CN112999341B - Edwardsiella tarda outer membrane protein vaccine and preparation and application thereof - Google Patents

Edwardsiella tarda outer membrane protein vaccine and preparation and application thereof Download PDF

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CN112999341B
CN112999341B CN202110226657.XA CN202110226657A CN112999341B CN 112999341 B CN112999341 B CN 112999341B CN 202110226657 A CN202110226657 A CN 202110226657A CN 112999341 B CN112999341 B CN 112999341B
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edwardsiella tarda
outer membrane
membrane protein
protein vaccine
vaccine
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李墨非
金成东
孙黎
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Institute of Oceanology of CAS
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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Abstract

The invention relates to the field of molecular vaccinology, in particular to an Edwardsiella tarda outer membrane protein vaccine and a preparation method and application thereof. The outer membrane protein vaccine contains an antigen of the outer membrane protein vaccine of Edwardsiella tarda shown by amino acid in SEQ ID No. 1. The recombinant protein vaccine constructed by the invention can protect the infection of the paralichthys olivaceus to Edwardsiella tarda, and induce the generation of specific antibodies, and the protein has obvious promotion effect on the immunocompetence of fish immune cells.

Description

Edwardsiella tarda outer membrane protein vaccine and preparation and application thereof
Technical Field
The invention relates to the field of molecular vaccinology, in particular to an Edwardsiella tarda outer membrane protein vaccine and a preparation method and application thereof.
Background
Edwardsiella tarda (Edwardsiella tarda) is a gram-negative pathogen that infects a variety of animals, including fish and mammals. The Edwardsiella tarda can cause Edwardsiellosis of marine cultured fishes, and serious economic loss is caused to the aquaculture industry of China. At present, a large number of cultured fishes have been reported to die in a large scale due to Edwardsiella tarda. Aiming at the devochism of Edwardsiella tarda, Korea has invented a commercial vaccine based on inactivated whole bacteria, and only 1 attenuated Edwardsiella tarda vaccine has obtained a new national veterinary certificate so far due to late related research in China, but the vaccine is not applied in a large scale. Therefore, development of other kinds of vaccines against edwardsiella tarda is not slow. Recombinant subunit vaccines developed based on bacterial outer membrane proteins are an effective approach. The subunit vaccine antigen protein is heterogeneously expressed in colon bacillus, and the recombined vaccine antigen protein is obtained after separation and purification, and can induce efficient specific immune response.
Disclosure of Invention
The invention aims to provide an Edwardsiella tarda outer membrane protein vaccine, and a preparation method and application thereof.
In order to realize the purpose, the invention adopts the technical scheme that:
an outer membrane protein vaccine of Edwardsiella tarda, which contains an outer membrane protein vaccine antigen of Edwardsiella tarda shown by amino acid in SEQ ID No. 1.
The outer membrane protein vaccine is a recombinant induction expression plasmid pETOmp1 containing an Edwardsiella tarda outer membrane protein vaccine antigen shown by amino acid in SEQ ID No. 1.
The recombinant plasmid pETOmp1 is connected to a commercial plasmid pET28a by homologous recombination by taking an amino acid sequence shown as SEQ ID No.1 as a template, so as to obtain a recombinant plasmid pETOmp 1.
Construction of the plasmid: the recombinant plasmid pETOmp1 is synthesized by using an amino acid sequence shown in SEQ ID No.1 as a template, and a base sequence is connected to pET28a after being synthesized by the company of Biotechnology engineering, Inc. (Shanghai), so that the recombinant plasmid pETOmp1 is obtained.
The outer membrane protein vaccine is prepared by mixing an outer membrane protein vaccine antigen of Edwardsiella tarda containing an amino acid shown in SEQ ID No.1 with an adjuvant.
The antigen is obtained by connecting the antigen of the outer membrane protein vaccine of Edwardsiella tarda shown by amino acid in SEQ ID No.1 to a commercial plasmid pET28a through homologous recombination, and obtaining a recombination induction expression plasmid pETOmp1 containing the antigen;
wherein, plasmid pETOmp1 is transformed into Escherichia coli BL21(DE3) to obtain transformant BL21/pETOmp1, Escherichia coli BL21/pETOmp1 is cultured overnight with 5ml LB liquid medium containing kanamycin (50. mu.g/ml), the overnight cultured bacterial liquid is transferred into 500ml LB liquid medium, cultured at 37 ℃, and cultured until the bacterial liquid reaches absorbance OD600After approximatively 0.5, IPTG was added to a final concentration of 1mM and incubated at 16 ℃ for 16 hours. And centrifugally collecting the cultured bacterial liquid, quickly freezing and unfreezing, adding a lysate after ultrasonic crushing, splitting at room temperature for 2 hours, centrifugally collecting a supernatant of the completely split bacterial liquid, and purifying the supernatant by a nickel column to obtain the recombinant protein with the amino acid sequence in the sequence table SEQ ID No.1 for subsequent use as a vaccine.
The recombinant protein prepared above is diluted to 400 μ g/ml with PBS, and then the diluted solution is mixed with adjuvant in equal volume, and used as recombinant protein vaccine for subsequent experiments.
The adjuvant is prepared by mixing the following components in a volume ratio of 2: 5 NaOH and Al2(SO4)3The mixture was suspended in PBS to a concentration of 8 mg/ml.
The concentration of NaOH is 5% by mass; al (Al)2(SO4)3The concentration is 5% by mass.
The application of the outer membrane protein vaccine of Edwardsiella tarda comprises the following steps: the outer membrane protein vaccine is applied to the preparation of a vaccine preparation for preventing and treating Edwardsiella tarda.
The invention has the following advantages:
1. high protection performance. The immune protection efficiency of the vaccine on Edwardsiella tarda reaches 75 percent.
2. The recombinant protein vaccine constructed by the invention can protect the infection of the Edwardsiella tarda to the paralichthys olivaceus without other commercial adjuvants, and induce the generation of specific antibodies, and the protein has a remarkable promoting effect on the immunocompetence of fish immune cells.
Drawings
FIG. 1 is a drawing of purified rOmp1 provided in an example of the inventionEtElectropherograms, lane 1, molecular weight standards, lane 2, rOmp1EtA protein.
Detailed Description
The present invention will be further described with reference to the following examples. The examples are intended to illustrate the invention, but not to limit it in any way.
The following methods were adopted in the conventional experimental methods involved in the examples of the present invention:
DNA sequence synthesis and plasmid ligation were performed by Biotechnology, Inc. (Shanghai);
2. plasmid extraction was carried out using the corresponding kit of "Omega Biotek, USA".
3. Plasmid transformation into E.coli was carried out by the Hanahan method (Sambrook and Russell: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press 2001).
Example 1
The antigen of the outer membrane protein vaccine of the Edwardsiella tarda is shown as an amino acid sequence in a sequence table SEQ ID No.1, the amino acid sequence is a section of sequence (NCBI serial number: ACY85305.1) on the Edwardsiella tarda genome, a base sequence except a signal peptide part is obtained through gene synthesis as an antigen sequence by analyzing a signal peptide sequence (the synthesis is completed by the biological engineering Co., Ltd (Shanghai)), and the translated amino acid sequence is as follows.
SEQ ID No.1：
MSSGDSTITLNYIQQSNGQVEKDLAGFKQITDQFIGSEHFGASTAPYRDAEGVALSYRYEFTDCWGVIGRLSYTGLRRGMQIRRGHNYGPGVPVLVDGRSRSQRWGIMAGPSYRVTDNLSLYGLAGASVDRLSWHIQVDDGANDVLGTALHTADQQLTRVSMAYAAGVQLNAGGYVLDFSYTGVGGDDRSHGFLVGVGLIF
(a) Sequence characteristics:
length: 201
Type: amino acid sequence
Chain type: single strand
Topology: linearity
(b) Type of molecule: protein
(c) Suppose that: whether or not
(d) Antisense: whether or not
(e) The initial source is: edwardsiella tarda TX1
(f) Specific name: omp1Et
The strain Edwardsiella tarda is preserved in China general microbiological culture Collection center (CGMCC), and the preservation number is as follows: CGMCC No.2330, classified and named Edwardsiella tarda (Edwardsiella tarda), preservation date: 2008, 1 month and 9 days. This strain is described in the' 201510096406.9 patent and filed as a certificate of the relevant deposit.
Example 2
Construction method of Edwardsiella tarda vaccine expression vector
Construction of recombinant plasmid pETOmp 1: the antigen base sequence of the Edwardsiella tarda outer membrane protein vaccine synthesized by the above-mentioned biological engineering Co., Ltd (Shanghai) is used as a template, and the above-mentioned template is connected into the commercial plasmid pET28a by means of homologous recombination so as to obtain the recombinant plasmid pETOmp 1. The LB liquid culture medium is used for culturing escherichia coli and comprises the following components in percentage by weight: 1.0% peptone, 0.5% yeast powder, 1.0% sodium chloride, 97.5% distilled water;
embodiment 3
Inducible expression and purification of vaccine proteins
Vaccine protein rOmp1EtInduced expression and purification of (1): plasmid pETOmp1 was transformed into E.coli BL21(DE3) (Beijing, all-grass Biotech Co., Ltd.), and the bacterial solution was cultured on LB solid medium containing kanamycin (50. mu.g/ml) for 18 to 24 hours, and one positive clone BL21/pETOmp1 was selected and obtained. BL21/pETOmp1 was cultured overnight in LB liquid medium containing kanamycin (50. mu.g/ml). 5ml of the overnight-cultured broth was added to 500ml of fresh LB liquid medium containing kanamycin (50. mu.g/ml), and the mixture was shaken on a shaker at 37 ℃ and 180rpm to OD600After approximatively 0.5, IPTG was added to a final concentration of 1mM and incubation was continued at 16 ℃ for 16 hours with shaking at 180 rpm. The cells were centrifuged at 6000g at 4 ℃ for 10 minutes to collect the cells cultured under the above conditions. 5ml of lysis buffer (10 mM NaH final concentration) was added to the cells2PO410mM Tris and 8M urea, pH 8.0), and gently shaken at room temperature for 1-2 hours until the bacterial suspension becomes clear. The supernatant was collected by centrifugation at 12000g at 4 ℃ for 30 minutes. The supernatant was recovered and purified by His Trap HP Columns (Qiagen, Germany), and the molecular weight of the purified protein was determined by SDS-PAGE (see FIG. 1) (electrophoresis conditions 80v after 25-30min, 120v electrophoresis for 1-1.5 h). The purified protein is verified to be recombinant antigen protein rOmp1 with an amino acid sequence in a sequence table SEQ ID No.1 by mass spectrometryEt。
Example 4
rOmp1EtUse of vaccines
Step 1) preparation of adjuvant and vaccine mixed liquor.
Preparing an adjuvant: mixing 5% (mass ratio) NaOH and 5% (mass ratio) Al2(SO4)3Mixing at a volume ratio of 2: 5. The mixture was centrifuged at 10,000g for 5 minutes, and the precipitate was collectedThe pellet was suspended in PBS to 8 mg/ml.
Preparing a vaccine mixed solution: purified rOmp1 from example 3 aboveEtDiluted to 400. mu.g/ml in PBS and the diluted vaccine protein was mixed with an equal volume of adjuvant.
Preparation of adjuvant control solution: PBS was mixed with adjuvant in equal volumes.
The PBS comprises the following components in percentage by weight: 0.8% NaCl, 0.02% KCl, 0.358% Na2HPO4·12H2O，0.024％NaH2PO4And the balance being water.
Step 2) immune application of the vaccine. 80 paralichthys olivaceus (each weighing about 10g) were randomly divided into 2 groups of 40. These 2 groups were named group a and group B, respectively. Each fish in group A was intraperitoneally injected with 100. mu.l of rOmp1 from step 1) aboveEtVaccine mixture, 100 μ l of adjuvant control solution of step 1) above was intraperitoneally injected into each fish of group B.
And 3) preparing the Edwardsiella tarda suspension. Culture of Edwardsiella tarda to OD in LB Medium6000.5, and then centrifuged (10000g, 4 ℃) for 2 minutes. The cells were collected and suspended in PBS to a final concentration of 4X 108CFU/ml。
And 4) detecting the immune protection effect of the vaccine.
On the 28 th day after the immunization injection in step 2), the 2 groups of fish of step 2) were injected intramuscularly with the Edwardsiella tarda suspension of the above step 3), and the injection amount per fish was 100. mu.l. In the following 15 days, the death of each group of fish was observed and recorded every day. After 15 days, the total number of deaths was counted for each group of fish: group A, 10 strips; group B, 40 strips. The relative immunoprotective efficiency (RPS) was calculated using the following formula: RPS 100 × (1-total mortality percentage in immunized/control group).
rOmp1 calculated according to the formulaEtThe immunoprotection efficiency was 75% respectively. Therefore, the obtained vaccine can effectively protect the paralichthys against the Edwardsiella tarda infection.
Step 5) detection of antibody titer
The serum preparation method comprises the following steps:
taking the vaccine group and the control group of the paralichthys olivaceus, wiping the fish body with 75% alcohol, and then extracting fish blood. Shaking at 4 deg.C for 1.5-2 hr, centrifuging at 4 deg.C, 600g, and 10 min. The supernatant was removed by filtration through a 0.22 μm filter and sterilized, and the serum was aliquoted and stored at-80 ℃ until use.
At 28 days after immunization in step 2) above, 5 fish from each group were tested for the level of vaccine-specific antibodies in serum by enzyme linked immunosorbent assay (ELISA). The results show that the titer of the antibody produced by the immunized histones is 27。
In summary, rOmp1EtIs a high-efficiency vaccine, and can generate high-efficiency serum antibody titer and immune protection effect.
Sequence listing
<110> oceanic research institute of Chinese academy of sciences
<120> Edwardsiella tarda outer membrane protein vaccine, preparation and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 201
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ser Ser Gly Asp Ser Thr Ile Thr Leu Asn Tyr Ile Gln Gln Ser
1 5 10 15
Asn Gly Gln Val Glu Lys Asp Leu Ala Gly Phe Lys Gln Ile Thr Asp
20 25 30
Gln Phe Ile Gly Ser Glu His Phe Gly Ala Ser Thr Ala Pro Tyr Arg
35 40 45
Asp Ala Glu Gly Val Ala Leu Ser Tyr Arg Tyr Glu Phe Thr Asp Cys
50 55 60
Trp Gly Val Ile Gly Arg Leu Ser Tyr Thr Gly Leu Arg Arg Gly Met
65 70 75 80
Gln Ile Arg Arg Gly His Asn Tyr Gly Pro Gly Val Pro Val Leu Val
85 90 95
Asp Gly Arg Ser Arg Ser Gln Arg Trp Gly Ile Met Ala Gly Pro Ser
100 105 110
Tyr Arg Val Thr Asp Asn Leu Ser Leu Tyr Gly Leu Ala Gly Ala Ser
115 120 125
Val Asp Arg Leu Ser Trp His Ile Gln Val Asp Asp Gly Ala Asn Asp
130 135 140
Val Leu Gly Thr Ala Leu His Thr Ala Asp Gln Gln Leu Thr Arg Val
145 150 155 160
Ser Met Ala Tyr Ala Ala Gly Val Gln Leu Asn Ala Gly Gly Tyr Val
165 170 175
Leu Asp Phe Ser Tyr Thr Gly Val Gly Gly Asp Asp Arg Ser His Gly
180 185 190
Phe Leu Val Gly Val Gly Leu Ile Phe
195 200

Claims (5)

1. An Edwardsiella tarda outer membrane protein vaccine is characterized in that: the outer membrane protein vaccine comprises an Edwardsiella tarda outer membrane protein vaccine antigen shown as an amino acid sequence in SEQ ID No. 1.
2. A method for preparing the edwardsiella tarda outer membrane protein vaccine according to claim 1, which is characterized in that: the antigen of the outer membrane protein vaccine of Edwardsiella tarda, which is shown by the amino acid sequence in SEQ ID No.1, is mixed with an adjuvant.
3. The method for preparing the edwardsiella tarda outer membrane protein vaccine according to claim 2, wherein: the adjuvant is prepared by mixing the following components in a volume ratio of 2: 5 NaOH and Al2(SO4)3 The precipitates were mixed, collected, and suspended in PBS to a concentration of 8 mg/ml.
4. The method for preparing the edwardsiella tarda outer membrane protein vaccine according to claim 3, wherein the vaccine comprises: the concentration of NaOH is 5% by mass; al (Al)2(SO4)3The concentration is 5% by mass.
5. The use of the edwardsiella tarda outer membrane protein vaccine of claim 1, wherein: the outer membrane protein vaccine is applied to the preparation of the medicine for preventing the Edwardsiella tarda infection.
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