CN115724923A

CN115724923A - Helicobacter pylori vaccine recombinant protein antigen NC-1, and preparation method and application thereof

Info

Publication number: CN115724923A
Application number: CN202211195643.7A
Authority: CN
Inventors: 刘开云; 李彦; 刘宇; 孙敏; 于婕
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2022-07-19
Filing date: 2022-09-29
Publication date: 2023-03-03
Anticipated expiration: 2042-09-29
Also published as: CN115724923B

Abstract

The invention provides a helicobacter pylori vaccine recombinant protein antigen NC-1, the amino acid sequence is shown as SEQ ID NO. 1. The present invention also provides a nucleic acid encoding the helicobacter pylori vaccine recombinant protein antigen NC-1 according to claim 1. The nucleic acid sequence is shown as SEQ ID NO. 2. The helicobacter pylori vaccine recombinant protein antigen NC-1 provided by the invention has the advantages of soluble expression, easiness in purification, high purity, simplicity and convenience in preparation method and the like, has remarkable economic benefits, has good immune protection effect by effectively stimulating an organism to generate immune response through animal experiments, and can be used as a vaccine candidate component for preventing helicobacter pylori infection.

Description

Helicobacter pylori vaccine recombinant protein antigen NC-1, and preparation method and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to preparation and application of Helicobacter Pylori (HP) recombinant protein NC-1.

Background

Helicobacter Pylori (HP) is a gram-negative bacterium, one of the most common bacterial pathogens that pose a health hazard to humans. The global infection rate is 19-88%, the annual recurrence rate is 1.75-2.4%, and the trend is gradually increased year by year, wherein the HP infection rate of developing countries is up to 90%, and the annual recurrence rate is far higher than that of developed countries. In HP-infected patients, 15% -20% of the patients develop more serious diseases, including duodenal ulcer, gastric ulcer, mucosa-associated tissue lymphoma and gastric adenocarcinoma. In addition, numerous studies have demonstrated that HP infection is also closely related to the development of several other diseases of the non-digestive system.

For HP, the world health organization classified it as a class i carcinogen as early as 1994, and the global consensus of kyoto also made its treatment as a primary prophylactic for gastric cancer. The treatment of HP is usually carried out by combining antibiotics. However, antibiotic therapy also has major disadvantages, such as: the treatment cost is expensive, the recurrence rate is high, and the patients with the serious chronic diseases and the like can not normally take the medicine due to counter factors such as the elderly, the children of the low age, the pregnant women, the patients with the serious chronic diseases and the like. In this case, only if infection is effectively prevented by means of immunization, and the transmission of HP is gradually blocked, the infection can be fundamentally controlled, so that the occurrence of related diseases is reduced.

It is known that while bacteria are in the process of maintaining and living, absorption of nutrients and excretion of metabolites are involved, and most of the molecules and compounds cannot directly enter and exit through cell membranes due to the existence of the cell membranes. This requires a large number of specific membrane proteins to perform transport and thus ensure their absorption and metabolism. Wherein sodium/calcium exchanger is an important bidirectional transport protein on cell membrane, and 3 Na are activated ⁺ Transport to the inside/outside of the cell while simultaneously transporting 1 Ca ²⁺ Transport to the extracellular/intracellular location, thereby maintaining the balance of physiological functions of the bacteria. Once the regulation function is lost, ca is generated ²⁺ Abnormal accumulation such as overload can cause serious damage to the bacteria. It is because of the importance of sodium/calpain in the survival of HP that it is widely distributed on the cell membrane and highly conserved.

Therefore, the partial structure of the protein is screened out by a bioinformatics technology 'reverse vaccinology' so as to develop the candidate antigen of the HP genetic engineering vaccine, and the candidate antigen has very important significance for preventing and treating the helicobacter pylori.

Disclosure of Invention

The invention aims to provide a helicobacter pylori vaccine recombinant protein antigen NC-1 and a preparation method thereof, which can effectively stimulate an organism to generate immune response, have good immune protection effect and have good clinical application prospect.

The invention provides a helicobacter pylori vaccine recombinant protein antigen NC-1, the amino acid sequence is shown as SEQ ID NO. 1.

The invention also provides a nucleic acid for encoding the helicobacter pylori vaccine recombinant protein antigen NC-1. In a preferred embodiment, the nucleic acid sequence is as set forth in SEQ ID NO 2.

The invention provides a preparation method of the helicobacter pylori vaccine recombinant protein antigen NC-1, which comprises the following steps:

1) Plasmid construction, prokaryotic expression

Connecting the recombinant protein antigen NC-1 nucleic acid into an expression vector plasmid, and transferring the connected plasmid into host bacteria for induced expression;

2) Breaking and centrifuging:

collecting expressed bacteria liquid, re-suspending and mixing the bacteria liquid with pH6.0-8.0, homogenizing under given pressure, centrifuging, and collecting supernatant;

3) And (3) purifying the supernatant sequentially by using a Ni affinity chromatographic column and an SP chromatographic column respectively to obtain the recombinant protein antigen NC-1.

In the step 1), the recombinant protein antigen NC-1 nucleotide sequence is screened out by reverse vaccinology; the expression vectors used are pET28a, pET22b, pET30a and the like; the host bacteria are E.coli BL21 (DE 3), AD494 (DE 3), origami B (DE 3) and the like; the induced expression condition is induced expression overnight at 16-37 ℃ and 150-220 rpm; 0.1-0.5mM IPTG was used for induction of expression.

In the step 2), the bacteria-breaking solution is 20-50mM PB, 0.15-0.5M NaCl and 0-25 mM imidazole with the pH value of 6.0-8.0; the bacteria breaking parameters are that the external circulation temperature is-4 to-2 ℃, the pressure is 600 to 700bar, the power is 20 to 30 percent, and 6 to 7 circulations are carried out; the centrifugation condition is 12000-17000g, 30-40min.

The Ni affinity chromatography column affinity purification operation in the step 3) comprises the following steps: firstly, balancing a Ni affinity chromatographic column by using solution A, then sampling filtered supernate, and performing gradient elution by using mixed solution of solution A and solution B in different proportions; the Ni affinity chromatography column uses Ni Sepharose High Performance (cytiva, cat # 17526802); the solution A is as follows: 20-50mM PB with pH of 6.0-8.0, 0.15-0.5M NaCl, 20-25 mM imidazole; the B solution is 20-50mM PB, 0.15-0.5M NaCl and 0.5-1M imidazole with pH value of 6.0-8.0.

The SP chromatography column affinity chromatography operation is: the target protein obtained after purification by a Ni affinity chromatographic column is obtained by firstly balancing an SP chromatographic column by using a solution C and then eluting the solution C and the solution D in different proportions; the SP column used was a packing material SP Sepharose High performance (cytiva, cat # 17108701); the solution C is: 20 to 50mM PB, pH6.0 to 8.0; the solution D is 20-50mM PB with pH6.0-8.0 and 0.5-1M NaCl.

The purity of the recombinant protein antigen NC-1 obtained by the preparation method is more than 95%, the molecular mass of the recombinant protein antigen NC-1 is predicted to be about 54.8KD through an amino acid sequence, and the isoelectric point is about 9.1 (obtained through the prediction of the amino acid sequence).

The invention further provides the application of the helicobacter pylori vaccine recombinant protein antigen NC-1 in preparing a helicobacter pylori recombinant subunit genetic engineering candidate vaccine. The purification method of the present invention mainly comprises Ni affinity purification and SP affinity chromatography, and the recombinant protein antigen NC-1 purified by the above method is detected by 15% SDS-PAGE, and presents a single target protein band, the molecular mass is about 54.8KD, and the protein purity is 98.12%. And (3) injecting the purified NC-1 and an aluminum phosphate adjuvant together to immunize BalB/C mice, and finding that the IgG titer of the serum antibody generated by the recombinant protein antigen NC-1 immunized mice reaches 1:128000; after the recombinant protein antigen NC-1 and LTs63K adjuvant are dripped into a nose to immunize a Balb/c mouse, the titer of antibody sIgA can be obviously improved, and mucosal immune response is induced to be generated. This proves that the recombinant protein antigen NC-1 obtained by the purification method of the invention can effectively stimulate the organism to generate higher immune response. And the immune protective ability evaluation experiment proves that the composition has good protective effect. Compared with the prior art, the invention has the following beneficial effects:

1. the helicobacter pylori vaccine recombinant protein antigen NC-1 is obtained by expression of escherichia coli genetic engineering, and animal experiments show that the helicobacter pylori vaccine recombinant protein antigen NC-1 can effectively stimulate an organism to generate immune response and has good immune protection effect; can be used as a candidate component of vaccine for preventing helicobacter pylori infection;

2. the preparation method of the helicobacter pylori vaccine recombinant protein antigen NC-1 provided by the invention is simple in process, and the obtained target protein antigen has the advantages of soluble expression, easiness in purification, high purity, good repeatability, high recovery rate and the like, and has remarkable economic benefits.

Drawings

FIG. 1 shows the result of double restriction enzyme identification of recombinant plasmid NC-1-Pet28 a; m: takara DL5000 DNA Marker; lane 1: plasmid pET28a-N-C-antiporter-1; lane 2: double enzyme digestion pET28a-N-C-antiporter-1; the identification revealed that the isolated fragments were about 5300bp and 1389bp.

FIG. 2 shows the result of NC-1 protein induction identification; lane 4: a whole bacterial liquid; lane 5: breaking the bacteria and clearing the supernatant; lane 6: breaking the bacteria and precipitating; m: thermo Scientific Protein miller; the identification result shows that the NC-1 protein is about 54.8KD and can be expressed in a soluble mode.

FIG. 3 shows the result of NI affinity chromatography; (a) Is (Bio-Rad, NGC QUEST 100 PLUS raw purification data plot); (b) is the result of electrophoresis; 1: sampling; 2: flow through; 3:5% of B-1;4:5% by weight of B-2;5:10% of B-1;6:10% of B-2;7:10% of B-3;8:15% by weight of B-1;9:20% by weight of B;10:30% of B-1;11:30% by weight of B-2;12:50% by weight of B; m: marker

FIG. 4 shows the results of SP chromatography; (a) Is (Bio-Rad, NGC QUEST 100 PLUS raw purification data graph); (b) is the result of electrophoresis; 1: sampling; 2: flow through; 3:35% by weight of D;4:45% by weight of D;5:55% by weight of D; m: marker

FIG. 5 shows the result of LTs63K electrophoretic identification; 1: sampling; 2: flow through; 3: eluting 1;4: eluting 2;5: eluting 3; m: marker;

FIG. 6 shows the IgG geometric mean titer of mice immunized with the recombinant protein antigen NC-1 (corresponding to an antibody titer of 1;

FIG. 7 shows the geometric mean titers of sIgA of vaginal lavage of mice after immunization with the recombinant protein antigen NC-1 (corresponding to antibody titer 1.

Detailed Description

So that the technical solutions of the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings, it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, belong to the present invention.

The strains and reagents used in the following examples are as follows:

(1) Helicobacter pylori strains:

helicobacter pylori is purchased from American ATCC (J99/Helicobacter pylori: (R))

700824))；

(2) Reagent:

1) Plasmid pET-28a was purchased from GE Healthcare Life Sciences;

2) Coli strain BL21 (DE 3) was purchased from Shanghai super research Biotech, inc.;

3) DNA Marker, restriction enzyme Nco I and Xho I, T4 DNA Ligase and protein Marker are Thermo Fisher products;

4) The plasmid extraction kit, the gel recovery kit, the bacterial genome extraction kit and the ultrathin recovery kit are products of Tiangen Biochemical technology (Beijing) Co.

Example 1: construction and identification of recombinant plasmid pET28a/NC-1 of NC-1 gene

Firstly, according to the whole genome sequence of helicobacter pylori, a reverse vaccinology theory is applied and a bioinformatics technology is used to screen out candidate antigen NC-1 (see WP _ 209612225.1sodium.

Coli preference codon optimization was performed based on the amino acid sequence of antigen NC-1 (SEQ ID NO: 1) (see Villalobos A, ness JE, gustafsson C, minshull J, govindarajan S.Gene Designer: a synthetic biology tool for constructing affinity DNA segments BMC Bioinformatics.2006) to obtain the nucleic acid sequence of interest, SEQ ID NO:3 (the first CCATGG and the last CTCGAG represent the cleavage site base sequence).

Synthesis of the nucleic acid sequence of interest: the target gene fragment was inserted into expression plasmid pET28a through Nco I and Xho I restriction sites, and the nucleic acid sequence (SEQ ID NO: 2) was completely identical as a result of the plasmid sequencing compared with the sequence information submitted for synthesis.

The synthetic plasmid was dissolved in 40. Mu.l sterile water, 2. Mu.l of transformed E.coli BL21 (DE 3) competent cells were taken, ice-washed for 30min, heat-shocked at 42 ℃ for 90s, and ice-washed rapidly for 3min. Adding 1ml SOC culture medium, mixing, and shaking in 37 deg.C shaker at 220rpm for 45min.

100 mul of the bacterial liquid was spread on Kana resistant LB plate and cultured in 37 ℃ incubator for 16h.

Then, the screening and the identification of the pET28a/NC-1/BL21 (DE 3) positive recombinant plasmid are carried out, and the method comprises the following steps:

(1) Selecting a single colony well separated on a transformation plate, inoculating the single colony in a Kana resistant LB culture medium, and carrying out shaking culture at 37 ℃ overnight;

(2) Extracting plasmids according to the specification of the plasmid extraction kit;

(3) Carrying out Nco I and Xho I double enzyme digestion on the extracted plasmid DNA at 37 ℃, wherein the enzyme digestion time is 2h; the double enzyme digestion reaction system is shown in table 1;

(4) The double restriction enzyme was detected by electrophoresis using 1.5% agarose gel (1.5 g agarose dissolved in 100ml TAE buffer), and the results are shown in FIG. 1, indicating that the construction of the recombinant plasmid was successful.

TABLE 1 double digestion reaction System

Reagent	Volume of mu.l
		10×K Buffer	1
0.1％BSA	1
		Xho I	0.4
Nco I	0.4
		Plasmids	2
Water,nuclease-free	Up to 10

Example 2: recombinant protein NC-1 is induced to express, purify and identify expression form in prokaryotic expression system-escherichia coli

Adding 100 μ l of overnight cultured pET28a/NC-1/BL21 (DE 3) bacterial liquid into 10mL LB culture medium with kanamycin + resistance, and culturing overnight at 37 ℃ and 220rpm in a shaking manner; separately, 200. Mu.l of overnight-cultured bacterial liquid was added to 20mL of LB medium resistant to kanamycin + and cultured at 37 ℃ for 2 hours at 220rpm, and when the cells were secondarily activated to OD600 of 0.8, 10. Mu.l of IPTG isopropyl-. Beta. -D-thiogalactoside was added to give a final concentration of 0.5mM, and the cells were induced to express overnight at 220rpm on a shaker at 16 ℃.

Taking out the bacteria liquid after induction expression, centrifuging for 15min at 8000g, discarding the supernatant, adding 3ml of bacteria breaking liquid (50mM PB,0.5M NaCl,25mM imidazole, pH 6.0), mixing, performing ice-bath ultrasonic lysis for 10min (stopping ultrasound for 6s at 5 s), centrifuging for 30min at 12000g at 4 deg.C, and separating the supernatant and precipitate.

Treating a sample: adding 3ml of bacteria breaking liquid into the sediment for resuspension, respectively taking 40 ul of the bacteria breaking liquid, the supernatant and the resuspended sediment, adding 10 ul of 5X protein loading buffer (Bio-engineering, product number: C508320-0010) into each 40 ul of the sediment, standing for 10min at 100 ℃ of a metal bath, and then centrifuging for 3min at 12000 g.

SDS-PAGE electrophoresis: 10 μ l of the treated lysate, supernatant and precipitate were subjected to SDS-PAGE at 15% concentration. After the electrophoresis, the gel was taken out, stained in Coomassie brilliant blue staining solution, destained in destaining solution (5% glacial acetic acid and 10% anhydrous ethanol aqueous solution), and scanned by using a gel scanning imaging system (ChemiDoc MP, bio-Rad), and the scanning results are shown in FIG. 2. The result shows that the target protein of pET28a/NC-1/BL21 (DE 3) has correct size and is soluble expression.

Example 3: preparation of recombinant protein antigen NC-1

Obtaining the recombinant protein NC-1 by amplification culture: 30mL of overnight-cultured pET28a/NC-1/BL21 (DE 3) bacterial solution was added to 3L Kana + resistant TB medium, and cultured at 37 ℃ and 220rpm for 3 hours, and when the OD600 was 1, 1.5mL of 1M IPTG was added to give a final concentration of 0.5mM, and expression was induced at 16 ℃ and 220rpm overnight. The induced bacterial liquid is centrifuged for 15min at 8000g to collect thalli, 160ml of bacteria breaking liquid (same as example 2) is added to resuspend the thalli, and the bacterial liquid is subjected to high-pressure homogenization and crushing (external circulation temperature-4 ℃, pressure 650bar, power 25% and 6 cycles). Then, the mixture was centrifuged at 12000g for 30min to obtain a supernatant.

Purifying the recombinant protein NC-1, comprising the following steps:

(1) Ni column affinity chromatography: taking supernatant of the bacteria-breaking liquid, and filtering the supernatant through a 0.45 mu m filter membrane for later use. Balancing Ni column affinity chromatography column using A liquid (50mM PB,0.15M NaCl,25mM imidazole, pH 6.0), taking the supernatant, loading the supernatant, using 5% B liquid (50mM PB,0.15M NaCl,0.5M imidazole, pH 6.0) +95% A liquid elution impurity, 10% B liquid +90% A liquid elution objective protein, 15% B liquid +85% A liquid elution objective protein, 20% B liquid +80% A liquid elution objective protein, 30% B liquid +70% A liquid elution objective protein, 50% B liquid +50% A liquid elution objective protein. The chromatogram of the Ni-column affinity column is shown in FIG. 3 (a), and the electrophoresis result of the Ni-column affinity column after SDS-PAGE is shown in FIG. 3 (b).

(2) SP column chromatography

60ml of the protein eluted in (1) was diluted to 600ml with 20mM PB (pH 6.0). Equilibrating the SP column with solution C, sampling, loading, washing with solution C, eluting with 35% solution D (50mM PB,1M NaCl, pH 6.0) +65% solution C, eluting with 45% solution D +55% solution C, eluting with 55% solution D +45% solution C, collecting the eluted target protein (i.e. protein antigen NC-1), and storing at 4 deg.C for future use. The chromatogram of SP column chromatography is shown in FIG. 4 (a), and the electrophoresis result of SDS-PAGE after SP column chromatography is shown in FIG. 4 (b), and the target protein with purity of 98.12% is obtained.

Example 4: immune and serum-specific antibody IgG detection in animals

(1) Recombinant protein antigen NC-1 and aluminum phosphate adjuvant combined immune mouse

Balb/C mice, female, 8-10 weeks old, were purchased from Jiangsu Jiejiaokang Biotech GmbH. The test solution is divided into an immunization group (recombinant protein antigen NC-1+ aluminum phosphate adjuvant), a negative control group (aluminum phosphate adjuvant) and a blank control group, wherein each group comprises 20 mice.

The process for immunizing mice by combining the recombinant protein antigen NC-1 with the aluminum phosphate adjuvant comprises the following steps:

1) For the first immunization, the volume ratio of 50 mu g of recombinant protein antigen NC-1 to aluminum phosphate adjuvant is 1:1 mix and inject bilaterally into the thigh muscle (100. Mu.l/mouse).

2) The second immunization, the second immunization is carried out on the 14 th day, and the injection amount and the immunization mode are the same;

3) The third immunization, which is carried out on the 21 st day, and the injection amount and the immunization mode are the same;

4) The fourth immunization, the third immunization on day 28, was performed in the same manner as the immunization.

5 days after the fourth immunization, orbital venous blood of Balb/C mice was collected, left to stand at 4 ℃ for 3 hours, centrifuged at 3000rpm for 5min to separate serum, and then subjected to Elisa to detect NC-1 specific IgG level changes, comprising the following steps:

1) Antigen coating: taking the coating solution to dilute the NC-1 purified protein prepared in the example 3 to 4 mu g/mL, then coating an ELISA plate according to 100 mu L/hole, and standing overnight at 4 ℃; the coating solution was 20mM PB,0.15M NaCl, pH 6.0.

2) And (3) sealing: sealing solution of 300 mu L/hole, incubating at 37 ℃ for 1h, washing the plate by PBST, and storing at 4 ℃ for later use; the blocking solution was 10mM PBS (pH 7.4) +1% BSA.

3) Diluting a specimen: sera were serially diluted in multiples starting from 1.

4) Sample adding: taking the coated ELISA plate, sequentially adding diluted serum and 100 mu L/hole, performing two repetitions of each sample, incubating at 37 ℃ for 1h, and then washing with PBST for 4 times; PBST wash 10mM PBS (pH7.4) +0.05% Tween-20.

5) Adding a secondary antibody: diluting HRP-labeled goat anti-mouse IgG (Bio-Industrial, cat: D110087-0100) in an antibody diluent 1 (10000) at 100. Mu.L/well, incubating at 37 ℃ for 30min, and then washing with PBST 4 times; antibody dilution 10mM PBS (pH7.4) +0.05% Tween-20+0.5% BSA.

6) Color development: adding 100 μ L/well substrate developer, incubating at 37 deg.C for 10min, and stopping addingMeasuring OD value on an enzyme labeling instrument at the wavelength of 450nm by using 50 mu L of the solution per hole; the color development liquid is TMB storage liquid: substrate buffer: 3% hydrogen peroxide = 10: 90: 1; dissolving TMB in DMSO, wherein the TMB stock solution is 1 mg/mL; the substrate buffer solution is 0.53mM (pH 5.0) of citric acid and Na ₂ HPO ₄ 100mM; the medium stop solution is 2 MH ₂ SO ₄ 。

7) And (5) judging a result: the A sample/A negative is more than or equal to 2.1 and is positive.

As a result: as shown in Table 2 and FIG. 6, the titer of the antibody IgG generated by the mice immunized with the recombinant protein antigen NC-1 reaches 1:128000; the geometric mean titer of the NC-1 immunized mice to the recombinant NC-1 is 1 73731.83, and the antibody positive rate reaches 100 percent after immunization, which shows that the recombinant protein antigen NC-1 can enable the immunized mice to generate antibodies in vivo.

TABLE 2 IgG geometric mean Titers

(2) Recombinant protein antigen NC-1 and LTs63K adjuvant combined nasal drop immunized mouse

Balb/C mice, female, 8-10 weeks old, purchased from Jiangsu Jiejiaokang Biotechnology GmbH, LTs63K, self-made (preparation method is shown in the literature: construction expression and property research of von Kangchi recombinant Escherichia coli heat-labile enterotoxin and mutants thereof, and B subunit thereof [ D ]. Chongqing university 2003), the preparation results are shown in FIG. 5, and the animal groups are shown in the following Table 3:

TABLE 3 grouping of NC-1 mice immunized in combination with LTs63K adjuvant

The process for immunizing mice by combining the recombinant protein antigen NC-1 with LTs63K adjuvant comprises the following steps:

1) First immunization, mixing 50 mug recombinant protein antigen NC-1 and 10 mug LTs63K adjuvant, gently mixing for 30min at 4 ℃ by a mixer, placing in an ice box for immunization for standby, sucking the prepared immunogen, and slowly dripping in a mouse nasal cavity: 7 μ L/side, total 14 μ L/mouse;

2) The second immunization, the second immunization is carried out on the 14 th day, and the injection dosage and the immunization mode are the same;

3) The third immunization, which is carried out on the 21 st day, and the injection dosage and the immunization mode are the same as those of the first immunization;

4) The fourth immunization, the third immunization on day 28, was performed at the same dose as the immunization schedule.

5 days after the fourth immunization, vaginal douches of Balb/C mice were collected with PBST (PBS containing 0.05% Tween 20), 75. Mu.L/time, 4 times, 300. Mu.L/mouse. Vortex for 1min after collection, then centrifuge for 3min at 12000g and take the supernatant for Elisa to detect NC-1 specific sIgA level changes.

1) Antigen coating: diluting NC-1 purified protein to 4 mug/mL by using coating solution, coating an ELISA plate according to 100 mug/hole, and standing overnight at 4 ℃; the coating solution was 20mM PB,0.15M NaCl, pH 6.0.

2) And (3) sealing: 300 mu L/hole of confining liquid, incubating at 37 ℃ for 1h, washing the plate by PBST, and storing at 4 ℃ for later use; the blocking solution was 10mM PBS (pH 7.4) +1% BSA.

3) Diluting a specimen: sera were serially diluted in multiple ratios from 1.

4) Sample adding: taking the coated ELISA plate, sequentially adding diluted serum and 100 mu L/hole, performing duplicate reaction on each sample, incubating at 37 ℃ for 1h, and then washing by PBST for 4 times; PBST wash 10mM PBS (pH7.4) + 0.05%;

5) Adding a secondary antibody: HRP-labeled goat anti-mouse IgA (Abcam, cat # Ab 97235) was diluted with antibody dilution 1:10000, 100. Mu.L/well, incubated at 37 ℃ for 30min, and washed 4 times with PBST; antibody dilution 10mM PBS (pH7.4) + 0.05%;

6) Color development: adding 100 mu L/hole of substrate color development solution, incubating at 37 ℃ for 10min, adding 50 mu L/hole of stop solution, and measuring OD value on an enzyme-linked immunosorbent assay (ELISA) instrument at a wavelength of 450 nm; the color development liquid is TMB storage liquid, substrate buffer liquid, 3% hydrogen peroxide =1090: 1; dissolving TMB in DMSO, wherein the TMB stock solution is 1 mg/mL; the substrate buffer solution is 0.53mM (pH 5.0) of citric acid and Na ₂ HPO4 100mM; the medium stop solution is 2 MH ₂ SO ₄ 。

As a result: as shown in table 4 and fig. 7, the recombinant protein antigen NC-1 immunized mice were tested to generate sIgA antibody titers of 1; the geometric mean titer of NC-1 immunized mice to the recombinant protein antigen NC-1 is 1; the antibody positive rate after immunization reaches 100%, which indicates that the recombinant protein antigen NC-1 can stimulate mice to generate mucosal immune response.

TABLE 4 geometric mean titre of vaginal lavage fluid sIgA of mice after NC-1 immunization

Example 5: evaluation of toxic attack protection capability of recombinant protein antigen NC-1 after immunization

And (3) evaluating the protective power of the recombinant protein antigen NC-1 after the ideological challenge on the mice 10 days after the last nasal drip immunization according to the following steps:

(1) Intragastric administration: performing challenge test on oral gavage helicobacter pylori J99 viable bacteria 10 days after last nasal drip immunization, wherein the dose of infection of each mouse is 2.0 × 10 ⁷ CFU, and recovering water and food 2h after intragastric administration.

(2) Plate culture: one week after gastric lavage, the killed mice were minced and placed in PBS buffer, vortexed for 3min, and then the washing stock solution and 10-fold dilution were applied to Skirrow plates containing 5% defibrinated sheep blood (Nanjing maojie organism) and 0.5% complex antibiotics (vancomycin 1.67mg/mL, polymyxin 0.0694mg/mL, trimethoprim 0.5mg/mL, amphotericin B0.2 mg/mL) (month shows protein 15g/L (Haibo organism), tryptone 2.5g/L (Oxoid), yeast extract 5g/L (Oxoid), sodium chloride 5g/L (cologne), pH7.4), and microaerophilic at 37 ℃ (5O) for 3min ₂ 、10％CO ₂ 、85％N ₂ ) And observing after culturing for 3 d.

(3) Detecting whether the Hp exists on the flat plate by combining the characteristic of the Hp colony, a rapid urease reagent, a microscopic examination and other modes so as to determine whether the mouse is infected with the Hp successfully; wherein, the vaccine protection rate = (control group infection positive rate-immune group infection positive rate)/control group infection positive rate 100%.

As a result: statistics of 20 mice plate culture conditions of the control group and the experimental group are shown in the following table, the 20 mice plate culture detection of the control group is positive, the infection rate is 100%, 12 mice plate culture of the 20 mice of the experimental group are positive, the infection rate is 60%, and then the protection efficiency of the vaccine is 40%.

Therefore, the recombinant protein antigen NC-1 prepared by the invention has good immunogenicity, can induce a mouse to generate stronger immune response, and can effectively inhibit the colonization of helicobacter pylori in the stomach of the mouse.

TABLE 5 mice post-immunization challenge Hp infection Positive Rate statistics

Mouse numbering	Control group	Experimental group	Mouse numbering	Control group	Experimental group
							1	+	+	11	+	+
2	+	-	12	+	-
						3	+	+	13	+	+
4	+	-	14	+	+
						5	+	+	15	+	+
6	+	-	16	+	-
						7	+	-	17	+	+
8	+	+	18	+	-
						9	+	+	19	+	+
10	+	-	20	+	+

Note that "+" indicates that the fast urease reagent and the microscopic examination are both positive, and "-" indicates that the fast urease reagent and the microscopic examination are negative

In conclusion, the invention screens out partial protein structures of the proteins, named NC-1 protein, by adopting a reverse vaccinology technology through a bioinformatics technology, recombines a protein expression vector into which a selected gene is cloned, performs large-scale expression in engineering bacteria, and prepares a genetic engineering subunit vaccine after purification. The vaccine has the advantages of simple process, low cost, strong operability and the like, and is one of the most promising candidate antigens of the Hp genetic engineering vaccine as an important component NC-1 protein in the HP membrane transporter.

It will be appreciated by those of ordinary skill in the art that the embodiments described herein are intended to assist the reader in understanding the principles of the invention and are to be construed as being without limitation to such specifically recited embodiments and examples. Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.

Claims

1. A helicobacter pylori vaccine recombinant protein antigen NC-1 is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A nucleic acid encoding the helicobacter pylori vaccine recombinant protein antigen NC-1 of claim 1.

3. The nucleic acid of claim 2, wherein the nucleic acid sequence is set forth in SEQ ID NO 2.

4. A method for preparing the helicobacter pylori vaccine recombinant protein antigen NC-1 according to claim 1, which comprises the steps of:

1) Plasmid construction, prokaryotic expression

Connecting the recombinant protein antigen NC-1 nucleic acid sequence of claim 2 or 3 into an expression vector plasmid, and transferring the connected plasmid into host bacteria for induced expression;

2) Breaking and centrifuging:

3) And purifying the supernatant by using a Ni affinity chromatographic column and an SP chromatographic column respectively in sequence to obtain the recombinant protein antigen NC-1.

5. The use of the recombinant protein antigen NC-1 of helicobacter pylori vaccine as claimed in claim 1 in the preparation of a helicobacter pylori recombinant subunit genetic engineering candidate vaccine.