CN116478953B

CN116478953B - Acinetobacter baumannii DlaT recombinant protein, preparation method and application

Info

Publication number: CN116478953B
Application number: CN202310700281.0A
Authority: CN
Inventors: 王宁; 李彦; 周杨杨; 石云; 游瑜; 陈凯; 孙向成; 张晓敏; 向传英; 谢雨; 杨红
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2023-06-14
Filing date: 2023-06-14
Publication date: 2023-09-12
Anticipated expiration: 2043-06-14
Also published as: CN116478953A

Abstract

The invention provides a Acinetobacter baumannii Dlat recombinant protein, a preparation method and application, and relates to the technical field of biology. The invention realizes high expression quantity of DlaT recombinant protein by constructing a prokaryotic system recombinant expression vector and recombinant engineering bacteria, is convenient for separation and purification, is efficient and safe, and can be directly matched with an adjuvant to prepare subunit vaccine for resisting Acinetobacter baumannii infection and related detection kit. The quality is controllable during the preparation, and the purification process is simple and quick. Meanwhile, the recombinant protein has higher immunoprotection in resisting the lethal infection of Acinetobacter baumannii, and is beneficial to the research and development of the Acinetobacter baumannii vaccine.

Description

Acinetobacter baumannii DlaT recombinant protein, preparation method and application

Technical Field

The invention relates to the technical field of biology, in particular to a Acinetobacter baumannii DlaT recombinant protein, a preparation method and application thereof.

Background

Acinetobacter baumannii (Acinetobacter baumannii) is a non-fermented gram-negative bacillus, widely exists in nature, hospital environment, human skin and respiratory tract, and has become the most common pathogenic bacteria for nosocomial infections. Due to the widespread use of antibiotics, the drug resistant form of Acinetobacter baumannii is increasingly severe, which has become one of the main pathogenic bacteria for nosocomial infections. Such infections are mainly manifested by ventilator-related lung inflammation, sepsis, meningitis, and burn-related skin and soft tissue infections. The occurrence of multiple resistant acinetobacter poses a greater risk for this infection, and multiple resistant acinetobacter baumannii exists in 80% of cases of ventilator-associated pneumonia obtained in hospitals worldwide. The development of new vaccines helps to address the threat of acinetobacter baumannii and the emergence of multiple acinetobacter baumannii. In the research progress of the Acinetobacter baumannii vaccine, no candidate Acinetobacter baumannii vaccine enters a clinical stage, and the safety and stability of the currently researched vaccine are also deficient, so that the development of a safe and stable vaccine is a problem which needs to be solved urgently at present.

The recombinant protein subunit vaccine has the advantages of definite components, controllable product purity, easiness in controlling the endotoxin content and impurity content of bacteria and the like, and the current research shows that the recombinant protein subunit vaccine can play a protective role in animal experiments as a potential vaccine. Therefore, there is a need to develop a recombinant protein against a. Baumannii infection, which is suitable for the development of a subunit vaccine of a. Baumannii recombinant protein.

Disclosure of Invention

The invention aims to provide a recombinant protein of Acinetobacter baumannii dihydrolipoamide transacetylase (dihydrolipoamide acyltransferase, dlat), a preparation method and application thereof. The DlaT recombinant protein has the advantages of simple and convenient production process, low time cost, low growth cost, controllable quality, high purity and good immunogenicity.

Dihydrolipoamide transacetylase (Dlat) as one of the main components in the bacterial pyruvate dehydrogenase complex has an important role in bacterial carbohydrate metabolism, blocking its function may affect bacterial survival, proliferation, and bacterial virulence and pathogenicity. The Acinetobacter baumannii Dlat is used as a recombinant protein subunit vaccine to carry out efficient and stable expression and purification, which not only lays a foundation for developing safe and efficient infection prevention or therapeutic vaccine, but also opens up a new thought for researching Acinetobacter baumannii vaccine.

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

the invention provides a Acinetobacter baumannii dihydrolipoamide transacetylase recombinant protein, which comprises Acinetobacter baumannii Dlat protein, wherein the amino acid sequence of the Acinetobacter baumannii Dlat protein is shown as SEQ ID NO. 1.

According to a preferred embodiment, the amino acid sequence of the recombinant protein is shown in SEQ ID NO. 3.

The invention also provides a polynucleotide for encoding the acinetobacter baumannii Dlat recombinant protein.

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

(1) Designing a PCR primer according to a nucleotide sequence of the acinetobacter baumannii encoding DlaT protein, taking the whole genome of the acinetobacter baumannii as a template, and carrying out PCR amplification on a target gene fragment encoding the DlaT protein according to the designed PCR primer, wherein the forward primer is as follows: 5 '-CGCGGATCCATGGCAACCGAAATTAAAGCA-3'; reverse primer: 5 '-TTATGCGGCCGCCTTAAAGATCAAGAATGAGTTTAGC-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 DlaT fusion protein containing the GST tag;

(3) Separating the target protein from the GST tag by using an enzyme digestion method to obtain the DlaT recombinant protein.

The invention also provides an expression vector which comprises a polynucleotide for encoding the acinetobacter baumannii DlaT recombinant protein, and the expression vector is pGEX-6P-2 plasmid.

The invention also provides a host cell comprising the expression vector.

According to a preferred embodiment, the host cell is E.coli BL21.

The invention also provides application of the acinetobacter baumanii Dlat recombinant protein in preparing a medicament for treating or preventing acinetobacter baumanii infection.

According to a preferred embodiment, the medicament is an acinetobacter baumannii vaccine.

The invention also provides application of the acinetobacter baumanii Dlat recombinant protein in preparation of an acinetobacter baumanii detection kit.

Based on the technical scheme, the Acinetobacter baumannii Dlat recombinant protein, the preparation method and the application have at least the following beneficial effects:

1. the Acinetobacter baumannii Dlat recombinant protein has the advantages of simple production process, low time cost, low growth cost and controllable quality, and the obtained protein has good solubility, maintains the original spatial conformation to the maximum extent, has high purity and has good immunogenicity.

2. The recombinant subunit vaccine prepared by utilizing the Acinetobacter baumannii Dlat recombinant protein provided by the invention has no toxic or side effect, can generate high-titer antibodies, and has a good immune protection effect on Acinetobacter baumannii infection. And the Acinetobacter baumannii Dlat recombinant protein subunit vaccine has definite components, safety and reliability.

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 DlaT gene fragment.

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

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

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

FIG. 5 is a graph showing the results of GST-Dlat and GST-tag-containing 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 purified DlaT recombinant protein after cleavage of GST-DlaT fusion protein using 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.

1. Strain

Acinetobacter baumannii strain ATCC17978 is provided by ACTT in the United states.

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.

The plasmid extraction kit, the gel recovery kit, the bacterial genome extraction kit and the chromogenic solution are the products of Tiangen company;

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

Example 1: cloning of Acinetobacter baumannii Dlat protein.

1. Firstly, searching a DlaT protein sequence (number: AIY 36145.1) of Acinetobacter baumannii dihydrolipoamide transacetylase in an NCBI protein database to obtain an amino acid sequence, wherein the amino acid sequence is shown as SEQ ID NO.1, and simultaneously, searching a DNA sequence for obtaining DlaT codes, and the DNA sequence is shown as SEQ ID NO. 2.

2. The PCR method is adopted to amplify the gene fragment of DlaT protein by taking the whole genome of Acinetobacter baumannii as a template, and the amplification steps are as follows:

1) PCR primers were designed based on the DlaT gene sequence of Acinetobacter baumannii, 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`- CGCGGATCCATGGCAACCGAAATTAAAGCA- 3`

BamH I

reverse primer (SEQ ID NO. 5):

5'-TTATGCGGCCGCCTTAAAGATCAAGAATGAGTTTAGC-3`

Not I

in this example, a DNA sequence SEQ ID NO.2 encoding the amino acid sequence of the DlaT protein shown in SEQ ID NO.1 was used as a target gene fragment for PCR amplification. However, it will be understood by those skilled in the art that any sequence derived from the DNA sequence shown in SEQ ID NO.2, in which a plurality of codons have been deleted at the amino terminus of the amino acid sequence corresponding to the encoded protein, may be selected as the desired gene fragment.

2) The stored Acinetobacter baumannii is taken out from the freezer at the temperature of minus 80 ℃ and coated on a TSA solid culture medium, cultured overnight at the temperature of 37 ℃, then single bacterial colony is selected and inoculated on a TSB liquid culture medium, and the culture is carried out for 8 hours at the temperature of 220rpm in a constant temperature shaking table, and the whole genome DNA of the Acinetobacter baumannii is extracted by referring to a bacterial genome extraction kit.

3) Taking the whole genome DNA of Acinetobacter baumannii as a template, and carrying out PCR amplification on DlaT protein gene fragments;

wherein, the PCR system:

component (A)	Volume of
		Acinetobacter baumannii whole genome DNA template (200 ng/. Mu.l)	3μl
Forward primer (1. Mu.M)	2μl
		Reverse primer (1. Mu.M)	3μl
2×High Fidelity PCR Master Mix	25μl
		Sterilizing double distilled water	17μl
Total volume of	50μl

PCR amplification reaction conditions: pre-denaturation at 95℃for 30s, denaturation at 95℃for 15s, annealing at 58℃for 15s, extension at 72℃for 1min12s,30 cycles, and complete extension at 72℃for 5min. After completion of the reaction, the PCR amplification results were detected by electrophoresis using 1% agarose gel, and the PCR amplification results are shown in FIG. 1. Wherein lane M: nucleic Acid (DNA) molecular weight standard (Marker: DL 5000); lane 1: PCR amplification product of DlaT gene fragment (1197 bp).

4) The DlaT PCR product was 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 DlaT PCR products, respectively, at 37℃for 3h.

Wherein, the enzyme digestion reaction system:

component (A)	Volume of
		BamH I	3μl
Not I	3μl
		Buffer	15μl
Plasmids or PCR products	10μl
		H ₂ O	19μl
Total volume of	50μl

2) pGEX-6P-2 plasmid and BamH I and Not I digested PCR products were recovered using a gel recovery kit.

3) Ligation and transformation.

The molar ratio of vector to exogenous fragment is generally 1: 2-10, designing the following connection reaction system.

Wherein, the connection reaction system:

component (A)	Volume of
		DNA Ligation Mix	5μl
Enzyme cutting recovery product of target gene	4.5μl
		PGEX-6P-2 enzyme digestion recovery product	0.5μl
Total volume of	10μl

Mixing, and connecting at 37deg.C for 30min.

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. 500 ul of LB blank medium is added, mixed evenly and placed in a shaking table at 37 ℃ for 1h with 200rp shaking.

Each tube was centrifuged at 4000rpm at room temperature for 5min, the 400 ul supernatant was discarded, 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 12h.

5) Screening and identifying pGEX-6p-2/DlaT recombinant expression plasmid.

(1) Negative control plates had no colonies present; positive control plates were full of colonies, indicating correct competent cell production and reliable results. Selecting well-separated colonies on a connection product conversion plate, inoculating the colonies into an Amp-resistant LB medium, and performing 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;

wherein, the double enzyme digestion reaction system:

BamH I	0.5μl
		Not I	0.5μl
Buffer	5μl
		plasmid(s)	5μl
H ₂ O	9μl
		Total volume of	20μl

Enzyme cutting at 37 ℃ for 1h;

(4) double digestion results were detected by 1% agarose gel electrophoresis, as shown in FIG. 2, lane M: nucleic Acid (DNA) molecular weight standard (Marker: TSJ102-1 kb DNA Ladder); lane 1: the identification results of the recombinant expression plasmid pGEX-6p-2/DlaT after enzyme digestion show 4000bp and 1197bp fragments separated after enzyme digestion, and the size of the fragment generated by enzyme digestion in lane 1 is the same as that of the target gene DNA fragment, which indicates that the construction of the pGEX-6p-2/DlaT recombinant expression plasmid is successful.

(5) pGEX-6p-2/DlaT recombinant expression plasmid is sent to Shanghai biological company for sequencing, and the sequencing is correct through comparison and is shown in figure 3, namely the sequence of the target gene fragment of the recombinant plasmid is correct.

Example 2: the Acinetobacter baumannii Dlat protein is induced to express, purify and identify expression forms in a prokaryotic expression system-escherichia coli.

1. The target protein induces expression.

1) 100 mu L of bacterial liquid of pGEX-6P-2-DlaT/BL21 (DE 3) with correct double enzyme digestion identification is added into 10mL of Amp-resistant TB culture medium, 100rpm is used for overnight culture at 37 ℃, 2mL of bacterial liquid cultured overnight is respectively added into 18mL of Amp-resistant TB culture medium (the rest bacterial liquid is stored in a refrigerator at 4 ℃) for standby), the culture is carried out at 37 ℃ for 2-3 hours, the rotating speed is 250rpm, when the bacterial liquid is activated for the second time until the OD600 is 0.8-1.2, IPTG is added to lead the final concentration to be 200 mu M (2 mL of bacterial liquid is taken before the addition, 12000rpm/min is carried out, and bacterial bodies are collected after centrifugation for 10min and are taken as before induction), and then the bacterial liquid is placed into a shaking table for induction expression at 16 ℃ for overnight induction.

2) Taking out the bacteria liquid after induced expression, centrifuging at 1000rpm for 2min, discarding the supernatant, adding 1mL PBS buffer solution for uniform mixing, performing ultrasonic lysis for 10min, performing ultrasonic treatment for 5s, suspending for 5s, preventing and treating protein overheating, collecting the liquid after ultrasonic treatment by using a 2mL EP tube, centrifuging at 14000rpm for 15min at 4 ℃ by using a high-speed centrifuge, collecting the supernatant, re-suspending the rest precipitate by using 1mL PBS buffer solution, and performing bacterial operation before induction. After the induction, 40ul of the supernatant and the precipitate were collected, 10ul of 5x SDS loading buffer was added, the mixture was deformed in a metal bath at 100℃for 5min, and after the denaturation was completed, 10ul of the denatured sample was taken, and SDS-PAGE was performed on the pre-induced protein, the post-induced protein, and the post-induced protein supernatant and the protein precipitate (FIG. 4). From the results of fig. 4, it can be seen that: lane M: protein molecular weight standard (Marker); lane "-": protein strips of the whole bacteria before the recombinant engineering bacteria are induced to express; lanes "+": after the recombinant engineering bacteria are induced to express at 16 ℃, GST-DlaT which is fusion protein containing GST tag and is expressed in the whole bacteria; lane "S": after the recombinant engineering bacteria are induced to express at 16 ℃, GST-DlaT fusion protein is obtained in supernatant. After induction, the target protein in the "+" lane is obviously expressed, the gray value of the target protein is analyzed by the ImageJ software to be the gray value of the whole lane protein, the protein content is calculated to be about 30% of the total protein, and the high expression of the recombinant protein is indicated. And protein expression can be mostly detected in bacterial lysates (lane S), indicating that the protein is expressed in a soluble form that facilitates maintenance of the protein' S conformation, function and subsequent purification and process expansion for large-scale vaccine production. The result shows that the recombinant protein GST-Dlat can be effectively expressed, and can be expressed in a soluble form in bacterial supernatant, and the arrow indicates the recombinant protein GST-Dlat, and the amino acid sequence of the recombinant protein GST-Dlat is shown as SEQ ID NO. 3. Meanwhile, the pGEX-6P-2-BL21 (DE 3) recombinant engineering bacteria obtained by the method have the advantages of easy culture, short purification period, safety, controllability and convenience in operation.

Example 3: preparation of DlaT recombinant protein antigen.

1. Amplifying and culturing to obtain fusion protein GST-DlaT.

pGEX-6P-2-DlaT/BL21 (DE 3) strain stored in-80℃refrigerator was inoculated on LB ampicillin resistant plate and cultured overnight at 37 ℃; single colonies were picked and inoculated in 100ml of ampicillin-resistant LB medium (100 ug/ml of ampicillin concentration), cultured overnight at 37℃and 200 rpm; adding activated 100ml of bacterial liquid into 2L of LB culture medium containing Amp resistance for secondary activation, culturing at 37 ℃ for 3-4 h until OD600 is 0.8, adding 1ml of IPTG (final concentration is 500 uM), placing in a shaking table at 16 ℃ for induction for 12h, centrifuging at 6000rpm for 20min to collect bacterial cells, adding 80ml of PBS to resuspension bacterial cells, circularly crushing for 5 cycles by a high-pressure homogenizing instrument (800 bar, flow rate 25), centrifuging at 12000rpm by using a floor type high-speed centrifuge for 30min, and collecting supernatant to combine with 4ml of glutathione-agarose gel 4B; a large number of GST-tagged dihydrolipoamide transacetylase fusion proteins were obtained.

2. And separating the target protein from the GST tag by using an enzyme digestion method to obtain the DlaT recombinant protein.

To the remaining about 4ml of glutathione-agarose gel 4B to which the target protein had been bound, 4ml of PBS and 120. Mu. L PreScission protease (PP enzyme) were added, and after centrifugation and aspiration of the supernatant, the supernatant was washed 2 times with 2ml of PBS, 10. Mu.L of the sample was denatured, and 10. Mu.L of the sample was subjected to SDS-PAGE, and the result was observed in a phase system, and DlaT protein having a molecular weight of about 43kDa and conforming to the expected protein molecular weight was obtained after the cleavage, and the result of the electrophoresis was shown in FIG. 5, lane M: protein molecular weight standard (Marker), lane 1 shows the target protein obtained from the supernatant after the recombinant engineering bacteria are induced to express at 16 ℃, lane 2 shows the fusion protein, target protein and GST protein after the first washing after the cleavage, lane 3 shows the target protein obtained from the gel beads after the second washing after the cleavage, and lane 4 shows the protein bound to the gel beads after the cleavage.

3. The buffer was replaced and the protein of interest was stored in PBS (ph=8.0) buffer.

4. And further purifying the DlaT recombinant protein by using an ion exchange column to obtain the DlaT recombinant protein with high purity.

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 filtered PBS (pH=8.0) solution; after filtering the samples stored in PBS (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 1 shows the purified DlaT recombinant protein after ion exchange column, and the protein purity of the DlaT recombinant protein is 97.8% as calculated by scanning the proportion of the grey value of the DlaT band of the target protein in the protein gel to the total grey value of the protein in the whole lane with Image J software.

5. Protein concentration was measured according to BCA method protein assay kit instructions, with a final measured concentration of 2.2mg/mL.

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

Inoculating Acinetobacter baumannii 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/OD600 was calculated and the bacterial solution was diluted to 1X 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, this dose was chosen for subsequent mouse model construction (table 1).

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%

Example 5: preparation of DlaT recombinant protein subunit vaccine.

The recombinant DlaT protein and different adjuvants are subjected to adsorption to perform a preliminary experiment, the adsorption effect is detected, the aluminum hydroxide adjuvant is found to have the best adsorption efficiency, and the follow-up main aluminum hydroxide is used as an adjuvant component to prepare subunit vaccine through adsorption. The method after fumbling is as follows: measuring an aluminum hydroxide adjuvant, adjusting the concentration to 5 mg/ml by using histidine diluent with pH of 6.0, and fully and uniformly mixing to obtain a solution A; diluting DlaT recombinant protein to 200 mug/mL by using histidine diluent with pH of 6.0 to obtain solution B; and taking the solution A and the solution B with equal volumes, and carrying out rotary suspension adsorption 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 vaccine diluent for the recombinant antigen solution.

Example 6: recombinant protein DlaT immunizes animals.

1. Animals were immunized.

1) For the first immunization, intramuscular injection was performed on the inner side of the thigh of the mouse with an insulin needle, the injection amount of each C57Bl/6 mouse was 200. Mu.L, and 100. Mu.L each of the left and right thighs was set with an adjuvant control group.

2) The second immunization is carried out on the 14 th day, the immune components are the same as those of the first immunization, the immune route is the same as that of the first immunization, and the immunization is intramuscular injected outside the thigh of the mouse;

3) Third immunization is carried out on the 21 st day, the immune components are the same as those of the first immunization, the immune route is the same as that of the first immunization, and the immunization is intramuscular injected into the inner side of the thigh of the mouse;

2. on day 7 after the third immunization, venous blood was collected from C57Bl/6 mice, serum was centrifuged after clotting, and the IgG humoral response level of the mice after immunization was detected by ELISA.

Example 7: ELISA detects EF-Ts recombinant protein specific antibodies.

1. Preparation of ELISA kit.

1) Preparing a reagent: coating liquid: 0.05 M carbonate buffer (NaHCO) ₃ 1.6g/L，Na ₂ CO ₃ 2.9g/L, pH 9.6); the wash solution was PBST (PBS+0.05% Tween-20, pH 7.4); blocking solution was PBS (pH 7.4) +3% BSA; the serum and antibody dilutions were PBS (pH 7.4) +1% BSA+0.05% Tween-20, and the stop solution was 2M H ₂ SO ₄ 。

2) Coating antigen: diluting the purified DlaT recombinant protein to 4 mug/ml by using coating liquid; adding diluted recombinant protein into an ELISA plate, and washing with a washing solution for 4 times after incubating overnight at 4 ℃ at 100 mu L/hole;

3) Closing: adding blocking solution into the ELISA plate after antigen coating, incubating for 2 hours at 37 ℃ with 100 mu L/hole, and washing for 4 times to obtain an ELISA reagent plate for detecting the antibody;

4) And matching with an HRP-marked rabbit anti-mouse IgG antibody and an antibody diluent, and obtaining the TMB chromogenic solution and the stop solution as the DlaT antibody detection kit.

2. Detection of DlaT recombinant protein-specific antibodies.

1) The serum after DlaT protein immunization and the serum of a control group are respectively subjected to multiple dilution, and the dilution gradient is set to be 1:1000, 1:5000, 1:25000 and 1:125000;

2) Taking a sealed ELISA plate in an ELISA detection kit, sequentially adding serum with different dilution concentrations, 100 mu L/hole, simultaneously setting a PBS blank hole, placing the PBS blank hole in an incubator at 37 ℃ for 2h, washing for 3 times, and drying;

3) Diluting HRP-labeled rabbit anti-mouse IgG antibody stock solution at a ratio of 1:10000 to prepare an antibody working solution;

4) Adding diluted antibody working solution, 100 mu L/hole, placing in an incubator at 37 ℃ for 45min, washing for 5 times, and drying;

5) Adding a substrate color development solution TMB,100 mu L/hole, and reacting at room temperature for 5-10 min in a dark place;

6) Adding 50 mu L/hole of stop solution, and placing on an enzyme-labeled instrument to measure OD450 value at the wavelength of 450 nm;

7) And (3) judging results: OD (optical density) _{Sample of} ∕OD _{Negative of} A positive value of ∈ 2.1.

Table 2: ELISA detection of recombinant protein post-immunization antibodies (OD 450)

Dilution factor	Adjuvant control group	Dlat immune group
			125000	0.045	0.471
25000	0.037	1.282
			5000	0.034	2.489
1000	0.034	3.579

Results: the DlaT protein ELISA detection kit and the detection method are successfully established, and can be used for detecting the DlaT protein specific antibody. The result of detecting DlaT protein antibody titer by using the kit shows that (table 2) the OD450 of the antibody produced by the DlaT protein antigen immunized mice is still as high as 0.471 after the dilution of 125000, and the antibody titer is higher than 1:125000 shows that the DlaT recombinant protein prepared by the invention can generate high-titer specific antibodies in immunized mice, and has good immunogenicity.

Example 8: the toxicity protection of DlaT recombinant protein immunized animals was determined by immunizing mice.

The same immunization protocol as in example 6 was followed by a step of challenge with live Acinetobacter baumannii bacteria by tail vein injection at a lethal dose of 3X 10 per mouse at day 14 after the third immunization ⁷ CFU, injection volume was 100 μl, observation was made for 7 days, and survival rates of each group of mice were counted. The results are shown in Table 3 below.

Table 3: animal immune protection results

Group of	Mouse (Only)	Immune component	Number of deaths	Mortality (%)	Protection ratio (%)
						DlaT protein immune group	10	DlaT+ Al(OH) ₃ Adjuvant	4	40	55.55
Adjuvant control group	10	Al(OH) ₃ Adjuvant	9	90	0

Table 3 shows the results of animal immunization experiments, mortality in the adjuvant control group was 90%, mortality in the Dlat vaccine immunization group was 40%, and immunoprotection was calculated according to the formula: [ immune protection rate= (control morbidity-inoculation morbidity)/control morbidity x 100% ], dlat protein immune group protection rate was 55.55%.

The DlaT recombinant protein prepared by the invention has high purity (as shown in figure 6, the purity of the protein obtained by calculation through imageJ software is 97.8%), good effect of protecting Acinetobacter baumannii infection, and in mouse experiments, the DlaT recombinant protein is supplemented with Al (OH) ₃ The immune protection rate of the adjuvant group is 55.55%, and the adjuvant has no toxic and side effects. Therefore, the dihydrolipoamide acyltransferase DlaT recombinant protein has good immunogenicity and can be excitedThe organism generates specific antibodies and can play a good role in immune protection on Acinetobacter baumannii infection.

The recombinant protein prepared by the invention can be used for preparing related kits with other related reagents such as detection antibodies, color developing agents, terminators and the like.

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. Acinetobacter baumannii @Acinetobacter baumannii) The application of the dihydrolipoamide transacetylase recombinant protein in preparing the medicine for preventing the infection of the acinetobacter baumannii is characterized in that the dihydrolipoamide transacetylase recombinant protein of the acinetobacter baumannii comprises an acinetobacter baumannii Dlat protein, the amino acid sequence of the acinetobacter baumannii Dlat protein is shown as SEQ ID NO.1, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 3.

2. The use according to claim 1, wherein the medicament is a acinetobacter baumannii vaccine.

3. The use of the acinetobacter baumannii dihydrolipoamide transacetylase recombinant protein of claim 1 in the preparation of an acinetobacter baumannii detection kit.