CN116715780A

CN116715780A - Recombinant pseudomonas aeruginosa nanoparticle protein and preparation method and application thereof

Info

Publication number: CN116715780A
Application number: CN202310683385.5A
Authority: CN
Inventors: 顾江; 魏玉洁; 张怡; 程新; 廖亚玲; 鲁东水; 李宇航; 高晨; 张月月; 曾浩; 邹全明
Original assignee: Third Military Medical University TMMU
Current assignee: Third Military Medical University TMMU
Priority date: 2023-06-09
Filing date: 2023-06-09
Publication date: 2023-09-08

Abstract

The invention provides a recombinant pseudomonas aeruginosa nanoparticle protein, a preparation method and application thereof, wherein the protein is composed of effective components of pseudomonas aeruginosa vaccine candidate antigens pcrV, oprI and PA heat shock protein GroEL, and can be applied to the preparation of pseudomonas aeruginosa nanoparticle vaccines. The repO-GroEL recombinant protein can induce animals to generate specific antibodies and cellular immune responses, and exert high-efficiency immune protection effects, and the speed and the protection efficiency of the induced antibody responses are obviously superior to those of monomeric pcrV and OprI fusion proteins.

Description

Recombinant pseudomonas aeruginosa nanoparticle protein and preparation method and application thereof

Technical Field

The invention relates to the technical field of pharmacy, in particular to a recombinant pseudomonas aeruginosa nanoparticle protein repO-GroEL, a preparation method and application thereof.

Background

In 2017 WHO issued a list of pathogens that preferentially developed new antibiotics, pseudomonas aeruginosa was listed as the "most critical" class, whose emergence and spread has become a serious public health problem, mainly for three reasons: first, PA is a conditional pathogen that can cause serious infections in patients with impaired immune function or disrupted natural defense systems, which is the primary cause of hospital-acquired pneumonia, and data indicate that the mortality rate of hospital-acquired pneumonia patients is around 13% to 50%. Second, PA has extremely strong resistance. At present, antibiotics are mainly used for clinical treatment, but the effect is poor, and the death rate of patients is still up to 40.2 percent. Third, with the use of antibiotics, strains of multi-and broadly-resistant pseudomonas aeruginosa are increasingly derived, and their widespread worldwide has posed a serious threat to public health. There are data showing that in some areas of europe, the united states, etc., the ratio of multi-drug and broadly drug resistant pseudomonas aeruginosa is 15% -30%, and the number of death from pseudomonas aeruginosa infection in 2019 is reported to be as high as 55.9 tens of thousands in the journal of lancets at month 11 of 2022. PA infection and its resistance have become a troublesome medical challenge.

Vaccines are an important means of preventing and controlling pseudomonas aeruginosa infection. To date, more than 60 candidate PA vaccines have been tested in animal or clinical human, only 11 PA vaccines have entered clinical stage, and these 11 PA vaccines can be divided into five major classes, namely Mucinous Extracellular Polysaccharides (MEP), flagella, outer membrane proteins, lipopolysaccharides and inactivated whole fungi, according to the antigen class, but only 3 PA candidate vaccines have entered clinical stage III, namely octavalent O-polysaccharide-exotoxin A conjugateHis-tagged outer membrane proteins fuse the OprF-OprI protein (IC 43) and the bivalent flagellin vaccine, but fail due to weak immunogenicity or poor safety, and efficient and safe antigen delivery vehicles may be an effective approach to solve this problem.

Nanoparticles are a good delivery system with the advantages of targeted delivery, good size adjustability and stability, and most importantly, their use as carriers to deliver antigens can enhance their immunogenicity and protective effects.

PcrV is one of the key proteins of the PA iii secretion system (T3 SS), located at the tip of the T3SS needle, controls secretion of effector proteins, and anti-PcrV antibodies resist toxins secreted by PA, thereby protecting the body from infection. Candidate vaccines based on PcrV antigens provide effective protection against PA infection in the lung and burn wounds. OprI is an outer membrane lipoprotein of PA, can promote the production of IL-12, initiate innate immunity and specific T cell responses, and has an adjuvant effect.

The self molecular chaperone protein GroEL of the PA has high conservation, and can spontaneously form a nano structure with good biocompatibility. Studies have shown that GroEL immunization is effective against Mycobacterium tuberculosis, edwardsiella tarda, salmonella typhi, shigella and other infections, and is a protective antigen for most bacteria.

Disclosure of Invention

Aiming at the hazard of pseudomonas aeruginosa, the invention provides a pseudomonas aeruginosa nanoparticle protein rePO-GroEL, which consists of effective components of pseudomonas aeruginosa vaccine candidate antigens pcrV, oprI and PA heat shock protein GroEL, and can be applied to the preparation of pseudomonas aeruginosa nanoparticle vaccines.

The invention firstly provides a recombinant pseudomonas aeruginosa nanoparticle protein which is formed by sequentially connecting repO, - (Linker) n-and GroEL, wherein repO is a pseudomonas aeruginosa pcrV and OprI fusion antigen, each Linker is independently selected from any one of SEQ ID NO:5GGGGS, SEQ ID NO:6GGSGG and SEQ ID NO:7YAPVDV when each occurrence, and n is 1, 2, 3 or 4, preferably 1;

the pseudomonas aeruginosa heat shock protein is GroES or GroEL.

In one embodiment according to the invention, the amino acid sequence of repO is SEQ ID NO 3.

In one embodiment according to the invention, the GroEL has the amino acid sequence SEQ ID NO. 4; groES has the amino acid sequence SEQ ID NO. 18.

In one embodiment according to the invention, the amino acid sequence is SEQ ID NO. 2 or SEQ ID NO. 16.

The invention also provides a coding gene of the recombinant pseudomonas aeruginosa nanoparticle protein, and the nucleotide sequence of the coding gene is SEQ ID NO. 1 or SEQ ID NO. 17.

In a further aspect, the invention provides a recombinant expression vector, which is characterized by comprising the coding gene of the recombinant pseudomonas aeruginosa nanoparticle protein and an expression plasmid; the expression plasmid is selected from any one of pGEX series vectors, pET series vectors or pQE series vectors, and is preferably pET-28a.

The invention further provides a recombinant strain for expressing the recombinant pseudomonas aeruginosa nanoparticle protein, which comprises the recombinant expression vector and host bacteria; the host strain is selected from any one of an escherichia coli XL1-blue strain, a BL21 series strain and an HMS174 series strain, and is preferably an escherichia coli BL21 strain.

The invention also provides a preparation method of the recombinant protein, which comprises the following steps:

1) Constructing a recombinant expression vector for expressing the coding gene of the recombinant pseudomonas aeruginosa nanoparticle protein by a DNA synthesis and subcloning method; preferably, the coding gene of the recombinant pseudomonas aeruginosa nanoparticle protein is SEQ ID NO. 1;

2) Transforming the recombinant vector obtained in the step 1) into host bacteria;

3) Inducing the transformed host bacteria to express recombinant protein;

4) Purifying the recombinant protein to obtain the recombinant pseudomonas aeruginosa nanoparticle protein.

The invention also provides application of the recombinant pseudomonas aeruginosa nanoparticle protein, the recombinant gene, the recombinant expression vector or the recombinant strain in preparing subunit vaccine for resisting pseudomonas aeruginosa.

The invention also provides a vaccine for preventing or treating pseudomonas aeruginosa infection, which contains the recombinant pseudomonas aeruginosa nanoparticle protein.

The technical scheme of the invention has the following beneficial effects:

1) The recombinant protein repO-GroEL of the invention is induced in a prokaryotic expression system-escherichia coli and expressed in a soluble form; the expression vector is connected with an amino acid sequence (His 6 tag) consisting of 6 histidine residue links, the expressed fusion protein contains a His tag, and the tag becomes a protein purification tag, so that the purification condition is mild, the steps are simple, and the purified protein can keep the spatial conformation and the biological activity to the maximum extent; the purity of the purified repO-GroEL recombinant protein is more than 93%;

2) According to the invention, a part of active fragments of pseudomonas aeruginosa encoded protein are selected, based on the isoelectric point, surface charge, hydrophobicity and other properties of the recombinant protein subunits, a connection mode to pcrV, oprI and GroEL is optimized, gly-Gly-Gly-Ser amino acid sequences are used as Linker connection, the biological activity of the two connected proteins is better reserved, and the probability of misfolding is reduced. The Linker has high glycine content and strong flexibility, can increase accessibility of epitopes, avoid misfolding and improve O-glycosylation efficiency.

3) The rePO-GroEL recombinant protein immunity can be independently assembled in vitro to form stable and uniform nano particles, the production process is simple, and the large-scale preparation can be realized;

4) The repO-GroEL recombinant protein can induce animals to generate specific antibodies and cellular immune responses, and exert high-efficiency immune protection effects, and the speed and the protection efficiency of the induced antibody responses are obviously superior to those of monomeric pcrV and OprI fusion proteins.

Drawings

FIG. 1 is a diagram showing PCR and plasmid identification of the regrind encoding gene. The left graph shows the amplification result of the regrind and linearized pET-28a (+) gene fragment with homologous sequence, and the right graph shows the PCR of pET-28a (+) -regrind/BL 21 bacterial liquid.

FIG. 2 is a diagram showing PCR and plasmid identification of the repO-GroEL encoding gene. The left image shows the amplification of repO-linker and linearized pET-28a (+) -regrinEL gene fragment with homologous sequence, and the right image shows the PCR identification of pET-28a (+) -repO-GroEL/BL21 bacterial liquid.

FIG. 3 is a diagram showing the analysis of repO-GroEL, reGroEL and repO SDS-PAGE. M: maker 1: rePO-GroEL 2:regraEL 3:rePO.

FIG. 4 is a graph showing the results of molecular sieve analyses of repO, repGroEL and repO-GroEL.

FIG. 5 is a graph showing the results of dynamic light scattering detection by regrind and rePO-GroEL.

FIG. 6 is a graph showing the results of the observation of regrind and rePO-GroEL electron microscopes. A is the result of a regrind electron microscope and B is the result of a rego-GroEL electron microscope (scale bars are 50 nm).

FIG. 7 is a graph showing the results of the humoral immune response induced by repO-GroEL. A is the result of detection of anti-rePO IgG antibody titers induced by rePO and rePO-GroEL, and B is the result of detection of anti-reGroEL IgG antibody titers induced by reGroEL and rePO-GroEL.

FIG. 8 is a graph showing the results of ELISPOT detection of IFN-gamma, IL-17A and IL-4 secreting cell numbers. A is a spot shooting image, B is a cell number statistical result

FIG. 9 is a graph of survival after infection with PA XN-1 in repO-immunized, repO-GroEL-immunized, repoEL-immunized and sham mice.

FIG. 10 is a graph showing the results of lung bacterial colonization after infection with PA XN-1 in repO-immunized, repO-GroES-immunized and sham mice.

FIG. 11 is a graph showing the amplification results of linker-repO gene and linearized pET-28a (+) -regroES gene.

FIG. 12 is a PCR characterization of pET-28a (+) -rePO-GroES/BL21 strain.

FIG. 13 is a diagram showing the results of SDS-PAGE analysis of repO, regroES and repO-GroES. Wherein M is: maker;1 is rePO;2 is regroES;3 is rePO-GroES;

FIG. 14 is a graph showing the molecular sieve analysis results of repO, regroES and repO-GroES.

FIG. 15 is a graph showing the results of dynamic light scattering detection by regrines and rePO-GroES.

FIG. 16 is a diagram of regrines and rePO-GroES electron microscopy; wherein A is a regrines electron microscope result diagram and B is a rego-GroES electron microscope result diagram (the scale bars are all 50 nm).

FIG. 17 is a graph showing the results of in vitro safety assessment of repO-GroES; wherein A is 10-80 mug/mL of repO-GroES hemolysis detection in PBS buffer (left), OD in supernatant after interaction of different concentrations of repO-GroES with erythrocytes ₅₄₅ (right); b is the cytotoxicity detection result of the rePO-GroES;

FIG. 18 is a graph showing the results of tissue sections of repO-GroES and PBS immunized mice;

FIG. 19 is a graph showing the results of detection of the humoral immune response induced by repO-GroES; a is anti-rePO IgG potency detection induced by rePO and rePO-GroES; b is a repO and repO-GroES induced anti-repO IgG antibody subtype assay.

FIG. 20 is a graph showing the results of ELISPOT detection of IFN-gamma, IL-17A and IL-4 secreting cell numbers. A is a spot shooting image; b is the cell count statistics.

FIG. 21 is a graph showing survival after infection with PA XN-1 in the repO-immunized, repO-GroES-immunized, reproES-immunized and blank mice.

FIG. 22 is a graph showing the results of changes in body weight of repO-immunized, repO-GroES-immunized and sham mice after PA XN-1 infection.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.

Unless otherwise specified, all reagents used in this example were analytically pure.

Example 1: construction of recombinant plasmids pET-28 a-regrinol and pET-28a-repO-GroEL

1. Test method

1.1 Gene fragment amplification

(1) Retrieval of GroEL Gene sequence in PA (PAO 1 Strain) from NCBI functional network (NC-002516.2: c4917124-4915481Pseudomonas aeruginosa PAO1)

(2) Primers for the regrind and rePO-GroEL encoding genes (synthesized by Shanghai bioengineering Co.), corresponding PCR templates and insertion sites were designed, respectively.

The recombinant plasmid is named pET-28a (+) -regrooEL, P1 and P2 are target fragment primers, and PAO1 bacterial liquid is target fragment template. V1 and V2 are vector primers, and the vector template is pET-28a (+). The insertion cleavage sites are Nde I and Xho I.

P1:5'-CGCGGCAGCCATATGATGGCTGCCAAAGAAGTTAAGTTCG-3'

(SEQ ID NO:8)；

P2:5'-GTGGTGGTGGTGCTCTTACATCATGCCGCCCATGCCGCCC-3'(SEQ ID NO:9)；

V1:5'-GAGCACCACCACCACCACCACTGAGATCCG-3'(SEQ ID NO:10)；

V2:5'-CATATGGCTGCCGCGCGGCACCAGGCCGCT-3'(SEQ ID NO:11)；

The recombinant plasmid is named pET-28a (+) -repO-GroEL, P3 and P4 are target fragment primers, the template is pGEX-6P-1-repO (Zhang Yiwen, etc., the pseudomonas aeruginosa recombinant PCrV-OprI vaccine protects the effect of the mice after fracture fixation postoperative infection, the army university of medical science, 2023, 45 (3)), V3 and V4 are vector primers, and the template is pET-28a (+) -regrEL. The insertion cleavage sites are Nde I and Xho I.

P3:(SEQ ID NO:12)

5'-CGCGGCAGCCATATGGAGCAGGAGGAACTGCTGGCCCTGT-3'

P4:(SEQ ID NO:13)

5'-TTCTTTGGCAGCCATGCTGCCGCCGCCGCCCTTGCGGCTG-3'

V3:(SEQ ID NO:14)

5'-GGCGGCGGCGGCAGCATGGCTGCCAAAGAAGTTAAGTTCGGCGAT-3'

V4:(SEQ ID NO:15)

5'-CATATGGCTGCCGCGCGGCACCAGGCCGCTGCTGTGATGATGATG-3'

(3) According to the prompt of the primer instruction, adding corresponding volume of I-grade water into the synthesized primer dry powder for dissolution, and mixing the reagents according to the following system:

after the system described above was applied, the EP tube was placed in a small centrifuge at 4000rpm and centrifuged for 30s.

(4) Then the EP tube is placed in a PCR instrument, and the parameters are set as follows: (1) 98 ℃, 68 ℃ at 65 ℃, 62 ℃, 59 ℃,15 s/3 ℃ at 72 ℃ for 1min (4)4 ℃ for 30 cycles of steps (1) - (4). After the procedure is completed, the product is taken out and stored in a refrigerator at 4 ℃.

(5) mu.L of the final product was mixed with 1. Mu.L of 10 XLoading Buffer, followed by addition to a nucleic acid gel immersed in 1 XTAE Buffer for gel electrophoresis. And placing the nucleic acid gel subjected to electrophoresis under an ultraviolet imaging system for observation and taking pictures.

(6) The nucleic acid gel was immersed in 1×TAE Buffer, the PCR products of the same target fragment were combined together and 10. Mu.L of 10×Loading Buffer was added, and after mixing, the mixture was added to the nucleic acid gel for gel electrophoresis. After electrophoresis, the PCR fragments were cut under an ultraviolet imaging system. The required colloid is weighed, the required colloid is put into a 1.5mL clean EP pipe together, binding buffer with the volume of 1000 times of the gram of the colloid is added, the unit is microlitres, the temperature is 50-60 ℃ and is heated until the colloid is dissolved, the dissolved solution is transferred into an adsorption column, the speed is 12000rpm, the centrifugation is carried out for 1 minute, the filtrate is discarded, and the process is repeated for 2 times. Adding 700 mu L Spw wash Buffer into an adsorption column, centrifuging at 12000rpm for 1min, idling for 2 min, placing the adsorption column into a clean 1.5mL EP tube, standing for 5 min, adding 15-30 mu L of an absorption Buffer into the center of the adsorption column when alcohol volatilizes completely, standing at room temperature for 2 min, centrifuging at 12000rpm for 1min, and collecting purified PCR fragments.

(7) To the purified PCR product, 1. Mu.L of Dpn I enzyme was added, and after gentle mixing, the mixture was incubated in an incubator at 37℃for 2 hours.

(8) The PCR product is cleaned and recovered by using a PCR purification kit, and the specific steps are as follows: and (3) blowing and uniformly mixing the equal volume of the solution 1 and the product after the gel cutting recovery, adding the equal volume of the uniformly mixed sample of the solution I into a DNA purification column, standing for 1 minute at room temperature, centrifuging at 13000rpm for 1 minute, and pouring out the liquid in a collection pipe. Adding 700 mu L of solution II into a DNA purification column, standing for 1min at room temperature, centrifuging for 1min, washing impurities, and pouring out the liquid in the collection tube. Then 500. Mu.L of solution II was added and centrifuged at 13000rpm for 1min to wash out impurities, the liquid in the collection tube was discarded, the procedure was repeated 1 time to remove the residual liquid and allow the residual ethanol to evaporate sufficiently. The DNA purification column is placed on a 1.5mL centrifuge tube, 50 mu L of solution III is added to the center of the column surface in the tube, the liquid is absorbed by the purification column, and the column is placed for 3 to 5 minutes and centrifuged at 13000rpm for 1 minute to obtain a gene fragment. mu.L of the final product was mixed with 1. Mu.L of 10 XLoading Buffer, followed by addition to a nucleic acid gel immersed in 1 XTAE Buffer for gel electrophoresis. And placing the nucleic acid gel subjected to electrophoresis under an ultraviolet imaging system for observation and taking pictures.

1.2 ligation of pET-28 a-regrinol and pET-28a (+) -repO-GroEL plasmids

The recovered PCR product was added to 600. Mu.L EP tube according to the following system, and incubated at 50℃for 15min to complete ligation:

TABLE 1In-Fusion system

1.3 transformation of recombinant plasmids pET-28 a-regrinol and pET-28a-repO-GroEL

1) mu.L of the fusion product was added to 50. Mu.L of DH 5. Alpha. Competent cells, respectively, and the mixture was gently mixed and placed on ice for 30min. The temperature of the metal bath is adjusted to 42 ℃ and heat shock is carried out for 90s, and the metal bath is taken out and put on ice for 3min.

2) 1mL of antibiotic-free LB culture solution is added into the fused product, and the mixture is cultured in a culture box at 37 ℃ for 40min to 1h.

3) Centrifuge 5000rpm,2min, aspirate 800. Mu.L of supernatant, blow by heavy swirling, apply 100. Mu.L of the desired fusion product to Kana-resistant solid LB medium, positive control to Amp-resistant solid LB medium, negative control to Kana-resistant solid medium, and invert overnight at 37 ℃.

4) The plate was removed from the incubator at 37℃and single colonies were picked up in 10mL LB medium containing Kana, placed in a shaker at 37℃and shaken at 180rpm overnight.

5) Bacterial solutions were taken out of the shaker, 1mL of each was aspirated, pET-28a (+) -regrino EL and pET-28a (+) -repO-GroEL recombinant plasmids were extracted according to the protocol using Plasmid Mini Kit I kit, and 2. Mu.L of each was aspirated and transformed into Ecoli BL21 (DE 3) competent cells, and the specific procedures were the same as 2) to 4).

1.4 identification of recombinant plasmids pET-28 a-regrinEL and pET-28a (+) -repO-GroEL

1) The plate was removed from the incubator at 37℃and single colonies were picked up in 10mL LB medium containing Kana, placed in a shaker at 37℃and shaken at 180rpm overnight.

2) The bacterial liquid was removed from the shaker, and 2mL of bacterial liquid was sent to Jin Kairui, inc. for sequencing and comparison. mu.L of bacterial liquid is sucked and mixed with 700 mu.L of glycerol, and the mixture is frozen and stored at the temperature of minus 80 ℃ for seed preservation. Bacterial liquid PCR was performed using pET-28 a-regrinEL/BL 21 and pET-28a (+) -repO-GroEL/BL21 as templates, and P1 and P2 or P3 and P4 as primers, respectively.

2. Results

In order to construct pET-28a (+) -regrooEL recombinant plasmid, pseudomonas aeruginosa PAO1 bacterial liquid and pET-28a (+) plasmid genome are respectively used as templates, primers are designed to amplify regrooEL gene fragments and linearized pET-28a (+) gene fragments with homologous sequences, and agarose gel electrophoresis detection results show that the sizes of the two fragments are respectively about 1600bp and 6000bp and are consistent with theoretical values. The regrinel coding gene fragment with the homologous sequence and the linearized pET-28a (+) gene fragment are connected by an In-Fusion gene Fusion method, 7 positive clones are selected, bacterial liquid PCR identification is carried out by using P3 and P4 as primers, the result shows that the obtained band size is about 1600bp, the size is consistent with the size of the regrinel gene fragment, and the sequencing result of the positive clone gene is consistent with the theory, and is shown In figure 1.

Corresponding primers are designed according to the base sequence of the pET-28a (+) -rePO-GroEL recombinant plasmid, and the pET-28a (+) -reGroEL plasmid genome and pGEX-6P-1-rePO plasmid genome are respectively used as templates, and a rePO-linker gene fragment with a homologous sequence and a linearized pET-28a (+) -reGroEL gene fragment are obtained through PCR amplification. The agarose gel electrophoresis detection result shows that the two fragments are respectively positioned near the bands of about 1000bp and 6000bp, which are consistent with the theoretical value. The two fragments are connected and 5 positive clones are selected, bacterial liquid PCR is carried out by taking P5 and P6 as primers, the obtained band size is positioned near 1000bp, the size is consistent with the size of the repO coding gene, and the sequencing of the positive clone gene completely accords with the theory, and the sequencing is shown in figure 2. The above results indicate that pET-28a (+) -regrinol and pET-28a (+) -repO-GroEL recombinant plasmids have been successfully constructed.

Example 2: preparation of repO-GroEL antigen

1. Test method

1.1 expression characterization of repO-GroEL

1) Taking out the pET-28a (+) -regrinL/BL 21 and pET-28a (+) -repO-GroEL/BL21 seed retaining bacterial solutions at the temperature of minus 80 ℃, respectively sucking 10 mu L bacterial solutions after the bacterial solutions return to room temperature, inoculating the bacterial solutions into 10mL LB culture solution containing Kana, placing the bacterial solutions in a shaking table, and culturing at the temperature of 37 ℃ and 180rpm for overnight.

2) The bacterial liquid was taken out, 200. Mu.L of the bacterial liquid was aspirated and inoculated into 20mL of LB medium containing Kana, and the mixture was placed in a shaker at 37℃and 180rpm to culture for 4 hours.

3) The bacterial liquid is taken out and placed in a shaking table at 16 ℃ and cooled at 180 rpm. After completion of the cooling, 4. Mu.L of 1MIPTG solution was added to 20mL of the bacterial liquid, and the mixture was incubated at 16℃and 180rpm overnight.

4) 50 mu L of the induced bacterial liquid is sucked into a 1.5mL EP tube, 10 mu L of 6 Xprotein loading buffer solution is added and mixed uniformly, and the mixture is boiled in a metal bath at 98-100 ℃ for 6-10 min.

5) Centrifuging the residual bacterial liquid, carrying out 12000rpm for 10min, discarding the supernatant, adding 2mL of PBS buffer solution for heavy swirling, placing in an ice-water mixture, and carrying out bacterial breaking by using an ultrasonic crusher, wherein the set parameters are as follows: 5s ultrasonic on, 6s ultrasonic off, total time length of 6min and power of 28%.

6) After completion of sonication, the supernatant and pellet were separated and 2mL of PBS buffer was added to the pellet for vortexing.

7) The supernatant and the precipitate were each pipetted 50. Mu.L into a 1.5mL EP tube, 10. Mu.L of protein loading buffer was added and the mixture was subjected to metal bath boiling at 98-100℃for 6-10 min.

8) The solutions in steps 3) and 7) were each pipetted into 10. Mu.L for SDS-PAGE detection.

1.2 expanded culture of pET-28a (+) -regrinEL/BL 21 and pET-28a (+) -repO-GroEL/BL21

1) 20. Mu.L of seed retaining bacteria solution of pET-28a (+) -regrinO/BL 21 and pET-28a (+) -repO-GroEL/BL21 were respectively aspirated, and added to 20mL of sterile LB medium containing Kana, and the mixture was cultured overnight at 150rpm and 37℃to perform primary activation.

2) Taking out the bacterial liquid, adding the bacterial liquid into 2L of sterile LB medium containing Kana, culturing for 4h at 180rpm and 37 ℃ and performing secondary activation to OD ₆₀₀ 1.0.

3) The bacterial solution was taken out and placed in a shaking table at 16℃and incubated at 180rpm for 1 hour, cooled, and 400. Mu.L of 1M IPTG solution was added to the solution and incubated overnight.

4) Taking out the induced bacterial liquid, centrifuging 1000g at 4 ℃ for 20min. The supernatant was discarded, the bacterial sludge was removed to a 50mL centrifuge tube, weighed and labeled on the side of the tube, and placed at-20 ℃ for cryopreservation.

1.3 purification of the repO-GroEL protein

1) 20mM PB buffer (pH 8.0) was added to the bacterial sludge at a volume 5 times the weight of the bacterial sludge, and the bacterial sludge was thoroughly vortexed. Placing the powder into a beaker, placing the beaker into an ice-water mixture for ultrasonic treatment, and using an ultrasonic crusher to set the parameters as follows: the ultrasonic switch is turned on for 8s and turned off for 9s, the total time is 15min, and the power is 38%.

2) The supernatant and pellet were separated by centrifugation at 12000rpm at 10℃and the same volume of PB buffer as in step (1) was added to the pellet and vortexed uniformly and 50. Mu.L was sampled in the EP tube.

3) Loading the supernatant onto Ni Bestarose FF gel beads, mixing uniformly, placing on a suspension instrument, combining for 40min at 4 ℃, catching the flow-through after combining is completed, sampling, flushing the filler by using PB buffer solution, and sampling.

4) Purification was performed according to the protocol written in the following table:

TABLE 2 regrinol and rePO-GroEL purification methods

5) Adding 10 mu L of 6 Xprotein buffer solution into the samples obtained in the step 1) and the step 3), uniformly mixing, and placing the mixture in a metal bath at 98-100 ℃ for 6-10 min for SDS-PAGE gel electrophoresis.

6) Opening an Avant Pure 150 purification instrument, installing a G25 chromatographic column, balancing primary water through an A pipe, respectively loading a regrinL protein solution and a repO-GroEL protein solution containing imidazole by using PBS buffer solution (pH 8.0) at a flow rate of 8mL/min, starting sample connection when the absorbance value of 280nm is more than 5mAU, and stopping sample connection when the absorbance value is reduced to 5mAU, so as to obtain the protein solution with imidazole removed. The purified regrinl and rePO-GroEL protein solutions were frozen at-80 ℃.

2. Results

The repO protein, repGroEL protein and repO-GroEL protein bands are near bands of standard molecular weights 33.0kDa, 60.0kDa and 95.0kDa, respectively. This result suggests that rePO proteins, reproel proteins and rePO-GroEL proteins have been successfully obtained, see fig. 3.

Example 3: physicochemical property detection of repO-GroEL protein

1. Test method

Molecular sieve chromatography of the rePO-GroEL protein

The Avant Pure 150 purification apparatus was turned on, a Surperose 6 column was installed, primary water was passed through the A tube and equilibrated with PBS buffer (pH 8.0), 500. Mu.L of regrind and repO-GroEL proteins were loaded, respectively, using a SuperLoop volume of 500. Mu.L, at a flow rate of 0.5mL/min. The front volume was recorded and a result map was derived.

1.2 particle size detection of repO-GroEL protein

mu.L of regrinE and repO-GroEL protein solutions were aspirated separately, placed in the loading well of a nanoparticle analyzer equipped with an argon ion laser emitter, repeatedly examined 3 times, and experimental data were recorded and saved.

1.3 electron microscopic observation of the repO-GroEL protein

0.01mg/mL regrinol and rePO-GroEL solutions were adsorbed onto a carbon support membrane, stained with 1% uranyl acetate, dried, and the samples were placed in Tecnai G2 for centering, adjusting appropriate parameters, and observed and photographed.

2. Results

The forward volumes of rePO, regrino and rePO-GroEL were 17.4mL, 12.7mL and 11.8mL, respectively (fig. 4). The molecular weights of rePO, reGroEL and rePO-GroEL were estimated to be about 35.5kDa, 420.8kDa and 665.7kDa, respectively, by standard protein curve regression analysis. Dynamic light scattering showed that regrino and rePO-GroEL formed particles with particle sizes of 22.7nm and 28.2nm, respectively (fig. 5), and electron microscopy showed that regrino and rePO-GroEL formed uniform nanoparticles (fig. 6). These results suggest that the regrin and rePO-GroEL proteins may have self-assembled into aggregates, whereas rePO exists in monomeric form.

Example 4: detection of rePO-GroEL induced immune response

1. Test method

1.1 Immunization

(1) 20 female BALB/c mice of SPF grade 16-18 g were randomly divided into 4 groups, which were blank, repO immune, regrino immune and repO-GroEL immune, respectively.

(2) Each dose contained 246 μg Al (OH) in a total volume of 100 μl ₃ An adjuvant. 6.32 mug regrinol was immunized with regrino-GroEL, 10 mug rego-GroEL was immunized with rego-GroEL, 3.68 mug rego was immunized with rego, the content of rego was kept consistent, the immunization was intramuscular injection, the total immunization was 3 times, and the immunization time was 0,7, 14 days.

1.2ELISA detection of antibody titers produced by repO-GroEL recombinant protein immunized mice

1) On the 6 th day after each immunization, tail vein blood collection is carried out on the immunized mice and the blank mice, and after collection, the mice are incubated for 1-2 h at 37 ℃. Serum and blood cells were separated by centrifugation at 8000rpm for 10 minutes. Blood cells were discarded, serum was stored at-20℃and labeled.

2) The rePO protein and GroEL protein solutions were diluted to 0.6 μg/mL using coating solution, respectively, and added to 96-well elisa plates. 100 μl per well was wrapped with a preservative film and left to stand at 4deg.C overnight.

3) And taking out the ELISA plate, and cleaning the coated ELISA plate by using an automatic plate cleaning machine. The plate washer parameters were set to wash 3 times, 300. Mu.L/well of PBST solution each time, and shaking for 5 seconds. And after cleaning, the board is patted dry on the absorbent paper.

4) Adding a sealing liquid into the ELISA plate, wrapping 250 mu L/hole with a preservative film, and placing the ELISA plate into a 37 ℃ incubator for incubation for 2 hours.

5) And taking out the ELISA plate, cleaning the ELISA plate by using a PBST solution, and drying the plate on absorbent paper after cleaning.

6) The collected serum was removed from-20 ℃ and rewarmed, and the serum was diluted with PBST solution by selecting the appropriate dilution factor. 100. Mu.L of PBST solution was added to each well starting from the second transverse row of the 96-well ELISA plate, and 200. Mu.L of diluted serum sample was added to each well of the first row. 100. Mu.L of the solution is sucked from the first row and added to the second row and gently blown for 15 to 18 times, taking care not to blow bubbles so as not to affect the experimental result, and the like, finally sucking 100. Mu.L and discarding, and placing the ELISA plate in a 37 ℃ incubator for incubation for 1h.

7) And taking out the ELISA plate, cleaning the ELISA plate by using a PBST solution, and drying the plate on absorbent paper after cleaning.

8) IgG antibodies were diluted at a dilution ratio of 1:7500 using PBST solution, added at 100 μl per well after mixing, and incubated for 45min at 37 ℃.

9) And taking out the ELISA plate, cleaning the ELISA plate by using a PBST solution, and drying the plate on absorbent paper after cleaning.

10 TMB color development solution was added to the ELISA plate at 100. Mu.L per well, incubated at 37℃for 8min, and treated in the absence of light.

11 50. Mu.L/well of stop solution was added to terminate the color development. The absorbance at 450nm was read within 15 min.

1.3 cell immunoassay

1) At 14 days after the last immunization, 5 mice were randomly selected from the rePO immune group, rePO-GroEL immune group and blank group, blood was collected by eye-drop method, infiltrated with alcohol lamp, dissected with left abdomen facing upward, and spleen was placed in 200 mesh cell sieve containing 2mL sterile PBS.

2) The spleen was thoroughly ground, the screened liquid was aspirated into a 15mL centrifuge tube, PBS solution was added, and the volume was made up to 5mL. Centrifuge at 1700rpm at 4℃for 10min. The supernatant was discarded, 5mL of red blood cell lysate was added, gently swirled, lysed at room temperature for 10min, followed by 5mL of PBS to terminate the lysis and mix well. Centrifuge at 1700rpm at 4℃for 10min. The supernatant was discarded, washed with 10mL of sterile PBS by vortexing, and the supernatant was discarded by centrifugation again. 10mL of PBS solution was added for vortexing. Mu.l were pipetted from each centrifuge tube into a cell counting plate, splenocytes were counted and recorded. The tube was centrifuged at 1700rpm for 10min and the supernatant was discarded. The amount of cells required for plating was calculated and splenocytes were diluted to the desired concentration using complete 1640 medium.

3) And (3) placing the pre-packaged plate of the kit, which is hermetically packaged by the aluminum film, into an ultra-clean workbench, tearing the package, and paying attention to marking. 200 μl of sterile PBS per well was added to the pre-coated plate, and after two minutes of standing, it was snapped out and repeated three times. Then 200. Mu.L of complete 1640 medium was added to each well, which was left to stand at room temperature for 30min and then snapped out.

4) mu.L of the repO peptide library was aspirated, added to 14250. Mu.L of complete 1640 medium, and the cell suspension at the adjusted concentration was added to each well of the experiment, 200. Mu.L per well. The concentration in 3 positive control wells was 5X 10 ⁵ Cells per well and 20 μl positive stimulus. 250. Mu.L of complete 1640 medium was added to 3 background negative control wells and the concentration was 5X 10 in experimental wells ⁵ Cells per well and 50. Mu.L of polypeptide solution were stimulated with a final polypeptide concentration of 10. Mu.g/mL and 5X 10 concentration was added to the experimental negative control wells ⁵ Cells per well and 50 μl of complete 1640 medium containing 5% DMSO. Covering the cover plate, placing the mixture into a furnace at 37 ℃ and 5% CO ₂ The medium was cultured for 3 days.

5) The plates were removed, the solution was subtracted, 200 μl of sterile PBS per well was added to the plates, allowed to stand for 2min, and the subtraction was repeated 5 times.

6) 100. Mu.L of IL-17A detection antibody, IL-4 detection antibody and IFN-gamma detection antibody were diluted to 0.25. Mu.g/mL, 1. Mu.g/mL and 1. Mu.g/mL in PBS containing 0.5% fetal bovine serum, respectively, and incubated at room temperature for 2 hours. Repeating the step (5).

7) The antibodies in the kit are diluted and mixed uniformly in PBS containing 0.5% of fetal bovine serum according to the proportion of 1:1000, 100 mu L of the antibody is added into each hole, the mixture is incubated for 1 hour at room temperature, and the step (5) is repeated.

8) The TMB color was filtered through a 0.45mm filter, 100. Mu.L of each well was added, incubated at room temperature until visible spotting occurred, rinsed extensively with tap water, the solution was rinsed clean, the plate was then air dried, and the next day was read and recorded.

2. Results

ELISA results showed that both anti-rePO IgG and anti-reGroEL IgG antibody titers produced by repO-GroEL immunized mice were significantly higher than in the blank (P < 0.01). anti-rePO IgG antibody titers produced by rePO-GroEL immunized mice were significantly higher than rePO immunized group (P < 0.05) at 3 detection time points on days 7, 21, 28. As immunization time increased, the repO-GroEL-induced anti-GroEL IgG antibody titers continued to rise, with no significant difference from the repoEL immunized group, see FIG. 7. The splenocyte numbers of IFN-. Gamma., IL-17A and IL-4 secreted by repO-GroEL immunized mice were significantly higher than those of repO immunized and empty (P < 0.05), see FIG. 8, suggesting that repO-GroEL significantly enhanced Th1, th2 and Th17 immune responses compared to repO monomer.

Example 5: evaluation of protective Effect of repO-GroEL immunization in mice model of pneumonia

1 test method

Survival observations after 1.1 lethal dose of PA-infected mice

1) After 5 days of final immunization, PAXN-1 broth was dipped in an ultra clean bench using an inoculating loop and streaked on LB solid medium containing Kana using a three-wire method. Placing into a constant temperature incubator at 37 ℃ for incubation for 15 hours, taking out the incubator the next day, and storing in a refrigerator at 4 ℃.

2) XN-1 single colonies were picked in 10mL of antibiotic-free LB medium, and a total of 5 single colonies were picked and placed in 5 Erlenmeyer flasks, respectively, at 37℃and 180rpm, and cultured overnight.

3) And (3) selecting a bottle of bacterial liquid with optimal growth vigor for secondary activation, sucking 200 mu L of bacterial liquid, adding the bacterial liquid into 20mL of antibiotic-free LB culture medium, and culturing at 37 ℃ for 3.5 hours at 220 rpm.

4) Selecting a bottle from the bacterial liquid in the step (3), detecting and recording the OD thereof ₆₀₀ Numerical values. The supernatant was discarded by centrifugation at 8500rpm for 5min, swirled again with normal saline, and the supernatant was discarded by centrifugation and repeated 2 times. Adding a certain volume of physiological saline to make the bacterial liquid OD ₆₀₀ Reaching 1.19-1.23, diluting the solution by 12 times, subpackaging the solution into 1.5mL EP pipes, and placing the EP pipes on ice.

5) The anesthetic is injected into the abdominal cavity of mice in immune group and blank group, after anesthesia, trachea cannula is performed, diluted bacterial liquid is injected into the lung of the mice, 20 mu L bacterial liquid (1×10) is injected into each mouse ⁶ CFU/mouse)。

6) Observations were made every 12h after challenge, and survival numbers were recorded for each group for 7 consecutive days.

1.2 sublethal dose of PA pulmonary bacterial colonization after mice infected

1) After 5 days of final immunization, single colonies were picked and subjected to secondary activation as described above.

2) Selecting a bottle of bacterial liquid from the bacterial liquid in the step 1), and detecting the OD (optical density) of the bacterial liquid ₆₀₀ Numerical values. Centrifuging to remove supernatant, swirling with physiological saline, centrifuging to remove supernatant, and repeating for 2 times. Adding a certain volume of physiological saline to make the bacterial liquid OD ₆₀₀ Reaching 1.19-1.23, diluting the mixture 16 times, and split charging the mixture into 1.5mL EP pipes.

3) The mice were anesthetized and then tracheal cannulated, and each mouse was pulmonary injected with 20. Mu.L XN-1 bacteria solution (2.4X10) ⁵ CFU/mouse)。

4) 24 hours after challenge with sublethal dose PAXN-1, 5 mice per group were randomly selected, bled by eyeball extraction, sacrificed by neck removal and infiltrated with 75% alcohol. Lungs from each group of mice were removed and placed in a sterile mill containing 1mL PBS for milling, after which the tissue homogenate was poured into a 1.5mL EP tube and placed on ice.

5) The lungs of each group of mice were homogenized by 20 μl with PBS solution at 1:10,1:100,1:1000,1:10000,1:100000 and 1:1000000, the remaining homogenates were stored at-20 ℃. After completion of dilution, 10. Mu.L of each dilution was added to the mixture containing 5X 10 ^-5 Square solid of g/mL KanaThe body culture medium was placed at an upper position and then moderately inclined, so that the bacterial liquid was slowly flowed down until no more was flowed down. Note that the bacterial liquid cannot stick to the plate wall. The lung homogenate from each mouse was repeated 3 times to incubate for 15h at 37℃with inversion, and the numbers were removed and recorded.

2 results

The lethal dose PA infection results showed 85% survival for the rePO-GroEL immunized group, 60% for the rePO immunized group, a significant statistical difference for both groups and higher than the blank group (P < 0.01), 15% for the rePO-el immunized mice, and a significant difference from the blank group, see fig. 9. The results of lung bacterial colonization of sublethal dose PA-infected mice showed that the number of bacteria in the rePO-GroEL immunized group was significantly less than that in the rePO immunized group and the blank group, see fig. 10. The results show that repO-GroEL, repO and regoEL have remarkable protection effects on PA infection, wherein the repO-GroEL has the best protection effect.

Example 6: construction of recombinant plasmid pET-28a-rePO-GroES

1. Test method

1.1 amplification of linker-rePO and pET-28 a-regrooES Gene fragments

(1) GroES amino acid sequence is SEQ ID NO. 18, and repO-GroEs gene sequence is SEQ ID NO. 17. Synthesis of the Gene and ligation of the sequence to the pET-28a (+) plasmid was synthesized by Jiangsu Style biotechnology Co. P1 and P2 are target fragment primers, the template is pGEX-6P-1-rePO, V1 and V2 are vector primers, and the template is pET-28a (+) -reGroES. The insertion sites are Nde I and Xho I to construct a recombinant plasmid pET-28a (+) -rePO-GroES.

P1:(SEQ ID NO:19)

5'-GCGGTCTTGGAAGATGGCGGCGGCGGCAGCGAGCAGGAGG-3'

P2:(SEQ ID NO:20)

5'-GTGGTGGTGCTCGAGTTACTTGCGGCTGGCTTTTTCCAGC-3'

V1:(SEQ ID NO:21)

5'-CTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAA-3'

V2:(SEQ ID NO:22)

5'-GCTGCCGCCGCCGCCATCTTCCAAGACCGCCAGGATCTCGCTCTC-3'

(2) According to the prompt of the primer instruction, adding corresponding volume of I-grade water into the synthesized primer dry powder for dissolution, and mixing the reagents according to the following system:

(3) Then the EP tube is placed in a PCR instrument, and the parameters are set as follows: (1) 98 ℃, 57 ℃ for 10s, 59 ℃, 61 ℃, 63 ℃,15s, 3 ℃ for 72 min, (4)4 ℃, steps (1) to (4) react for 30 cycles, and after the procedure is finished, the product is taken out and stored in a refrigerator at 4 ℃.

(4) mu.L of the final product was mixed with 1. Mu.L of 10 XLoading Buffer, followed by addition to a nucleic acid gel immersed in 1 XTAE Buffer for gel electrophoresis. And placing the nucleic acid gel subjected to electrophoresis under an ultraviolet imaging system for observation and taking pictures.

(5) The nucleic acid gel was immersed in 1×TAE Buffer, the PCR products of the same target fragment were combined together and 10. Mu.L of 10×Loading Buffer was added, and after mixing, the mixture was added to the nucleic acid gel for gel electrophoresis. After electrophoresis, the PCR fragments were cut under an ultraviolet imaging system. The required colloid is weighed, the required colloid is put into a 1.5mL clean EP pipe together, binding buffer with the volume of 1000 times of the gram of the colloid is added, the unit is microlitres, the temperature is 50-60 ℃ and is heated until the colloid is dissolved, the dissolved solution is transferred into an adsorption column, the speed is 12000rpm, the centrifugation is carried out for 1 minute, the filtrate is discarded, and the process is repeated for 2 times. Adding 700 mu L Spw wash Buffer into an adsorption column, centrifuging at 12000rpm for 1min, idling for 2 min, placing the adsorption column into a clean 1.5mL EP tube, standing for 5 min, adding 15-30 mu L of an absorption Buffer into the center of the adsorption column when alcohol volatilizes completely, standing at room temperature for 2 min, centrifuging at 12000rpm for 1min, and collecting purified PCR fragments.

(6) To the purified PCR product, 1. Mu.L of Dpn I enzyme was added, and after gentle mixing, the mixture was incubated in an incubator at 37℃for 2 hours.

(7) The PCR product is cleaned and recovered by using a PCR purification kit, and the specific steps are as follows: and (3) blowing and uniformly mixing the equal volume of the solution 1 and the product after the gel cutting recovery, adding the equal volume of the uniformly mixed sample of the solution I into a DNA purification column, standing for 1 minute at room temperature, centrifuging at 13000rpm for 1 minute, and pouring out the liquid in a collection pipe. Adding 700 mu L of solution II into a DNA purification column, standing for 1min at room temperature, centrifuging for 1min, washing impurities, and pouring out the liquid in the collection tube. Then 500. Mu.L of solution II was added and centrifuged at 13000rpm for 1min to wash out impurities, the liquid in the collection tube was discarded, the procedure was repeated 1 time to remove the residual liquid and allow the residual ethanol to evaporate sufficiently. The DNA purification column is placed on a 1.5mL centrifuge tube, 50 mu L of solution III is added to the center of the column surface in the tube, the liquid is absorbed by the purification column, and the column is placed for 3 to 5 minutes and centrifuged at 13000rpm for 1 minute to obtain a gene fragment. mu.L of the final product was mixed with 1. Mu.L of 10 XLoading Buffer, followed by addition to a nucleic acid gel immersed in 1 XTAE Buffer for gel electrophoresis. And placing the nucleic acid gel subjected to electrophoresis under an ultraviolet imaging system for observation and taking pictures.

1.2 ligation of pET-28a (+) -rePO-GroES plasmid

TABLE 1In-Fusion system

1.3 transformation of recombinant plasmid pET-28a-rePO-GroES

(1) mu.L of the fusion product and 2. Mu.L of pET-28a (+) -regroES recombinant plasmid solution were added to 50. Mu.L of DH 5. Alpha. Competent cells, respectively, and the mixture was gently mixed and placed on ice for 30min. The temperature of the metal bath is adjusted to 42 ℃ and heat shock is carried out for 90s, and the metal bath is taken out and put on ice for 3min.

(2) 1mL of antibiotic-free LB culture solution is added into the fused product, and the mixture is cultured in a culture box at 37 ℃ for 40min to 1h.

(3) Centrifuge 5000rpm,2min, aspirate 800. Mu.L of supernatant, blow by heavy swirling, apply 100. Mu.L of the desired fusion product to Kana-resistant solid LB medium, positive control to Amp-resistant solid LB medium, negative control to Kana-resistant solid medium, and invert overnight at 37 ℃.

(4) The plate was removed from the incubator at 37℃and single colonies were picked up in 10mL LB medium containing Kana, placed in a shaker at 37℃and shaken at 180rpm overnight.

(5) Extracting bacterial liquid from a shaking table, respectively sucking 1mL, extracting pET-28a (+) -regrines and pET-28a (+) -repO-GroES recombinant plasmids according to the specification method by using a Plasmid Mini Kit I kit, respectively sucking 2 mu L of recombinant plasmids and transforming the recombinant plasmids into Ecoli BL21 (DE 3) competent cells, and carrying out the specific steps in the same way as (2) - (4).

1.4 identification of recombinant plasmid pET-28a (+) -repO-GroES

(1) The plate was removed from the incubator at 37℃and single colonies were picked up in 10mL LB medium containing Kana, placed in a shaker at 37℃and shaken at 180rpm overnight.

(2) The bacterial liquid was removed from the shaker, and 2mL of bacterial liquid was sent to Jin Kairui, inc. for sequencing and comparison. mu.L of bacterial liquid is sucked and mixed with 700 mu.L of glycerol, and the mixture is frozen and stored at the temperature of minus 80 ℃ for seed preservation. And (3) performing bacterial liquid PCR by taking the bacterial liquid as a template and taking P1 and P2 as primers.

2. Results

The linker-rePO gene fragment and the linearized pET-28a (+) -regrooES gene fragment with homologous sequences are obtained by PCR amplification, and the two fragments are respectively positioned at about 1000bp and 6000bp and are consistent with theoretical values by agarose gel electrophoresis detection, which is shown in figure 11. The linker-repO gene fragment with homologous sequence is connected with the linearized pET-28a (+) -regrooES gene fragment by an In-Fusion gene Fusion technology, positive clones are selected, bacterial liquid PCR identification is carried out by taking P1, P2, V1 and V2 as primers, the result shows that the obtained band sizes are about 1000bp and 6000bp respectively, the theoretical sizes of the linker-repO gene fragment and the pET-28a (+) -regrooES gene fragment are consistent, the result of the sequencing of the positive clone gene is completely consistent with the theory, and the result indicates that the pET-28a (+) -repO-GroES recombinant plasmid is successfully constructed.

Example 7: preparation of repO-GroES antigen

1. Test method

1.1 identification of expression of repO-GroES

(1) Taking out the pET-28a (+) -regrines/BL 21 and pET-28a (+) -repO-GroES/BL21 seed retaining bacterial solutions at the temperature of minus 80 ℃, respectively sucking 10 mu L bacterial solutions after the bacterial solutions return to room temperature, inoculating the bacterial solutions into 10mL LB culture solution containing Kana, placing the bacterial solutions in a shaking table, and culturing at the temperature of 37 ℃ and 180rpm overnight.

(2) The bacterial liquid was taken out, 200. Mu.L of the bacterial liquid was aspirated and inoculated into 20mL of LB medium containing Kana, and the mixture was placed in a shaker at 37℃and 180rpm to culture for 4 hours.

(3) The bacterial liquid is taken out and placed in a shaking table at 16 ℃ and cooled at 180 rpm. After completion of the cooling, 4. Mu.L of 1MIPTG solution was added to 20mL of the bacterial liquid, and the mixture was incubated at 16℃and 180rpm overnight.

(4) 50 mu L of the induced bacterial liquid is sucked into a 1.5mL EP tube, 10 mu L of 6 Xprotein loading buffer solution is added and mixed uniformly, and the mixture is boiled in a metal bath at 98-100 ℃ for 6-10 min.

(5) Centrifuging the residual bacterial liquid, carrying out 12000rpm for 10min, discarding the supernatant, adding 2mL of PBS buffer solution for heavy swirling, placing in an ice-water mixture, and carrying out bacterial breaking by using an ultrasonic crusher, wherein the set parameters are as follows: 5s ultrasonic on, 6s ultrasonic off, total time length of 6min and power of 28%.

(6) After completion of sonication, the supernatant and pellet were separated and 2mL of PBS buffer was added to the pellet for vortexing.

(7) The supernatant and the precipitate were each pipetted 50. Mu.L into a 1.5mL EP tube, 10. Mu.L of protein loading buffer was added and the mixture was subjected to metal bath boiling at 98-100℃for 6-10 min.

(8) And (3) sucking 10 mu L of each of the solutions in the steps (3) and (7), and performing SDS-PAGE detection.

1.2 expanded culture of pET-28a (+) -regrines/BL 21 and pET-28a (+) -repO-GroEL/BL21

(1) 20. Mu.L of seed retaining bacteria solution of pET-28a (+) -reGroES/BL21 and pET-28a (+) -rePO-GroES/BL21 were respectively aspirated, and added to 20mL of sterile LB medium containing Kana, and cultured overnight at 150rpm and 37℃to perform primary activation.

(2) Taking out the bacterial liquid, adding the bacterial liquid into 2L of sterile LB medium containing Kana, culturing for 4h at 180rpm and 37 ℃ and performing secondary activation to OD ₆₀₀ 1.0.

(3) The bacterial solution was taken out and placed in a shaking table at 16℃and incubated at 180rpm for 1 hour, cooled, and 400. Mu.L of 1M IPTG solution was added to the solution and incubated overnight.

(4) Taking out the induced bacterial liquid, centrifuging 1000g at 4 ℃ for 20min. The supernatant was discarded, the bacterial sludge was removed to a 50mL centrifuge tube, weighed and labeled on the side of the tube, and placed at-20 ℃ for cryopreservation.

1.3 purification of the repO-GroES protein

(1) 20mM PB buffer (pH 8.0) was added to the bacterial sludge at a volume 5 times the weight of the bacterial sludge, and the bacterial sludge was thoroughly vortexed. Placing the powder into a beaker, placing the beaker into an ice-water mixture for ultrasonic treatment, and using an ultrasonic crusher to set the parameters as follows: the ultrasonic switch is turned on for 8s and turned off for 9s, the total time is 15min, and the power is 38%.

(2) The supernatant and pellet were separated by centrifugation at 12000rpm at 10℃and the same volume of PB buffer as in step (1) was added to the pellet and vortexed uniformly and 50. Mu.L was sampled in the EP tube.

(3) Loading the supernatant onto Ni Bestarose FF gel beads, mixing uniformly, placing on a suspension instrument, combining for 40min at 4 ℃, catching the flow-through after combining is completed, sampling, flushing the filler by using PB buffer solution, and sampling.

(4) Purification was performed according to the protocol written in the following table:

TABLE 3 regrones and rePO-GroES purification methods

(5) And (3) adding 10 mu L of 6 Xprotein buffer solution into the samples obtained in the step (1) and the step (3), uniformly mixing, and then placing the mixture in a metal bath at 98-100 ℃ for 6-10 min for SDS-PAGE gel electrophoresis.

(6) Opening an Avant Pure 150 purification instrument, installing a G25 chromatographic column, balancing primary water through an A pipe, respectively loading a regrines and a rePO-GroES protein solution containing imidazole by using PBS buffer solution (pH 8.0) at a flow rate of 8mL/min, starting sample connection when the absorbance value of 280nm is more than 5mAU, and stopping sample connection when the absorbance value is reduced to 5mAU, thus obtaining the protein solution with imidazole removed. The purified regroES and repO-GroES protein solutions were frozen at-80 ℃.

2. Results

RePO protein, reGroES protein and rePO-GroES (amino acid sequence is SEQ ID NO: 16). Protein bands were around bands of standard molecular weights 33.0kDa, 12.0kDa and 45.0kDa, respectively. This result suggests that rePO proteins, reproes proteins and rePO-GroES proteins have been successfully obtained, see fig. 13.

Example 8: physicochemical property detection of repO-GroES protein

1. Test method

Molecular sieve chromatography of the rePO-GroES protein

The Avant Pure 150 purification apparatus was turned on, a Surperose 6 column was installed, primary water was passed through the A tube and equilibrated with PBS buffer (pH 8.0), and 500. Mu.L of regrines and repO-GroES proteins were loaded, respectively, using a SuperLoop volume of 500. Mu.L, at a flow rate of 0.5mL/min. The front volume was recorded and a result map was derived.

1.2 particle size detection of repO-GroES protein

mu.L of regrines and rePO-GroES protein solutions were aspirated separately, placed in the loading well of a nanoparticle analyzer equipped with an argon ion laser emitter, repeatedly examined 3 times, and experimental data were recorded and saved.

1.3 electron microscope observations of the repO-GroES protein

0.01mg/mL regrones and rePO-GroES solutions were adsorbed onto carbon support membranes, stained with 1% uranyl acetate, dried, and the samples were placed in Tecnai G2 for centering, adjusting appropriate parameters, and observed and photographed.

2. Results

The peak volumes of rePO, reGroES and rePO-GroES were 17.4ml, 16.8ml and 13.0ml, respectively, as analyzed by Superose 6 molecular sieve chromatography. Curve regression analysis of the standard proteins, estimated that the repO, regroES and repO-GroES proteins had molecular weights of about 35.5kDa, 70.0kDa and 315.5kDa, respectively, see FIG. 14. The dynamic irradiation results showed that regrones and rePO-GroES were better dispersed, with average diameters of about 10.2nm and 28.2nm, respectively, as shown in fig. 15. Uniform regrines and rePO-GroES particles were also visible under electron microscopy, with particle sizes of about 10.5nm and 28.1nm, respectively, see fig. 16. The above results indicate that the present study successfully prepared rePO proteins, reGroES and rePO-GroES nano self-assembled granule proteins.

Example 9: safety detection of repO-GroES protein

1 test method

1.1 cell hemolysis and CCK-8

(1) A blank group of mice was randomly selected for venous blood sampling at 150. Mu.L, centrifuged at 1500rpm for 15min, and red blood cells were purified and dispersed in PBS buffer.

(2) mu.L of erythrocytes were placed in different concentrations of 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 40. Mu.g/mL and 80. Mu.g/mL repO-GroES protein solution, the negative control was erythrocytes untreated in PBS, the positive control was erythrocytes treated with 1% Triton X-100, and incubated at 37℃for 1h. Centrifugation at 1500rpm for 15min and analysis of 540nm absorbance of each supernatant using an enzyme labelling apparatus.

(3) 100. Mu.L of 1X10 concentration was added to a 96-well plate ⁶ Individual/mL DC2.4 cell suspension, plates were incubated at 37℃with 5% CO ₂ The cells were pre-cultured in an incubator for 4 hours and observed under a microscope for cell adhesion. If the cell adhesion accounts for more than two thirds of the culture surface, the number of cells is large, the experiment can be continued, otherwise, the culture is needed again.

(4) 100. Mu.L of repO-GroES protein solution of different concentrations were added to the plates at final concentrations of 50. Mu.g/mL, 100. Mu.g/mL and 200. Mu.g/mL, respectively. And a negative control group and a background negative control group are arranged, wherein the negative control group is formed by mixing a cell suspension with 100 mu L of sterile PBS, and the background control group is a complete 1640 culture medium. The plates were placed at 37℃with 5% CO ₂ Incubate in incubator for 24 hours.

(5) mu.L of CCK-8 solution was added to each well of the plate. The plates were placed at 37℃with 5% CO ₂ Incubate in incubator for 1.5 hours. Absorbance at 450nm was detected using a microplate reader.

1.2 tissue section observations

After 7 days of final immunization, 2 mice were randomly selected from the rePO-GroES immunized and blank groups, sacrificed by cervical removal, dissected, and heart, liver, spleen, lung and kidney were removed and immersed in 4% paraformaldehyde solution. One pathologist performs slicing, and after slicing, the slicing is observed and photographed under a microscope, and scoring is performed, and the higher the scoring is, the more serious.

2 results

The results show that no hemolysis reaction occurs in the concentration of 0-80 mu g/mL of repO-GroES, OD ₅₄₅ The values of the cells were not higher than the positive control, and were similar to the negative control, the DC2.4 cell viability was maintained at 120% or higher at a concentration of repO-GroES of 50-200. Mu.g/mL, as shown in FIG. 17. No significant abnormalities were seen in heart, liver, spleen, lung and kidney tissue sections of rePO-GroES immunized and sham mice, see figure 18. The above results suggest that the repO-GroES protein is safe.

Example 10: detection of rePO-GroES induced immune response

1. Test method

1.2 Immunization

16-18 g SPF female BALB/c mice were randomly divided into 4 groups of 22 animals each containing 246 μg of Al (OH) per vaccine dose ₃ Adjuvant, 3.68 μg of repO was immunized with repO in the repO group, 1.58 μg of repGroES was immunized with repO-GroES in the repO-GroES group, and 5.26 μg of repO-GroES was immunized with repO, keeping the repO content consistent. Each dose was 100. Mu.L, and the blank was equal volume PBS. Intramuscular immunization was performed for 0,7, 14 days for a total of 3 times.

1.2ELISA detection of antibody titres produced by repO-GroES recombinant protein immunized mice

1.2.1 anti-repO IgG antibody titre detection

(1) On the 6 th day after each immunization, tail vein blood collection is carried out on the immunized mice and the blank mice, and after collection, the mice are incubated for 1-2 h at 37 ℃. Serum and blood cells were separated by centrifugation at 8000rpm for 10 minutes. Blood cells were discarded, serum was stored at-20℃and labeled.

(2) The rePO protein solution was diluted to 0.6 μg/mL using the coating solution and added to a 96-well elisa plate. 100 μl per well was wrapped with a preservative film and left to stand at 4deg.C overnight.

(3) And taking out the ELISA plate, and cleaning the coated ELISA plate by using an automatic plate cleaning machine. The plate washer parameters were set to wash 3 times, 300. Mu.L/well of PBST solution each time, and shaking for 5 seconds. And after cleaning, the board is patted dry on the absorbent paper.

(4) Adding a sealing liquid into the ELISA plate, wrapping 250 mu L/hole with a preservative film, and placing the ELISA plate into a 37 ℃ incubator for incubation for 2 hours.

(5) And taking out the ELISA plate, cleaning the ELISA plate by using a PBST solution, and drying the plate on absorbent paper after cleaning.

(6) The collected serum was removed from-20 ℃ and rewarmed, and the serum was diluted with PBST solution by selecting the appropriate dilution factor. 100. Mu.L of PBST solution was added to each well starting from the second transverse row of the 96-well ELISA plate, and 200. Mu.L of diluted serum sample was added to each well of the first row. 100. Mu.L of the solution is sucked from the first row and added to the second row and gently blown for 15 to 18 times, taking care not to blow bubbles so as not to affect the experimental result, and the like, finally sucking 100. Mu.L and discarding, and placing the ELISA plate in a 37 ℃ incubator for incubation for 1h.

(7) And taking out the ELISA plate, cleaning the ELISA plate by using a PBST solution, and drying the plate on absorbent paper after cleaning.

(8) IgG antibodies were diluted at a dilution ratio of 1:7500 using PBST solution, added at 100 μl per well after mixing, and incubated for 45min at 37 ℃.

(9) And taking out the ELISA plate, cleaning the ELISA plate by using a PBST solution, and drying the plate on absorbent paper after cleaning.

(10) TMB color development was added to the ELISA plate at 100. Mu.L per well, incubated at 37℃for 8min, and the incubation was carefully performed in the absence of light.

(11) The color development was stopped by adding 50. Mu.L/well of stop solution. The absorbance at 450nm was read within 15 min.

1.2.2 antibody subtype potency detection

(1) Serum 7 days after the last immunization was taken out from-20 ℃ and returned to room temperature, and then subjected to gradient dilution in the same manner as above.

(2) HRP-labeled IgG with PBST solution ₁ 、IgG _2a And IgG _2b The secondary antibodies were diluted at a dilution ratio of 1:7500, mixed well, added with 100. Mu.L per well, incubated at 37℃for 45min, and washed.

(3) TMB color development was added to the ELISA plate at 100. Mu.L per well, incubated at 37℃for 8min, and the incubation was carefully performed in the absence of light.

(4) The color development was stopped by adding 50. Mu.L/well of stop solution. The absorbance at 450nm was read within 15 min.

1.3 cell immunoassay

(1) At 14 days after the last immunization, 5 mice were randomly selected from the rePO immune group, the rePO-GroES immune group and the blank group, blood was collected by eye-picking, infiltrated with an alcohol lamp, dissected with the left abdomen facing upward, and the spleens were removed and placed in a 200 mesh cell sieve containing 2mL of sterile PBS.

(2) The spleen was thoroughly ground, the screened liquid was aspirated into a 15mL centrifuge tube, PBS solution was added, and the volume was made up to 5mL. Centrifuge at 1700rpm at 4℃for 10min. The supernatant was discarded, 5mL of red blood cell lysate was added, gently swirled, lysed at room temperature for 10min, followed by 5mL of PBS to terminate the lysis and mix well. Centrifuge at 1700rpm at 4℃for 10min. The supernatant was discarded, washed with 10mL of sterile PBS by vortexing, and the supernatant was discarded by centrifugation again. 10mL of PBS solution was added for vortexing. Mu.l were pipetted from each centrifuge tube into a cell counting plate, splenocytes were counted and recorded. The tube was centrifuged at 1700rpm for 10min and the supernatant was discarded. The amount of cells required for plating was calculated and splenocytes were diluted to the desired concentration using complete 1640 medium.

(3) And (3) placing the pre-packaged plate of the kit, which is hermetically packaged by the aluminum film, into an ultra-clean workbench, tearing the package, and paying attention to marking. 200 μl of sterile PBS per well was added to the pre-coated plate, and after two minutes of standing, it was snapped out and repeated three times. Then 200. Mu.L of complete 1640 medium was added to each well, which was left to stand at room temperature for 30min and then snapped out.

(4) mu.L of the repO peptide library was aspirated, added to 14250. Mu.L of complete 1640 medium, and the cell suspension at the adjusted concentration was added to each well of the experiment, 200. Mu.L per well. The concentration in 3 positive control wells was 5X 10 ⁵ Cells per well and 20 μl positive stimulus. 250. Mu.L of complete 1640 medium was added to 3 background negative control wells and the concentration was 5X 10 in experimental wells ⁵ Cells per well and 50. Mu.L of polypeptide solution were stimulated with a final polypeptide concentration of 10. Mu.g/mL and 5X 10 concentration was added to the experimental negative control wells ⁵ Cells per well and 50 μl of complete 1640 medium containing 5% DMSO. Covering the cover plate, placing the mixture into a furnace at 37 ℃ and 5% CO ₂ The medium was cultured for 3 days.

(5) The plates were removed, the solution was subtracted, 200 μl of sterile PBS per well was added to the plates, allowed to stand for 2min, and the subtraction was repeated 5 times.

(6) 100. Mu.L of IL-17A detection antibody, IL-4 detection antibody and IFN-gamma detection antibody were diluted to 0.25. Mu.g/mL, 1. Mu.g/mL and 1. Mu.g/mL in PBS containing 0.5% fetal bovine serum, respectively, and incubated at room temperature for 2 hours. Repeating the step (5).

(7) The antibodies in the kit are diluted and mixed uniformly in PBS containing 0.5% of fetal bovine serum according to the proportion of 1:1000, 100 mu L of the antibody is added into each hole, the mixture is incubated for 1 hour at room temperature, and the step (5) is repeated.

(8) The TMB color was filtered through a 0.45mm filter, 100. Mu.L of each well was added, incubated at room temperature until visible spotting occurred, rinsed extensively with tap water, the solution was rinsed clean, the plate was then air dried, and the next day was read and recorded.

2. Results

anti-rePO IgGs titers generated by rePO-GroES immunized mice were significantly higher than rePO immune group (P) at all 4 detection time points<0.05 With IgG as rePO-GroES induced IgG subtype ₁ Mainly, and IgG ₁ 、IgG _2a And IgG _2b Are all significantly more potent than the rePO immune group (P<0.01 See fig. 19). To further analyze the level of rePO-GroES cell immunity, mouse spleen cells were taken 14 days after the last immunization and examined for their cell numbers secreting IFN- γ, IL-17A and IL-4. The results show that the number of cells secreting IFN-gamma, IL-17A and IL-4 in the spleen of the mice of the repO-GroES immunized group is significantly higher than that of the repO immunized group (P<0.05 Among them, repO-GroES can significantly increase Th17 response (P)<0.01 See fig. 20). The above results indicate that rePO-GroES significantly enhances Th2 and Th17 immune response compared to rePO monomers.

Example 11: evaluation of protective Effect of repO-GroES immunization in mice model of pneumonia

1 test method

Survival observations after 1.1 lethal dose of PA-infected mice

(1) After 5 days of final immunization, PAXN-1 broth was dipped in an ultra clean bench using an inoculating loop and streaked on LB solid medium containing Kana using a three-wire method. Placing into a constant temperature incubator at 37 ℃ for incubation for 15 hours, taking out the incubator the next day, and storing in a refrigerator at 4 ℃.

(2) XN-1 single colonies were picked in 10mL of antibiotic-free LB medium, and a total of 5 single colonies were picked and placed in 5 Erlenmeyer flasks, respectively, at 37℃and 180rpm, and cultured overnight.

(3) And (3) selecting a bottle of bacterial liquid with optimal growth vigor for secondary activation, sucking 200 mu L of bacterial liquid, adding the bacterial liquid into 20mL of antibiotic-free LB culture medium, and culturing at 37 ℃ for 3.5 hours at 220 rpm.

(4) Selecting a bottle from the bacterial liquid in the step (3), detecting and recording the OD thereof ₆₀₀ Numerical values. The supernatant was discarded by centrifugation at 8500rpm for 5min, swirled again with normal saline, and the supernatant was discarded by centrifugation and repeated 2 times. Adding a certain volume of physiological saline to make the bacterial liquid OD ₆₀₀ Reaching 1.19-1.23, diluting the solution by 12 times, subpackaging the solution into 1.5mL EP pipes, and placing the EP pipes on ice.

(5) The anesthetic is injected into the abdominal cavity of mice in immune group and blank group, after anesthesia, trachea cannula is performed, diluted bacterial liquid is injected into the lung of the mice, 20 mu L bacterial liquid (1×10) is injected into each mouse ⁶ CFU/mouse)。

(6) Observations were made every 12h after challenge, and survival numbers were recorded for each group for 7 consecutive days.

1.2 weight changes after sub-lethal dose of PA infection in mice

(1) After 5 days of final immunization, single colonies were picked and subjected to secondary activation as described above.

(2) Selecting a bottle of bacterial liquid from the bacterial liquid in the step (1), and detecting the OD (optical density) of the bacterial liquid ₆₀₀ Numerical values. Centrifuging to remove supernatant, swirling with physiological saline, centrifuging to remove supernatant, and repeating for 2 times. Adding a certain volume of physiological saline to make the bacterial liquid OD ₆₀₀ Reaching 1.19-1.23, diluting the mixture 16 times, and split charging the mixture into 1.5mL EP pipes.

(3) The mice were anesthetized and then tracheal cannulated, and each mouse was pulmonary injected with 20. Mu.L XN-1 bacteria solution (2.4X10) ⁵ CFU/mouse)。

(4) After challenge, observations were made every 12h, and each group of mice was weighed and recorded for 84 hours in succession.

2 results

The lethal dose PA infection results showed 80% survival of the rePO-GroES immunized group, 60% of the rePO immunized group, and significant statistical differences were found in both groups and higher than in the blank group (P < 0.05), whereas mice immunized with repos had no significant differences from the blank group, see figure 21. The result of the weight change of the mice infected with the PA with sublethal dose shows that the weight change of the mice in the immune group and the blank group is a trend of decreasing first and then rising, the weight change amplitude of the repO-GroES immune group is obviously lower than that of the repO immune group and the blank group (P < 0.05), the weight change amplitude of the repO immune group and the blank group is not obviously different, and the result shows that the repO-GroES has obvious protection effect on PA infection and is superior to that of the repO monomer, and the repoES has no obvious protection effect.

While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.

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Claims

1. A recombinant pseudomonas aeruginosa nanoparticle protein, characterized in that it is composed of rePO, - (Linker) n-and pseudomonas aeruginosa heat shock proteins connected in sequence, wherein rePO is pseudomonas aeruginosa PcrV and OprI fusion antigen, linker is each independently selected from any one of SEQ ID NO:5GGGGS, SEQ ID NO:6GGSGG and SEQ ID NO:7YAPVDV for each occurrence, n is 1, 2, 3 or 4, preferably 1;

the pseudomonas aeruginosa heat shock protein is GroEL or GroES.

2. The recombinant pseudomonas aeruginosa nanoparticle protein of claim 1, wherein the amino acid sequence of rePO is SEQ ID No. 3.

3. The recombinant pseudomonas aeruginosa nanoparticle protein according to claim 1, characterized in that the amino acid sequence of GroEL is SEQ ID No. 4; the amino acid sequence of GroES is SEQ ID NO. 18.

4. A recombinant pseudomonas aeruginosa nanoparticle protein according to any of claims 1-3, characterised in that the amino acid sequence is SEQ ID No. 2 or SEQ ID No. 16.

5. The recombinant pseudomonas aeruginosa nanoparticle protein encoding gene according to any one of claims 1-4, wherein the nucleotide sequence is SEQ ID No. 1 or SEQ ID No. 17.

6. A recombinant expression vector comprising a gene encoding a recombinant pseudomonas aeruginosa nanoparticle protein according to claim 5, and an expression plasmid; the expression plasmid is selected from any one of pGEX series vectors, pET series vectors or pQE series vectors, and is preferably pET-28a.

7. A recombinant strain for expressing the recombinant pseudomonas aeruginosa nanoparticle protein according to any one of claims 1-4, comprising the recombinant expression vector of claim 6 and a host bacterium; the host strain is selected from any one of an escherichia coli XL1-blue strain, a BL21 series strain and an HMS174 series strain, and is preferably an escherichia coli BL21 strain.

8. A method for producing the recombinant protein according to claim 1, comprising the steps of:

1) Constructing a recombinant expression vector for expressing the coding gene of the recombinant pseudomonas aeruginosa nanoparticle protein according to any one of claims 1-4 by a DNA synthesis and subcloning method; preferably, the coding gene of the recombinant pseudomonas aeruginosa nanoparticle protein is SEQ ID NO. 1;

3) Inducing the transformed host bacteria to express recombinant protein;

4) Purifying the recombinant protein to obtain the recombinant pseudomonas aeruginosa nanoparticle protein according to any one of claims 1-4.

9. Use of the recombinant pseudomonas aeruginosa nanoparticle protein of claims 1-4, the recombinant gene of claim 5, the recombinant expression vector of claim 6, or the recombinant strain of claim 7 in the preparation of subunit vaccines against pseudomonas aeruginosa.

10. A vaccine for preventing or treating infection by pseudomonas aeruginosa comprising the recombinant pseudomonas aeruginosa nanoparticle protein of claims 1-4.