CN109293750B

CN109293750B - Pseudomonas aeruginosa vaccine recombinant protein SBP and preparation method and application thereof

Info

Publication number: CN109293750B
Application number: CN201811382091.4A
Authority: CN
Inventors: 郭刚; 郑艮梅; 杨念; 周璐; 罗莉
Original assignee: Chongqing Ailibi Biological Technology Co ltd
Current assignee: Chongqing Ailibi Biological Technology Co ltd
Priority date: 2018-11-20
Filing date: 2018-11-20
Publication date: 2021-08-24
Anticipated expiration: 2038-11-20
Also published as: CN109293750A

Abstract

The invention belongs to the technical field of bacterial antigens, and discloses a pseudomonas aeruginosa vaccine recombinant protein SBP, a preparation method and application thereof; the nucleotide sequence is SEQ ID NO 1; the amino acid sequence is SEQ ID NO 2; the recombinant expression vector comprises a nucleotide sequence SEQ ID NO 1. The invention adopts pGEX-6p-1 vector to construct recombinant expression plasmid, which expresses recombinant protein SBP, pGEX is a vector for expressing fusion protein constructed by Smith and Johnson in 1987, and is mainly characterized in that the vector is connected with glutathione-S-transferase (GST) with molecular weight of 26kDa, the expressed fusion protein contains a GST label, and the label is a mark for protein purification. Compared with other fusion vectors, the pGEX series vectors can ensure that the purified protein can keep the spatial conformation and the immunogenicity to the maximum extent.

Description

Pseudomonas aeruginosa vaccine recombinant protein SBP and preparation method and application thereof

Technical Field

The invention belongs to the technical field of bacterial antigens, and particularly relates to a pseudomonas aeruginosa vaccine recombinant protein SBP, a preparation method and an application thereof.

Background

Currently, the current state of the art commonly used in the industry is such that: pseudomonas aeruginosa (Pseudomonas aeruginosa, PA) is commonly known as Pseudomonas aeruginosa, is a Pseudomonas in non-zymocyte, widely distributed in the nature, normal human skin, intestinal tract and respiratory tract, commonly exists in hospital wards and medical appliances, and is one of the most common conditional pathogenic bacteria in clinic. At present, the bacterium has become one of the pathogenic bacteria with highest separation rate of mechanical ventilation related pneumonia (VAP) in ICU ward, burn and war wound infection in the world. PA infection can occur in any part and tissue of human body, such as burn or wound part, middle ear, cornea, urethra and respiratory tract, etc., and can also cause systemic infection such as endocarditis, gastroenteritis, pneumonia and even septicemia, etc., and the mortality rate of the systemic infection is more than 20%. In addition, the drug resistance problem of PA is gradually highlighted due to abuse of antibiotics and the like, pan-resistant pseudomonas aeruginosa (PDR-PA) and multi-drug resistant pseudomonas aeruginosa (MDR-PA) appear, and the separation rate of drug resistant PA is increased year by year. The drug resistance is becoming more serious day by day, and Chinese drug-resistant bacteria monitoring network (CHINET) data shows that the annual drug resistance rate of PA to imipenem and meropenem in 2017 is 23.6% and 20.9% respectively, while the drug resistance rate of strains detected by patients with ventilator-associated pneumonia to the imipenem and meropenem is higher. For the above reasons, PA was listed as the first most critical grade in the global antibiotic-resistant bacteria priority list issued by WHO in 2017. Due to the high infection rate and the rising drug resistance rate of PA in clinic, the search for new 'non-antibiotic therapy' is urgent,

in summary, the problems of the prior art are as follows: due to abuse of antibiotics and the like, the drug resistance problem of PA is gradually highlighted, and pan-drug-resistant pseudomonas aeruginosa (PDR-PA) and multi-drug-resistant pseudomonas aeruginosa (MDR-PA) appear, and the separation rate of drug-resistant PA is increased year by year. Starting from the immunology perspective, the development of safe and effective vaccines becomes the most ideal choice, but no pseudomonas aeruginosa vaccine on the market is available at present. The key point of vaccine research is to find out an antigen with good immunogenicity and immune protection effect.

The difficulty and significance for solving the technical problems are as follows: the genetic engineering subunit vaccine is prepared by cloning pathogenic factors of pathogens or genes of mutants thereof into a protein expression vector, carrying out large-scale expression of recombinants in engineering bacteria, and purifying. The vaccine has the advantages of simple process, low cost, strong operability and the like, and becomes an important direction for developing the vaccine. The key to the development of genetically engineered vaccines is how to screen for good protective antigenic molecules from the thousands of pathogen proteomes. The research group of the inventor adopts a reverse vaccinology technology to screen out the recombinant protein SBP from more than five thousand open reading frames coded by the whole genome of the pseudomonas aeruginosa, and finds that the recombinant protein SBP has good immunogenicity and immune protection effect, which has important significance for the next step of developing immune regulation means such as pseudomonas aeruginosa vaccines and the like.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a pseudomonas aeruginosa vaccine recombinant protein SBP, a preparation method and application thereof.

The invention is realized by the pseudomonas aeruginosa vaccine recombinant protein SBP, the nucleotide sequence of the pseudomonas aeruginosa vaccine recombinant protein SBP is SEQ ID NO. 1; the amino acid sequence is SEQ ID NO. 2.

Another object of the invention is to provide a recombinant expression vector containing the recombinant protein SBP of the Pseudomonas aeruginosa vaccine, wherein the recombinant expression vector contains a nucleotide sequence SEQ ID NO. 1.

Another object of the present invention is to provide a host bacterium into which the recombinant expression vector is introduced.

The invention also aims to provide an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, an aluminum monostearate adjuvant, MF59, a complete Freund's adjuvant, an incomplete Freund's adjuvant and a mycobacterial BCG adjuvant which are matched with the recombinant protein SBP of the pseudomonas aeruginosa vaccine.

The invention also aims to provide a preparation method of the pseudomonas aeruginosa vaccine recombinant protein SBP, which comprises the following steps:

step one, adding 400 mu L of pGEX-6p-1-SBP/XL-1blue bacterial liquid stored in a refrigerator at 4 ℃ for later use into 20mL of LB culture medium containing Amp resistance for primary activation, culturing at 200rpm at 37 ℃ for 5-6 h, adding 10mL of the primary activated bacterial liquid into 1000mL of LB culture medium containing Amp resistance for secondary activation, and culturing at 37 ℃ for 3-4 h until OD600 is 1.0;

step two, adding 200 mu LIPTG, placing the mixture in a shaking table at 16 ℃ for overnight induction, centrifuging at 12000rpm for 15min to collect thalli, adding 50mL lysis buffer to resuspend the thalli, carrying out ultrasonic lysis on bacterial liquid for 3min, and combining the collected supernatant with 10mL Glutathione Sepharose4B gel beads for combining with GST fusion protein;

and step three, adding 10mL of PBS and 2PreScission protease into the remaining 10mL of protein-bound Glutathione Sepharose4B, performing vertical rotary digestion for 5h at room temperature, centrifuging and sucking the supernatant, taking 20 mu L of sample for denaturation treatment, loading 10 mu L of sample for protein electrophoresis, and observing the result under a phase system, wherein the molecular weight of the SBP protein subjected to enzyme digestion is 26kDa and is consistent with the molecular weight of the expected protein.

In summary, the advantages and positive effects of the invention are: through a reverse vaccinology technology and an immunoproteomics technology, the ABC transfer complex-binding protein (SBP) coded by a PA gene (PA5505) is found to have good immunogenicity and immune protection effects for the first time from more than five thousand proteins of pseudomonas aeruginosa, and the specific expression is that the geometric mean titer of the antibody titer generated by an SBP immune mouse is 1:47200, and the antibody positive rate reaches 100%; in addition, SBP immunization showed protection rates as high as 73.3% in a lethal PA-infected pneumonia model. These data indicate that SBP is a good candidate antigen for a PA genetically engineered vaccine. The recombinant protein is cloned and expressed by adopting a genetic engineering technology, is convenient to separate and purify, can be directly matched with adjuvants (such as an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, an aluminum monostearate adjuvant, MF59, a complete Freund adjuvant, an incomplete Freund adjuvant, a mycobacteria BCG adjuvant and the like) for use, and is suitable for injection immunization.

The invention adopts pGEX-6p-1 vector to construct recombinant expression plasmid, which expresses recombinant protein SBP, pGEX is a vector for expressing fusion protein constructed by Smith and Johnson in 1987, and is mainly characterized in that the vector is connected with glutathione-S-transferase (GST) with molecular weight of 26kDa, the expressed fusion protein contains a GST label, and the label is a mark for protein purification. Compared with other fusion vectors, the pGEX series vectors have the advantages of mild purification conditions, simple steps and no need of adding a denaturant, so that the spatial conformation and the immunogenicity of the purified protein can be kept to the maximum extent.

The gene engineering recombinant SBP protein has the following advantages:

1) the recombinant SBP protein can induce animals to generate specific antibodies and has immune protection effect; the subunit vaccine prepared by the recombinant SBP protein can be used for immunization through a subcutaneous (intramuscular) injection way, and an organism is stimulated to generate a high-titer IgG antibody. Animal experiments prove that the SBP-induced immune response has a good protection effect on resisting PA infection.

2) The recombinant SBP protein can be expressed in a prokaryotic expression system, namely escherichia coli, and has low cost and high yield;

3) when pGEX vector series are selected, SBP recombinant protein is expressed in a fusion protein form; the expression vector is connected with glutathione-S-transferase (GST) with the molecular weight of 26kDa, and the expressed fusion protein contains a GST label which becomes a protein purification label, so that the purification condition is mild, the steps are simple, and a denaturant is not required to be added, so that the purified SBP protein can keep the spatial conformation and the immunogenicity to the maximum extent.

Drawings

FIG. 1 is a flow chart of the preparation method of the recombinant protein SBP of the Pseudomonas aeruginosa vaccine provided by the embodiment of the invention.

FIG. 2 is a schematic diagram showing the PCR amplification result of the SBP gene fragment provided in the embodiment of the present invention;

in the figure: lane 1: nucleic Acid (DNA) molecular weight standards (Marker), from top to bottom, are: 4500. 3000, 2000, 1200, 800, 500, 200 bp; (ii) a Lane 2: the target gene fragment SBP (. about.720 bp).

FIG. 3 is a schematic diagram of the double restriction enzyme digestion identification result of the recombinant plasmid pGex-6p-1-SBP provided by the embodiment of the invention;

in the figure: lane 1: nucleic Acid (DNA) molecular weight standards (Marker), from top to bottom, are: 4500. 3000, 2000, 1200, 800, 500, 200 bp; lane 2-6: and (3) identifying the recombinant expression plasmid pGEX-6p-1-SBP after enzyme digestion, wherein the fragment separated after enzyme digestion is about 4000bp and about 720 bp.

FIG. 4 is a schematic diagram of the result of the SBP-induced identification of the protein provided by the embodiment of the present invention;

in the figure: lane 1: protein molecular weight standard (Marker), from top to bottom size: 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, 10 kDa; lane 2: GST filler bound to the expressed induction ultrasound supernatant; lane 3: supernatant obtained after enzyme digestion by PP enzyme; lane 4: GST filler after being cut by PP enzyme.

FIG. 5 is a schematic diagram of the electrophoresis result of the purified protein SBPSDS-PAGE provided by the embodiment of the invention;

in the figure: lane 1: protein molecular weight standard (Marker), from top to bottom size: 130kDa, 100kDa, 70kDa, 55kDa, 40kDa, 35kDa, 25kDa, 15kDa, 10 kDa; lane 2: purified SBP protein.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The drug resistance problem of PA is gradually prominent due to abuse of antibiotics and the like, and the problems that pan-drug-resistant pseudomonas aeruginosa (PDR-PA) and multi-drug-resistant pseudomonas aeruginosa (MDR-PA) occur, and the separation rate of drug-resistant PA is increased year by year. The recombinant protein is cloned and expressed by adopting a genetic engineering technology, is convenient to separate and purify, can be directly matched with adjuvants (such as an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, an aluminum monostearate adjuvant, MF59, a complete Freund adjuvant, an incomplete Freund adjuvant, a mycobacteria BCG adjuvant and the like) for use, and is suitable for injection immunization.

The following detailed description of the principles of the invention is provided in connection with the accompanying drawings.

The recombinant protein SBP of the pseudomonas aeruginosa vaccine provided by the embodiment of the invention removes a signal peptide and a transmembrane region of the protein, intercepts an SBP fragment (Ala22-Phe260) and has an amino acid sequence of SEQ ID NO. 2.

The invention also provides a recombinant expression vector for expressing the SBP recombinant protein, which comprises a nucleotide sequence SEQ ID NO 1 for encoding the SBP recombinant protein.

The present invention also provides a host bacterium into which the recombinant expression vector constructed as described above has been introduced.

The recombinant protein is cloned and expressed by adopting a genetic engineering technology, is convenient to separate and purify, can be directly matched with adjuvants (such as an aluminum hydroxide adjuvant, an aluminum phosphate adjuvant, an aluminum monostearate adjuvant, MF59, a complete Freund adjuvant, an incomplete Freund adjuvant, a mycobacteria BCG adjuvant and the like) for use, and is suitable for injection immunization.

As shown in FIG. 1, the preparation method of the recombinant protein SBP of the Pseudomonas aeruginosa vaccine provided by the embodiment of the invention comprises the following steps:

s101: adding 400 mu L of pGEX-6p-1-SBP/XL-1blue bacterial liquid stored in a refrigerator at 4 ℃ for later use into 20mL of LB culture medium containing Amp resistance for primary activation, culturing at 200rpm at 37 ℃ for 5-6 h, adding 10mL of the primary activated bacterial liquid into 1000mL of LB culture medium containing Amp resistance for secondary activation, and culturing at 37 ℃ for 3-4 h until OD600 is 1.0;

s102: adding 200 μ LIPTG (final concentration of 200 μ M) and placing in a shaking table at 16 ℃ for overnight induction, centrifuging at 12000rpm for 15min to collect thalli, adding 50mL lysine buffer (same as example 2) to resuspend the thalli, subjecting the bacterial liquid to ultrasonic lysis for 3min (200V), collecting supernatant, and combining with 10mL Glutathione Sepharose4B (GE company) gel beads (beads) for binding GST fusion protein;

s103: 10mL of PBS and 2PreScissionprotease (PP enzyme, GE corporation) were added to the remaining 10mL of protein-bound Glutathione Sepharose4B, and after the mixture was vertically spun at room temperature for 5 hours, 20. mu.L of the supernatant was collected by centrifugation and denatured, 10. mu.L of the supernatant was subjected to protein electrophoresis (same as above), and the molecular weight of the cleaved SBP protein was 26kDa, which corresponds to the molecular weight of the expected protein, as observed under the phase system.

The application of the principles of the present invention will now be described in further detail with reference to specific embodiments.

The strains and various reagents used in the invention are as follows:

1. pseudomonas aeruginosa strains

Pseudomonas aeruginosa International Standard strain PA01 supplied by ATCC in the United states (

BAA-47^TM) The clinical strain PAXN-1 is from China center for type culture Collection (accession number: CCTCC M2015730).

2. Reagent

The plasmid pGEX-6p-1 and the Escherichia coli strain XL-1blue are stored as the unit where the inventor is located, and the prime STAR HS DNA Polymerase, the DNA Marker, the restriction enzymes BamH I and EcoR I and the protein Marker are products of Takara corporation;

the plasmid extraction kit and the gel recovery kit are products of American Omega company;

the bacterial genome extraction kit, the ultrathin recovery kit and the developing solution are products of Tiangen corporation;

t4DNA Ligase is a product of Fermentas corporation;

Glutathione-Sepharose 4B is a product of GE Healthcare, USA.

Example 1: cloning of SBP Gene and construction of recombinant plasmid pGEX-6P-1-SBP

1. Firstly, according to the SBP protein full-length gene sequence of the pseudomonas aeruginosa PA01, structural analysis is carried out by using biological information software, and the SBP target gene segment needing to be amplified is determined.

2. According to the analysis result, the target gene fragment of SBP is amplified from the PA01 genome by adopting a PCR method, and the amplification steps are as follows:

1) PCR primers were designed as follows, SEQ ID NO: 3-4 (base sequence of restriction enzyme site underlined)

2) And (3) taking out the preserved pseudomonas aeruginosa strain PA01 from a freezer at-80 ℃, coating the pseudomonas aeruginosa strain PA01 on an LB solid culture medium, culturing at 37 ℃ overnight, picking out a single colony, inoculating the single colony in an LB liquid culture medium, culturing for 8 hours, and extracting a PA01 genome according to a bacterial genome extraction kit.

3) PCR amplification of SBP gene fragment by using PA01 whole genome DNA as template

And (3) PCR system:

PCR amplification reaction conditions of pre-denaturation at 98 ℃ for 5min, denaturation at 98 ℃ for 30s, annealing at 52 ℃ for 40s, extension at 72 ℃ for 90s, 30 cycles, and complete extension at 72 ℃ for 10 min. After the reaction, the PCR amplification result was detected using 1% agarose gel, and is shown in FIG. 2.

4) The SBPPCR product was recovered using a gel recovery kit.

And 3, identifying and cloning a PCR product, comprising the following steps:

1) BamH I and EcoR I digestion of pGEX-6p-1 plasmid and SBPPCR product

An enzyme digestion reaction system:

the enzyme was cleaved at 37 ℃ for 2 h.

2) The pGEX-6p-1 plasmid and the PCR product digested with BamH I and EcoR I were recovered using an ultra-thin recovery kit.

3) Ligation and transformation

Measuring the nucleic acid concentration of the SBP enzyme digestion recovery product by an ultraviolet spectrophotometer: 32 ng/. mu.l, the concentration of the nucleic acid of the product recovered by pGEX-6p-1 enzyme digestion: 63 ng/. mu.l.

Connecting a reaction system:

mixing, and connecting at 16 deg.C for 2 h.

4) Taking 3 tubes of escherichia coli XL1-blue competent cells from a refrigerator at the temperature of-80 ℃, and adding pGEX-6p-1 plasmid into the first tube to serve as a positive control; adding DNA ligation product into the second tube; the third tube was used as a negative control without the addition of exogenous DNA. Ice-cooling for 50min, heating in 42 deg.C metal bath for 90s, and rapidly cooling in ice bath for 2 min. Adding 600ul LB blank medium, mixing, and shaking 220rp in 37 deg.C shaking table for 1 h.

Each tube was centrifuged at 5000rpm for 3min at room temperature, 300ul of the supernatant was discarded, the cells were resuspended, and 200. mu.l of the suspension was spread on Amp-resistant LB plates. The plate was placed upside down in an incubator at 37 ℃ and incubated for 24 hours.

5) Screening and identification of pGEX-6p-1-SBP positive recombinant plasmid

Negative control plate has no bacterial colony; the positive control plate is full of colonies, so that the competent cells are correctly prepared, and the result is credible. Selecting well-separated colonies on the transformation plate, inoculating the colonies in an Amp resistant LB culture medium, and carrying out shaking culture at 37 ℃ overnight;

② plasmid extraction: reference is made to the plasmid extraction kit instructions;

carrying out double enzyme digestion on the plasmid DNA by using BamH I and EcoR I;

a double enzyme digestion reaction system:

carrying out enzyme digestion for 2h at 37 ℃;

fourthly, detecting the double digestion result by 1 percent agarose gel electrophoresis, wherein the result is shown in figure 3, and the lane 2 sample is pGEX-6p-1-SBP recombinant plasmid which is successfully constructed;

fifthly, the pGEX-6p-1-SBP recombinant plasmid is sent to Shanghai's chemical company for sequencing, the sequencing result is compared with GeneBank sequence information, and the result shows that the nucleotide sequences of the two sequences are completely the same.

Example 2: induction expression, purification and identification of expression form of recombinant fusion protein SBP in prokaryotic expression system-escherichia coli

SBP inducible expression

1) Adding 100 mu L of overnight cultured pGEX-6p-1-SBP/XL-1blue bacterial liquid into 10mL of Amp + resistant LB culture medium, carrying out overnight culture at 180rpm and 37 ℃, respectively adding 400 mu L of overnight cultured bacterial liquid into 20mL of Amp + resistant LB culture medium (the rest bacterial liquid is stored in a refrigerator at 4 ℃ for later use), culturing at 37 ℃ for 2-3 h at 200rpm, carrying out secondary activation until OD600 is 0.6-0.8, adding 4 mu L (1mol/L) of IPTG to enable the final concentration to be 200 mu M, and then carrying out shake table induced expression at 30 ℃ for induced expression for 3 h.

2) Taking out the bacteria liquid after induction expression, centrifuging at 12000rpm for 5min, discarding the supernatant, adding 1ml lysis buffer (20mM PB, pH 7.2, 250 ml NaCl), mixing, performing ultrasonic lysis for 3min (30 s/time ultrasonic lysis for 6 times), centrifuging at 14000rpm for 15min at 4 ℃, and separating the supernatant and the precipitate.

2. Treating the supernatant

After 3 washes of glutaminone Sepharose4B20 μ l with PBS, the prepared supernatant was added to glutaminone Sepharose4B and combined for 1h at room temperature. After centrifugation at 14000rpm for 3min at 4 ℃, washed 2 times with PBS-0.25% Tween 20 and once with PBS. To the bound Glutathione Sepharose4B was added 20. mu.L of 2 XProte buffer, boiled for 5min, and centrifuged at 14000rpm for 3min.

Cleavage of SBP target proteins

The same method for preparing the target protein binding Glutathione Sepharose4B filler, adding 100 u LPBS and 20 u LPreScissionprotease (PP enzyme, GE company), at room temperature vertical rotation enzyme digestion for 5h, centrifuging and absorbing the supernatant, taking 10u L sample for denaturation treatment, and loading 5u L for protein electrophoresis. The remaining filler was washed 3 times with 100. mu.L of PBS, and then 10. mu.L of the sample was denatured and subjected to protein electrophoresis at 5. mu.L.

SDS-PAGE electrophoresis

Pouring 5% concentrated glue into offset plate, adding distilled water to flatten the glue, standing at room temperature for 30min for solidification, pouring out the upper layer of distilled water, pouring 10% separation glue, immediately inserting comb, and standing at room temperature for 30min for solidification. The treated samples were subjected to SDS-PAGE by taking 5. mu.L of each sample. The voltage is firstly electrophoresed for 30min at 80v, then adjusted to 180v, and after electrophoresis is carried out for 1-2 h, the gel is taken out, is placed in Coomassie brilliant blue staining solution for oscillation staining, is placed in destaining solution for oscillation destaining, and then the result is observed under an imaging system, and PGEX-6p-1-SBP/XL-1blue is expressed in a soluble form at 30 ℃ (figure 4, lane 3).

Example 3: preparation of SBP antigens

1. Amplifying culture to obtain protein

Adding 400 μ L of pGEX-6p-1-SBP/XL-1blue bacterial liquid reserved in a refrigerator at 4 ℃ into 20mL of LB culture medium containing Amp resistance for primary activation, culturing at 200rpm and 37 ℃ for 5-6 h, adding 10mL of the once activated bacterial liquid into 1000mL of LB culture medium containing Amp resistance for secondary activation, culturing at 37 ℃ for 3-4 h until OD600 is 1.0, adding 200 μ L of LIPTG (final concentration is 200 μ M) and placing in a shaker at 16 ℃ for overnight induction, centrifuging at 12000rpm for 15min to collect bacterial cells, adding 50mL of lysis buffer (same as example 2) for re-suspension, performing ultrasonic lysis on the bacterial liquid for 3min (200V), and collecting the supernatant and 10mL of Gluthione Sepharose4B (GE) gel beads (beads) for binding GST fusion protein.

2. Separating target protein and GST label by enzyme digestion method to obtain SBP target protein

10mL of PBS and 2PreScissionprotease (PP enzyme, GE Co.) were added to the remaining 10mL of protein-bound Glutathione Sepharose4B, and after the mixture was vertically spun at room temperature for 5 hours, 20. mu.L of the supernatant was collected by centrifugation and denatured, 10. mu.L of the supernatant was applied to the mixture and subjected to protein electrophoresis (see above), and the molecular weight of the cleaved SBP protein was 26kDa, which corresponds to the molecular weight of the expected protein, as observed in the phase system, as shown in FIG. 5.

Example 4: immunization of animals

1) The first immunization, dilution of SBP antigen with PBS, addition of Al (OH) at a concentration of 1mg/mL₃(ii) a Injecting with No. 5 semi-type needle, bilateral thigh muscle, and 100 μ L injection amount of each BALB/C mouse, and setting positive control group, negative control group and blank control group;

2) the second immunization, the second immunization is carried out on the 14 th day, the immunization components are the same, the amount of the protein antigen injected is the same as that of the first immunization, and the immunization approaches are the same;

3) the third immunization, the third immunization is carried out on day 21, the immunization components are the same, the amount of the protein antigen injected is the same as that of the first immunization, and the immunization way is the same as that of the first immunization;

example 5: detection of antibodies

Blood from BALB/C mice was collected 7 and 14 days after the third immunization, and the antigen-specific IgG response level after the mice immunization was measured by ELISA.

1. Preparation of liquids

1) Preparation of coating liquid: weighing Na₂CO₃1.6g，NaHCO₃2.9g in 1L ddH₂O, adjusting the pH to 9.6 by using a pH meter;

2) preparing a sealing liquid: 1g bovine serum V, dissolved in 100mL of antibody diluent (1: 100);

3) preparing an antibody diluent: dissolving phosphate in 1L ddH₂O, adding 500 mu L of Tween 20, and adjusting the pH to 7.4 by using a pH meter;

4) preparation of a washing solution: diluting with antibody;

5) a color developing solution (TMB) which is a product of Tiangen corporation;

6) stop solution (2 MH)₂SO₄) The preparation of (1): 22.2mL of concentrated sulfuric acid was poured into 177.8mL of ddH₂And (4) in O.

ELISA detection of antibody titer produced by SBP recombinant protein immunized mice

1) And diluting the purified SBP recombinant protein by using a coating solution to obtain: 1ug/mL, 5ug/mL, 10 ug/mL;

2) coating: adding the recombinant protein diluent into an enzyme label plate, washing for 3 times by using a washing solution after overnight standing at 4 ℃, wrapping by using a preservative film after air drying, and placing in a refrigerator at 4 ℃ for later use;

3) and (3) sealing: adding 200 mu L of confining liquid into an ELISA plate per hole, placing the ELISA plate in an incubator at 37 ℃ for 2 hours, and washing for 3 times;

4) diluting the serum at an equal ratio of 1:1000, 1:2000, 1:4000 and 1: 8000;

5) taking the sealed enzyme label plate, sequentially adding diluted serum with the concentration of 100 mu L/hole, placing the plate in an incubator at 37 ℃ for 1h, washing for 3 times, and drying in the air;

6) adding an HRP-labeled goat anti-mouse IgG antibody preservative solution, diluting 1: 5000, preparing an antibody working solution;

7) adding diluted antibody working solution at a concentration of 100 μ L/well, placing in an incubator at 37 deg.C for 40min, washing for three times, and air drying;

8) adding 100 mu L/hole of substrate color development liquid (TMB), and reacting for 5min at room temperature in a dark place;

9) addingStopping solution (2 MH)₂SO₄) Immediately placing the sample on an enzyme-labeling instrument and measuring the OD value at the wavelength of 450 nm;

10) and (5) judging a result: a. the_{Sample (I)}/A_{Negative of}The value ≧ 2.1 is positive (the negative control is the serum 1: 1000-fold dilution before mouse immunization).

As a result: the geometric mean titer of the antibody titer generated by the mice immunized with the SBP protein antigen is 1: 47200; the antibody positive rate of 7 days after the last immunization reaches 100%, which indicates that the SBP recombinant protein constructed by the invention has good immunogenicity.

Example 6: challenge protection evaluation of SBP recombinant protein animal immunity

The challenge protection evaluation of the SBP recombinant protein animal immunity is carried out according to the reference documents: gao Chen et al Clin Immunol2017, 183, 354-. Briefly, 10-14 days after the last SBP immunization, PAXN-1 bacterial liquid is prepared and the concentration is adjusted to 1.5 × 10 by using physiological saline¹⁰CFU/mL, infected by nasal drip after anesthetizing the mice with isoflurane, with an infection rate of 20 μ L per mouse, and the same dose of physiological saline (NS) was used as a blank control. Observing mouse death every 1 day after infection, wherein the observation period is 7 days, and the rest animals are treated with CO after the observation period is over₂Inhalation euthanized. The survival rate of each group of mice was counted. The results are shown in Table 1.

TABLE 1 protective Effect of challenge after immunization of mice with SBP recombinant proteins

Table 1 shows: the survival rates of the negative control group and the blank control group were 16.7% and 10.0%, respectively, and the recombinant fusion protein SBP plus Al (OH)₃The survival rate of the adjuvant group was 73.3%, and the SBP protection rate was 70.3% as calculated by the formula. Therefore, the SBP recombinant protein has good immunogenicity, can induce an organism to generate immune response, can play a role in protecting PAXN-1 infection, and can be supplemented with an aluminum adjuvant to prepare a subunit vaccine for preventing pseudomonas aeruginosa infection.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Chongqing Ailidi Biotech Co., Ltd

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Leu Pro Ser Gly Ala Thr Val Val Ile Pro Asn Asp Ala Thr Asn Gly

100 105 110

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

115 120 125

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

130 135 140

Lys Asn Ile Lys Ile Arg Glu Leu Glu Ala Ala Thr Leu Pro Arg Val

145 150 155 160

Leu Thr Gln Val Asp Met Ala Leu Ile Asn Thr Asn Tyr Ala Leu Glu

165 170 175

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

180 185 190

Ser Pro Tyr Val Asn Ile Leu Val Ala Arg Pro Asp Asn Lys Asp Ser

195 200 205

Asp Ala Met Gln Lys Leu Ala Lys Ala Leu His Ser Ala Glu Ile Lys

210 215 220

Gln Phe Ile Gln Glu Lys Tyr Lys Gly Ala Val Val Pro Ala Phe

225 230 235

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgcggatccg ccgagtccct caccgtc 27

<210> 4

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccggaattcg aacgccggta ccaccg 26

Claims

1. A preparation method of a recombinant protein SBP of a pseudomonas aeruginosa vaccine is characterized in that the nucleotide sequence of the recombinant protein SBP of the pseudomonas aeruginosa vaccine is SEQ ID NO. 1; the amino acid sequence is SEQ ID NO 2;

the preparation method of the recombinant protein SBP of the pseudomonas aeruginosa vaccine comprises the following steps:

and step three, adding 10mL PBS and 2mL PreScission protease into the remaining 10mL protein-bound Glutathione Sepharose4B, performing vertical rotary enzyme digestion for 5h at room temperature, centrifuging and sucking supernatant, taking 20 mu L of sample for denaturation treatment, loading 10 mu L of sample for protein electrophoresis, and observing the result under a phase system, wherein the molecular weight of the SBP protein subjected to enzyme digestion is 26kDa and is consistent with the molecular weight of the expected protein.