CN113388033A - Identification method of largemouth bass-derived aeromonas hydrophila immunogenic outer membrane protein - Google Patents
Identification method of largemouth bass-derived aeromonas hydrophila immunogenic outer membrane protein Download PDFInfo
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Abstract
The invention discloses a method for identifying an immunogenic outer membrane protein of an aeromonas salmoides source aeromonas hydrophila, and successfully identifies an immunogenic outer membrane protein of the aeromonas salmoides, wherein the identified immunogenic outer membrane protein of the aeromonas salmoides source aeromonas salmoides is AHA _3793(Genebank Number: A0KPP 0). The identification method specifically comprises the following steps: firstly, preparing anti-aeromonas hydrophila serum of micropterus salmoides, identifying the outer membrane protein of the aeromonas hydrophila with the anti-aeromonas salmoides serum by using an anti-micropterus salmoides IgM antibody as a tool and combining with mass spectrometry, then obtaining corresponding recombinant outer membrane protein through prokaryotic expression, and finally analyzing the reaction of the recombinant outer membrane protein and the anti-aeromonas salmoides serum of micropterus salmoides through western blotting to screen the outer membrane protein. The establishment of the identification method and the successfully identified aeromonas hydrophila immunogenic outer membrane protein provide a new idea and a new method for developing an aeromonas hydrophila subunit vaccine aiming at micropterus salmoides.
Description
Technical Field
The invention relates to the field of aquaculture, in particular to an identification method of an immunogenic outer membrane protein of micropterus salmoides.
Technical Field
The largemouth bass is an important economically cultured fish in China, has the advantages of fast growth, strong adaptability, no muscle stabbing, delicious meat quality and the like, and is deeply loved by consumers. In recent years, the breeding industry of the largemouth bass in China develops rapidly, and with the continuous development of the breeding in a large-scale and intensive direction, the water quality of the breeding environment and the breeding water area deteriorates gradually, so that diseases frequently occur, especially the outbreaks of diseases such as enteritis, gill rot disease, rhabdovirus disease and the like cause the largemouth bass breeding industry to be disastrous. The enteritis is mainly caused by infecting the largemouth bass by aeromonas, and the aeromonas hydrophila is an important member of the aeromonas and also an important pathogenic bacterium causing the enteritis of the largemouth bass.
At present, the prevention and control of aquaculture diseases mainly depend on antibiotics, and the abuse of the antibiotics causes the problems of bacterial drug resistance, drug residues, diffusion in the environment and the like. In order to promote the green aquaculture, relevant fishery departments begin to popularize the immune prevention and control technology of aquatic diseases. The application of the vaccine for prevention and control can not only effectively control the occurrence of infectious diseases, but also avoid the problem of drug resistance of pathogenic microorganisms caused by long-term use of the drug, and become one of the important technical means for realizing healthy aquaculture. Subunit vaccines are vaccines made using active components of pathogenic microorganisms, and are widely used because of their unique advantages. Currently, many reports exist on the research of subunit vaccines of the aeromonas hydrophila, but the strains and serotypes of the aeromonas hydrophila in aquaculture are numerous, so that aiming vaccines must be developed for the aeromonas hydrophila from different sources. However, although the aeromonas hydrophila is an important pathogenic bacterium of the micropterus salmoides, no report exists on the research of the subunit vaccine of the aeromonas salmoides.
The outer membrane protein plays an important role in infecting a host by the aeromonas hydrophila and is also an important target spot for screening subunit vaccines. The screening of the immunogenic protein aiming at different hosts aiming at the outer membrane protein of the aeromonas hydrophila is also beneficial to screening and developing subunit vaccines from the outer membrane protein of the aeromonas hydrophila. However, at present, no research aiming at screening immunogenic protein of the micropterus salmoides exists for the micropterus salmoides, and the screening and the development of the micropterus salmoides subunit vaccine are restricted.
Disclosure of Invention
In view of the above-mentioned disadvantages and the above-mentioned needs, the applicant has developed a method for identifying an immunogenic outer membrane protein of Aeromonas salmoides of Lateolabrax origin, and succeeded in identifying an immunogenic outer membrane protein of Aeromonas salmoides to solve the above problems.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for identifying an immunogenic outer membrane protein of an aeromonas salmoides source aeromonas hydrophila comprises the following steps:
s1: preparing anti-aeromonas hydrophila serum of the largemouth bass;
s2: identifying the immunogenic outer membrane protein of the Micropterus salmoides-derived aeromonas hydrophila;
s3: prokaryotic expression of the outer membrane protein of the Micropterus salmoides-derived aeromonas hydrophila;
s4: and (3) verifying the immunogenicity outer membrane protein of the micropterus salmoides.
Further, in the above method for identifying an immunogenic outer membrane protein of an aeromonas salmoides-derived aeromonas hydrophila, the step S1 of preparing the anti-aeromonas salmoides serum comprises the following steps:
taking and streaking aeromonas hydrophila strain separated to diseased micropterus salmoides on an LB solid culture medium, culturing at the constant temperature of 28 ℃ for 24h, flushing the aeromonas hydrophila on an LB plate by using 0.1M PBS, centrifuging at 8000rpm for 10min, collecting thalli, centrifuging and washing for 3 times by using PBS, adjusting the bacterial concentration to be 1 multiplied by 107cfu/mL, inactivating for 72h by using 0.5% formalin solution at 4 ℃, and then detecting whether the inactivation is successful by plate streaking;
taking inactivated aeromonas hydrophila to immunize the micropterus salmoides, and preparing the anti-aeromonas hydrophila serum of the micropterus salmoides by the following specific operations:
(1) uniformly mixing 200 mu L of inactivated aeromonas hydrophila with equivalent Freund's complete adjuvant, and performing intraperitoneal injection on the immunized micropterus salmoides;
(2) after 2 weeks, 200 mu L of inactivated aeromonas hydrophila is taken and evenly mixed with equivalent Freund incomplete adjuvant, and the immune micropterus salmoides is injected into the abdominal cavity;
(3) after 1 week, 200 μ L of inactivated aeromonas hydrophila is taken and injected into the abdominal cavity of the immunized micropterus salmoides;
(4) after 1 week, 200 μ L of inactivated aeromonas hydrophila is taken and injected into the abdominal cavity of the immunized micropterus salmoides;
(5) after 1 week, narcotizing micropterus salmoides with MS-222, collecting blood from tail vein, placing in 10ml centrifuge tube, standing at room temperature for 1h, and standing overnight at 4 deg.C;
(6) and (3) centrifuging at the temperature of 4 ℃ for 15min at 5000rpm, collecting supernatant, namely the prepared anti-aeromonas hydrophila serum of the micropterus salmoides, and analyzing the binding capacity of the prepared anti-aeromonas hydrophila serum of the micropterus salmoides and the aeromonas hydrophila by ELISA (enzyme-Linked immuno sorbent assay) by using an anti-micropterus salmoides IgM antibody as a tool.
Further, in the above method for identifying an immunogenic outer membrane protein of an aeromonas salmoides derived aeromonas salmoides, the step S2 of identifying an immunogenic outer membrane protein of an aeromonas salmoides derived aeromonas salmoides comprises the following steps: the culture was carried out as described in S1 to obtain Aeromonas hydrophila, and the reaction of the anti-Aeromonas hydrophila serum of Pericarpus salmoides with Aeromonas hydrophila was analyzed by western blotting using anti-Periopterus salmoides IgM antibody as a tool.
Further, in the identification method of the immunogenic outer membrane protein of the aeromonas salmoides of micropterus salmoides, the identification of the immunogenic outer membrane protein of the aeromonas salmoides of micropterus in the step S2 specifically comprises the following steps:
(1) adding 5 × Loading Buffer (15 μ L) into 60 μ L Aeromonas hydrophila, mixing, metal-bath decocting for 10min, adding into sample hole, and performing SDS-PAGE gel electrophoresis;
(2) after electrophoresis is finished, transferring the whole protein of the aeromonas hydrophila to a PVDF membrane in an electrotransfer mode, placing the PVDF membrane in 5% BSA, and sealing for 2h at 37 ℃;
(3) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in anti-Aeromonas hydrophila serum of Lateolabrax, diluting with PBS (1: 500), incubating at 28 deg.C for 3h, and using the negative serum of Lateolabrax as control instead of the anti-Aeromonas hydrophila serum of Lateolabrax;
(4) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in rabbit anti-Lateolabrax japonicus IgM antibody (PBS dilution, 1:10000), and incubating at 37 deg.C for 1 h;
(5) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in HRP-labeled goat anti-rabbit IgG antibody (PBS diluted 1:10000), and incubating at 37 deg.C for 1 h;
(6) washing the PVDF membrane for 3 times (5 min each time) by PBS-T, then developing by using a DAB developing kit, developing for 5-10min in a dark place, washing by using distilled water, stopping developing, then photographing and recording.
According to the result of western blotting, cutting a corresponding protein band on SDS-PAGE gel electrophoresis gel, carrying out LS-MS mass spectrometry, and then screening the aeromonas hydrophila outer membrane protein from the mass spectrometry result.
Further, in the identification method of the immunogenic outer membrane protein of the micropterus salmoides-derived aeromonas hydrophila, the step S3 of prokaryotic expression of the outer membrane protein of the micropterus salmoides-derived aeromonas salmoides specifically comprises the following steps:
according to the result of S2, the sequence of the corresponding protein was obtained from Genbank, primers were designed using Primer 5.0, and BamH I and Hind III cleavage sites were added to the upstream and downstream primers, respectively. Then, after PCR amplification and enzyme digestion, connecting the plasmid to pET-28a to construct a recombinant expression vector, and transferring the recombinant expression vector into BL21 competent cells (E.coli) after sequencing verification is correct; and (3) taking a positive transformant, inoculating the positive transformant to an LB liquid culture medium, carrying out shaking culture at 37 ℃ and 200rpm of a shaking table until the OD600 value reaches 0.6, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, continuing culturing for 6 hours, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel electrophoresis to verify the expression, collecting the thallus, and carrying out ultrasonic disruption. Then purifying with Ni ion affinity chromatographic column to obtain recombinant protein, and analyzing the protein purification condition by SDS-PAGE gel electrophoresis.
Further, in the method for identifying the immunogenic outer membrane protein of the aeromonas salmoides derived aeromonas salmoides, the S4 verification of the immunogenic outer membrane protein of the aeromonas salmoides derived aeromonas salmoides comprises the following steps: the recombinant outer membrane protein obtained in S3 is used as an antigen, an anti-micropterus salmoides IgM antibody is used as a tool, the reaction capacity of the recombinant outer membrane protein and anti-Aeromonas hydrophila serum of micropterus salmoides is analyzed through western blotting, and the immunogenic outer membrane protein of the micropterus salmoides is verified.
Further, in the identification method of the immunogenic outer membrane protein of the aeromonas salmoides of micropterus salmoides, the verification of the immunogenic outer membrane protein of the aeromonas salmoides of micropterus salmoides of S4 specifically comprises the following steps:
(1) taking 40 mu L of recombinant aeromonas hydrophila outer membrane protein, adding 15 mu L of 5 × Loading Buffer, uniformly mixing, boiling for 10min in a metal bath, adding into a sample Loading hole, and performing SDS-PAGE gel electrophoresis;
(2) after electrophoresis is finished, transferring the recombinant aeromonas hydrophila outer membrane protein to a PVDF membrane in an electrotransfer mode, placing the PVDF membrane in 5% BSA, and sealing for 2h at 37 ℃;
(3) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in anti-Aeromonas hydrophila serum of Lateolabrax, diluting with PBS (1: 500), incubating at 28 deg.C for 3h, and using the negative serum of Lateolabrax as control instead of the anti-Aeromonas hydrophila serum of Lateolabrax;
(4) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in rabbit anti-Lateolabrax japonicus IgM antibody (PBS dilution, 1:10000), and incubating at 37 deg.C for 1 h;
(5) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in HRP-labeled goat anti-rabbit IgG antibody (PBS diluted 1:10000), and incubating at 37 deg.C for 1 h;
(6) washing the PVDF membrane for 3 times (5 min each time) by PBS-T, then developing by using a DAB developing kit, developing for 5-10min in a dark place, washing by using distilled water, stopping developing, then photographing and recording.
Further, the identification method of the immunogenic outer membrane protein of the micropterus salmoides aeromonas salmoides is used for preparing the micropterus salmoides aeromonas salmoides subunit vaccine.
The invention has at least the following beneficial effects: the method comprises the steps of preparing anti-aeromonas hydrophila serum of the micropterus salmoides, identifying the immunogenic outer membrane protein of the aeromonas hydrophila aiming at the micropterus by using an anti-micropterus salmoides IgM antibody as a tool and combining western blotting with mass spectrometry, and successfully obtaining the immunogenic outer membrane protein of the aeromonas salmoides source aeromonas salmoides aiming at the micropterus salmoides through western blotting verification, thereby being beneficial to development and development of the subunit vaccine of the aeromonas salmoides source aeromonas salmoides.
Drawings
FIG. 1 shows the binding of the anti-serum of Lateolabrax japonicus to Aeromonas hydrophila in the ELISA assay of the examples;
FIG. 2 is a graph showing the reaction between the anti-Aeromonas hydrophila serum of Lateolabrax and the Aeromonas hydrophila in Western blotting analysis in the example. Lane 1: SDS-PAGE result of aeromonas hydrophila whole bacterial protein; lane 2: the immunoblotting result of the combination of the aeromonas hydrophila and the anti-aeromonas hydrophila serum of the largemouth bass; lane 3: negative control;
FIG. 3 is an example of SDS-PAGE analysis of recombinant expression of Aeromonas hydrophila outer membrane proteins. A: AHA _ 3973; b: AHA _ 4200. Lane M: a protein Marker; lane 1: positive transformants before induction; lane 2: inducing positive transformant; lane 3: purified outer membrane protein.
FIG. 4 is a graph showing the reaction between recombinant Aeromonas hydrophila outer membrane protein and anti-Aeromonas hydrophila serum of Lateolabrax in Western blotting analysis in the examples. A: AHA _ 3973; b: AHA _ 4200. Lane M: a protein Marker; lane 1: SDS-PAGE analysis of purified outer membrane proteins; lane 2: the recombinant outer membrane protein and the anti-aeromonas hydrophila serum of the largemouth bass are subjected to immunoblotting; lane 3: and (5) negative control.
Detailed Description
The invention will be further illustrated by the following specific examples, which are given by way of illustration only and not by way of limitation, and the reagents described are commercially available without further description.
Examples
An immunogenic outer membrane protein for identifying an aeromonas salmoides-derived aeromonas hydrophila, wherein the identified immunogenic outer membrane protein of the aeromonas salmoides-derived aeromonas salmoides: AHA _3793(Genebank Number: A0KPP 0).
The identification of the immunogenic outer membrane protein of the aeromonas hydrophila for the largemouth bass comprises the following steps:
s1: preparation of anti-aeromonas hydrophila serum of Lateolabrax.
Streaking and inoculating an aeromonas hydrophila strain separated to diseased micropterus salmoides on an LB solid culture medium, culturing at the constant temperature of 28 ℃ for 24h, flushing the aeromonas hydrophila on an LB plate by using 0.1M PBS, centrifuging at 8000rpm for 10min, collecting thalli, centrifuging and washing for 3 times by using PBS, adjusting the concentration of the bacteria to 1 × 107cfu/mL, inactivating by using 0.5% formalin solution at the temperature of 4 ℃ for 72h, and then detecting whether the inactivation is successful or not by streaking on the plate.
Taking inactivated aeromonas hydrophila to immunize the micropterus salmoides, and preparing the anti-aeromonas hydrophila serum of the micropterus salmoides by the following specific operations:
(1) uniformly mixing 200 mu L of inactivated aeromonas hydrophila with equivalent Freund's complete adjuvant, and performing intraperitoneal injection on the immunized micropterus salmoides;
(2) after 2 weeks, 200 mu L of inactivated aeromonas hydrophila is taken and evenly mixed with equivalent Freund incomplete adjuvant, and the immune micropterus salmoides is injected into the abdominal cavity;
(3) after 1 week, 200 μ L of inactivated aeromonas hydrophila is taken and injected into the abdominal cavity of the immunized micropterus salmoides;
(4) after 1 week, 200 μ L of inactivated aeromonas hydrophila is taken and injected into the abdominal cavity of the immunized micropterus salmoides;
(5) after 1 week, narcotizing micropterus salmoides with MS-222, collecting blood from tail vein, placing in 10ml centrifuge tube, standing at room temperature for 1h, and standing overnight at 4 deg.C;
(6) and (3) centrifuging at the temperature of 4 ℃ for 15min at 5000rpm, collecting supernatant, namely the prepared anti-aeromonas hydrophila serum of the micropterus salmoides, and analyzing the binding capacity of the prepared anti-aeromonas hydrophila serum of the micropterus salmoides and the aeromonas hydrophila by ELISA (enzyme-Linked immuno sorbent assay) by using an anti-micropterus salmoides IgM antibody as a tool.
S2: identification of largemouth bass source aeromonas hydrophila immunogenic outer membrane protein
Obtaining the aeromonas hydrophila by culturing as S1, and analyzing the reaction of the anti-aeromonas hydrophila serum of the largemouth bass and the aeromonas hydrophila by western blotting by using an anti-largemouth bass IgM antibody as a tool, wherein the specific operation is as follows:
(1) adding 5 × Loading Buffer (15 μ L) into 60 μ L Aeromonas hydrophila, mixing, metal-bath decocting for 10min, adding into sample hole, and performing SDS-PAGE gel electrophoresis;
(2) after electrophoresis is finished, transferring the whole protein of the aeromonas hydrophila to a PVDF membrane in an electrotransfer mode, placing the PVDF membrane in 5% BSA, and sealing for 2h at 37 ℃;
(3) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in anti-Aeromonas hydrophila serum of Lateolabrax, diluting with PBS (1: 500), incubating at 28 deg.C for 3h, and using the negative serum of Lateolabrax as control instead of the anti-Aeromonas hydrophila serum of Lateolabrax;
(4) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in rabbit anti-Lateolabrax japonicus IgM antibody (PBS dilution, 1:10000), and incubating at 37 deg.C for 1 h;
(5) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in HRP-labeled goat anti-rabbit IgG antibody (PBS diluted 1:10000), and incubating at 37 deg.C for 1 h;
(6) washing the PVDF membrane for 3 times (5 min each time) by PBS-T, then developing by using a DAB developing kit, developing for 5-10min in a dark place, washing by using distilled water, stopping developing, then photographing and recording.
According to the result of western blotting, cutting a corresponding protein band on SDS-PAGE gel electrophoresis, sending the protein band to Shanghai biological engineering Limited company for LS-MS mass spectrometry, and then screening the aeromonas hydrophila outer membrane protein from the mass spectrometry result.
S3: prokaryotic expression of Micropterus salmoides-derived aeromonas hydrophila outer membrane protein
According to the result of S2, the sequence of the corresponding protein was obtained from Genbank, primers were designed using Primer 5.0, and BamH I and Hind III cleavage sites were added to the upstream and downstream primers, respectively. And then, after PCR amplification and enzyme digestion, connecting the plasmid to pET-28a to construct a recombinant expression vector, and transferring the recombinant expression vector into BL21 competent cells (E.coli) after the sequencing verification is correct.
And (3) taking a positive transformant, inoculating the positive transformant to an LB liquid culture medium, carrying out shaking culture at 37 ℃ and 200rpm of a shaking table until the OD600 value reaches 0.6, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, continuing culturing for 6 hours, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel electrophoresis to verify the expression, collecting the thallus, and carrying out ultrasonic disruption. Then purifying with Ni ion affinity chromatographic column to obtain recombinant protein, and analyzing the protein purification condition by SDS-PAGE gel electrophoresis.
S4: verification of largemouth bass source aeromonas hydrophila immunogenic outer membrane protein
The recombinant outer membrane protein obtained in S3 is used as an antigen, an anti-micropterus salmoides IgM antibody is used as a tool, the reaction capacity of the recombinant outer membrane protein and anti-Aeromonas hydrophila serum of micropterus salmoides is analyzed through western blotting, and the immunogenic outer membrane protein of the Aeromonas salmoides of micropterus salmoides source is verified, and the specific operation is as follows:
(1) taking 40 mu L of recombinant aeromonas hydrophila outer membrane protein, adding 15 mu L of 5 × Loading Buffer, uniformly mixing, boiling for 10min in a metal bath, adding into a sample Loading hole, and performing SDS-PAGE gel electrophoresis;
(2) after electrophoresis is finished, transferring the recombinant aeromonas hydrophila outer membrane protein to a PVDF membrane in an electrotransfer mode, placing the PVDF membrane in 5% BSA, and sealing for 2h at 37 ℃;
(3) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in anti-Aeromonas hydrophila serum of Lateolabrax, diluting with PBS (1: 500), incubating at 28 deg.C for 3h, and using the negative serum of Lateolabrax as control instead of the anti-Aeromonas hydrophila serum of Lateolabrax;
(4) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in rabbit anti-Lateolabrax japonicus IgM antibody (PBS dilution, 1:10000), and incubating at 37 deg.C for 1 h;
(5) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in HRP-labeled goat anti-rabbit IgG antibody (PBS diluted 1:10000), and incubating at 37 deg.C for 1 h;
(6) washing the PVDF membrane for 3 times (5 min each time) by PBS-T, then developing by using a DAB developing kit, developing for 5-10min in a dark place, washing by using distilled water, stopping developing, then photographing and recording.
As shown in figure 1, ELISA analysis shows that the anti-Aeromonas hydrophila serum of Lateolabrax has high affinity with Aeromonas hydrophila, and provides an important tool for subsequent identification of the immunogenic outer membrane protein of the Aeromonas hydrophila of Lateolabrax.
As shown in FIG. 2 and Table 2, western blotting analysis showed that there were 5 bands successfully developed on the whole protein of Aeromonas hydrophila, the molecular weights of the proteins were 40kDa, 37kDa, 30kDa, 24kDa and 20kDa, and the outer membrane proteins AHA _3793 and AHA _4200 were identified by mass spectrometry. The mass spectrometry results are shown in table 1.
TABLE 1 Mass Spectrometry identification results
As shown in FIG. 3, the Aeromonas hydrophila outer membrane proteins AHA _3793 and AHA _4200 were successfully prokaryotic expressed and purified, and the molecular weights of the recombinant AHA _3793 and AHA _4200 were about 45kDa and 42kDa, respectively, and the purities after purification were good.
As shown in fig. 4, western blotting analysis showed that recombinant AHA _3793 could react with micropterus salmoides anti-aeromonas hydrophila serum, but recombinant AHA _4200 could not react with micropterus salmoides anti-aeromonas hydrophila serum, indicating that AHA _3793 is an aeromonas hydrophila immunogenic outer membrane protein for micropterus salmoides.
In conclusion, by preparing the anti-aeromonas hydrophila serum of the micropterus salmoides, using an anti-micropterus salmoides IgM antibody as a tool, identifying the immunogenic outer membrane protein of the aeromonas hydrophila aiming at the micropterus salmoides by adopting western blotting combined with mass spectrometry, and then successfully obtaining the immunogenic outer membrane protein of the aeromonas salmoides source aeromonas hydrophila aiming at the micropterus salmoides through western blotting verification, which is beneficial to the development and development of the subunit vaccine of the aeromonas salmoides source aeromonas salmoides.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that modifications can be made by those skilled in the art without departing from the principle of the present invention, and these modifications should also be construed as the protection scope of the present invention.
Claims (8)
1. A method for identifying an immunogenic outer membrane protein of an aeromonas hydrophila of a largemouth bass source is characterized by comprising the following steps:
s1: preparing anti-aeromonas hydrophila serum of the largemouth bass;
s2: identifying the immunogenic outer membrane protein of the Micropterus salmoides-derived aeromonas hydrophila;
s3: prokaryotic expression of the outer membrane protein of the Micropterus salmoides-derived aeromonas hydrophila;
s4: and (3) verifying the immunogenicity outer membrane protein of the micropterus salmoides.
2. The method for identifying an immunogenic outer membrane protein of Aeromonas salmoides of Lateolabrax origin according to claim 1, wherein the step S1 of preparing the serum of Aeromonas salmoides of Lateolabrax includes the following steps:
taking and streaking aeromonas hydrophila strain separated to diseased micropterus salmoides on an LB solid culture medium, culturing at the constant temperature of 28 ℃ for 24h, flushing the aeromonas hydrophila on an LB plate by using 0.1M PBS, centrifuging at 8000rpm for 10min, collecting thalli, centrifuging and washing for 3 times by using PBS, adjusting the bacterial concentration to be 1 multiplied by 107cfu/mL, inactivating for 72h by using 0.5% formalin solution at 4 ℃, and then detecting whether the inactivation is successful by plate streaking;
taking inactivated aeromonas hydrophila to immunize the micropterus salmoides, and preparing the anti-aeromonas hydrophila serum of the micropterus salmoides by the following specific operations:
(1) uniformly mixing 200 mu L of inactivated aeromonas hydrophila with equivalent Freund's complete adjuvant, and performing intraperitoneal injection on the immunized micropterus salmoides;
(2) after 2 weeks, 200 mu L of inactivated aeromonas hydrophila is taken and evenly mixed with equivalent Freund incomplete adjuvant, and the immune micropterus salmoides is injected into the abdominal cavity;
(3) after 1 week, 200 μ L of inactivated aeromonas hydrophila is taken and injected into the abdominal cavity of the immunized micropterus salmoides;
(4) after 1 week, 200 μ L of inactivated aeromonas hydrophila is taken and injected into the abdominal cavity of the immunized micropterus salmoides;
(5) after 1 week, narcotizing micropterus salmoides with MS-222, collecting blood from tail vein, placing in 10ml centrifuge tube, standing at room temperature for 1h, and standing overnight at 4 deg.C;
(6) and (3) centrifuging at the temperature of 4 ℃ for 15min at 5000rpm, collecting supernatant, namely the prepared anti-aeromonas hydrophila serum of the micropterus salmoides, and analyzing the binding capacity of the prepared anti-aeromonas hydrophila serum of the micropterus salmoides and the aeromonas hydrophila by ELISA (enzyme-Linked immuno sorbent assay) by using an anti-micropterus salmoides IgM antibody as a tool.
3. The method according to claim 1, wherein said step S2 of identifying the immunogenic outer membrane protein of Aeromonas salmoides includes the following steps:
the culture was carried out as described in S1 to obtain Aeromonas hydrophila, and the reaction of the anti-Aeromonas hydrophila serum of Pericarpus salmoides with Aeromonas hydrophila was analyzed by western blotting using anti-Periopterus salmoides IgM antibody as a tool.
4. The method according to claim 3, wherein the step S2 of identifying the immunogenic outer membrane protein of the Aeromonas salmoides is implemented by the steps of:
(1) adding 5 × Loading Buffer (15 μ L) into 60 μ L Aeromonas hydrophila, mixing, metal-bath decocting for 10min, adding into sample hole, and performing SDS-PAGE gel electrophoresis;
(2) after electrophoresis is finished, transferring the whole protein of the aeromonas hydrophila to a PVDF membrane in an electrotransfer mode, placing the PVDF membrane in 5% BSA, and sealing for 2h at 37 ℃;
(3) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in anti-Aeromonas hydrophila serum of Lateolabrax, diluting with PBS (1: 500), incubating at 28 deg.C for 3h, and using the negative serum of Lateolabrax as control instead of the anti-Aeromonas hydrophila serum of Lateolabrax;
(4) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in rabbit anti-Lateolabrax japonicus IgM antibody (PBS dilution, 1:10000), and incubating at 37 deg.C for 1 h;
(5) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in HRP-labeled goat anti-rabbit IgG antibody (PBS diluted 1:10000), and incubating at 37 deg.C for 1 h;
(6) washing the PVDF membrane for 3 times (5 min each time) by PBS-T, then developing by using a DAB developing kit, developing for 5-10min in a dark place, washing by using distilled water, stopping developing, then photographing and recording;
according to the result of western blotting, cutting a corresponding protein band on SDS-PAGE gel electrophoresis gel, carrying out LS-MS mass spectrometry, and then screening the aeromonas hydrophila outer membrane protein from the mass spectrometry result.
5. The method for identifying an immunogenic outer membrane protein of Aeromonas salmoides of Lateolabrax origin according to claim 1, wherein the step S3 of prokaryotic expression of the outer membrane protein of Aeromonas salmoides of Lateolabrax origin comprises the following steps:
obtaining the sequence of the corresponding protein from Genbank according to the result of S2, designing a Primer by using Primer 5.0, and adding BamH I and Hind III enzyme cutting sites in the upstream and downstream primers respectively; then, after PCR amplification and enzyme digestion, connecting the plasmid to pET-28a to construct a recombinant expression vector, and transferring the recombinant expression vector into BL21 competent cells (E.coli) after sequencing verification is correct; taking a positive transformant, inoculating the positive transformant to an LB liquid culture medium, carrying out shaking culture at 37 ℃ and 200rpm of a shaking table until the OD600 value reaches 0.6, adding IPTG (isopropyl-beta-thiogalactoside) for induction expression, continuously culturing for 6h, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to verify the expression, collecting thalli, and carrying out ultrasonic disruption; then purifying with Ni ion affinity chromatographic column to obtain recombinant protein, and analyzing the protein purification condition by SDS-PAGE gel electrophoresis.
6. The method for identifying the immunogenic outer membrane protein of the Aeromonas salmoides of Lateolabrax origin according to claim 1, wherein the step of verifying the immunogenic outer membrane protein of the Aeromonas salmoides of Lateolabrax origin by S4 comprises the following steps: the recombinant outer membrane protein obtained in S3 is used as an antigen, an anti-micropterus salmoides IgM antibody is used as a tool, the reaction capacity of the recombinant outer membrane protein and anti-Aeromonas hydrophila serum of micropterus salmoides is analyzed through western blotting, and the immunogenic outer membrane protein of the micropterus salmoides is verified.
7. The method for identifying the immunogenic outer membrane protein of the Aeromonas salmoides of Lateolabrax origin according to claim 6, wherein the step of verifying the immunogenic outer membrane protein of the Aeromonas salmoides of S4 comprises the following steps:
(1) taking 40 mu L of recombinant aeromonas hydrophila outer membrane protein, adding 15 mu L of 5 × Loading Buffer, uniformly mixing, boiling for 10min in a metal bath, adding into a sample Loading hole, and performing SDS-PAGE gel electrophoresis;
(2) after electrophoresis is finished, transferring the recombinant aeromonas hydrophila outer membrane protein to a PVDF membrane in an electrotransfer mode, placing the PVDF membrane in 5% BSA, and sealing for 2h at 37 ℃;
(3) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in anti-Aeromonas hydrophila serum of Lateolabrax, diluting with PBS (1: 500), incubating at 28 deg.C for 3h, and using the negative serum of Lateolabrax as control instead of the anti-Aeromonas hydrophila serum of Lateolabrax;
(4) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in rabbit anti-Lateolabrax japonicus IgM antibody (PBS dilution, 1:10000), and incubating at 37 deg.C for 1 h;
(5) washing PVDF membrane with PBS-T for 3 times (5 min each time), placing in HRP-labeled goat anti-rabbit IgG antibody (PBS diluted 1:10000), and incubating at 37 deg.C for 1 h;
(6) washing the PVDF membrane for 3 times (5 min each time) by PBS-T, then developing by using a DAB developing kit, developing for 5-10min in a dark place, washing by using distilled water, stopping developing, then photographing and recording.
8. The use of the method for identifying an immunogenic outer membrane protein of an Aeromonas salmoides-derived Aeromonas salmoides according to any one of claims 1 to 7 in the preparation of a subunit vaccine of the Aeromonas salmoides-derived Aeromonas salmoides.
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