CN104861048A - Acinetobacter baumanniihy pothetical protein A1S-1462 protein and preparation method and application thereof - Google Patents

Abstract

The invention relates to an A1S-1462 recombinant protein and a preparation method and application thereof. The recombinant protein comprises A1S-1462 mature peptide, and the amino acid sequence of the recombinant protein is shown as SEQ ID NO. 3. The recombinant protein is high in expression amount, easy to separate and purify, efficient and safe, can be directly used with an adjuvant, and is used for the preparation of an acinetobacter baumannii infection resistant subunit vaccine and a related detection kit. Confirmed by animal experiments, the genetic engineering recombinant subunit vaccine has good acinetobacter baumannii infection resistant immune protection effect, lays a foundation for the further study on combined vaccines and multicomponent fusion vaccines, and plays an important role for development and application of prevention and control vaccines and diagnostic kits.

Description

Acinetobacter baumannii hypothetical protein A1S_1462 protein and its preparation method and application

technical field

The invention relates to the field of biotechnology, in particular to the hypothetical protein A1S_1462 protein of Acinetobacter baumannii and its preparation method and application.

Background technique

Acinetobacter baumannii (Acinetobacter baumannii) is a non-fermenting gram-negative bacillus that widely exists in nature and is an opportunistic pathogen. This bacterium is an important pathogenic bacterium of nosocomial infection, mainly causing respiratory tract infection, and can also cause bacteremia, urinary tract infection, secondary meningitis, surgical site infection, ventilator-associated pneumonia, etc. Domestic data show that Acinetobacter baumannii accounts for more than 70% of clinically isolated Acinetobacter. The resistance rate of Acinetobacter baumannii to the third and fourth generation cephalosporins has reached 63.0% to 89.9%. The resistance rate to four aminoglycosides (amikacin, gentamicin, netilmicin, tobramycin) and ciprofloxacin reached 96.3%. Most of the current strains in my country are sensitive to imipenem, meropenem, Acinetobacter cephalosporin baumannii, petone/sulbactam, and polymyxin B, but they are less effective in the treatment of respiratory tract infections.

Because existing antibiotics are difficult to effectively treat Acinetobacter baumannii infection, and new antibiotics that can be used to treat Acinetobacter baumannii infection are not available in the short term, the treatment of infectious diseases caused by Acinetobacter baumannii It has become increasingly complex. In recent years, relevant pathogenic microorganism research students in the medical field have also conducted related research on the prevention and treatment of Acinetobacter baumannii. Michael J.McCnnell et al. from Spain used the mouse model of acute sepsis to evaluate the active and passive immunity of the inactivated Acinetobacter baumannii vaccine; at the same time, Michael J.McCnnell et al. The outer membrane protein vaccine of Acinetobacter baumannii has been studied, and the results suggest that OMC can stimulate T cells to secrete IFN-γ and activate Th1 and Th2 helper B cells to secrete IgG antibodies, all of which have good immune protection for mice. However, the above-mentioned whole-bacteria vaccines and outer-membrane complex vaccines have complex components and contain a large amount of endotoxins, which have relatively large toxic and side effects on the body. Therefore, it is an inevitable trend to develop a quality-controllable, safe and effective Acinetobacter baumannii vaccine .

Contents of the invention

The object of the present invention is to provide a protein A1S_1462 of Acinetobacter baumannii and its preparation method and application. The protein can be applied to the preparation of subunit vaccines against Acinetobacter baumannii infection and related detection kits.

Technical scheme of the present invention is as follows:

The recombinant protein of Acinetobacter baumannii A1S_1462 comprises the mature peptide of A1S_1462, and the amino acid sequence of the mature peptide is shown in SEQ ID NO.1.

The amino acid sequence of the recombinant protein is shown in SEQ ID NO.5.

The recombinant protein is expressed through the fusion of the tag protein GST, and the tag protein is fused to the N-terminus of the A1S_1462 protein.

The amino acid sequence of the recombinant protein also includes immunogenic variants with deletion, substitution, and/or addition of 1-20 amino acid residues at the amino-terminal and/or carboxy-terminal of SEQ ID NO.1, the variant is at least 80% identical to the amino acid sequence.

The recombinant protein is formed by adding five amino acids of GPLGS to the amino terminal of the mature peptide after fusion expression and digestion.

A polynucleotide encoding A1S_1462 recombinant protein.

The preparation method of A1S_1462 recombinant protein, comprises the following steps:

1) Design PCR primers as follows:

forward primer

5'-CGC GGATCC ATGGCACCTGTAAAAGAACAAAAAAT-3'

BamH Ⅰ

reverse primer

5'-TTAT GCGGCCGCCTTACTTTTTTTGTAGCTGCG -3'

Not Ⅰ

2) using the primers designed in step 1) to amplify the target gene fragment encoding the A1S_1462 protein by PCR;

3) The gene fragment obtained in step 2) is cloned into an expression vector, and then transformed into a host bacterium;

4) Inducing the transformed host bacteria to express the A1S_1462 recombinant protein;

5) Purify the recombinant protein.

An expression vector or a host cell, comprising a polynucleotide encoding the recombinant protein of claim 1 or a host cell.

The vector is a pGEX-6P-2 expression vector; the host cell is Escherichia coli, and the Escherichia coli is XL1-Blue.

Antibody against A1S_1462 protein.

Application of A1S_1462 recombinant protein in preparation of medicament for preventing or treating Acinetobacter baumannii infection.

Application of A1S_1462 recombinant protein in preparation of Acinetobacter baumannii detection kit.

The A1S_1462 protein of the present invention can be naturally produced or artificially introduced mutations or variations (including but not limited to, substitutions, deletions and/or additions) in SEQ ID NO: 1, 3 or 5, without affecting the biological characteristics of A1S_1462 . Therefore, in the present invention, "A1S_1462" is intended to include the full-length A1S_1462 protein, its mature peptide and its variants, including the polypeptide shown in SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:5 and its native or An artificial variant that retains the biological properties of A1S_1462, ie is highly immunogenic. And, when describing the sequence fragment of A1S_1462, it not only includes the sequence fragment of the polypeptide shown in SEQ ID NO: 1, 3 or 5, but also includes the corresponding sequence fragment in the natural or artificial variant of the polypeptide.

According to the present invention, when used in the context of a protein/polypeptide, the term "variant" refers to a protein whose amino acid sequence has one or more (e.g. 1-10 or 1-5 or 1-3) amino acid differences (e.g. conservative amino acid substitutions), or have at least 60%, 80%, 85%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, or 99% identity, and it retains the essential properties of the reference protein/polypeptide. In the present invention, the essential property of the protein/polypeptide (for example, the A1S_1462 protein of the present invention) may refer to strong immunogenicity.

The above "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by an adenine, or both a position in each of the polypeptides is occupied by lysine), then the molecules are identical at that position. "Percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions match. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, comparisons are made when two sequences are aligned for maximum identity.

As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological activity of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions for amino acid residues with amino acid residues that have similar side chains, e.g., are physically or functionally similar (e.g., have similar size, shape, charge, chemical properties, including Substitution of residues with the ability to form covalent or hydrogen bonds, etc.).

According to the present invention, the term "immunogenicity" refers to the ability to stimulate the body to form specific antibodies or sensitize lymphocytes. It not only refers to the characteristics that antigens can stimulate specific immune cells to activate, proliferate, and differentiate immune cells, and finally produce immune effect substances such as antibodies and sensitized lymphocytes, but also means that after antigens stimulate the body, the immune system of the body can form antibodies or Specific immune response of sensitized T lymphocytes.

In the present invention, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably.

In view of the fact that the full-length A1S_1462 protein contains a signal peptide, the signal peptide corresponds to the amino acid sequence of positions 1 to 20, in order to keep the structure and function of A1S_1462 from major changes to the greatest extent, the present invention also provides a fusion A protein comprising a mature peptide of the A1S_1462 protein, a variant thereof or a fragment thereof, and a tag protein. Preferably, the tag protein is GST; preferably, the tag protein is fused to the N-terminal of the mature peptide of the A1S_1462 protein. The above fusion protein is digested to obtain A1S_1462 recombinant protein; preferably, when the tag protein is GST, the enzyme used for digestion is PreScission protease (PP enzyme), and the A1S_1462 recombinant protein obtained by enzyme digestion increases five amino acid residues GPLGS.

In another aspect, the present invention relates to polynucleotides encoding the A1S_1462 protein, fusion proteins and variants thereof of the present invention and vectors containing the polynucleotides.

Vectors that can be used for insertion of a polynucleotide of interest include, but are not limited to, cloning vectors and expression vectors. In one embodiment, the vector is, for example, a plasmid, cosmid, phage, cosmid, and the like.

In another aspect, the present invention also relates to a host cell comprising the above polynucleotide or vector. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cells of the invention may also be cell lines.

The present invention provides a method for obtaining A1S_1462 recombinant protein, which comprises the following steps:

1. Utilize the recombinant host cell to express the fusion protein of the present invention;

2. recovering the fusion protein;

3. Digest the recovered fusion protein, and recover the A1S_1462 recombinant protein from the digested product;

Preferably, when the tagged protein is GST, the enzyme used for digestion is PreScission protease (PP enzyme).

In a preferred embodiment, the step (1) of the method for obtaining the A1S_1462 recombinant protein of the present invention also includes:

(i) designing a forward primer and a reverse primer according to the nucleic acid sequence encoding the A1S_1462 protein;

(ii) using the forward primer and reverse primer designed in step (i), amplify the gene fragment encoding the A1S_1462 protein by PCR;

(iii) cloning the gene fragment obtained in step (ii) into an expression vector, and then transforming into a host cell;

(iv) Inducing the transformed host cells to express the A1S_1462 fusion protein.

The present invention preferably adopts pGEX-6p-2 plasmid to construct recombinant prokaryotic cells (such as Escherichia coli) to express A1S_1462 fusion protein, and its main feature is that the amino terminus of the expressed fusion protein is connected with a 26kDa GST tag, which can be as a marker for protein purification. Compared with other fusion vectors, the pGEX series vectors have mild purification conditions, simple steps, and do not require the addition of denaturants, so that the purified protein can maintain its spatial conformation and immunogenicity to the greatest extent.

The nucleotide sequences of the forward primer and the reverse primer of the present invention are respectively SEQ ID NO.7 and SEQ ID NO.8, and the host cell used is Escherichia coli XL-1 bule.

The invention also provides the application of A1S_1462 recombinant protein in the preparation of subunit vaccine against Acinetobacter baumannii.

The present invention also provides an application of the A1S_1462 recombinant protein in the preparation of a detection kit for Acinetobacter baumannii.

The present invention also provides an antibody against A1S_1462 protein or a fusion protein comprising A1S_1462 protein.

The invention adopts genetic engineering technology to clone and express the hypothetical recombinant protein of the protective antigen, which has a high expression level, is convenient for separation and purification, and is highly efficient and safe. It is assumed that the recombinant protein can be used directly with adjuvants (such as Al(OH) ₃ adjuvant, AlPO ₄ adjuvant, MF59, AS03, AS04, incomplete Freund's adjuvant, complete Freund's adjuvant, etc.), preferably AlPO ₄ Adjuvants are used for intramuscular immunization.

The expression method of the genetically engineered recombinant protein of the present invention has the following 6 advantages:

1. Neither the A1S_1462 protein nor the hypothetical protein has been used in the field of recombinant subunit vaccines;

2. The expression plasmid of the putative protein is induced and expressed in the prokaryotic expression system (Escherichia coli), with high expression level and controllable quality and safety;

3. Select the pGEX-6p-2 expression vector, assuming that the recombinant protein is expressed in the form of a fusion protein, maintaining its original spatial conformation to the greatest extent;

4. The expressed fusion protein contains a GST tag, which becomes a marker for protein purification, which makes the purification conditions mild, the steps are simple, and no denaturant is required, so that the purified protein can maintain its spatial conformation to the greatest extent and immunogenicity;

5. Assume that the expression rate of the recombinant protein is about 30%, and the purity of the purified recombinant protein is greater than 95%;

6. Assume that the recombinant protein can induce animals to produce specific antibodies.

The subunit vaccine prepared by using the hypothetical recombinant protein of the present invention can be immunized through subcutaneous (muscle) injection to stimulate the body to produce high titer IgG antibody and cellular immune response. It is also confirmed by animal experiments that the genetically engineered recombinant monovalent subunit vaccine has a good immune protection effect against Acinetobacter baumannii infection. It lays the foundation for further research on combined vaccines and multi-subunit fusion vaccines, and plays an important role in the development and application of prevention and control vaccines and diagnostic kits.

Description of drawings

Fig. 1 is the PCR amplification result of hypothetical gene fragment, wherein, swimming lane M: nucleic acid (DNA) molecular weight standard (Marker); Swimming lane 1-3: the PCR amplification product of hypothetical basic gene fragment (0.9kb);

Fig. 2 is the enzyme digestion identification result of expression vector pGEX-6p-2-A1S_1462: wherein, swimming lane M: nucleic acid (DNA) molecular weight standard (Marker); Identification results after digestion, lanes 1-4 all represent the 4kb and 0.9kb fragments separated after digestion;

Figure 3 shows the results of protein expression induced at different temperatures: among them, lane M: protein molecular weight standard (Marker); lane 1: the fusion protein obtained in the supernatant after recombinant engineering bacteria were induced to express at 30°C; lane 2: recombinant After the engineered bacteria were induced to express at 16°C, the fusion protein was not obtained in the supernatant;

Fig. 4 shows the fusion protein contained in the supernatant obtained after recombinant engineering bacteria were induced for 3 hours overnight at 30°C, wherein, swimming lane M: protein molecular weight standard (Marker); swimming lanes 1-3: recombinant engineering bacteria induced at 30°C Finally, the GST-tagged fusion protein was obtained in the supernatant.

Figure 5 shows the result of enzymatic digestion of fusion proteins containing GST tags, wherein, lane M: protein molecular weight standard (Marker); lane 1: hypothetical recombinant protein obtained before enzyme digestion; lane 2: hypothetical recombinant protein obtained after enzyme digestion; Lane 3: After enzyme digestion, the obtained hypothetical recombinant protein; Lane 4: After enzyme digestion, the target protein non-specifically bound to glutathione-sepharose 4B and specifically bound to glutathione-agarose Enzyme and GST tags on gel 4B;

Fig. 6 is a graph of prediction results of signal peptide of A1S_1462 protein using the online signal peptide analysis software http://www.cbs.dtu.dk/services/SignalP-4.0/, and the results show that 1-20 amino acids are the signal peptide sequence.

Fig. 7 Comparison results of the DNA sequence of the recombinant expression vector and the hypothetical protein after sequencing, the results show that the DNA sequence is completely consistent with the theory.

Detailed ways

The bacterial strain used in the present invention and various reagents are as follows:

1. Strains

The international standard strain of Acinetobacter baumannii 17978 was provided by the American ATCC;

2. Reagents

Plasmids pGEX-6p-2 (purchased from GE), pET-22b (purchased from Novagen) and Escherichia coli strain XL-1blue (purchased from Protin) were preserved by the applicant's Department of Microbiology;

primeSTAR HS DNA Polymerase, DNA Marker, DNA Ligation Mix, restriction endonuclease BamH I and Not I, protein Marker are products of Dalian TakaRa Company;

Plasmid extraction kit and gel recovery kit are products of Omega Company in the United States;

Bacterial genome extraction kit, ultra-thin recovery kit and chromogenic solution are products of Tiangen Company;

Glutathione Sepharose 4B is a product of GE Healthcare in the United States.

Example 1: Cloning of hypothetical proteins from Acinetobacter baumannii

1. Firstly, according to the gene sequence of Acinetobacter baumannii 17978 standard strain A1S_1462, bioinformatics software was used for structural analysis, and the analysis results are shown in Figure 6, so as to determine the gene fragment of the hypothetical protein to be amplified.

2. According to the analysis results, the PCR method was used to amplify the gene fragment of the hypothetical protein using the whole genome of Acinetobacter baumannii 17978 as a template. The amplification steps were as follows:

1) Design the PCR primers as follows, which are SEQ ID NO: 7-8 (the base sequence of the enzyme cutting site is underlined)

Forward primer PA1S1462B1: SEQ ID NO.7

5'-CGC GGATCC ATGGCACCTGTAAAAGAACAAAAAAT-3'

BamH Ⅰ

Reverse primer PA1S1462N2: SEQ ID NO.8

5'-TTAT GCGGCCGCCTTACTTTTTTTGTAGCTGCG -3'

Not Ⅰ

In this embodiment, the DNA sequence SEQ ID NO.2 encoding the hypothetical protein amino acid sequence shown in SEQ ID NO.1 is used as the target gene fragment for PCR amplification.

2) Take out the preserved Acinetobacter baumannii 17978 strain from the -80°C freezer and spread it on the special LB solid medium, culture it overnight at 37°C, then pick a single colony and inoculate it in the LB liquid medium for 8 hours , refer to the bacterial genome extraction kit to extract the whole genome.

3) Using the whole genome DNA of Acinetobacter baumannii 17978 as a template to amplify the hypothetical protein gene fragment by PCR

PCR system:

Template (179ng/μl) 1μl PA1S1462B1 (1μM) 2μl PA1S1462N2 (1μM) 2μl Taq enzyme 0.5μl dNTP 4μl Buffer 10μl Sterilized double distilled water 30.5μl total capacity 50μl

The PCR amplification reaction conditions were pre-denaturation at 98°C for 40s, denaturation at 94°C for 10s, annealing at 58°C for 40s, extension at 72°C for 1min, 30 cycles, and complete extension at 72°C for 10min. After the reaction was completed, 1% agarose gel was used to detect the PCR amplification result, and the PCR amplification result is shown in FIG. 1 .

4) Recover hypothetical PCR products using a gel recovery kit.

3. Identification and cloning of PCR products, the steps are as follows:

1) Digest pGEX-6P-2 plasmid and hypothetical PCR product with BamH I and Not I, and digest at 37°C for 3 hours.

Enzyme digestion reaction system:

Bam H I 3μl Not I 3μl 10×K Buffer 3μl 0.1%BSA 6μl Product 45μl total capacity 60μl

2) Use the ultra-thin recovery kit to recover the pGEX-6P-2 plasmid and the PCR product digested by BamH I and Not I.

3) Connection and Transformation

Measure the nucleic acid concentration of the target gene enzyme digestion and recovery product by ultraviolet spectrophotometer: 56ng/μl, pGEX-6P-2 enzyme digestion recovery product nucleic acid concentration: 48ng/μl, according to the molar ratio of vector and foreign source fragments, the general ratio is 1:2- 10. Design the following connection reaction system.

Connection reaction system:

DNA Ligation Mix 6μl Recovered product of target gene enzyme digestion 3μl PGEX-6P-2 enzyme digestion product 3μl total capacity 12μl

Mix well and connect at 16°C for 1h.

4) Take 3 tubes of Escherichia coli XL-1blue competent cells from the -80°C refrigerator, add pGEX-6P-2 plasmid to the first tube as a positive control; add DNA ligation products to the second tube; add no exogenous DNA to the third tube , as a negative control. Ice bath for 30min, heat shock in 42℃ metal bath for 90s, rapid ice bath for 2min. Add 600 μl LB blank medium, mix well, and shake at 200rp in a shaker at 37°C for 1h.

Each tube was centrifuged at 5000 rpm for 5 min at room temperature, discarded 400 μl of supernatant, resuspended bacteria, and spread 100 μl on Amp-resistant LB plate. Plates were placed upside down in a 37°C incubator for 24 hours.

5) Screening and identification of pGEX-6p-2/A1S_1462 positive recombinant plasmid

① There are no colonies on the negative control plate; the positive control plate is full of colonies, indicating that the competent cells are made correctly and the results are credible. Pick well-separated colonies on the transformation plate, inoculate them in Amp-resistant LB medium, and culture overnight at 37°C with shaking;

②Plasmid extraction: refer to the instructions of the plasmid extraction kit;

③ Plasmid DNA was digested with BamH I and Not I;

Double enzyme digestion reaction system:

Bam H I 0.5μl Not I 0.5μl 10×K Buffer 0.5μl 0.1%BSA 1μl recombinant plasmid 8μl total capacity 12.5μl

Enzyme digestion at 37°C for 2 hours;

④ 1% agarose gel electrophoresis to detect the results of double enzyme digestion, the results are shown in Figure 2, it can be seen that the samples in lanes 1-4 are successfully constructed pGEX-6p-2/A1S_1462 recombinant plasmids;

⑤ The pGEX-6p-2/A1S_1462 recombinant plasmid was sent to Bao Biological Company for sequencing. The comparison results of the sequencing results are shown in Figure 7. It can be seen that the sequence of the target gene fragment of the recombinant plasmid is correct.

Example 2: Induced expression, purification and identification of expression form of hypothetical protein of Acinetobacter baumannii-17978 in prokaryotic expression system-Escherichia coli

1. Induced expression of target protein

1) Take 100 μL of the pGEX-6P-2-A1S_1462/XL-1blue bacterial solution that was correctly identified by double enzyme digestion and add it to 10 mL of Amp-resistant TB medium. Cultivate overnight at 100 rpm at 37°C. Take 2 ml of the overnight cultured bacterial solution and add it to 18 mL In the Amp-resistant TB medium (the rest of the bacterial solution was stored in a 4°C refrigerator for later use), cultured at 37°C for 2-3h at a rotation speed of 250rpm. The final concentration was 200 μM, and then placed on a shaker to induce expression at 30°C for 3 hours, and then induced expression at 16°C overnight.

2) Take out the bacterial solution after induced expression, centrifuge at 1000rpm for 2min, discard the supernatant, add 1mL of PBS buffer to mix, ultrasonically lyse for 3min, then centrifuge at 14000rpm at 4°C for 15min, and collect the supernatant.

2. Processing of Supernatant

Take 20 μl of glutathione-sepharose 4B, wash with PBS 3 times, add the prepared supernatant to glutathione-sepharose 4B, rotate overnight at 4°C for binding (or room temperature for 1h) . After centrifugation at 5000 rpm for 3 min at 4°C, wash twice with PBS-0.25% Tween 20 and once with PBS. Add 5 μl of 5× protein loading buffer to the bound glutathione-sepharose 4B, add for 5 minutes, and centrifuge at 10,000 rpm for 2 minutes.

3. For SDS-PAGE electrophoresis, pour 5% concentrated gel into the gel plate, add distilled water to flatten the gel, leave it at room temperature for 30 minutes to solidify, drain the upper layer of distilled water, pour 10% separating gel, and insert immediately Put on a comb and let it solidify at room temperature for 30 minutes.

4. Take 10 μL of the processed supernatant samples and load them for SDS-PAGE electrophoresis. The voltage was electrophoresed at 80v for 30 minutes, and then adjusted to 200V. After electrophoresis for 45 minutes, the gel was taken out, placed in Coomassie brilliant blue staining solution for oscillation and staining, and then placed in decolorization solution for oscillation and decolorization, and the results were observed under the imaging system. The results are shown In Figure 3, PGEX-6P-2-A1S_1462/XL-1blue can only express a hypothetical protein containing a GST tag with a molecular weight of about 60kDa only at 30°C, and the recombinant protein is in the supernatant of sonication, so The recombinant protein is a soluble protein under induction at 30 DEG C, and the expression level of the target protein is high, and its purity reaches more than 95%.

Example 3: Preparation of hypothetical protein antigens

1. Amplify culture to obtain protein

Take 400 μL of the spare pGEX-6P-2-A1S_1462/XL-1blue bacterial solution stored in the refrigerator at 4°C and add it to 100mL LB medium containing Amp resistance for one activation, culture at 200rpm at 37°C overnight, add to 2L containing Amp Perform secondary activation in resistant LB medium, culture at 37°C for 3-4h to OD600 of 1.2, add 420ml of IPTG (final concentration: 200uM) and induce in a shaker at 30°C for 3h, then centrifuge at 6000rpm for 5min to collect the bacteria , after adding 80ml of PBS to resuspend the bacteria, the bacteria solution was ultrasonically lysed for 30 minutes, and the supernatant collected by centrifugation as above was combined with 4ml of glutathione-Sepharose 4B; then SDS-PAGE gel electrophoresis was performed, and the results were shown in Figure 4, it can be seen that a large number of hypothetical fusion proteins containing GST tags were obtained in the examples.

2. Use the enzyme digestion method to separate the target protein from the GST tag to obtain the hypothetical protein

Add 4ml PBS and 120μL PreScission protease (PPase) to the remaining 4ml of glutathione-Sepharose 4B that has been bound to the target protein, rotate the enzyme vertically at 4°C overnight, centrifuge to absorb the supernatant, and wash with 2ml PBS After washing twice, take 10 μL of the sample after denaturation treatment, load 10 μL of the sample for protein electrophoresis (the method is the same as above), observe the results in a phase-forming system, and obtain a hypothetical protein molecular weight of about 34 kDa after enzyme digestion, which is consistent with the expected protein molecular weight. , the electrophoresis results are shown in Figure 5. Lane 1 represents the target protein obtained in the supernatant after digestion due to the separation of the target protein from the GST label bound to the gel beads; Lane 2 represents the first wash of the gel after digestion The target protein obtained from the beads; Lane 3 represents the target protein obtained by washing the gel beads for the second time after enzyme digestion; Lane 4 represents the target protein non-specifically bound to the gel beads and specifically bound to the filler after enzyme digestion Enzyme and GST tags on.

3. Replace the buffer, and store the target protein in histidine buffer (10 μm histidine, pH 6.0).

4. The protein concentration was determined by BCA method, and the concentration was 4.0 mg/mL.

Example 4: Establishment of a standard quantitative curve for Acinetobacter baumannii (International Standard Strain 17978) for infection

Inoculate the strain on the MH plate and incubate at 37°C for 24 hours; pick a single colony on the plate, inoculate it in MH liquid medium, place it in a constant temperature shaker at 37°C for 6 hours, and then centrifuge at 6000rpm for 10min to collect the bacteria. Wash the bacteria twice with normal saline; then dilute the bacteria 10 times and 1.25 times, and measure the absorbance (OD600) of each bacteria solution at 600nm under the ultraviolet spectroscopic system, and take 100ul of each dilution of the bacteria solution Spread on MH plates, incubate at 37°C for 24 hours, and then count the colonies; draw a standard quantitative curve based on the number of colonies on each plate and the OD600 value of the bacterial solution.

Results: The formula of the standard curve is Y=3.012X+0.0051 (10 ⁹ CFU/ml), and the correlation coefficient is 0.9998.

Embodiment 5: Construction of sepsis animal model

1. Inoculate the strain on the MH plate and incubate at 37°C for 24 hours; pick a single colony on the plate, inoculate it in the MH liquid medium, place it in a constant temperature shaker at 37°C for 6 hours, collect the bacteria, and Use the standard curve formula for quantification, then dilute (or concentrate) the bacterial solution to different concentration groups of 2.0×10 ⁹ CFU/mL, 2.1×10 ⁹ CFU/mL, and 4×10 ⁹ CFU/mL, and then use the bacterial solution of each group BALB/C mice (100 μl/only) of 6-8 weeks of age and body weight of 18-20 g were injected intraperitoneally to carry out systemic infection, and a normal saline control group was set at the same time, and the mortality rate of mice in each group was counted for 7 days after observation;

2. After infection, count the amount of bacterial colonization by colony counting method every 24 hours (observation for 7 days): randomly select 3 mice from each infection group and control group, and use the eyeball enucleation method to take 100 μl of blood samples from the mice Apply on a flat plate, place at 37°C, and count the number of clones after 24 hours; after taking blood samples, kill the mice, soak them in 75% alcohol for disinfection, take them out, fix their limbs, dissect them, take out the spleen, kidney, and liver, Place in 1mL sterile PBS, homogenize in a clean glass homogenizer, take 100μl of homogenate and dilute according to the ratio of 1:10, 1:100, 1:1000; take 100μL for each dilution and lightly smear Spread on solid medium, place at 37°C, incubate for 24 hours, and count the colonies.

The results are shown in Table 1:

Table 1 Determination of infectious dose and lethal dose of Acinetobacter baumannii-17978

The mortality rate of mice in the 2.0×10 ⁸ CFU dose group was 0 within 7 days; the mouse mortality rate in the 2.2×10 ⁸ CFU dose group was 20% within 48 hours; the mouse mortality rate in the 4.2×10 ⁸ CFU dose group was 90% within 48 hours; It can be seen that the infectious dose of Acinetobacter baumannii-17978 was 2.2×10 ⁸ CFU, and the sublethal dose was 4.2×10 ⁸ CFU.

3. Colonization of BALB/C mice with Acinetobacter baumannii-17978 in blood and various organs:

The bacterial count in the lungs reached its peak at 24 hours after infection, and the maximum colonization amount reached 2.0×10 ⁴ CFU/ml. The bacterial count in the lungs began to decrease at 48 hours, and no bacteria were detected in the lungs at 72 hours; the bacterial counts in the kidneys after infection It reached the peak at 24 hours, and the maximum colonization amount reached 1.0×10 ³ CFU/ml. The amount of bacteria in the lungs began to decrease at 48 hours, and no bacteria were detected in the lungs at 72 hours; colonization in the blood, spleen, heart, and liver after infection No bacteria were detected; the blood, spleen, kidney, and liver of control mice showed zero bacterial colonization.

The above results were used to evaluate the survival rate of mice and the amount of bacterial colonization in the main organs of blood, spleen, kidney and liver, which is the success of the Acinetobacter baumannii single subunit vaccine and the Acinetobacter baumannii subunit fusion vaccine. It laid the foundation for the development and study of the pathogenesis of Acinetobacter baumannii infection.

Embodiment 6: the detection of immune animal and antibody

1. Immunization of animals

1) For the first immunization, physically mix the hypothetical protein antigen with AlPO ₄ adjuvant, adjust the antigen concentration to 200 μg/ml with protein preservation solution, and place it at 4°C for overnight spin adsorption to make a vaccine; use a No. 5 half-type needle, double Side groin injection, each BALB/C mouse injection volume is 150 μ l, and set negative control group (AlPO ₄ adjuvant group) and blank control group (protein preservation solution group);

2) The second immunization, the second immunization was carried out on the 14th day, the immune components were the same as above, the injection volume was the same as that of the first immunization, and the immunization route was the same as above;

3) The third immunization, the third immunization was carried out on the 21st day, the immune components were the same as above, the injection volume was the same as that of the first immunization, and the immunization route was the same as above;

2. On the 7th and 14th day after the third immunization, the blood of BALB/C mice was collected, and the level of IgG humoral response after immunization was detected by ELISA.

1) Preparation of liquid

① Preparation of coating solution: Weigh 1.6g of NaHCO ₃ and 2.9g of Na ₂ CO ₃ , dissolve in 1L ddH ₂ O, and adjust the pH to 9.6 with a pH meter;

② Preparation of blocking solution: 1g bovine serum Ⅴ, dissolved in 100mL antibody diluent (1:100);

③Preparation of antibody diluent: dissolve phosphate in 1L ddH ₂ O, add 500μL Tween 20, and adjust the pH to 7.4 with a pH meter;

④ Preparation of washing solution: Weigh 2.42g Tris and dissolve in 1L ddH ₂ O, then add 500μL Tween 20, and adjust the pH to 7.4 with a pH meter;

⑤ Chromogenic solution (TMB) is a product of Tiangen Company;

⑥ Preparation of stop solution (2M H ₂ SO ₄ ): Pour 22.2 mL of concentrated sulfuric acid into 177.8 mL of ddH ₂ O.

2) ELISA detection of the antibody titer produced by hypothetical recombinant protein immunized mice

① Dilute the purified hypothetical recombinant protein to 10 μg/mL with coating solution;

② Coating: add recombinant protein dilution to the microplate, 200 μL/well, wash 3 times with washing solution after overnight at 4 °C;

③Sealing: Add 100 μL/well of blocking solution to the ELISA plate, place in a 37°C incubator for 2 hours, and wash 3 times;

④ Dilute the serum at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000;

⑤ Take the sealed ELISA plate, add diluted serum in sequence, 100 μL/well, place in a 37°C incubator for 30 minutes, wash 3 times, and air dry;

⑥ Dilute the HRP-labeled goat anti-mouse IgG, IgG1, IgG2a antibody preservation solution at 1:5000 to make antibody working solution;

⑦ Add diluted antibody working solution, 100 μL/well, place in a 37°C incubator for 1 hour, wash three times, and air dry;

⑧Add 100 μL/well of substrate chromogenic solution (TMB), and react for 5 minutes at room temperature in the dark;

⑨ Add the stop solution (2M H ₂ SO ₄ ), and immediately place it on a microplate reader to measure the OD value at a wavelength of 450nm;

⑩ Judgment of results: A _sample A _negative value ≧ 2.1 is positive (negative control is mouse pre-immune serum).

Result: detect the antibody titer that hypothetical protein antigen immunizes mice and produce and reach 1:256000; The positive rate of antibody on the 7th day after immunization reaches 90%, and the positive rate of antibody on the 7th day after immunization reaches 100%; Explain that the present invention constructs It is hypothesized that the recombinant protein is capable of producing antibodies in immunized mice.

Example 7: Determination of the challenge protection of hypothetical recombinant protein immunized animals by immunizing mice

Same as the immunization scheme in Example 6, after the third immunization of the mice, a lethal dose was used on the 14th day, and the live bacteria of Acinetobacter baumannii-17978 were injected into the tail vein for the challenge experiment, and each BALB/C mouse was injected with the bacteria The liquid volume was 4×10 ⁸ CFU, observed for 10 days, and the survival rate of mice in each group was counted. The results are shown in Table 2.

Table 2

Table 2 shows: for four animal immunization tests (each experiment is 10 mice) result, the result in the table shows that the average immune protection rate of negative control group and blank control group is 16.66% and 10% respectively, assuming recombinant protein auxiliary The average immune protection rate of the AlPO ₄ adjuvant group was 53.33%.

Therefore, the hypothetical recombinant protein of the present invention has good immunogenicity, can play an immunoprotective role against Acinetobacter baumannii-17978 infection, can induce the body to produce an immune response, and can be prepared, for example, with aluminum adjuvant Vaccines are used to prevent Acinetobacter baumannii infection.

Through the above examples, those skilled in the art can obviously use the recombinant protein prepared by the present invention and other related reagents, such as coating reagents, detection antibodies, chromogenic reagents, terminators, etc., to prepare related kits by using common technical knowledge in the field , such as detection kits, used to diagnose Acinetobacter baumannii infection, determine the prognosis, etc.

Those skilled in the art can use the hypothetical recombinant protein of the present invention for any other applicable application.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the implementation scope of the present invention; if it does not depart from the spirit and scope of the present invention, any modification or equivalent replacement of the present invention shall be covered by the claims of the present invention. within the scope of protection.

Claims (12)

1. A recombinant protein of Acinetobacter baumannii A1S_1462, characterized in that: comprising A1S_1462 mature peptide, the amino acid sequence of the mature peptide is as shown in SEQ ID NO.1. 2. The protein according to claim 1, characterized in that: the amino acid sequence of the recombinant protein is as shown in SEQ ID NO.3. 3. The recombinant protein according to claim 1, characterized in that: the recombinant protein is expressed through the fusion of a tag protein GST, and the tag protein is fused to the N-terminus of the A1S_1462 protein. 4. The recombinant protein according to claim 1, characterized in that: the amino acid sequence of the recombinant protein also includes amino-terminal and/or carboxy-terminal deletions, substitutions, and/or additions of 1- An immunogenic variant of 20 amino acid residues having at least 80% identity to said amino acid sequence. 5. The recombinant protein according to claim 1, characterized in that: the recombinant protein is formed by adding five amino acids of GPLGS to the amino terminal of claim 1 after fusion expression and digestion. 6. A polynucleotide encoding the A1S_1462 recombinant protein of claim 1. 7. the preparation method of A1S_1462 recombinant protein described in claim 1, is characterized in that, comprises the following steps: 1) Design PCR primers as follows: forward primer 5'-CGC GGATCC ATGGCACCTGTAAAAGAACAAAAAAT-3' BamH Ⅰ reverse primer 5'-TTAT GCGGCCGCCTTACTTTTTTTGTAGCTGCG -3' Not Ⅰ 2) using the primers designed in step 1) to amplify the target gene fragment encoding the A1S_1462 protein by PCR; 3) The gene fragment obtained in step 2) is cloned into an expression vector, and then transformed into a host bacterium; 4) Inducing the transformed host bacteria to express the A1S_1462 recombinant protein; 5) Purify the recombinant protein. 8. An expression vector or host cell, characterized in that it comprises the polynucleotide or host cell encoding the recombinant protein of claim 1. 9. The vector or host cell according to claim 7, characterized in that: the vector is a pGEX-6P-2 expression vector; the host cell is Escherichia coli, and the Escherichia coli is XL1-Blue. 10. An antibody against the A1S_1462 protein of claim 1. 11. The application of the A1S_1462 recombinant protein according to claim 1 in the preparation of a medicament for preventing or treating Acinetobacter baumannii infection. 12. The application of the A1S_1462 recombinant protein according to claim 1 in the preparation of a detection kit for Acinetobacter baumannii.