CN108359683B - Edwardsiella tarda outer membrane protein OmpA with immune protection effect - Google Patents
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Abstract
The invention discloses an application of Edwardsiella tarda outer membrane protein OmpA with immune protection in subunit vaccine, wherein the recombinant protein is prepared by a prokaryotic expression method, and the method specifically comprises the following steps: taking an Edwardsiella tarda ET-CL genome sequence as a template, and carrying out PCR amplification on ompA gene; constructing a pMD18-T-ompA vector, and carrying out sequence determination on an ompA gene; constructing a recombinant expression vector pET-32 a-ompA; transforming the recombinant expression vector into DH5 alpha competent cells to obtain positive clones, extracting plasmids and sequencing; prokaryotic expression of the recombinant protein is carried out; analyzing the expression condition of the recombinant protein by SDS-PAGE; western blot verification is carried out on the recombinant protein. The invention obtains a large amount of purified recombinant protein of the outer membrane protein OmpA of the edwardsiella tarda by a prokaryotic expression method, and further evaluates the immunogenicity of the recombinant protein and the protection of a model animal by using an animal experiment, which indicates that the recombinant protein can be used for developing the subunit vaccine and the nucleic acid vaccine target of the edwardsiella tarda.
Description
Technical Field
The invention relates to the technical field of biological engineering, in particular to an Edwardsiella tarda outer membrane protein OmpA with an immune protection effect.
Background
Edwardsiella tarda (Edwardsiella tarda, E.tarda) is one of the common pathogenic bacteria in aquaculture, and can cause infection and morbidity of various seawater and freshwater fishes, so that high mortality, systemic infection and septicemia are caused, the overall immunity of fish herds is reduced, and the aquaculture development is seriously influenced. At present, E.tarda becomes one of the most important pathogens of flounder, flounder and eel in China, and causes great loss to aquaculture. In addition, the E.tarda can cause various types of human infection, can cause septicemia seriously and endanger life, and therefore has important public health significance for the research of the bacterium.
The vaccine is widely used in livestock and poultry breeding, has good control effect on epidemic diseases, and provides reference for controlling the aquatic epidemic diseases. Tarda, subunit vaccine and live vector vaccine studies based on protective antigen screening studies have been the focus of research. Research shows that outer membrane proteins (Omps) have excellent immunogenicity, can induce organisms to generate very strong immune response, and shows very good protection effect when animals are immunized by using outer membrane protein crude extracts. The invention utilizes a prokaryotic expression system to express and purify the outer-membrane protein main component OmpA (outer-membrane protein A), and the protective power of the recombinant protein on model animals is evaluated through animal experiments, which shows that the recombinant protein can be used for developing Edwardsiella tarda subunit vaccines and nucleic acid vaccine targets.
Disclosure of Invention
The invention aims to provide an Edwardsiella tarda outer membrane protein OmpA with immune protection function, and particularly obtains an Edwardsiella tarda outer membrane protein OmpA recombinant protein by a prokaryotic expression method, so as to further develop an Edwardsiella tarda subunit vaccine.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of the outer membrane protein OmpA of Edwardsiella tarda with immune protection function in subunit vaccine.
Further, the preparation method of the edwardsiella tarda outer membrane protein OmpA with the immune protection effect comprises the following specific steps:
(1) performing PCR amplification on ompA gene by taking an Edwardsiella tarda ET-CL genome sequence as a template and P1 and P2 as primers; the reaction system for PCR amplification is 20. mu.L, wherein 2 XTaq PCR Mix is 10. mu.L, each of primers P1 (20. mu.M) and P2 (20. mu.M) is 1. mu.L, template is 1. mu.L, ddH2O7 mu L, the reaction program of PCR amplification is pre-denaturation at 94 ℃ for 5min, 30s at 94 ℃, 30s at 54 ℃, 60s at 72 ℃, 25 cycles and 10min at 72 ℃; wherein the primers P1 and P2 are:
P1：5’-ATGAAAAAAACAGCGATCGCA-3’；SEQ ID NO：1；
P2：5’-TTAAGCCTGCGGCTGAGAAACTTC-3’；SEQ ID NO：2；
(2) constructing a pMD18-T-ompA vector, and carrying out sequence determination on an ompA gene;
(3) carrying out amplification culture on the strain with the correct sequencing result, extracting a plasmid, carrying out PCR amplification on an ompA gene by taking pMD18-T-ompA as a template and P3(20 mu M) and P4(20 mu M) as primers to obtain a purified target gene; the reaction system for PCR amplification is 20 μ L, wherein 2 XTaqPCR Mix 10 μ L, primers P3 and P4 are 1 μ L each, template 1 μ L, ddH2O7 mu L, the reaction program of PCR amplification is pre-denaturation at 94 ℃ for 5min, 30s at 94 ℃, 30s at 53 ℃, 60s at 72 ℃, 30 cycles and 10min at 72 ℃; wherein the primers P3 and P4 are:
(4)P3：5’-GGAATTCGCTCCGAAAGACGACACCTGG-3’，EcoRⅠ；SEQ ID NO：3；
(5)P4：5’-ACGCGTCGACAGCCTGCGGCTGAGAAACTTC-3’，SalⅠ；SEQ ID NO：4；
wherein the underlined part is the cleavage site;
(6) carrying out double enzyme digestion on the purified target gene and the pET-32a vector by using restriction enzymes EcoRI and SalI respectively to construct a recombinant expression vector pET-32 a-ompA;
(7) transforming the recombinant expression vector into DH5 alpha competent cells, selecting a single clone to perform PCR and double enzyme digestion verification to obtain a positive clone, extracting a plasmid, and sequencing;
(8) respectively transforming BL21 engineering bacteria into the recombinant expression vector pET-32a-ompA and the empty vector pET-32a to perform prokaryotic expression of recombinant protein;
(9) analyzing the expression condition of the recombinant protein by SDS-PAGE;
(10) western blot verification is carried out on the recombinant protein by taking His-Mab as a primary antibody and HRP-IgG as a secondary antibody.
The invention has the beneficial effects that: the invention obtains a large amount of purified recombinant protein of the outer membrane protein OmpA of the Edwardsiella tarda by a prokaryotic expression method, and further evaluates the immunogenicity of the recombinant protein and the protection of a model animal by utilizing an animal experiment, thereby further developing the subunit vaccine and the nucleic acid vaccine target of the Edwardsiella tarda.
Description of the drawings:
FIG. 1 shows the homology analysis of ompA gene of different strains in example 1 of the present invention;
FIG. 2 is a PCR amplification of ompA gene in example 2 of the present invention;
m.dl2000plus Marker; ompA gene amplification;
FIG. 3 is an SDS-PAGE analysis of the expression of recombinant proteins in E.coli in example 4 of the present invention;
m. protein Marker; pET-32a-ompA/BL21 thalli lysate; pET-32a/BL21 empty control;
FIG. 4 shows the expression of His-OmpA in E.coli in Western blot analysis in example 4 of the present invention;
pET-32a-ompA/BL21 thalli lysate; pET-32a/BL21 empty control;
FIG. 5 is a solubility analysis of the recombinant protein in example 4 of the present invention;
m. protein Marker; pET-32a-ompA/BL21 thalli lysate;
pET-32a-ompA/BL21 thalli lysate supernatant; 3, precipitating a bacterial lysate of pET-32a-ompA/BL 21;
FIG. 6 is an SDS-PAGE of the target protein purified in example 4 of the present invention;
m. protein Marker; 1, precipitation of bacterial lysate of pET-32a-ompA/BL 21; 2. purifying the recombinant protein;
FIG. 7 shows identification of recombinant proteins by Westernblot analysis positive sera in example 4 of the present invention;
1. a recombinant protein; 2. negative control (His- σ C reovirus capsid protein);
FIG. 8 shows the immunoprotection effect of the recombinant protein of example 5 of the present invention on mice;
FIG. 9 is a graph showing the change in the antibody level in the serum of the immunized mouse in example 5 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the attached drawings, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention.
Example 1 sequence analysis of Edwardsiella tarda ompA Gene
Primers were designed based on the ompA gene of the e.tarda reference strain ET-1 genomic sequence (CP001135.1) registered on GenBank, primers P1 and P2 are:
P1：5’-ATGAAAAAAACAGCGATCGCA-3’；SEQ ID NO：1；
P2：5’-TTAAGCCTGCGGCTGAGAAACTTC-3’；SEQ ID NO：2；
extracting an ET-CL genome by using a bacterial genome extraction kit, and carrying out PCR amplification on an ompA gene by using the ET-CL genome as a template; wherein the reaction system for PCR amplification is 20 μ L, wherein 2 XTaq PCR Mix is 10 μ L, primers P1(20 μ M) and P2(20 μ M) are 1 μ L each, template is 1 μ L, ddH2O7 mu L; PCR amplification reaction procedure: pre-denaturation at 94 ℃ for 5min, at 94 ℃ for 30s, at 54 ℃ for 30s, at 72 ℃ for 60s, for 25 cycles, at 72 ℃ for 10 min; recovering the target gene, connecting pMD18-T vector, and setting 10 mu L system: 4.5 mu L of target gene, 0.5 mu L of pMD18-T Vector and 5 mu L of 2 Xconnect buffer are connected at 16 ℃ overnight; the ligation product was transformed into DH5 α competent cells; single colonies were picked for PCR detection by BiotechSequence determination, and the results of alignment analysis of the sequencing results by referring to the E.tarda reference strain in GenBank and the ompA gene sequences of other Enterobacteriaceae bacteria, the results are shown in FIG. 1, the ompA gene is highly conserved among Edwardsiella members, and the homology with Aeromonas is high.
Example 2 Edwardsiella tarda ompA Gene amplification
Basic structural characteristics of OmpA protein of E.tarda are analyzed by using a UniProt website, specific amplification primers P3 and P4 are designed according to a sequencing result, an OmpA signal peptide part is removed, and the OmpA protein signal peptide influences protein expression.
Amplifying and culturing the PMD18-T-ompA strain with correct sequencing result in example 1, extracting plasmids, and carrying out PCR amplification on the ompA gene by taking the plasmids as a template and P3(20 mu M) and P4(20 mu M) as primers to obtain a target fragment (shown in figure 2) with the size of 990bp, and purifying the target gene; wherein the reaction system for PCR amplification is 20 mu L, wherein 2 xTaq PCR Mix is 10 mu L, each of the primers P3 and P4 is 1 mu L, the template is 1 mu L, and ddH2O7 mu L, the reaction program of PCR amplification is pre-denaturation at 94 ℃ for 5min, 30s at 94 ℃, 30s at 53 ℃, 60s at 72 ℃, 30 cycles and 10min at 72 ℃; wherein the primers P3 and P4 are:
P3：5’-GGAATTCGCTCCGAAAGACGACACCTGG-3’，EcoRⅠ；SEQ ID NO：3；
P4：5’-ACGCGTCGACAGCCTGCGGCTGAGAAACTTC-3’，SalⅠ；SEQ ID NO：4；
in which the underlined part represents the cleavage site.
Example 3 construction of pET-32a-ompA recombinant expression vector
Carrying out double enzyme digestion on the purified target gene and the pET-32a vector by using EcoRI and SalI respectively to construct a recombinant expression plasmid pET-32a-ompA, transforming DH5 alpha competent cells, carrying out ice bath for 30min, carrying out hot bath for 90s at 42 ℃ and carrying out ice bath for 5 min; adding 1mL LB liquid culture medium, rejuvenating at 37 deg.C for 1h, coating on a plate containing 100 μ g/mL final concentration of ampicillin, and culturing at 37 deg.C overnight; selecting a single clone for PCR and double enzyme digestion verification, wherein the double enzyme digestion system is 30 mu L: recombinant plasmid/pET-32 a 25. mu.L, EcoRI/SalI restriction enzyme 1. mu.L each, 10 XGreen Buffer 3. mu.L, 37 ℃ 1h, positive clone extraction plasmid sequence determination, ensure no mutation and frame shift.
EXAMPLE 4 prokaryotic expression of recombinant proteins
Respectively transforming the recombinant vector pET-32a-ompA and the empty vector into BL21 engineering bacteria, carrying out ice bath for 30min, carrying out hot bath for 90s at 42 ℃ and carrying out ice bath for 5 min; 1mL of LB liquid medium was added, rejuvenated at 37 ℃ for 1 hour, spread on a plate containing 100. mu.g/mL of ampicillin at a final concentration, and incubated overnight at 37 ℃. Single colonies were picked and inoculated in liquid LB medium containing 100. mu.g/L ampicillin and shake-cultured at 37 ℃ to OD600The value is 0.6-0.8, IPTG is added to the final concentration of 0.5mmol/L, shake culture is carried out for 4 h-6 h at 37 ℃, thalli are collected by centrifugation, and PBS buffer solution is used for washing for 3 times; adding protein sample buffer solution, mixing, boiling for 10min, centrifuging, and collecting supernatant as thallus lysate.
Performing SDS-PAGE identification, adjusting voltage to 80V after sample loading, adjusting voltage to 120V after the sample passes through separation gel and concentrated gel separation line, taking out the film, performing Coomassie brilliant blue staining for 1min, and boiling and decolorizing with distilled water for multiple times; as a result, as shown in FIG. 3, a band of the target protein appeared at 58 kD.
The expression of the target protein is further confirmed by a Western blot method. After SDS-PAG electrophoresis, taking out the film, transferring the protein to an NC film by using a film transfer instrument (80V 1 h); after the membrane transfer is finished, taking out the NC membrane, and washing the NC membrane for 3 times and 5 min/time by PBST; sealing 5% skimmed milk powder at room temperature for 1h, washing with PBST for 3 times, 5 min/time; diluting His-Mab with 5% skimmed milk powder at a ratio of 1:1000 as primary antibody, and incubating at room temperature for 1 h; PBST washing 3 times, 5 min/time; diluting HRP-IgG as a secondary antibody by 5 percent of skimmed milk powder according to a ratio of 1:5000, and incubating for 30min at room temperature; PBST washing 3 times, 5 min/time; adding developing solution, keeping out of the sun for 5min, and finally performing luminescence development; the results are shown in FIG. 4, and further validation shows that the recombinant protein OmpA is successfully expressed.
Centrifuging to collect thallus, adding protein sample buffer solution, placing in ice box, performing ultrasonic treatment for 20min for 3s, and pausing for 3 s; centrifuging after cracking, and collecting supernatant and precipitate; SDS-PAGE analysis showed that the recombinant protein was mainly present in the pellet and expressed as inclusion bodies, as shown in FIG. 5.
Purifying the inclusion body by a urea extraction method, dissolving the sediment after ultrasonic cracking in 1mL of water according to per gram (wet weight), and centrifuging at a high speed for 15min at 4 ℃; resuspend the pellet with 1mL of 0.1mol/LTris-Cl (pH8.5) containing 1M urea, and centrifuge at 4 ℃ for 15min at high speed; the supernatant was collected to obtain a relatively pure target protein, and the protein concentration was measured to be 0.56mg/mL, as shown in FIG. 6.
The recognition of the recombinant protein by the positive serum is verified by Western blot with E.tarda-infected mouse positive serum (1: 100) as a primary antibody and HRP-IgG (1:5000) as a secondary antibody, and the result is shown in FIG. 7, and the E.tarda-infected mouse positive serum can specifically recognize the recombinant OmpA protein.
Example 5 recombinant protein immunoprotection assay
40 healthy Kunming mice were randomly divided into two groups of 20 mice each. Intramuscular injection of 30. mu.g of recombinant protein OmpA (Freund's complete adjuvant emulsification) for the first immunization, secondary immunization (Freund's incomplete adjuvant emulsification) after 2 weeks, and boosting immunization with Freund's incomplete adjuvant emulsified protein after 30 days; control groups were injected with an equal volume of PBS (emulsified in freund's adjuvant). 48d two groups of mice were inoculated with 5LD in the abdominal cavity50(2.0×107cfu) Edwardsiella tarda ET-CL bacterial liquid, observing for 4d after infection, and counting the death number. The results showed that the control mice all died, 9 mice in the immunized group died, and the remaining mice gradually recovered to health after experiencing a short mental depression and no food intake. The results are shown in FIG. 8, which shows that the recombinant protein OmpA has certain protection power after immunizing mice, and the protection rate is 55%.
Collecting blood of mice and separating serum from infraorbital sinus at different time points before and after immunization of mice, detecting titer of antibodies in serum of mice by using an indirect ELISA method and using negative serum OD450Adding 3 times of standard deviation to the average value to serve as a negative and positive judgment standard, calculating a critical value to be 0.160, and judging the serum to be detected to be positive when the OD value is larger than the critical value; and when the OD value of the serum to be detected is less than the critical value, judging the serum to be negative. The indirect ELISA detection of the sera collected for 4 times is carried out, and the result is shown in FIG. 9, which indicates that the recombinant protein immunized mice can produce high-level specific antibodies and reach the highest level after the boosting immunization.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
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Claims (4)
1. The application of Edwardsiella tarda flagellin OmpA with immune protection in preparing subunit vaccine is characterized in that the preparation method of the subunit vaccine is as follows:
（1）performing PCR amplification by taking an Edwardsiella tarda ET-1 genome sequence as a template and P1 and P2 as primersompAA gene; wherein the primers P1 and P2 are:
P1：5’-ATGAAAAAAACAGCGATCGCA-3’ SEQ ID NO：1；
P2：5’-TTAAGCCTGCGGCTGAGAAACTTC-3’ SEQ ID NO：2；
(2) construction of pMD18-T-ompACarrier, pairompASequencing the gene;
(3) amplifying and culturing the strain with correct sequencing result, extracting the plasmid, and performing pMD18-T-ompAAs template, P3 and P4 as primers, PCR amplificationompAGene, obtaining purified target gene; wherein the primers P3 and P4 are:
P3：5’-GGAATTCGCTCCGAAAGATAACACCTGG-3’，EcoRⅠ；SEQ ID NO：3；
P4：5’-ACGCGTCGACAGCCTGCGGCTGAGTAACTTC-3’，SalⅠ；SEQ ID NO：4；
(4) using restriction endonucleasesEcoRI andSali, respectively carrying out double enzyme digestion on the purified target gene and the pET-32a vector to construct a recombinant expression vector pET-32a-ompA；
(5) Transforming the recombinant expression vector into DH5 alpha competent cells, selecting a single clone to perform PCR and double enzyme digestion verification to obtain a positive clone, extracting a plasmid, and sequencing;
(6) recombinant expression vector pET-32a-ompAAnd the empty vector pET-32a are respectively transformed into BL21 engineering bacteria to carry out prokaryotic expression of recombinant protein;
(7) analyzing the expression condition of the recombinant protein by SDS-PAGE;
(8) carrying out Western blot verification on the recombinant protein;
(9) purifying the inclusion body by a urea extraction method, dissolving the sediment after ultrasonic cracking in 1mL of water per gram, and centrifuging at a high speed at 4 ℃ for 15 min; resuspending the pellet in 1mL of 0.1mol/L Tris-Cl pH8.5 containing 1M urea, and centrifuging at 4 deg.C for 15 min; taking the supernatant to obtain a relatively pure target protein OmpA;
(10) emulsifying the target protein OmpA obtained in the step (9) by using a Freund complete adjuvant to obtain a subunit vaccine;
the specific steps for converting the BL21 engineering bacteria in the step (6) are as follows: respectively adding 5 μ L of recombinant vector pET-32a-ompAAnd 5 mul of empty vector pET-32a are added into 100 mul of BL21 engineering bacteria; performing ice bath for 30min, performing heat bath at 42 ℃ for 90s, and performing ice bath for 5 min; after adding 1mL of liquid LB medium and rejuvenating at 37 ℃ for 1 hour, 5. mu.L of each was spread on solid LB plates with a final concentration of ampicillin of 100. mu.g/mL, inverted and incubated overnight at 37 ℃.
2. The use of Edwardsiella tarda flagellin OmpA with immune protection effect in the preparation of subunit vaccine according to claim 1, wherein the reaction system of PCR amplification in step (1) is 20 μ L, 2 xTaq PCR Mix 10 μ L, 20 μ M P11 μ L, 20 μ M P21 μ L, template 1 μ L, ddH2O7. mu.L, the reaction program of PCR amplification is pre-denaturation at 94 ℃ for 5min, 30s at 94 ℃, 30s at 54 ℃, 60s at 72 ℃, 25 cycles and 10min at 72 ℃.
3. The use of Edwardsiella tarda flagellin OmpA with immune protection effect in the preparation of subunit vaccine according to claim 1, wherein the reaction system of PCR amplification in step (3) is 20 μ L, 2 xTaq PCR Mix 10 μ L, 20 μ M P31 μ L, 20 μ M P41 μ L, template 1 μ L, ddH2O7. mu.L, the reaction program of PCR amplification is pre-denaturation at 94 ℃ for 5min, 30s at 94 ℃, 30s at 53 ℃, 60s at 72 ℃, 30 cycles and 10min at 72 ℃.
4. The use of Edwardsiella tarda flagellin OmpA with immune protection effect in the preparation of subunit vaccine according to claim 1, wherein the Western blot in step (8) verifies His-Mab as primary antibody and HRP-IgG as secondary antibody.
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