CN116474081B

CN116474081B - Acinetobacter baumannii vaccine and preparation method thereof

Info

Publication number: CN116474081B
Application number: CN202310723357.1A
Authority: CN
Inventors: 石云; 杨红; 张晓敏; 王宁; 李彦; 陈凯; 游瑜; 孙向成; 周杨杨; 向传英; 谢雨
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2023-06-19
Filing date: 2023-06-19
Publication date: 2023-09-12
Anticipated expiration: 2043-06-19
Also published as: CN116474081A

Abstract

The invention provides an acinetobacter baumannii vaccine and a preparation method thereof, and relates to the technical field of biology. The vaccine comprises a Acinetobacter baumannii nucleoside diphosphate kinase NDK recombinant protein, and the amino acid sequence of the Acinetobacter baumannii nucleoside diphosphate kinase NDK recombinant protein is shown as SEQ ID NO. 3. The vaccine prepared by the NDK recombinant protein can be used for immunization through an intramuscular injection way, and the organism is stimulated to generate high-titer antibodies. Animal experiments prove that the genetic engineering recombinant monovalent vaccine has good immune protection effect on Acinetobacter baumannii infection. The method lays a foundation for further research of combined vaccines and multi-subunit vaccines, and plays an important role in development and application of vaccines and diagnostic kits.

Description

Acinetobacter baumannii vaccine and preparation method thereof

Technical Field

The invention relates to the field of biotechnology, in particular to an acinetobacter baumannii vaccine and a preparation method thereof.

Background

Acinetobacter baumannii is a widely occurring non-fermented gram-negative bacillus, and as an opportunistic pathogen, can cause hospital-or community-acquired diseases such as meningitis, pneumonia (hospital-or community-acquired), bacteremia, endocarditis, urinary Tract Infection (UTI), skin and soft tissue infection, and the like. Hospital acquired infections caused by acinetobacter baumannii are reported to be 21%, 24.4%, 46.9% in the united states, korea and norway, respectively. ICU acquired pneumonia of 36% of asia is associated with acinetobacter baumannii infection. The detection rate of the drug-resistant Acinetobacter baumannii in the intensive care disease area is 78.3% which is far higher than that of other disease areas. In addition to hospital acquired infections, community acquired infections caused by acinetobacter baumanii are also increasing. The Acinetobacter baumannii drug resistance is serious, and a monitoring report of the drug resistance of the bacteria in China in 2021 shows that the detection rate of the carbapenem drug resistance Acinetobacter baumannii in 2021 is 54.3 percent, which is increased by 0.6 percent compared with 53.7 percent in 2020, and still maintains a higher level. Acinetobacter baumannii has been listed in the important research list of drug-resistant bacteria and antibacterial drug resistance of the World Health Organization (WHO) due to the increase of multi-drug resistant strains and high morbidity and mortality.

Because of the popularity and outbreak of multi-drug resistant Acinetobacter baumannii, the existing antibacterial drugs cannot effectively treat the infection caused by the strains, the development period of new antibacterial drugs is long, and the drug resistance is easy to generate, so that in order to treat the diseases related to the drug resistant Acinetobacter baumannii infection, new therapeutic drugs and strategies are urgently needed, a vaccine for preventing the Acinetobacter baumannii infection is developed, and the immune response is induced by organisms, so that the method is an effective method for treating the drug resistant Acinetobacter baumannii.

Disclosure of Invention

The invention aims to provide an Acinetobacter baumannii vaccine and a preparation method thereof, and subunit vaccine prepared by using the NDK recombinant protein can be used for immunization through an intramuscular injection way to excite organisms to generate IgG antibodies and cellular immune responses, so that the Acinetobacter baumannii vaccine has good immune protection effect on Acinetobacter baumannii infection.

Nucleoside diphosphate kinase (Nucleoside Diphosphate Kinase, NDK) is an enzyme involved in nucleotide metabolism in acinetobacter baumannii. The invention utilizes reverse vaccinology to screen potential protective antigen library of Acinetobacter baumannii, which comprises NDK protein, the protein is composed of 143 amino acids, the amino acid sequence is shown as SEQ ID NO.1, and the coding DNA sequence is shown as SEQ ID NO. 2. The invention designs an acinetobacter baumannii vaccine by utilizing the 1 st-143 th amino acid sequence of NDK protein.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the Acinetobacter baumannii vaccine provided by the invention comprises Acinetobacter baumannii nucleoside diphosphate kinase NDK recombinant protein, and the amino acid sequence of the Acinetobacter baumannii nucleoside diphosphate kinase NDK recombinant protein is shown as SEQ ID NO. 3.

According to a preferred embodiment, the acinetobacter baumannii NDK recombinant protein at least comprises the amino acid sequence of positions 1-143 of the NDK protein, and the amino acid sequence is shown as SEQ ID No. 1.

The invention also provides a preparation method of the Acinetobacter baumannii vaccine, which comprises the following steps:

(1) Designing a PCR primer according to the nucleotide diphosphate kinase NDK protein gene sequence of Acinetobacter baumannii, taking the whole genome DNA of the Acinetobacter baumannii as a template, and carrying out PCR amplification on a target gene of the NDK protein amino acid sequence of the Acinetobacter baumannii according to the designed PCR primer;

wherein, the forward primer of the designed PCR primer: 5'-CGCGGATCCATGGCAATTGAACGTACTTTGTCT-3', reverse primer: 5'-TTATGCGGCCGCTTAACGAGTGCGTGGGCAG-3';

(2) Cloning the PCR amplification product obtained in the step (1) to an expression vector containing a GST tag, and converting the PCR amplification product into a prokaryotic expression system to perform induction expression on the NDK fusion protein containing the GST tag;

(3) Separating the target protein from the GST tag by using an enzyme digestion method to obtain the NDK recombinant protein of the Acinetobacter baumannii;

(4) Purifying the NDK recombinant protein obtained in the step (3);

(5) And adsorbing the purified NDK recombinant protein with an adjuvant to form the Acinetobacter baumannii vaccine.

According to a preferred embodiment, in step (5), the adjuvant comprises Al (OH) ₃ Adjuvant, alPO ₄ Adjuvants, MF59, AS03, AS04, incomplete freund's adjuvant or complete freund's adjuvant.

Based on the technical scheme, the Acinetobacter baumannii vaccine and the preparation method thereof have at least the following beneficial effects:

(1) The NDK protein is used in the field of Acinetobacter baumannii vaccines, and the prepared Acinetobacter baumannii vaccine can effectively excite organisms to cause protective immune response, so that lethal infection of Acinetobacter baumannii is resisted.

(2) In the preparation method of the invention, the expression plasmid of the NDK protein is induced to express in a prokaryotic expression system (escherichia coli), and the expression is high, and the quality is safe and controllable. pGEX-6p-2 expression vector is selected, so that the NDK recombinant protein is expressed in the form of fusion protein, and the original spatial conformation of the NDK recombinant protein is maintained to the maximum extent.

(3) The fusion protein expressed by the preparation method provided by the invention contains a GST tag, and the GST tag can be used as a marker for protein purification, so that the purification conditions are mild, the steps are simple, and no denaturant is needed to be added, and therefore, the purified protein can keep the spatial conformation and immunogenicity to the greatest extent; and the expression rate of the NDK fusion protein is about 25% (as shown in FIG. 4, the gray value of the target protein is analyzed by Image Lab software, the proportion of the gray value of the target protein (shown by an arrow) in a lane "+" to the total gray value of the target protein in the whole "+" lane is calculated), and the purity of the purified NDK protein is 95% (as shown in FIG. 6, the result of the proportion of the gray value of the target protein of the NDK in the NDK lane to the total gray value of the target protein is calculated by Image Lab software analysis).

(4) The NDK recombinant protein prepared by the invention can induce animals to generate specific high-titer antibodies.

Subunit vaccine prepared by the NDK recombinant protein can be immunized by an intramuscular injection way, and the organism is stimulated to generate high-titer specific IgG antibodies. Animal experiments prove that the genetic engineering recombinant monovalent subunit vaccine has good immune protection effect on Acinetobacter baumannii infection. The method lays a foundation for further research of combined vaccines and multi-subunit recombinant vaccines, and plays an important role in development and application of vaccines and diagnostic kits.

Drawings

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

FIG. 1 is a diagram showing the result of PCR amplification of NDK gene.

FIG. 2 is a graph showing the results of the cleavage assay for recombinant expression plasmid pGEX-6 p-2/NDK.

FIG. 3 is a diagram showing the result of DNA sequence comparison between recombinant expression plasmid pGEX-6p-2/NDK sequencing and target protein.

FIG. 4 is a graph showing the results of induction of expression of NDK-GST fusion protein at 16 ℃.

FIG. 5 is a graph showing the results of purification of fusion proteins obtained from the supernatant after inducible expression of recombinant engineering bacteria at 16 ℃.

FIG. 6 is a graph showing the results of recombinant engineering bacteria induced at 16℃to obtain NDK recombinant protein after cleavage of NDK-GST fusion protein and purification by ion exchange column.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.

The following describes the technical scheme of the invention.

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

1. strain

Acinetobacter baumannii strain ATCC17978 was purchased from ACTT, USA.

2. Reagent(s)

Plasmid pGEX-6p-2 (purchased from GE company), E.coli strain BL21 (DE 3) (purchased from Optimaceae company);

2X High Fidelity PCR Master Mix, DNA Marker, DNA Ligation Mix purchased from Beijing engine biology company; restriction enzymes BamH I and Not I were purchased from NEB Inc. of the United states; protein markers are products of bure corporation.

Plasmid extraction kit, gel recovery kit, bacterial genome extraction kit, ultrathin recovery kit and developing solution are products of Tiangen company;

glutathione-sepharose Glutathione Sepharose B is a Shanghai Biotechnology company product.

Example 1: cloning of Acinetobacter baumannii NDK protein.

1. First, the NDK of Acinetobacter baumannii strain is searched in NCBI website protein library to obtain a protein sequence (GenBank: EZF 14775.1), and the protein sequence is shown as SEQ ID NO. 1. And according to the coding gene, the NDK gene sequence is shown as SEQ ID NO. 2.

2. Analyzing the property of the protein by bioinformatics software, expressing the full length of the protein, and amplifying the gene of the NDK protein by using the whole genome of Acinetobacter baumannii as a template by adopting a PCR method, wherein the amplification steps are as follows:

1) The PCR primers were designed as follows, SEQ ID NO.4 and SEQ ID NO.5, respectively;

(underlined shows the base sequence of the cleavage site)

Forward primer: SEQ ID NO.4

5'-CGCGGATCCATGGCAATTGAACGTACTTTGTCT-3'，

BamH I

Reverse primer: SEQ ID NO.5

5'-TTATGCGGCCGCTTAACGAGTGCGTGGGCAG-3'，

Not I

In this example, a DNA sequence encoding the amino acid sequence of the NDK protein shown in SEQ ID NO.1, which is shown in SEQ ID NO.2, was used as a target gene for PCR amplification.

2) The stored Acinetobacter baumannii ATCC17978 strain is taken out from a freezer at the temperature of 80 ℃ below zero, recovered on a TSA solid culture medium by a three-wire method, cultured overnight at the temperature of 37 ℃, then picked up and inoculated on a TSB liquid culture medium, placed in a constant temperature shaking table for culturing for 8 hours at the speed of 220rpm, and the whole genome is extracted by referring to a bacterial genome extraction kit.

3) The whole genome DNA of Acinetobacter baumannii is used as a template for PCR amplification of the NDK protein gene.

Wherein, the PCR system:

stencil (200 ng/. Mu.l)	2μl
		Forward primer (1. Mu.M)	1μl
Reverse primer (1. Mu.M)	1μl
		2×High Fidelity PCR Master Mix	20μl
Sterilizing double distilled water	16μl
		Total volume of	40μl

PCR amplification reaction conditions: pre-denaturation at 95℃for 30s, denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 1 min,30 cycles, and complete extension at 72℃for 5min. After completion of the reaction, the PCR amplification results were detected using 1% agarose gel and are shown in FIG. 1, in which lane M: a nucleic acid (DNA) molecular weight standard (Marker); lane 1: PCR amplified product of NDK gene (432 bp).

4) NDK PCR products were recovered using a gel recovery kit.

3. The PCR products were identified and cloned as follows:

1) BamH I and Not I cut pGEX-6P-2 plasmid and NDK PCR products respectively;

wherein, the enzyme digestion reaction system:

BamH I	2μl
		Not I	2μl
10× CutSmart Buffer	4μl
		plasmids or PCR products	1μg
Total volume (with sterile deionized water)	40μl

And the enzyme digestion is carried out for 1h at 37 ℃.

2) The digested pGEX-6P-2 plasmid and the digested PCR products were recovered using a gel recovery kit.

3) Ligation and transformation.

The concentration of nucleic acid of the target gene enzyme digestion recovery product is measured by an ultraviolet spectrophotometer, and the general ratio of the vector to the exogenous DNA is 1:2-10, designing the following connection reaction system.

And (3) connecting a reaction system:

T4 DNA Ligase	0.5μl
		enzyme cutting recovery product of target gene	4.3μl
PGEX-6P-2 enzyme digestion recovery product	0.7μl
		10×ligation buffer	1μl
Sterile deionized water	3.5μl
		Total volume of	10μl

Mixing, and connecting at 16deg.C for 1 hr.

4) Taking 3 tubes of escherichia coli BL21 (DE 3) competent cells from a refrigerator at the temperature of minus 80 ℃, and adding pGEX-6P-2 plasmid into the first tube to serve as a positive control; adding the DNA ligation product to the second tube; the third tube was not added with exogenous DNA and served as a negative control. Ice bath for 30min, hot impact at 42 ℃ for 90s in metal bath, and rapid ice bath for 2min. 600 μl of LB blank medium was added, mixed well and placed in a shaking table at 37℃for 1h with shaking at 200 rpm.

Each tube was centrifuged at 5000rpm at room temperature for 5min, 400. Mu.l of the supernatant was discarded, and the cells were resuspended and 100. Mu.l was plated on Amp-resistant LB plates. The plates were placed upside down in a 37℃incubator for 24h.

5) Screening and identifying pGEX-6p-2/NDK recombinant expression plasmids;

(1) negative control plates had no colonies present; positive control plates were full of colonies, indicating correct competent cell production and reliable results. Picking single colony well grown on a conversion plate, inoculating the single colony to an Amp resistance LB culture medium, and carrying out shake culture at 37 ℃ overnight;

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

(3) the plasmid DNA is subjected to BamHI and NotI double enzyme digestion;

double cleavage reaction system:

BamH I	1μl
		Not I	1μl
10×CutSmart Buffer	2μl
		plasmid(s)	1μg
Total volume (with sterile deionized water)	20μl

Enzyme cutting at 37 ℃ for 1h;

(4) double digestion results were detected by 1% agarose gel electrophoresis and are shown in FIG. 2, wherein lane M: a nucleic acid (DNA) molecular weight standard (Marker); lane 1: representing the identification result of recombinant expression plasmid pGEX-6p-2/NDK after enzyme digestion, wherein the fragment size after enzyme digestion is 4954bp and 432bp; the lane 1 sample is pGEX-6p-2/NDK recombinant expression plasmid which is successfully constructed;

(5) the pGEX-6p-2/NDK recombinant expression plasmid is sent to Shanghai biological company for nucleic acid sequencing, and the sequencing result completely accords with the target protein nucleic acid sequence through comparison, namely the sequence of the target gene fragment of the pGEX-6p-2/NDK recombinant expression plasmid is correct, as shown in figure 3.

Example 2: the Acinetobacter baumannii NDK protein is induced to be expressed and purified in a prokaryotic expression system, namely escherichia coli, and the expression form is identified.

1. The target protein induces expression.

1) Taking 100 mu L of two recombinant engineering strains pGEX-6P-2-NDK/BL21 (DE 3) bacterial liquid with correct double enzyme digestion identification, adding the bacterial liquid into 10ml of Amp-resistant TB culture medium, culturing overnight at 220rpm and 37 ℃, respectively taking 2ml of the bacterial liquid cultured overnight, adding the bacterial liquid cultured overnight into 18ml of Amp-resistant TB culture medium (the rest bacterial liquid is stored in a refrigerator at 4 ℃ for standby), culturing for 2-3 hours at 37 ℃, rotating at 250rpm, and when the bacterial liquid is activated for the second time until the OD600 is 0.8-1.2, adding 10 mu L of IPTG to make the final concentration of the bacterial liquid 500 mu M, and then placing the bacterial liquid in a shaking table for induction expression and the bacterial liquid cultured overnight at 16 ℃ for induction expression.

2) Taking out the bacterial liquid after induction expression, centrifuging at 10000rpm for 2min, discarding the supernatant, adding 1ml PBS buffer solution, mixing, performing ultrasonic lysis for 3min, centrifuging at 14000rpm at 4 ℃ for 15min, and collecting the supernatant.

SDS-PAGE electrophoresis, pouring 5% concentrated gel into offset plate, adding distilled water, flattening, standing at room temperature for 30min to solidify, pouring upper distilled water, pouring 10% separating gel, immediately inserting comb, standing at room temperature for 30min to solidify.

3. And (5) taking 10 mu L of the treated supernatant samples respectively for loading, and performing SDS-PAGE electrophoresis. The voltage is firstly 80V for 30min, then 200V is regulated, after electrophoresis for 45min, the gel is taken out, and is placed in coomassie brilliant blue staining solution for oscillation staining, and then placed in decolorizing solution for oscillation decolorizing, and then the result is observed under an imaging system, and the result is shown in fig. 4, lane M: protein molecular weight standard (Marker), lane "-": representing protein bands in the whole bacteria before the induced expression of the recombinant engineering bacteria; lanes "+": after the recombinant engineering bacteria are induced to express at 16 ℃, NDK-GST fusion protein is obtained in the whole bacteria, the arrow shows the band position of the target fusion protein, and the molecular weight is about 42.29 kD. Lane "S": after the recombinant engineering bacteria are induced to express at 16 ℃, NDK-GST fusion protein is obtained in bacterial strain supernatant. The results show that PGEX-6P-2-NDK/BL21 (DE 3) has the ability to express the correct GST-tagged NDK protein (NDK-GST fusion protein) at 16℃and that the recombinant proteins are expressed in soluble form in the sonicated supernatant. (the amino acid sequence is shown as SEQ ID NO. 3).

Example 3: preparation of NDK recombinant proteins.

1. And (5) carrying out amplification culture to obtain the protein.

pGEX-6P-2-NDK/BL21 (DE 3) strain stored in a refrigerator at-80 ℃ is inoculated on LB ampicillin resistance plates respectively, and cultured overnight at 37 ℃; picking single colony, inoculating to 50ml LB culture medium, culturing at 37 deg.C and 220rpm for overnight; adding 10ml of activated bacterial liquid into 1L of LB culture medium for secondary activation, culturing at 37 ℃ for 3-4 hours until OD600 is 0.8, adding 500 mu L of IPTG (final concentration is 500 mu M), placing in a shaking table at 16 ℃ for 12 hours, centrifuging at 6000rpm for 20 minutes to collect bacterial cells, adding 100ml of PBS to resuspend the bacterial cells, performing ultrasonic lysis for 30 minutes at 12000rpm, centrifuging for 30 minutes, and combining the collected supernatant with 10ml of GST filler at 4 ℃ overnight; a large number of GST-tagged NDK fusion proteins were obtained.

The LB solid and liquid culture medium in the step contains 100mg/ml ampicillin;

GST packing in this step is known as GST 4FF agarose purification resin.

2. Separating the target protein from the GST tag by using an enzyme digestion method to obtain an NDK recombinant protein;

adding 4ml PBS and 120 mu L PreScission protease (PP enzyme) into the rest about 4ml glutathione-agarose gel 4B combined with NDK-GST fusion protein, carrying out vertical rotary enzyme digestion at 4 ℃ overnight, centrifuging to absorb supernatant, washing 2 times with 2ml PBS respectively, taking 10 mu L of sample for denaturation treatment, loading 10 mu L of sample for SDS-PAGE protein electrophoresis, observing the result under an imaging system, obtaining NDK protein with the molecular weight of about 15kDa after enzyme digestion, conforming to the expected protein molecular weight, wherein the electrophoresis result is shown in fig. 5, lane M is a protein molecular weight standard (Marker), and lane 1 is a whole bacterium after recombinant engineering bacteria are induced to express at 16 ℃; lane 2 is the protein bound to the GST filler after overnight binding (mainly GST-NDK fusion protein and partial hybrid protein); lane 3 is the protein collected after cleavage (mainly the NDK recombinant protein of interest) and lane 4 is the protein remaining on the GST filler after elution (mainly the enzyme and the GST tag remaining after cleavage).

3. And further purifying the NDK recombinant protein by using an ion exchange column to obtain the high-purity NDK recombinant protein.

Taking out the ion exchange column, connecting and installing, flushing the ion exchange column by deionized water for 5 column volumes, and fully balancing the column by using a filtered 20mM PB (pH=8.0) solution; after filtering the samples stored in 20mM PB (ph=8.0) buffer, the samples were slowly loaded to allow for adequate protein binding; washing the exchange column with a solution containing 10mM NaCl and 20mM NaCl to remove the impurity protein; the target protein was eluted using a linear method, and the effluent was collected for electrophoresis detection, as shown in fig. 6, lane M: protein molecular weight standard (Marker), lane NDK shows the purified NDK recombinant protein after ion exchange column, and Image Lab software analysis shows that the target protein gray value in lane NDK accounts for 95% of the total protein gray value, i.e. the target protein purity is 95%.

4. The buffer was replaced and the target protein was stored in PBS buffer (pH 6.0).

The BCA assay measures recombinant protein concentration at about 0.45 mg/ml.

Example 4: and (6) constructing a mouse Acinetobacter baumannii infection model.

Inoculating Acinetobacter baumannii strain to a TSA solid culture medium plate by a three-wire method, and incubating for 16 hours at a constant temperature of 37 ℃; single colonies were picked up on plates, inoculated into 10ml TSB liquid medium and placed in a shaking table at 37℃under shaking at 220 rpm. After 5 hours, the cells were collected, diluted 15-fold with sterile PBS, and the OD600 was measured at 1.5X10 ⁹ CFU/ml/OD ₆₀₀ Calculating, diluting the bacterial liquid to 1×10 ⁸ CFU/ml、2×10 ⁸ CFU/ml、3×10 ⁸ CFU/ml，4×10 ⁸ Three different concentrations of CFU/ml were then intravenously injected into the tail veins (100. Mu.L/mouse) of C57Bl/6 female mice. Mice of 6-8 weeks of age and body weight of 18-20g are most preferred. Each group of 5 mice was set up, and 7 days of continuous observation was performed and mortality was counted for each group of mice. Finally, the infection dose of Acinetobacter baumannii is 3 multiplied by 10 ⁷ CFU/mouse, the dose was selected for subsequent mouse model construction.

Table 1: determination of lethal dose of Acinetobacter baumannii

Dose of infection (CFU/mouse)	Mouse (Only)	Number of deaths in seven days (only)	Mortality rate of
				1 x 10 ⁷	5	0	0
2 x 10 ⁷	5	2	40%
				3 x 10 ⁷	5	5	100%
4 x 10 ⁷	5	5	100%

The results are evaluated by an animal model aiming at the survival rate of mice, and a foundation is laid for successful development of the Acinetobacter baumannii vaccine and research on pathogenesis of Acinetobacter baumannii infection.

Example 5: and (3) preparing an NDK recombinant protein vaccine.

The adsorption of the NDK recombinant protein and different adjuvants is carried out to make a preliminary experiment, and the adsorption effect is detected, so that Al (OH) is found ₃ The adsorption efficiency is best, and the subsequent main Al (OH) ₃ As an adjuvant component, a vaccine was prepared by adsorption. The method after fumbling is as follows: the concentration of the NDK recombinant protein (i.e. antigen) is regulated to 200 mug/ml, the aluminium hydroxide adjuvant is measured, the histidine diluent with pH of 6.0 is used for regulating the concentration to 5 mg/ml, the mixture is fully and uniformly mixed, and the equal volumes of the NDK recombinant protein (i.e. antigen) solution and the adjuvant solution are taken to be rotationally suspended and adsorbed for 1 hour at the temperature of 4 ℃ to obtain the vaccine. The same conditions were used to prepare an adjuvant control formulation without antigen by substituting the recombinant protein (antigen) solution with a vaccine diluent.

Example 6: NDK recombinant proteins immunize animals.

For the first immunization, the vaccine preparation containing NDK protein was injected with insulin needle, two-sided thigh muscle injection of C57Bl/6 mice, 200. Mu.l each, 100. Mu.l each of hind limb thigh, and negative control group (Al (OH) was set ₃ An adjuvant group); the second immunization is carried out on the 14 th day, the immune components are the same, the injection quantity is the same as that of the first immunization, and the immunization route is the same; the third immunization was performed on day 21, the same immunization composition, the same injection as the first immunization, and the same immunization route.

Example 7: and (5) detecting the antibody titer of the recombinant NDK protein.

1. Sample: on day 7 after the third immunization of the recombinant NDK vaccine, blood was collected from immunized C57Bl/6 mice, and IgG humoral response levels after immunization of the mice were detected by ELISA.

ELISA detection reagent preparation.

(1) Preparing a coating liquid: weighing NaHCO ₃ 1.6g，Na ₂ CO ₃ 2.9g in 1L ddH ₂ O, adjusting the pH to 9.6 by a pH meter;

(2) preparing a sealing liquid: 1g of bovine serum albumin in 100ml of antibody diluent (1:100);

(3) preparation of antibody dilution: phosphate was dissolved in 1L ddH ₂ O, 500 μl Tween 20 was added, and the pH was adjusted to 7.4 with a pH meter;

(4) preparation of the washing liquid: 2.42g Tris was weighed out in 1L ddH ₂ O, 500 μl Tween 20 was added, and the pH was adjusted to 7.4 with a pH meter;

(5) color development liquid (TMB) is a product of Tiangen corporation;

(6) termination liquid (2M H) ₂ SO ₄ ) Is prepared from the following steps: 22.2ml of concentrated sulfuric acid are poured into 177.8ml of ddH ₂ O.

ELISA for detecting antibody titer generated by mice immunized with NDK recombinant protein.

(1) Diluting the purified NDK recombinant protein to 4 mug/ml by using coating liquid;

(2) coating: adding diluted NDK recombinant protein into an ELISA plate, and washing with a washing solution for 4 times after 100 μl/hole is over night at 4deg.C;

(3) closing: adding 100 μl/hole of sealing solution into the ELISA plate, incubating at 37deg.C for 2 hr, and washing for 4 times;

(4) serum was diluted 1:1000, 1:5000, 1:25000, 1:125000 fold;

(5) taking a sealed ELISA plate, sequentially adding diluted serum and 100 mu l/hole, placing the ELISA plate in an incubator at 37 ℃ for 2 hours, washing for 4 times, and drying;

(6) HRP-labeled goat anti-mouse IgG antibody stock solution was prepared at 1:10000, diluting to prepare an antibody working solution;

(7) adding diluted antibody working solution, placing 100 μl/hole in an incubator at 37deg.C for 45min, washing for 4 times, and drying;

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

(9) adding stop solution (2M H) ₂ SO ₄ ) Immediately placing the sample on an enzyme-labeled instrument to measure an OD450 value at a wavelength of 450 nm;

and (3) judging the result: a is that _{Sample of} ∕A _{Negative of} A value of ∈ 2.1 was positive, A was absorbance OD600 (negative control was adjuvant immunized).

TABLE 2 ELISA detection of antibody titers (OD 450) produced by NDK recombinant protein immunized mice

Dilution factor	Adjuvant group immune serum	NDK immune serum
			125000	0.034	0.111
25000	0.034	0.346
			5000	0.036	0.965
1000	0.047	1.925

The results are shown in table 2, and the antibody titer generated by NDK recombinant protein immunized mice is greater than 1:125000; the NDK recombinant protein constructed by the invention can stimulate mice to generate high-titer NDK specific antibodies, which indicates that the NDK has good immunogenicity and is a better vaccine candidate antigen.

Example 8: and determining the immune protection effect of the NDK recombinant protein through an immune toxicity attack experiment.

Following the immunization protocol of example 6, a challenge experiment was performed by administering a lethal dose of Acinetobacter baumannii live bacteria via the tail vein on day 7 after the third immunization of mice, with an amount of bacteria injected into each C57Bl/6 mouse of 3X 10 ⁷ CFU, 7 days of observation, survival of each group of mice was counted. The results are shown in Table 3.

TABLE 3 immunoprotection effects of recombinant proteins

Group of	Immune vaccine component	Mouse (Only)	Number of dead mice (Only)	Mortality (%)	Protection ratio (%)
						NDK recombinant protein immunization	Recombinant protein+Al (OH) ₃ Adjuvant	10	5	50	44.44
Adjuvant immune control group	Al(OH) ₃ Adjuvant	10	9	90	0

Table 3 shows animal immune test results, showing that the death rate of the adjuvant control group is 90%, the death rate of the NDK vaccine immune group is 50%, and the immune protection rate is calculated according to the formula: [ immunoprotection rate= (control morbidity-vaccinated morbidity)/control morbidity x 100% ], immunoprotection rate of NDK protein immunized group was 44.44%.

Therefore, the NDK recombinant protein has good immunogenicity, can play an immunoprotection role on Acinetobacter baumannii infection, can induce organisms to generate immune response, and can be used for preparing subunit vaccine for preventing the Acinetobacter baumannii infection by being assisted with an adjuvant such as aluminum hydroxide adjuvant.

The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. The Acinetobacter baumannii vaccine is characterized by comprising Acinetobacter baumannii nucleoside diphosphate kinase NDK recombinant protein, and the amino acid sequence of the Acinetobacter baumannii nucleoside diphosphate kinase NDK recombinant protein is shown as SEQ ID NO. 3.

2. The acinetobacter baumannii vaccine of claim 1, wherein said acinetobacter baumannii nucleoside diphosphate kinase NDK recombinant protein comprises at least amino acid sequence 1-143 of NDK protein, said amino acid sequence being shown in SEQ ID No. 1.

3. The method for preparing the acinetobacter baumanii vaccine as claimed in claim 1, comprising the following steps:

(1) Designing a PCR primer according to a nucleotide diphosphate kinase NDK protein gene sequence of Acinetobacter baumannii, taking whole genome DNA of Acinetobacter baumannii as a template, and carrying out PCR amplification on a target gene of the nucleotide diphosphate kinase NDK protein amino acid sequence of the Acinetobacter baumannii according to the designed PCR primer;

(2) Cloning the PCR amplification product obtained in the step (1) to an expression vector pGEK-6p-2 containing GST tag, and converting the PCR amplification product into a prokaryotic expression system to perform induction expression of the NDK fusion protein containing the GST tag;

(3) Separating the target protein from the GST tag by using an enzyme digestion method to obtain a nucleoside diphosphate kinase NDK recombinant protein of Acinetobacter baumannii;

(4) Purifying the NDK recombinant protein obtained in the step (3);

4. The method for producing a Acinetobacter baumannii vaccine according to claim 3, wherein in the step (5)Wherein the adjuvant comprises Al (OH) ₃ Adjuvant, alPO ₄ Adjuvants, MF59, AS03, AS04, incomplete freund's adjuvant or complete freund's adjuvant.