continued development of a safe and effective helicobacter pylori vaccine.

Helicobacter pylori, an important loop of which is adhesion during colonization. A number of H.pylori adhesion factors have been reported. Helicobacter pylori crosses the gastric mucus layer and adheres to host epithelial cells, possibly involving sialidases.

Sialidases (sialidases), also known as neuraminidases, are membrane proteins distributed on the surface of bacteria. Sialidases cleave oligosaccharides on the surface of host cells, exposing underlying recessive receptors, enhancing adhesion to tissues, and have a function which has been widely studied in Streptococcus pneumoniae and reported as a vaccine component for Streptococcus pneumoniae.

Helicobacter pylori has 1400 or more ORFs, and by analyzing each protein of helicobacter pylori and comparing the functions of the proteins analyzed by other bacteria in the NCBI database, it was found that the above proteins are also present on the helicobacter pylori envelope.

Therefore, the development and development of new helicobacter pylori vaccines using sialidases as candidate helicobacter pylori vaccine antigens is a problem that needs to be solved by those skilled in the art.

Disclosure of Invention

In view of this, the present invention provides a sialidase for use in gastric colonization against helicobacter pylori.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

a sialidase for resisting helicobacter pylori colonization in stomach has amino acid sequence shown in SEQ ID NO. 1.

The invention provides a gene for encoding the sialidase of claim 1, and the nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The invention provides a recombinant expression vector containing the gene.

Preferably: the recombinant vector also comprises a His tag sequence and a gene encoding a pro-lytic protein; genes encoding the pro-lysin include GST and MBP.

The invention also provides: the construction method of the recombinant expression vector comprises the following steps:

1) Amplifying helicobacter pylori genome DNA by using an F234/R234 primer pair to obtain a gene segment for encoding sialidase; the nucleotide sequence of F234 is shown as SEQ ID NO. 3;

the nucleotide sequence of R234 is shown as SEQ ID NO. 4;

2) Inserting a gene fragment encoding sialidase into a backbone vector to obtain a recombinant expression vector; the multiple cloning site of the backbone vector was BamHI/EcoRI.

Preferably: the skeleton carrier is a skeleton carrier containing His tag sequences and genes for encoding the pro-lysin; the construction method of the skeleton carrier containing His tag sequence and the gene for encoding the pro-lysoprotein comprises the following steps:

performing PCR amplification on the pMAL-c2X template by using the F29b-1/R502 primer pair to obtain an MBP gene fragment; PCR amplification is carried out on pGEX-6P-1 template by using F502/R29b-07 primer pair to obtain GST gene fragment;

the nucleotide sequence of F29b-1 is shown as SEQ ID NO. 5;

the nucleotide sequence of R502 is shown as SEQ ID NO. 6;

the nucleotide sequence of F502 is shown in SEQ ID NO. 7;

the nucleotide sequence of R29b-07 is shown as SEQ ID NO. 8;

performing PCR amplification by using the MBP gene fragment and the GST gene fragment as templates and using an F29b-2/R29b-07 primer pair to obtain His (Tag) -MBP-GST fragments;

the nucleotide sequence of F29b-2 is shown as SEQ ID NO. 9;

his (Tag) -MBP-GST fragment is inserted into pET29b plasmid to obtain recombinant expression vector containing His Tag sequence and two genes encoding the pro-lysoprotein.

Preferably: the inserted multiple cloning site of pET29b plasmid is NedI/BamHI.

The invention also provides a recombinant strain containing the gene or the recombinant vector.

The invention also provides an application of the sialidase obtained by recombinant expression of the recombinant strain or the sialidase in preparation of medicines or biological products for detecting, preventing and/or treating helicobacter pylori infection.

Preferably: biological products include vaccines.

Compared with the prior art, the invention discloses the sialidase for resisting the colonization of helicobacter pylori in the stomach, and the obtained technical effect is that the recombinant protein of helicobacter pylori sialidase is cloned and expressed by adopting a genetic engineering technology, the expression level of the supernatant is high, the separation and purification steps are simple and convenient, and the immune titer is high and the protection is provided. The sialidase recombinant protein can be directly combined with mucosal adjuvant LT (B) ₅ Is matched with the medicine for oral immunization.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram showing construction of pET29b-6 plasmid provided by the invention.

FIG. 2 is a diagram showing the result of PCR for identifying His (Tag) -MBP-GST gene fragment plasmids provided by the invention; m: DNA molecular weight standard; 1, a step of; his (Tag) -MBP-GST gene fragment (1803 bp) PCR product.

FIG. 3 is a diagram showing the result of high-fidelity PCR amplification of the sialidase gene fragment provided by the invention; m: DNA molecular weight standard; 1, a step of; the sialidase gene fragment (429 bp) was PCR-product.

FIG. 4 is a graph showing the result of the PCR identification of recombinant pET29b-sialidase/BL21 (DE) 3; m: DNA molecular weight standard; 1, a step of; the sialidase gene fragment (429 bp) was PCR-product.

FIG. 5 is a chart showing the affinity chromatography of the primary purification of the sialidase/BL21 (DE) 3 engineering bacterium strain supernatant. The first peak is the loading peak of the bacterial strain supernatant, the second, third and fourth peaks are the A2 buffer solution washing impurity protein peaks, the fifth peak is the A3 buffer solution washing impurity protein peak, and the sixth peak is the A4 buffer solution eluting target protein peak.

FIG. 6 is a diagram showing the results of induction expression and supernatant purification after bacterial disruption of the sialidase/BL21 (DE) 3 recombinant engineering bacteria provided by the invention, wherein lane 1 is a protein molecular mass standard (Thermo Fisher, 26616); lane 2 is sialidase/BL21 (DE) 3 recombinant engineering bacteria whole bacteria; lane 3 is sialidase/BL21 (DE) 3 recombinant engineering bacterium-disrupted supernatant; lane 4 is sialidase/BL21 (DE) 3 recombinant engineering bacteria pellet; lane 5 is the first pass of sialidase/BL21 (DE) 3 recombinant engineering bacterium supernatant purification; lane 6 is sialidase/BL21 (DE) 3 recombinant engineering bacterium supernatant purification second pass; lane 7 is sialidase recombinant protein with a pro-lytic tag; lane 8 is a PP enzyme digested sialidase recombinant protein containing a pro-lytic tag; lane 9 is sialidase recombinant protein.

FIG. 7 is a graph showing that sialidase recombinant proteins provided by the invention immunize Balb/c mice, and saliva IgA (1:4) and serum IgG (1:800) detect positive turnover rate.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The embodiment of the invention discloses sialidase for resisting the colonization of the stomach by helicobacter pylori, which is helicobacter pylori outer membrane protein, and the transmembrane region is 152aa-169aa.

The recombinant expression vector in the examples comprises a nucleotide sequence encoding Sialidase (Sialidase) recombinant protein, a His (Tag) sequence, a prolamin sequence and a plasmid sequence, preferably pET29b, the His (Tag) sequence being 7 histidines (His), the prolamin sequence being divided into GST and MBP (maltose binding protein).

Example 1

Construction of pET29b-6 double-tag dissolution promoting vector

His (Tag), MBP, GST (glutathione transferase) gene clone and connection

The amino acid sequence of the His (Tag) protein is selected from (His His His His His His His) SEQ ID NO.10, and the nucleotide sequence (catcatcatcatcatcatcat) is shown as SEQ ID NO.11.

And the device comprises a main body and a plurality of auxiliary bodies, wherein the main body is provided with a plurality of auxiliary holes, and the auxiliary holes are respectively arranged on the main body and the auxiliary bodies.

GST gene is derived from pGEX-6P-1 plasmid (Changshayou Bao Biotechnology Co., ltd.), the protein amino acid sequence of GST is selected from% the SerLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeedge-LeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLeLe-LeLeLeLeLeLeLeLe-LeLeLe-LeLeLeLeLe-Le-LeLeLeLeLe-Le-LeLeLe-Le-LeLeLeLeLe-Le- -Le- -Le-Th-Le-Th- -the nucleotide sequence (tcccctatactaggttattggaaaattaagggccttgtgcaacccactcgacttcttttggaatatcttgaagaaaaatatgaagagcatttgtatgagcgcgatgaaggtgataaatggcgaaacaaaaagtttgaattgggtttggagtttcccaatcttccttattatattgatggtgatgttaaattaacacagtctatggccatcatacgttatatagctgacaagcacaacatgttgggtggttgtccaaaagagcgtgcagagatttcaatgcttgaaggagcggttttggatattagatacggtgtttcgagaattgcatatagtaaagactttgaaactctcaaagttgattttcttagcaagctacctgaaatgctgaaaatgttcgaagatcgtttatgtcataaaacatatttaaatggtgatcatgtaacccatcctgacttcatgttgtatgacgctcttgatgttgttttatacatggacccaatgtgcctggatgcgttcccaaaattagtttgttttaaaaaacgtattgaagctatcccacaaattgataagtacttgaaatccagcaagtatatagcatggcctttgcagggctggcaagccacgtttggtggtggcgaccatcctccaaaatcggatctggaagttctgttccaggggcccctg) is shown as SEQ ID NO.15.

According to the design principle of the primer, designing a corresponding primer, and adding an enzyme cutting site. Primer sequences are shown in Table 1 below:

TABLE 1

His (Tag), MBP, GST Gene ligation

The gene sequence was amplified by high-fidelity PCR (KOD-Plus-Neo) from Toyo-yo Shanghai Biotechnology Co., ltd.

The high-fidelity PCR system is as follows: 1 μl of template DNA, 5 μl of 10 XPCR Buffer, 5 μl of dNTPs, 3 μl of magnesium sulfate, 1 μl of each of the upstream and downstream primers, 1 μl of high fidelity enzyme, and sterile water was added to a total volume of 50 μl.

High fidelity PCR procedure: after the reaction system is prepared, the mixture is uniformly mixed by oscillation and then is put into a PCR amplification instrument. Pre-denaturation at 94℃for 2min, denaturation at 98℃for 10 sec, annealing at 62℃for 30s, extension at 68℃for 35 cycles (depending on the size of the gene fragment of interest).

And (3) carrying out agarose gel electrophoresis recovery on the product after the high-fidelity PCR, wherein an agarose gel recovery kit (Shanghai JieRui bioengineering Co., ltd.) is adopted for gel recovery. The operation procedure is as follows:

(1) 1.5% agarose gel was prepared with 1×TAE;

(2) And adding the PCR amplification sample into a 10×loading buffer according to the ratio of 10:1, and blowing and mixing uniformly by using a pipetting gun.

(3) All samples are added into gel holes, a switch is turned on, electrophoresis condition parameters are set to 220V and 30min, and electrophoresis is started;

(4) And (3) scanning the gel after electrophoresis by using a gel scanning system, and comparing whether the target DNA exists or not and whether the band size accords with the corresponding theoretical value.

(5) The band of interest on agarose gel was excised in a UV analyzer and transferred to a 1.5mL EP tube.

(6) Performing adsorption column balancing treatment: 200. Mu.L Buffer CBS was added to the column, centrifuged at 12000rpm for 1min, the waste liquid in the collection tube was discarded, and the column was returned to the collection tube.

(7) The gel containing the fragment of interest will be excised, either estimated or accurately weighed. 150 mu L Binding Solution of agarose gel with concentration of 1.5% is added every 100mg, water bath is carried out for 5-10 min at 50-60 ℃, and the mixing is carried out intermittently and slightly reversely and evenly every 2-3 min until the gel blocks are completely melted.

(8) The above mixed solution was transferred to an adsorption column fitted with a 2mL collection tube, left at room temperature for 2min, centrifuged at 600 rpm for 1min at room temperature, the adsorption column was taken out, and the waste liquid in the collection tube was poured out.

(9) The column WAs replaced in the collection tube, 500. Mu.L WA Solution WAs added, and the mixture WAs centrifuged at 12000rpm for 1min at room temperature to discard the waste liquid in the collection tube.

(10) The column was replaced in the collection tube, 500. Mu.L Wash Solution was added, and the mixture was centrifuged at 12000rpm for 1min at room temperature, and the waste liquid in the collection tube was discarded. Repeating once.

(11) Putting the adsorption column back into the collecting tube again, centrifuging at 12000rpm at room temperature for 1min, opening the cover of the adsorption column, and standing at room temperature for 5-10 min or at 50 ℃ for 3-5 min to thoroughly remove Wash Solution.

(12) Placing the adsorption column into a clean 1.5mL collecting pipe, adding 30-50 mu LELUTION buffer into the center of the membrane in a suspended manner, covering a cover, placing at 37 ℃ for 2min, centrifuging at 12000rpm for 1min, and obtaining the liquid in the centrifuge tube as the solution containing the target DNA fragment. And (5) after the label is written, preserving at the temperature of minus 20 ℃ for standby.

High-fidelity PCR is carried out by taking pMAL-c2X plasmid as a template and F29b-1 and R502 as primers, and the product is named as MBP-1.

The pGEX-6P-1 plasmid is used as a template, F502 and R29b-07 are used as primers for high-fidelity PCR, and the product is named GST-1.

High-fidelity PCR is carried out by taking MBP-1 and GST-1 as templates and taking F29b-2 and R29b-07 as primers, wherein the product is named His (Tag) -MBP-GST, and the size of the His (Tag) -MBP-GST gene is 1803bp.

His (Tag) -MBP-GST Gene was ligated to pET29b plasmid

His (Tag) -MBP-GST gene and pET29b are respectively subjected to double enzyme digestion, and enzyme digestion sites are NedI/BamHI. NedI/BamHI was from Bao Ri doctor Material technology (Beijing) Inc. The procedure for the enzyme digestion was as follows:

(1) PCR products 10. Mu.L, 10 XKBuffer 4. Mu.L, nde I, bamHI enzymes 2. Mu.L each, and sterile water 22. Mu.L.

(2) pET29b plasmid 10. Mu.L, 10 XKBuffer 4. Mu.L, nde I, bamHI enzyme 2. Mu.L each, sterile water 22. Mu.L.

(3) After the enzyme cutting system is prepared, the enzyme cutting system is subjected to vibration and uniform mixing, and then is placed into a dry type thermostat, the temperature is set to be 30 ℃, and the reaction is carried out for 3 hours at constant temperature.

(4) The double enzyme-cut products are respectively subjected to 1% agarose gel electrophoresis, and the results are observed under an imaging system after the electrophoresis is finished. The operation is the same as above.

And cutting the gel to recover the required fragment, and then carrying out agarose gel recovery, wherein the agarose gel recovery operation is the same as that described above.

The double digested His (Tag) -MBP-GST gene was ligated to pET29b plasmid by ligase, the ligated product was designated pET29b-6, and ligase (Ligation high) was derived from Toyo-yo Shanghai Biotechnology Co. The ligation procedure and operation were as follows:

(1) 3 mu L of target DNA; 3. Mu.L of carrier; 3. Mu.L of ligase. After shaking and mixing, the mixture was put into a PCR amplification apparatus and connected at 16℃for 3 hours.

(2) The PET29b-6 plasmid was constructed as shown in FIG. 1.

Example 2

Transformation of DH5a with pET29b-6 plasmid

Preparation of E.coli DH5a competent cells:

(1) E.coli DH5a strain frozen at-80℃was picked up in a super clean bench with a pipette and cultured overnight in 3mL LB liquid medium. The LB liquid medium consists of: yeast extract 0.500%, tryptone 1.000%, sodium chloride 1.000% and water as solvent.

(2) Sucking 400 mu L of overnight cultured bacterial liquid into 100mL of LB liquid, and culturing the bacteria until OD ₆₀₀ The value is about 0.4.

(3) Placing the bacterial liquid on ice, pouring the bacterial liquid into two sterilized 50mL centrifugal barrels on average, and centrifuging the bacterial liquid, wherein the centrifugal parameters are as follows: the temperature is 8 ℃, and the centrifugal force is 10000g for 10min.

(4) Pouring out the supernatant after centrifugation, adding 30mL of 0.1M calcium chloride into a centrifugal barrel, blowing and mixing uniformly, standing on ice for 30min, and centrifuging, wherein the centrifugation parameters are as follows: the temperature is 8 ℃, and the centrifugal force is 10000g for 10min.

(5) After centrifugation, the supernatant was discarded. The above steps are repeated once more.

(6) The centrifuge bowl was placed on ice, 2.5mL of 30% glycerol and 0.1M calcium chloride were added to the centrifuge bowl, respectively, and the mixture was blown and mixed.

(7) The well-mixed competent cells were separated into 1.5mL EP tubes at 100. Mu.L, respectively. After the label is written, the label is put into a refrigerator with the temperature of minus 80 ℃ for preservation.

The ligated product was transferred into E.coli DH5a competent cells, which were derived from Takara doctor technology (Beijing) Co., ltd. The transformation system and procedure were as follows:

(1) DH5a competent cells were removed from the-80℃refrigerator and thawed on ice.

(2) The ligation products were all added to thawed DH5a competent cells and the competent cells were left on ice for 30min.

(3) And (5) carrying out heat shock at 42 ℃ for 45s, and standing on ice for 2min.

(4) The transformation product was added to 500. Mu.L of LB liquid, and cultured at 37℃with shaking at 220rpm for 1 hour, which was culture 1.LB liquid medium was the same as above.

(5) 100. Mu.L of culture 1 was plated on LB agar plates and incubated overnight at 37 ℃. LB agar plates were composed of: yeast extract 0.500%, tryptone 1.000%, sodium chloride 1.000%, agar powder 1.750%, kanamycin 0.001.5% and water as solvent.

Colonies were picked on overnight LB agar plates for ordinary PCR identification, with primers F29b-2, R29b-07, taKaRa Ex Taq from Takara doctor technology (Beijing) Inc.

The general PCR identification procedure was as follows:

(1) 1 mu L of bacterial suspension is used as a template; 13.3 mu L of sterile water; 1 mu L of each of the upstream and downstream primers; buffer 2. Mu.L; dNTP 1.6. Mu.L; taq enzyme 0.1. Mu.L.

(2) And (5) after the reaction system is uniformly mixed by vibration, putting the mixture into a PCR amplification instrument. Pre-denaturation at 95℃for 2min, denaturation at 98℃for 10s, annealing at 60℃for 30s, extension at 72℃for 120s,35 cycles, and complete extension at 72℃for 2min.

Mixing 10 mu L of colony PCR amplification solution with 1 mu L of 10 XLoadingbuffer, performing 1.5% agarose gel electrophoresis with electrophoresis parameters of 220V and 30min, and observing the result under an imaging system after electrophoresis is finished.

Colony PCR identified positive bacteria were inoculated in LB-1 medium, followed by shaking culture at 37℃and 220rpm overnight, which was culture 2. The LB-1 medium consists of: yeast extract 0.500%, tryptone 1.000%, sodium chloride 1.000%, kanamycin 0.001.5% and water as solvent.

Seed conservation is carried out on the culture 2, and the seed conservation parameters are the culture 2:30% glycerol was 1:1 and the storage conditions were-80 ℃.

Identification of pET29b-6 plasmid (containing His (Tag) -MBP-GST Gene)

And (3) taking the culture 2 for plasmid extraction, wherein the recombinant plasmid DNA extraction kit is from Shanghai Jieli bioengineering Co. The plasmid extraction procedure was as follows:

(1) DNA adsorption column balancing treatment: 200 μL buffer CBS was added to Plasmid Recovery Column, centrifuged at 12000rpm for 1min, the waste liquid in the collection tube was discarded, and Plasmid Recovery Column was replaced in the collection tube for further use.

(2) Taking 2-4 mL of bacterial liquid cultured overnight in LB culture medium, centrifuging at 12000rpm for 1min, and discarding the supernatant.

(3) 250. Mu.LSolution I was added and the bacteria were well suspended with a gun head or shaker.

(4) 250 mu L of Solution II is added, and the mixture is immediately and gently and fully turned over and mixed up and down for 6 to 8 times, so that the thalli are fully cracked until a transparent egg white-like Solution is formed.

(5) Adding 350 mu L Solution III, gently and fully turning up and down, mixing for 8-10 times, and standing at room temperature for 2-5 min. Centrifuge at 12000rpm for 10min.

(6) The supernatant was centrifuged at 12000rpm for 1min at Plasmid Recovery Column, and the waste liquid in the collection tube was discarded.

(7) 500 mu L W Solution was added and centrifuged at 12000rpm for 1min at room temperature to pour out the waste liquid from the collection tube.

(8) Add 500. Mu.L Wash Solution, centrifuge at 12000rpm for 1min, pour out waste liquid from the collection tube. Repeating once.

(9) Centrifugation at 12000rpm for 2min, wash Solution was removed thoroughly. Then the cover is opened and the mixture is left at room temperature for a plurality of minutes to thoroughly dry the residual rinse liquid in the adsorption column.

(10) Plasmid Recovery Column was placed in a clean 1.5ml centrifuge tube, 50-100. Mu.L of an addition Buffer was added to the center of the membrane, and the mixture was left at 37℃for 2 minutes. Centrifuging at 12000rpm for 2min, wherein the liquid in the centrifuge tube is the solution containing the target plasmid. Writing number at-20deg.C, and storing.

The extracted plasmid is subjected to ordinary PCR identification, and the primers are F29b-2 and R29b-07, and the identification system and the identification program are the same.

10. Mu.L of the plasmid PCR amplification solution and 1. Mu.L of 10 XLoadingbuffer were mixed and subjected to 1.5% agarose gel electrophoresis with electrophoresis parameters of 220V and 30min, and after the electrophoresis was completed, the results were observed under an imaging system, and the results are shown in FIG. 2.

The conforming plasmid was sequenced by ordinary PCR, and the sequencing primers were the T7 promoter and T7 terminator, sequencing units were from Biotechnology (Shanghai) Inc.

The sequencing result is compared with the original sequence, and the coincidence rate is 100%.

The sequenced plasmid was stored at-20deg.C for further use.

Example 3

Sialidase gene construction

Sialidase Gene cloning

Helicobacter pylori SS1 strain is from Chinese food and drug inspection institute.

Helicobacter pylori cultures are referred to conditions, formulations and methods of operation conventional in the art.

Extracting whole genome from helicobacter pylori culture, and storing the extracted product at-20deg.C. The bacterial genome extraction kit is derived from Shanghai JieRui bioengineering Co.

The amino acid sequence (GluArgGluAsnAlaIleLysSerGlyThrLysLysSerPhePheAsnLysAlaAsnPro ThrGluGluAsnLysProGluProThrProLysProGluGluLysProLysGluGlnAsp LysGlnGluLysGluAlaIleLysGluAsnProAsnThrIleTyrIleIleProLysLys AspIleTrpValGluValIleAspLeuAspGluLysLysAsnSerPheGlnLysValPhe LysLysAsnTyrSerLeuGluThrLysAsnHisArgLeuLeuLeuArgPheGlyHisGly HisLeuSerLeuLysAsnAsnHisGlnGluGlnAspTyrAsnAspSerLysThrArgArg PheLeuTyrGluProAsnLysGlyLeuThrLeuIleAsnGluAlaGlnTyrLysGluLeuGlnGln) of the sialidase is shown as SEQ ID NO.1.

The nucleotide sequence (gagcgagaaaatgctattaaatccggcactaaaaagagttttttcaataaagctaatcctacagaagaaaacaagccagagccaacgcctaaaccagaagaaaaaccaaaagaacaagacaagcaagaaaaagaagcgatcaaagaaaatcctaataccatttatattatccctaaaaaagatatttgggtagaagtgattgatttggatgagaaaaaaaattcctttcaaaaggtttttaaaaaaaattattctttagaaaccaaaaaccaccgcttgttgttgcgttttgggcatgggcatcttagtcttaaaaacaaccatcaagaacaagattataacgacagcaaaactaggcggtttttatacgagccaaataaaggcttaacgctcatcaacgaggcccaatacaaagaactccagcaatga) is shown as SEQ ID NO.2.

According to the design principle of the primers, designing corresponding primers of the sialidase gene, and adding enzyme cutting sites BamHI and EcoRI. Primer sequences are shown in Table 2 below:

TABLE 2

F234	CGGGATCCGAGCGAGAAAATGCTATT as shown in SEQ ID NO.3
		R234	GGAATTCTCATTGCTGGAGTTCTTT as shown in SEQ ID NO.4

The gene sequence is amplified by adopting a high-fidelity PCR method, the size of the Sialidase gene is 429bp, and the high-fidelity PCR enzyme, the high-fidelity system and the procedures are the same.

The high-fidelity product is subjected to agarose gel electrophoresis with the concentration of 1.5%, the electrophoresis parameters are 220V and 35min, and the result is observed under an imaging system after the electrophoresis is finished, and the result is shown in figure 3.

When the high-fidelity PCR fragment is consistent with the original sequence fragment in size, agarose gel recovery is carried out, and the gel recovery operation is the same as that described above.

Sialidase gene was ligated to pET29b-6 plasmid

Double enzyme digestion is carried out on the Sialidase gene and the pET29b-6 respectively, and enzyme digestion sites are BamHI and EcoRI. The enzyme digestion system and the procedure are the same, and the sources of the enzymes are the same.

The double enzyme-cut products are respectively subjected to 1% agarose gel electrophoresis, and the results are observed under an imaging system after the electrophoresis is finished.

The desired fragment was recovered by cutting the gel and the agarose gel recovery was performed as described above.

The double digested Sialidase gene was ligated to pET29b-6 plasmid using ligase, and the source of the ligase, the cleavage system and the procedure were as described above.

pET29b-6-Sialidase transformation DH5a

And transferring the connected product into escherichia coli DH5a competent cells, and obtaining the escherichia coli DH5a competent cells, a transformation system and the same procedures.

The transformation product was added to 500. Mu.L of LB liquid, and cultured at 37℃with shaking at 220rpm for 1 hour, which was culture 3.LB medium was the same as above.

100. Mu.L of culture 3 was plated on LB agar plates and cultured overnight at 37 ℃. LB agar plates were composed as above.

Colonies were picked on overnight LB agar plates for ordinary PCR identification, taKaRa Ex Taq source, ordinary PCR identification system and procedure as above.

Colony PCR identified positive bacteria were inoculated in LB-1 medium, followed by shaking culture at 37℃and 220rpm overnight, which was culture 4.LB-1 medium was the same as above.

Seed conservation is carried out on the culture 4, and the seed conservation parameters are the culture 4:30% glycerol was 1:1 and the storage conditions were-80 ℃.

Overnight culture 4 was taken and plasmid was extracted and designated pET29b-6-Sialidase. Recombinant plasmid DNA extraction kit and plasmid extraction procedure were as above.

The extracted plasmid is subjected to ordinary PCR identification, and the primers are F234 and R234, and the identification system and the identification program are the same.

Taking 10 mu L of plasmid identification common PCR amplification solution and 1 mu L of 10×loading buffer, mixing, performing 1.5% agarose gel electrophoresis with electrophoresis parameters of 220V and 30min, and observing the result under an imaging system after electrophoresis is finished.

The conforming plasmid identified by ordinary PCR is preserved at-20 ℃ for standby.

pET29b-6-Sialidase transformation BL21 (DE) 3

The pET29b-6-Sialidase plasmid is transferred into competent cells of escherichia coli BL21 (DE) 3, and the competent cells of escherichia coli BL21 (DE) 3 are derived from the physical technologies of Bao Ri doctor (Beijing) Co., ltd, and the transformation system and the program are referred to the specification.

The transformation product was added to 500. Mu.L of LB liquid, and cultured at 37℃with shaking at 220rpm for 1 hour, which was culture 5.LB medium was the same as above.

100. Mu.L of culture 5 was plated on LB agar plates and cultured overnight at 37 ℃. LB agar plates were composed as above.

The colony PCR amplification solution (10. Mu.L) and 10×loadingbuffer (1. Mu.L) were mixed and subjected to 1.5% agarose gel electrophoresis with electrophoresis parameters of 220V and 30min, and after electrophoresis, the results were observed under an imaging system, as shown in FIG. 4.

Colony PCR identified positive bacteria were inoculated in LB-1 medium, followed by shaking culture at 37℃and 220rpm overnight, which was culture 6.LB-1 medium was the same as above.

The seed retention was performed on culture 6, with seed retention parameters being overnight culture: 30% glycerol was 1:1 and the storage conditions were-80 ℃.

Sialidase E.coli fermentation

pET29b-6-Sialidase escherichia coli inoculation culture

And taking out the constructed engineering bacteria of pET29b-6-Sialidase escherichia coli from an ultralow temperature refrigerator at the temperature of minus 80 ℃, inoculating the engineering bacteria into an LB-1 culture medium, and culturing at the constant temperature of 37 ℃ and 220rpm for overnight. LB-1 medium was the same as above.

Amplified culture of pET29b-6-Sialidase Escherichia coli

Taking out the overnight cultured engineering bacteria, inoculating the engineering bacteria to a TB culture medium at an inoculating proportion of 1.5%, co-inoculating 4L, and culturing at a constant temperature of 220rpm for 3 hours at 37 ℃. The composition of the TB medium is: 0.2312% of monopotassium phosphate, 1.2540% of dipotassium phosphate, 2.4000% of yeast extract, 1.2000% of tryptone, 0.4000% of glycerol, 0.0500% of defoamer and water as solvent.

Inducible expression of pET29b-6-Sialidase by escherichia coli

At the end of the expansion culture, the induction temperature was set at 15℃and 0.08mM IPTG was added for induction of expression for 20 hours.

pET29b-6-Sialidase escherichia coli thallus collection

After induction, adopting a centrifugal machine to centrifugally collect thalli, wherein centrifugal parameters are as follows: 10000g,8 ℃ for 10min, and placing the thalli at-80 ℃ for standby after centrifugation. The yield of the wet bacteria is 11.77g/L.

Sialidase recombinant protein purification

Bacteria breaking

Taking 10-100 g of thallus obtained by fermentation according to the mass (g): buffer A1 was added at a volume (mL) ratio of 1:15, and the bacterial cell-containing buffer was subjected to shearing suspension at 4℃using a shearing machine. Buffer A1 consists of 1.17% sodium chloride, 0.03% sodium carbonate, 0.18% sodium bicarbonate, pH8.0 and water as solvent.

RO water was used to flush the high pressure homogenizer (AH-1500, ATS Industrial systems Co., ltd.) pipeline. And opening a low-temperature refrigerating system to pre-cool for later use. And (3) adding the precooled suspension into a high-pressure homogenizer, breaking bacteria for 7-8 times under the condition of 480-520 bar, and taking a whole bacteria sample.

The liquid after the sterilization was put into a centrifuge bowl, centrifuged at 10,000 g for 30min at 8℃and the supernatant was collected as supernatant 1.

The supernatant 1 was filtered using a vacuum pump, and the filtered supernatant was collected for use, which was supernatant 2, and then a supernatant sample was taken.

The pellet was suspended in buffer A1, and after suspension, the sample was taken and designated as pellet.

Ni filler affinity chromatography purification of recombinant protein containing double-tag dissolution-promoting Sialidase

Conditions of affinity chromatography:

(1) An instrument system: APPS200D purification System (Lisui technologies (Suzhou Co., ltd.)

(2) And (3) filling: ni-NTA

(3) Purification column specification: 50mm by 200mm

(4) Column loading volume: 100mL of

The buffer A1 was used to equilibrate the packing to electrical conductivity and UV balance.

Loading supernatant 2 at a low temperature of 4 ℃ at a loading flow rate of 10mL/min, taking flow through after loading 1 column volume, which is flow through 1.

Washing for the first time:

(1) After the sample loading is finished, taking a flow through, namely, a flow through 2.

(2) Washing with buffer A1 was performed at a volume of 1 to 2 column volumes and a flow rate of 15mL/min.

And (3) washing for the second time: after washing with buffer A1, washing with buffer A2 until the UV value is less than 100mAu and the flow rate is 15mL/min. Buffer A2 consisted of: sodium chloride 2.92%, sodium carbonate 0.03%, sodium bicarbonate 0.18%, imidazole 0.136%, pH8.0, and water as the solvent.

And (3) washing for the third time: after washing with buffer A2, washing with buffer A3 until the UV value is less than 70mAu and the flow rate is 15mL/min. Buffer A3 consisted of: sodium chloride 1.17%, sodium carbonate 0.03%, sodium bicarbonate 0.18%, imidazole 0.340%, pH8.0, and water as the solvent.

Eluting: after washing with buffer A3, elution was performed with buffer A4 at a flow rate of 15mL/min. The sample collection was started when the uv value was greater than 250mAu and stopped when the uv value was less than 250 mAu. Buffer A4 consisted of: sodium chloride 1.17%, sodium carbonate 0.03%, sodium bicarbonate 0.18%, imidazole 1.700%, pH8.0, and water as solvent. The sample is a recombinant protein containing double-tag dissolution-promoting Sialidase, and is taken for subsequent SDS-PAGE identification, and is named as sample 1.

The Ni filler affinity chromatography purification of the recombinant protein containing double-tag dissolution-promoting Sialidase is shown in figure 5.

Recombinant protease cleavage containing double-tag dissolution-promoting Sialidase

Double-tag-containing, pro-solubilized Sialidase recombinant proteins were diluted 5-fold with buffer A1.

The diluted recombinant protein containing double-tag dissolution-promoting Sialidase is subjected to centrifugal ultrafiltration concentration by a 10KD ultrafiltration tube under the conditions of 4000g and 15min.

The PP enzyme is taken out from the refrigerator with ultralow temperature of-80 ℃ and thawed for standby at the temperature of 4 ℃.

Adding 10-30 mLPP enzyme into the concentrated recombinant protein containing double-tag dissolution-promoting Sialidase, and performing enzyme digestion at 4 ℃ overnight.

Samples were taken after overnight digestion and subsequently identified by SDS-PAGE, which is sample 2.

Sialidase recombinant protein Ni filler affinity chromatography purification

The packing was equilibrated with buffer A1 to electrical conductivity and uv balance. The instruments, packing and purification columns used in this step are the same as above.

And (3) loading the double-tag-containing dissolution-promoting Sialidase recombinant protein solution subjected to enzyme digestion at a low temperature of 4 ℃ overnight, wherein the loading flow rate is 10mL/min.

The sample collection was started when the uv value was greater than 10 mAu.

After the loading was completed, the loading buffer A1 was continued at a loading flow rate of 10mL/min.

The sample collection was stopped when the uv value was less than 10 mAu.

Sialidase recombinant protein concentration

The Sialidase recombinant protein samples were concentrated by centrifugation ultrafiltration using a 10KD ultrafiltration tube at 4000g for 15min.

Concentrating Sialidase recombinant protein to 5-15 mL, and storing in a-80 ℃ ultralow temperature refrigerator for standby.

A sample of the concentrated Sialidase recombinant protein was subjected to subsequent SDS-PAGE identification, which was sample 3.

SDS-PAGE identification of Sialidase recombinant protein

15% SDS-PAGE was performed.

Taking 40 mu L of whole bacteria, supernatant, sediment, flow-through 1, flow-through 2, sample 1, sample 2 and sample 3, respectively, adding 10 mu L of 5x SDS-PAGE Loading Buffer, and boiling water for 5min.

After taking the above boiling water bath, SDS-PAGE was performed at 10. Mu.L per well, and the Protein Marker loading was 3. Mu.L.

After the sample addition is finished, the voltage is firstly regulated to 80V, and electrophoresis is carried out for 15min; and then the voltage is regulated to 220v, and the electrophoresis is carried out for about 40 min.

After electrophoresis, the gel is dyed and decolored by adopting a Coomassie brilliant blue method.

The results were observed under an imaging system after the decoloring was completed, and the results are shown in fig. 6.

Sialidase recombinant protein assay

Protein content was determined by the Fu Lin Fen method.

And estimating and detecting the concentration of the sample according to an SDS-PAGE electrophoresis chart of the Sialidase recombinant protein.

The concentration of the Sialidase recombinant protein is 6.01mg/mL, the correlation coefficient R2 = 0.9907 of the standard curve, and the quality control recovery rate is 95%.

Example 4

Sialidase vaccine oral immunized animals

Oral administration gastric lavage immune animal

1. Experimental animals: female Balb/c mice of 6 weeks of age, 90, 18 g.+ -. 2g. Mice were grouped by random grouping after purchase, 10 mice/cage. Immune group 30, infected group 30, blank group 30. Immunization experiments were performed after 1 day of transitional feeding.

2. Sialidase vaccine composition: sialidase antigen 1mg/mL, LT (B) ₅ 1mg/mL, solvent: sodium chloride 1.17%, sodium carbonate 0.03%, sodium bicarbonate 0.18%, glycerol 5mL/100mL, pH 8.0.

3. Infection group (LT (B) ₅ Adjuvant control group) immunization composition: LT (B) ₅ 1mg/mL, solvent: sodium chloride 1.17%, sodium carbonate 0.03%, sodium bicarbonate 0.18%, glycerol 5mL/100mL, pH 8.0.

4. Oral lavage immunization procedure: total immunization was performed 3 times, and the immunization time points were 0 day, 7 days, and 28 days.

5. Before immunization, the feed and water are required to be cut off 24 hours in advance.

6. Oral lavage immunization:

(1) The helicobacter pylori vaccine is taken out from the temperature of minus 80 ℃ before immunization and is placed in a refrigerator at the temperature of 4 ℃ for thawing for standby.

(2) Sucking Sialidase vaccine with sterile syringe at an immunization dose of Sialidase antigen 1 mg/LT (B) ₅ 1 mg/piece.

(3) The mice were given 1mL of vaccine 3 times per immunization, with a time interval of 20min per oral gavage.

(4) And recovering the water after 2 hours from the end of immunization.

(5) The immunization protocol was identical for each immunization run.

(II) oral gavage infection with helicobacter pylori after last immunization

Oral gavage helicobacter pylori SS1 live bacteria are carried out on the 10 th day after the last immunization for toxicity test, and the infection dose of each mouse is 4 multiplied by 10 ⁶ CFU。

Saliva and blood sample collection after last immunization

1. Mouse saliva sample collection

Saliva was collected from the mice on days 10 and 38 after the end of the last immunization.

Mice need to eat and water for 24 hours before saliva collection.

Before collecting saliva of a mouse, 5mg/mL pilocarpine 20uL is required to be injected into the abdominal cavity of the mouse, and the collected saliva is placed at the temperature of minus 80 ℃ for standby.

2. Blood sample collection in mice

Mice were collected on day 10, and on day 32, from tail venous blood after termination of the last immunization.

3. Mice need to eat and water for 24 hours before saliva collection.

4. Standing the collected blood at room temperature for 4h, centrifuging for 2min with 3000g, absorbing supernatant, repeating the above steps, and standing the separated serum at-80deg.C.

Saliva IgA and serum IgG sample Elisa indirect method detection

1. Elisa detection preparation

The composition of the sealing liquid is as follows: 0.01M PBS,1.5%BSA the solvent is water.

The composition of the PBST washing solution is as follows: 0.01M PBS,0.05mL/100 mLTwen-20, water as solvent.

The antibody dilutions consisted of: 0.01M PBS,0.05mL/100 mLTwen-20, 0.5% BSA, and water as solvent.

The substrate buffer consisted of: disodium hydrogen phosphate 1.4%, citric acid monohydrate 1.5%, and water as the solvent.

The 2M sulfuric acid composition is as follows: concentrated sulfuric acid 11.22mL/100mL, and water as solvent.

1mg/mLTMB consists of: TMB 0.15%, solvent DMSO.

The color development liquid comprises the following components: 1mg/mLTMB: substrate buffer: the volume ratio of the 30% hydrogen peroxide is 100:900:1.

Sialidase antigen coated elisa plate: the ELISA plate was coated with 2ug/mL of the immunizing antigen at 37℃for 2h, and then washed three times with PBST wash. 300. Mu.L/well blocking solution was added to the above ELISA plate and placed in a refrigerator at 4℃and blocked overnight. And then the ELISA plate is washed three times by PBST washing liquid, the ELISA plate is named as an ELISA plate 1, and the ELISA plate is put into a refrigerator with the temperature of 4 ℃ for standby.

2. Serum IgG sample Elisa assay

Serum samples were diluted 1:800 with antibody dilutions, 100 μl/well was added to elisa plate 1, incubated 45min at 37 ℃, and washed three times with pbst wash, designated elisa plate 2.

The goat anti-mouse IgG secondary antibody is diluted by an antibody diluent 1:10000, 100 mu L/hole is added into an ELISA plate 2, 37 ℃ and incubated for 45min, and PBST washing liquid washes the plate three times, and the ELISA plate is named as an ELISA plate 3.

Adding the color development solution into the ELISA plate 3 at 100 μL/well, incubating at 37deg.C for 15min, and adding 2M H at 50 μL/well ₂ SO ₄ Termination solution, this plate is designated as plate 4.

Placing the ELISA plate 4 into an ELISA analyzer, and selecting OD ₄₅₀ And (5) detecting, and storing the detection data and carrying out subsequent analysis.

3. Saliva IgA sample Elisa assay

Saliva samples were diluted 1:4 with antibody dilutions, 100. Mu.L/well was added to ELISA plate 1, incubated 45min at 37℃and washed three times with PBST wash, designated ELISA plate 5.

The goat anti-mouse IgA secondary antibody is diluted with an antibody diluent 1:5000, 100 mu L/hole is added to an ELISA plate 5, incubation is carried out for 45min at 37 ℃, and the plate is washed three times by a PBST washing solution, wherein the ELISA plate is named as an ELISA plate 6.

Adding the color development solution into the ELISA plate 6 at 100 μL/well, incubating at 37deg.C for 15min, and adding 2M H at 50 μL/well ₂ SO ₄ Termination solution, this plate is designated as plate 7.

Placing the ELISA plate 7 into an ELISA analyzer, and selecting OD ₄₅₀ And (5) detecting, and storing the detection data and carrying out subsequent analysis.

4. Saliva IgA and serum IgG potency detection results

And (3) judging a detection result: a positive value of > 2.1 was defined for the samples (immune group)/negative (infected group). Immune group titer detection positive mice/immune group mice x 100% were defined as positive turnover rate.

(1) Saliva IgA potency detection results: the saliva IgA (1:4) titer detection positive turnover rate at the endpoint of the mice immune group protection rate determination was 40%, as shown in FIG. 7.

(2) Serum IgG titer assay results: the serum IgG (1:800) titer detection positive turnover rate at the endpoint of the mouse immune group protection rate determination is 70%, as shown in FIG. 7.

(3) The Sialidase vaccine has good immunogenicity, can induce organisms to generate immune response, and provides support for the Sialidase vaccine to be used for eliminating the colonization of helicobacter pylori in the stomach.

Sialidase vaccine protection results

1. The protection rate of Sialidase vaccine was calculated by detecting the colonization rate of helicobacter pylori infection of each group of mice by plate culture at day 38 after the end of the last immunization. Protection = (number of immunized groups x infection rate of infected groups-number of immunized groups infected groups)/(number of immunized groups x infection rate of infected groups) ×100/100.

2. Helicobacter pylori plating refers to conditions, formulations and methods of operation conventional in the art.

3. Helicobacter pylori plate culture criterion:

(1) Colony morphology identification criteria: characteristic bacterial colonies with the diameter of 0.1-0.5 mm and the transparent needle tip sample size grown on the flat plate are judged to be positive to helicobacter pylori, and the positive bacterial colonies are indicated by "+" signs; if no colony grows on the plate or is not in the above-mentioned form, it is judged that helicobacter pylori is negative, and the result is indicated by "-" sign; when bacteria suspected of helicobacter pylori colony morphology were grown on the plates, they were indicated by "x".

(2) Fast urease assay identification criteria: dropping a proper amount of urease solution on a flat colony, and judging that helicobacter pylori is positive if the colony turns red, wherein the colony is marked with a "+" sign; if the colony urea thixotropic reaction does not change into red or does not change color, the colony is judged to be negative to helicobacter pylori, and the colony is indicated by a "-" sign; when the color of the colony urea thixotropic enzyme reaction changes to be not obvious in red, the colony is judged to be suspected helicobacter pylori, and the colony is expressed by the 'x'.

(3) Rapid gram staining microscopic identification criteria: microscopic examination is carried out on colony morphology and the suspected rapid urease experiment, and the microscopic examination standard is as follows: when the bacteria are purple and have spiral bent shapes and blunt ends, the bacterial colony is judged to contain helicobacter pylori, and the helicobacter pylori is represented by a "+" sign; on the contrary, if the microscopic bacteria are not purple and have no spiral bend and blunt end, the microscopic bacteria are judged to be negative to helicobacter pylori, and the helicobacter pylori is indicated by a "-" sign; if this detection is not performed, it is indicated by a "/" number.

4. Helicobacter pylori plate culture criterion: when one or more than one colony grows on the flat plate, the colony is identified, a rapid urease experiment and rapid gram staining microscopic examination are carried out, and when the colony is identified and the rapid urease experiment are positive or the rapid gram staining microscopic examination is positive, the colony is determined to be helicobacter pylori, and the success of infection and colonization of the helicobacter pylori in the stomach of the mouse is determined; when the colony morphology identification and the rapid urease experiment are negative or the rapid gram staining microscopic examination is negative, the colony is judged not to be helicobacter pylori, and meanwhile, the helicobacter pylori infection and colonization in the stomach of the mouse are judged to be unsuccessful.

5. The results of the implantation rate detection for the Sialidase vaccine immunization groups of 3 consecutive rounds (10 animals/round) of animal protection experiments are shown in tables 3 to 5 below:

TABLE 3 Table 3

TABLE 4 Table 4

TABLE 5

6. Since the identification of H.pylori colony morphology and the rapid urease experiment can be directly performed from the culture plate, rapid gram staining microscopy was not performed.

7. Based on the plate culture results, the average planting rates of each group of continuous 3-round animal protection experiments are shown in the following table 6:

TABLE 6

Group of	Fixed planting rate
		Immunization group
	70％(21/30)
		Infection group	93.3％(28/30)
Blank control group	0％(0/30)

According to the result of the planting percentage, the protection rate is shown in the following table 7:

TABLE 7

Group of	Protection rate
		Immunization group	25％(7/28)
Infection group	0％(0/28)
		Blank control group	0％(0/30)

As can be seen from the above table, the average protection rate of 3 rounds in the immunized group was 25%, and the average protection rate of 3 rounds in the infected group was 0%.

Therefore, the Sialidase recombinant protein has good immunogenicity, can induce mice to generate mucosal immune response, can eliminate the colonization of helicobacter pylori SS1 strain in the stomach of the mice, and can be used for preventing helicobacter pylori infection by preparing a helicobacter pylori subunit vaccine from a mucosal adjuvant and the Sialidase recombinant protein.

Through the embodiment, the double-promotion-dissolution tag can express a large amount of outer membrane Sialidase recombinant protein in supernatant, has simple fermentation and purification steps, and the obtained Sialidase recombinant protein has high yield and strong immunogenicity, and can be further applied to the prevention of helicobacter pylori infection.

In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Yi Gong Yi Miao Biotech Co Ltd

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Glu Glu Lys Pro Lys Glu Gln Asp Lys Gln Glu Lys Glu Ala Ile Lys

35 40 45

Glu Asn Pro Asn Thr Ile Tyr Ile Ile Pro Lys Lys Asp Ile Trp Val

50 55 60

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

65 70 75 80

Lys Lys Asn Tyr Ser Leu Glu Thr Lys Asn His Arg Leu Leu Leu Arg

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Phe Gly His Gly His Leu Ser Leu Lys Asn Asn His Gln Glu Gln Asp

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Tyr Asn Asp Ser Lys Thr Arg Arg Phe Leu Tyr Glu Pro Asn Lys Gly

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Leu Thr Leu Ile Asn Glu Ala Gln Tyr Lys Glu Leu Gln Gln

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His His His His His His His

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catcatcatc atcatcatca t 21

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Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys Gly

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

20 25 30

Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe Pro

35 40 45

Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala His

50 55 60

Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr

65 70 75 80

Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala

85 90 95

Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala

100 105 110

Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr

115 120 125

Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys

130 135 140

Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu

145 150 155 160

Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr

165 170 175

Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly Leu

180 185 190

Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp Thr

195 200 205

Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala Met

210 215 220

Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys Val

225 230 235 240

Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys

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Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn

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

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Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala Leu

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305 310 315 320

Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln Met

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gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca acatcgacac cagcaaagtg 720

aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc gttcgttggc 780

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Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly

35 40 45

Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu

50 55 60

Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn Met

65 70 75 80

Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu Gly

85 90 95

Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys

100 105 110

Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu Met

115 120 125

Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn Gly

130 135 140

Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp Val

145 150 155 160

Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu Val

165 170 175

Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu

180 185 190

Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr

195 200 205

Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu Phe

210 215 220

Gln Gly Pro Leu

225

<210> 15

<211> 684

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tcccctatac taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg 60

gaatatcttg aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg 120

cgaaacaaaa agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt 180

gatgttaaat taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg 240

ttgggtggtt gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat 300

attagatacg gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat 360

tttcttagca agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca 420

tatttaaatg gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt 480

gttttataca tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa 540

cgtattgaag ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg 600

cctttgcagg gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg 660

gaagttctgt tccaggggcc cctg 684

Claims

1. A sialidase for use in gastric colonization against helicobacter pylori, wherein the amino acid sequence of the sialidase is shown in SEQ ID No. 1.

2. A gene encoding the sialidase of claim 1, wherein the nucleotide sequence of said gene is shown in SEQ ID No. 2.

3. A recombinant expression vector comprising the gene of claim 2.

4. The recombinant vector according to claim 3, further comprising a His-tag sequence, a gene encoding a prolamin; the genes encoding the pro-lysin include GST and MBP.

5. A method of constructing the recombinant expression vector of claim 3, comprising the steps of:

1) Amplifying helicobacter pylori genome DNA by using an F234/R234 primer pair to obtain a gene segment for encoding sialidase;

The nucleotide sequence of the F234 primer is shown as SEQ ID NO. 3;

the nucleotide sequence of the R234 primer is shown as SEQ ID NO. 4;

2) Inserting the gene segment encoding sialidase into a backbone vector to obtain a recombinant expression vector; the multicloning site of the skeleton vector is BamHI/EcoRI.

6. The method of construction according to claim 5, wherein the backbone vector is a backbone vector comprising a His-tag sequence and a gene encoding a prolamin;

the construction method of the framework vector containing the His tag sequence and the gene for encoding the pro-lysin comprises the following steps:

the nucleotide sequence of the F29b-1 primer is shown as SEQ ID NO. 5;

the nucleotide sequence of the R502 primer is shown in SEQ ID NO. 6;

the nucleotide sequence of the F502 primer is shown in SEQ ID NO. 7;

the nucleotide sequence of the R29b-07 primer is shown as SEQ ID NO. 8;

B. performing PCR amplification by using the MBP gene fragment and the GST gene fragment as templates and using an F29b-2/R29b-07 primer pair to obtain His (Tag) -MBP-GST fragments;

The nucleotide sequence of the F29b-2 primer is shown as SEQ ID NO. 9;

C. inserting the His-MBP-GST fragment into pET29b plasmid to obtain a sequence containing His tag and two codes

Recombinant expression vector of the gene of the prolamin.

7. The construction method according to claim 6, wherein the inserted multicloning site of pET29b plasmid is NedI/BamHI.

8. A recombinant strain comprising the gene of claim 2 or the recombinant vector of claim 3.

9. Use of a sialidase obtained by recombinant expression of the recombinant strain of claim 8 or the sialidase of claim 1 for the preparation of a biologic for preventing helicobacter pylori infection.

10. The use according to claim 9, wherein the biologic is a vaccine.