Vibrio outer membrane protein A multi-epitope antigen and preparation method and application of recombinant antigen of vibrio outer membrane protein A multi-epitope antigen

Abstract

The invention discloses a preparation method and application of a vibrio outer membrane protein A multi-epitope antigen and a recombinant antigen thereof, and is characterized in that a protein multi-epitope antigen sequence synthesized by outer membrane proteins of vibrio splendidus, vibrio parahaemolyticus and vibrio alginolyticus is designed by utilizing a multi-epitope technology, and the amino acid sequence of the protein multi-epitope antigen sequence is shown as SEQ ID NO.4; the preparation method of the multi-epitope recombinant antigen comprises the following steps: synthesizing a multi-epitope gene according to a multi-epitope antigen amino acid sequence, connecting the multi-epitope gene to a pET-28a (+) vector, transforming a plasmid containing the multi-epitope gene into a protein expression type competent cell, screening out positive clones, and performing protein expression; the method comprises the following steps: carrying out protein purification by using affinity chromatography to obtain a multi-epitope recombinant antigen, further preparing a multi-epitope antibody by using the multi-epitope recombinant antigen, and detecting whether a to-be-detected sample solution contains vibrio or not by using the multi-epitope antibody as a labeled protein and using a trace thermophoresis technology, so that the method has the advantages of high detection speed and strong specificity.

Description

Technical Field

[0001] The invention relates to a Vibrio outer membrane protein multi-epitope antigen, in particular to a Vibrio outer membrane protein A multi-epitope antigen and a preparation method and application of a recombinant antigen thereof. Background Art

[0002] Vibriosis is a bacterial disease caused by Vibrio bacteria. This disease occurs frequently around the world. Its outbreak not only causes huge economic losses to the aquaculture of economic animals such as marine aquaculture fish, crustaceans and shellfish, but also leads to large-scale deaths of wild marine fish, crustaceans, shellfish and sea cucumbers. It is one of the most harmful bacterial diseases in marine aquaculture. Therefore, the research and prevention of vibriosis and its pathogens are the core issues for disease workers at home and abroad. It is reported that Vibrio splendidus, Vibrio alginolyticus and Vibrio parahaemolyticus are the main pathogens of shrimp, shellfish, sea cucumber and marine fish, often causing symptoms such as inflammation of the body surface and decolorization and congestion of the liver. Infected sea cucumbers can cause the intestinal wall to become brittle and a large amount of yellow-white mucus or pus in the intestinal cavity, which is a symptom of "enteritis". Due to the high pathogenicity of aquatic pathogens, rapid and sensitive detection and early warning of marine pathogens are imperative for aquatic and human health.

[0003] Outer membrane proteins (Omps) in Gram-negative bacteria are associated with a variety of properties, including membrane biogenesis, transport across the cell membrane, and signaling. These proteins consist of a transmembrane β-barrel as well as periplasmic and extracellular loops. Many Omps are known to play a role in host-pathogen interactions and host immune responses, and have good antigenicity and surface exposure, making them the best choice as antigens. OmpA is one of the most important outer membrane proteins of Gram-negative bacteria. Due to the high copy number and surface exposure of OmpA proteins in pathogenic bacteria, studies have shown that OmpA actively participates in a variety of pathogenic effects, including adhesion to mucosal surfaces, serum resistance, invasion, antimicrobial peptide import, and host cell activation. In addition, the surface exposure of OmpA proteins makes them targets of the immune system, so that they can activate innate and adaptive immune mechanisms. Therefore, surface exposure and high copy number enable OmpA proteins to be candidates with high antigenicity.

[0004] Epitope, also known as antigenic determinant, is a chemical group that exists on the surface of an antigen and has a special structure that determines the specificity of the antigen. A single antigen molecule can have one or more different epitopes, and each epitope has only one antigen specificity. Therefore, the epitope is the target structure recognized by immune cells and is also the basis for the specificity of the immune response. Its nature, number and spatial configuration determine the specificity of the antigen.

[0005] Microscale Thermophoresis (MST) is a simple, rapid and accurate method for quantitatively detecting the interaction between biomolecules. It can measure the movement of molecules in a microscopic temperature gradient field, and detect changes in the molecular hydration layer, charge and size, thereby quantitatively detecting the interaction between biomolecules. When conducting an MST experiment, the sample is heated by an infrared laser to generate a microscopic temperature gradient field, and then the directional movement of molecules is monitored and quantified by covalently bound fluorescent dyes. The scope of application includes the binding behavior of small molecules, the interaction between proteins and the interaction between protein complexes. The use of bioinformatics technology and online prediction software analysis can predict the surface antigen epitopes of biomolecules, and then use multi-epitope antibodies to achieve rapid detection of multiple Vibrio pathogens. At present, there is no public research report on the design and expression of multi-epitope antigens of the outer membrane protein OmpA of three common aquatic pathogens, Vibrio splendidus, Vibrio alginolyticus and Vibrio parahaemolyticus, and the detection of Vibrio in combination with micro-thermophoresis technology. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a preparation method and application of a Vibrio outer membrane protein A multi-epitope antigen and a recombinant antigen thereof with fast detection speed and strong specificity.

[0007] The technical solution adopted by the present invention to solve the above technical problems is: a Vibrio outer membrane protein A multi-epitope antigen, using multi-epitope technology to design a multi-epitope antigen sequence synthesized from the outer membrane proteins of Vibrio splendidus, Vibrio parahaemolyticus and Vibrio alginolyticus, and its amino acid sequence is shown in SEQ ID NO.4.

[0008] The present invention also provides a method for preparing a Vibrio outer membrane protein A multi-epitope recombinant antigen, which specifically comprises the following steps: converting the amino acid sequence of the Vibrio outer membrane protein A multi-epitope antigen into a nucleotide sequence, synthesizing the Vibrio outer membrane protein A gene and cloning it into a pET-19T cloning vector, then enzymatically ligating it to a pET-28a(+) expression vector, transforming a plasmid containing the Vibrio outer membrane protein A gene into a protein expression-type competent cell BL21(DE3), screening out positive clones and performing protein expression, and purifying the protein by affinity chromatography to obtain the Vibrio outer membrane protein A multi-epitope recombinant antigen.

[0009] Further, the specific steps are as follows: converting the amino acid sequence of the Vibrio outer membrane protein A multi-epitope antigen into a nucleotide sequence, synthesizing the Vibrio outer membrane protein A gene and cloning it into a pET-28a(+) cloning vector, then enzymatically ligating it to the pET-28a(+) expression vector, transforming the plasmid containing the Vibrio outer membrane protein A gene into a protein expression competent cell BL21(DE3) to screen out positive clones, inoculating the positive clones into an Escherichia coli liquid culture medium and culturing them at 37°C and 120r / min until the bacterial solution OD reaches 0. 600 When the pH value is 0.4-0.6, IPTG with a final concentration of 0.5 mM is added for induction at 37°C for 5 hours to promote the massive expression of multi-epitope antigens; after the induction, centrifuge at a speed of 10000 r / min for 5 minutes, discard the Escherichia coli liquid culture medium, resuspend the bacteria with 10 times buffer solution, and use a nano high-pressure homogenizer to crush at low temperature under a pressure of 55-65 MPa, centrifuge at 4°C and 10000 r / min for 20 minutes, pour the supernatant into a nickel column for 1-2 hours, wash the impurities with 70 mM imidazole until the protein peak drops to a stable state, replace with 500 mM imidazole, collect the eluate, and obtain the Vibrio outer membrane protein A multi-epitope recombinant antigen, wherein the buffer solution formula is 25 mM Tris, 150 mM NaCl, pH=8.0.

[0010] The present invention also provides an application of the Vibrio outer membrane protein A multi-epitope recombinant antigen obtained by the above preparation method in preparing multi-epitope antibodies. The steps of the multi-epitope antibody preparation method are as follows: the Vibrio outer membrane protein A multi-epitope recombinant antigen is mixed with Freund's complete adjuvant for the first immunization, and then mixed with Freund's incomplete adjuvant for three subsequent immunizations, mice are immunized by intraperitoneal injection, and blood is collected after 4 immunizations to obtain immune mouse serum, namely, multi-epitope antibodies.

[0011] The present invention also provides a method for detecting Vibrio in a sample solution to be tested using the multi-epitope antibody obtained by the above preparation method, wherein the multi-epitope antibody is used as a marker protein and micro-thermophoresis technology is used to detect Vibrio in the sample solution to be tested, and the specific steps are as follows:

[0012] (1) Mix equal volumes of Lysine RED-NHS II dye and NHS labeling buffer, and mix them evenly with a pipette to obtain a dye solution with a final concentration of 300 μM;

[0013] (2) After mixing a multi-epitope antibody with a concentration of 10 μM and a dye solution with a concentration of 300 μM at a volume ratio of 9:1, gently pipette to mix, and place in a dark place at room temperature for 30 minutes to obtain a dye-antibody protein labeling reaction solution;

[0014] (3) Transfer 100 μL of the dye-antibody protein labeling reaction solution to the resin in the center of the balanced protein purification column. After the liquid completely enters the resin, add 550 μL of binding buffer to the column. After elution, add another 450 μL of binding buffer to the column, and collect the eluate to obtain the calibrated antibody protein.

[0015] (4) The calibrated antibody protein is added to the sample solution containing Vibrio to be tested, and the red light detection program in the micro-thermophoresis instrument and the MO-K022 capillary are used to detect at a fluorescence intensity of 400-800 A.U. The type of Vibrio in the sample to be tested is determined based on the affinity between the antibody and the antigen.

[0016] Furthermore, the binding buffer in step (3) is a 0.01 M phosphate buffer with a pH of 7.4.

[0017] Furthermore, in step (4), when the affinity signal-to-noise ratio displayed on the microthermophoresis instrument is 9.7, the sample liquid to be tested contains Vibrio alginolyticus, when the affinity signal-to-noise ratio is 7.2, the sample liquid to be tested contains Vibrio splendens, and when the affinity signal-to-noise ratio is 5.3, the sample liquid to be tested contains Vibrio parahaemolyticus.

[0018] Compared with the prior art, the advantages of the present invention are: the preparation method and application of a Vibrio outer membrane protein A multi-epitope antigen and its recombinant antigen of the present invention, the outer membrane protein A of three common aquatic pathogens, namely, Vibrio splendidus, Vibrio parahaemolyticus and Vibrio alginolyticus, is selected as the target of multi-epitope antigen design, and the multi-epitope antigen of three Vibrio is designed. The outer membrane protein A is an important antigen on the surface of bacteria, and it is highly conservative. The multi-epitope antigen is further used to construct and express a recombinant plasmid, and a multi-epitope recombinant antigen with protein induction activity is obtained. The recombinant protein is used to immunize mice to prepare polyclonal antibodies. The micro-thermophoresis instrument is used to quickly determine whether the Vibrio in the test liquid is Vibrio splendidus, Vibrio parahaemolyticus and Vibrio alginolyticus through the determination of intermolecular interactions, providing the most advanced technology and method for vaccine development and protein-based detection technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The amino acid optimization scores of the multi-epitope antigens of the outer membrane protein A of three Vibrio species are shown. The horizontal axis is the number of amino acid positions and the vertical axis is the epitope score value.

[0020] Figure 2 The electrophoresis results of the recombinant expression and purification process of the multi-epitope antigen of Vibrio outer membrane protein A, where M: protein marker, 1: protein before IPTG induction, 2: protein 1h after IPTG induction, 3: protein 3h after IPTG induction, 4: protein 5h after IPTG induction, 5: purified protein;

[0021] Figure 3Western Blot verification of Vibrio outer membrane protein A multi-epitope antibody;

[0022] Figure 4 The results of using MST to detect the affinity of antibodies against three Vibrio species with the same multiple epitopes, where A is Vibrio alginolyticus, B is Vibrio splendidus, and C is Vibrio parahaemolyticus. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with reference to the accompanying drawings.

[0024] Specific Example 1: Preparation of protein multi-epitope antigen sequences synthesized from outer membrane protein A of three Vibrio species.

[0025] The protein sequences of three Vibrio species, namely, Vibrio splendidus, Vibrio alginolyticus and Vibrio parahaemolyticus, were obtained from the NCBI database, and the common proteins were obtained using the data perspective technology. The outer membrane protein A (abbreviated as OmpA) common to the three Vibrio species was selected as the target, and the amino acid sequences of the three Vibrio outer membrane protein A synthesized from NCBI were downloaded respectively (SEQ ID NO.1-NO.3), and the epitope prediction tool was used to screen and determine the dominant epitope sites of multi-epitope fusion expression. Figure 1 As shown, the amino acid position score of the Vibrio outer membrane protein OmpA epitope, the horizontal axis is the number of amino acid positions, and the vertical axis is the epitope score. The higher the score, the higher the antigenicity, the more likely it is to produce antibodies, represented by yellow; the smaller the value, the less likely it is to form an epitope, represented by green. The antigenic epitopes of the three Vibrio species were screened according to the score value, and the sequences with high antigenicity were connected using a flexible peptide (sequence GGGGS). After further optimization using an epitope sequence prediction tool, the Vibrio outer membrane protein Omp A multi-epitope antigen was obtained, and its amino acid sequence is shown in SEQ ID NO.4, and its molecular weight is about 50kDa. The amino acid sequence of the synthetic outer membrane protein A of Vibrio splendens is shown in SEQ ID NO.1: MMKR PYLLLSALTITLCACSSQNEEPYIETPEAEQISDLQDDDNDGVVNARDTCPGTPASSLVDNEGCGETVSSQEVRQLNILFANDSYEVNPIFSDQITTMAEFLETYQSASIEIQGYASKVGSNEYNLDLSKRRANNVQDELLSNGIEPERVIIVGYGEERLENEGDDATSHALNRKVTATVIGLSEQIVEEWTIFTTLEK.

[0026] The amino acid sequence of the synthetic outer membrane protein A of Vibrio alginolyticus is shown in SEQ ID NO.2: MQKQEHVV FKRAKVYSHDESSHSGAWKVAFADFMIALMALFLVLWVMQVVDKEERKAIVAHLHSSSVFDKSYGNPFDTSQSISPIDLAQDSSVPSKHNSNHVVSSYFKGDGDGPEIDSLIPGNFDTQEQLAALAKVIEQMTAQINAQGNVNVTVTPQGLRIVLQDDYKQHMFSRGGAELTPFFEDLLLALAPVFHRVNNPLIISGHTDAIPFKKRFGRQSNWALSASRADVARNTLVEGGMPDDRVMQVTGMSDRALLNPDEPDSSENRRIELFVLTTPAAKVLETLFGNHQESELQKAKDKAEFNQPVIRQEVINYSGDTATSEVKIQEL.

[0027] The amino acid sequence of the synthetic outer membrane protein A of Vibrio parahaemolyticus is shown in SEQ ID NO.3: MQKQEHV VFKRAKAHGHDEPHGGAWKVAFADFMIALMALFLVLWVMQVVDKEERKAIVAHLHSSSVFDKSYGNPFDTSQSISPIDLAQDSSVPSKHNSNHVVSSYFQGDGDGPEINSLVPGTFDTQEQLAALAKVIEEMTAQINAQGNVNVTVTPQGLRIVLQDDYKQHMFSRGGAELTPFFEDLLLALAPLFEQVTNPLIISGHTDAIPFKKRFGRQSNWALSASRADVARKTLVEGGMPDDRVMQVTGMSDRALLNPDEPDSSENRRIELFILTTPAAKVLETLFGNQDDSELQKAKQKAEFNQPVIRQEVIRYSADSETQEAKIQAL.

[0028] The amino acid sequence of the multi-epitope antigen sequence synthesized by the outer membrane protein A of Vibrio splendens, Vibrio parahaemolyticus and Vibrio alginolyticus is shown in SEQ ID NO.4: AHSSSVFDKSYGNPFDTSQSISPIDLAQDSSVPSKHNSNHFKGDGDGPEIDSLIPGNFDTQEQNAQGNVNVTDYKQHMFSRGGAPFKKRFGRQSNWAGMPDDSSEDRALLNPDEPDSSEPAAKFGNHQESELQKDKAEFNQPVIRQEVINYSGDTATSEVKGGGGSIANAEEGAFSEYQPSDSSENLGMNQSNQHNLSPTDSTGAEGESKPIASNATAEGRAKNNDAAGGGGSGEKGNRNNNNKWNSENAPWSN ELYTKDNRFKDAARLPKLSFSQARDFNFGQKQLSVKQSDDNVGSQLANSAGQAKHGARYPIASNNTSAGQAKRPEVKQPQQEKVGGGGSSNQIRDDNLADWEPADAP EYDEGSPHQREIAQPQSPNLKNCDGDYSAFSNLIAPEKSRAAKNRQLSDQGMRNENALPTAQERVGGGGSAIAEDEYDYRAFPTAEQIADLQDEDNDGNRDLCPGTP.

[0029] Specific example 2: Construction of Vibrio OmpA multi-epitope recombinant antigen.

[0030] The amino acid sequence of the Vibrio OmpA multi-epitope antigen synthesized in the specific example 1 was converted into a nucleotide sequence, and the nucleic acid sequence was sent to Shanghai Shenggong Biotechnology Co., Ltd. to synthesize the Vibrio OmpA gene and clone it into the pET-19T cloning vector. Then, it was cloned into the pET-28a (+) connection vector after BamHI / XhoI restriction digestion, and the plasmid containing the Vibrio OmpA gene was transformed into a DH5α plasmid and cloned into a pET-28a (+) connection vector. + )-resistant LB liquid culture medium at 37°C, and the plasmid was extracted with a plasmid extraction kit, the concentration was determined with a micro-UV spectrophotometer, and PCR amplification (amplification primers were M13 universal primers and conventional PCR amplification system) was used to confirm that the Vibrio OmpA gene had been transformed into the plasmid.

[0031] The plasmid containing the Vibrio OmpA gene was transformed into protein expression competent cells BL21 (DE3), positive clones were screened and protein expression was performed, and protein purification was performed using affinity chromatography. Samples were retained at each step of inducing protein expression for 15wt% SDS-PAGE analysis. The results are shown in Figure 2As shown, there is a single protein band between 50 and 70 kDa, and its molecular weight is very close to the predicted theoretical molecular weight. At the same time, the positive clones were inoculated into E. coli liquid culture medium and cultured at 37°C and 120 r / min until the bacterial liquid OD 600 When the pH value was 0.4-0.6, IPTG (isopropyl-β-D-thiogalactoside) was added at a final concentration of 0.5 mM and induced at 37°C for 5 h to promote the massive expression of Vibrio OmpA multi-epitope antigens. After the induction, centrifuge at 10000r / min for 5min, discard the Escherichia coli liquid culture medium, resuspend the bacteria with 10 times buffer solution (25mMTris, 150mM NaCl, pH 8.0), and crush them at low temperature under a pressure of 55-65MPa with a nano high-pressure homogenizer. Centrifuge at 4°C, 10000r / min for 20min, pour the supernatant into a nickel column for 1-2h, wash the impurities with 70mM imidazole, and replace it with 500mM imidazole to collect the eluate containing multi-epitope antigens after the protein peak on the protein purification system of deAKTA of General Electric (GE) of the United States has dropped to a stable level, and obtain the Vibrio OmpA multi-epitope recombinant antigen. The protein concentration of the antigen detected by the BCA kit is >3mg / ml.

[0032] Specific example 3: Preparation of multi-epitope antibodies against Vibrio outer membrane protein OmpA.

[0033] The Vibrio OmpA multi-epitope recombinant antigen solution with a concentration of 1g / L prepared in Specific Example 2 was intraperitoneally injected into eight four-week-old female mice to prepare polyclonal antibodies. The Vibrio OmpA multi-epitope recombinant antigen solution was mixed with Freund's complete adjuvant for the first immunization, and the mice were immunized at an antigen dose of 100μg / mouse. The Vibrio OmpA multi-epitope recombinant antigen solution was mixed with Freund's incomplete adjuvant for three subsequent immunizations, and immunized at an antigen dose of 75μg / mouse, with an interval of 7d for each immunization. After the third immunization, blood was collected from the tail for antibody titer detection, and the test result showed a titer of 1:32000. The fourth immunization was performed according to the antibody titer result. The mice were fasted for 24h after immunization for 3d, and blood was collected from the eyeballs after anesthesia. The mixture was left to stand at 4°C overnight, and the upper serum was drawn after centrifugation to obtain the Vibrio OmpA multi-epitope antibody, which was then packaged and frozen in a -80°C refrigerator for standby use.

[0034] Bromophenol blue dye was added to the immune mouse serum samples obtained and boiled for Western Bolt verification analysis. The results are as follows: Figure 3 As shown, the serum produced specific bands, proving that the polyclonal antibody had good reactivity with the constructed multi-epitope antigen. The titer of the artificial polypeptide antibody was subsequently determined to be 1:32000 by indirect ELISA, and Western Blot was used to prove the consistency of the antibody and antigen.

[0035] Specific embodiment 4: Microthermophoresis technique is used to detect the interaction between antigen and antibody.

[0036] Add the prepared 5 μL lysine RED-NHS second-generation dye and 5 μL NHS labeling buffer to a 1.5 mL brown centrifuge tube, gently pipette and mix evenly, obtain a 10 μL dye solution with a final concentration of 300 μM, continue to add 90 μL of the Vibrio OmpA multi-epitope antibody prepared in step 3 with a concentration of 10 μM to the centrifuge tube, and gently blow and mix with a pipette. The total volume at this time is 100 μL, and it is kept in a dark place at room temperature for 30 minutes. Transfer the 100 μL dye-antibody protein labeling reaction solution to the resin in the center of the balanced protein purification column, and do not let the solution touch the inner wall of the column. When the liquid completely enters the resin, take 550 μL binding buffer (0.01M phosphate buffer with a pH of 7.4) and add it to the column, collect it with a 1.5 mL centrifuge tube, and then take 450 μL binding buffer and add it to the column, collect it with a new 1.5 mL centrifuge tube, and obtain the calibrated antibody protein. Single colonies of Vibrio splendens, Vibrio parahaemolyticus, Vibrio alginolyticus, etc. were selected and inoculated into 2216E liquid culture medium and cultured overnight at 28°C and 110 r / min. MST detection was performed using the calibrated antibody protein.

[0037] The MST detection process is as follows: The affinity of the multi-epitope antibody to the three Vibrio species is detected by the Binding Check program in the MO.Control software. Figure 4 The results of affinity test of antibodies with the same multi-epitopes against three Vibrio species using MST, where A is Vibrio alginolyticus, B is Vibrio splendens, and C is Vibrio parahaemolyticus, with 4 parallels for each group; Capillary Scans from left to right provide information about the overall fluorescence intensity of each capillary and the adsorption of molecules to the capillary wall; MST Traces overview provides feedback on the aggregation of target molecules and the photobleaching characteristics of the sample, showing the fluorescence intensity of each capillary over time, reflecting the MST signal of the fluorescent target molecule and the complex; Signal-to-Noise is used to evaluate the binding quality of the receptor and ligand, and a signal-to-noise ratio greater than 5 is desirable.

[0038] The results are as follows Figure 4As shown in the figure, when three kinds of Vibrio were detected, firstly, the detection results of Capillary Scans and MST Traces showed that the curves were smooth without abnormal fluctuations, indicating that the sample in the capillary was aggregated; secondly, the fluorescence value of the labeled antibody protein was between 200-2000, and the fluorescence value of the parallel sample fluctuated within 20%, and the infrared laser inside the instrument successfully caused a micro-temperature gradient and thermal swimming phenomenon when irradiating the sample in the capillary, and a typical MST curve appeared, indicating that the samples we detected met the instrument detection standards in terms of activity and fluorescence; finally, we tested the affinity of the antibody to bacteria through the Signal to Noise step, and the final results showed that the affinity signal-to-noise ratios of Vibrio alginolyticus, Vibrio splendidus, and Vibrio parahaemolyticus with the multi-epitope antibody were 9.7, 7.2, and 5.3, respectively, and the affinity signal-to-noise ratios of Vibrio alginolyticus, Vibrio splendidus, and Vibrio parahaemolyticus with the antibody were all greater than 5, indicating that there was an interaction between the labeled protein and the ligand bacteria, indicating that the artificial multi-epitope antibody prepared and synthesized by us can bind to the target bacteria.

[0039] In summary, the results of the detection of the interaction between the three Vibrio antigens and Vibrio OmpA antibodies showed that the antibody obtained using multi-epitope technology can fully bind to Vibrio. The binding strength is ranked from high to low: Vibrio alginolyticus, Vibrio splendidus, and Vibrio parahaemolyticus. If the concentration of the antigens is increased, their interaction with the antibodies will be more obvious.

[0040] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A Vibrio outer membrane protein A multi-epitope antigen, characterized in that: The multi-epitope antigen sequence synthesized from the outer membrane proteins of Vibrio splendidus, Vibrio parahaemolyticus and Vibrio alginolyticus was designed by using multi-epitope technology, and its amino acid sequence is shown in SEQ ID NO.

4.

2. A method for preparing a multi-epitope recombinant antigen of Vibrio outer membrane protein A, characterized in that The specific steps are: converting the amino acid sequence of the Vibrio outer membrane protein A multi-epitope antigen described in claim 1 into a nucleotide sequence, synthesizing the Vibrio outer membrane protein A gene and cloning it into the pET-19T cloning vector, then enzyme-cutting and connecting it to the pET-28a(+) expression vector, transforming the plasmid containing the Vibrio outer membrane protein A gene into protein expression-type competent cells BL21(DE3), screening positive clones and expressing proteins, and purifying the proteins by affinity chromatography to obtain the Vibrio outer membrane protein A multi-epitope recombinant antigen.

3. The method for preparing a Vibrio outer membrane protein A multi-epitope recombinant antigen according to claim 2, characterized in that The specific steps are as follows: converting the Vibrio outer membrane protein A multi-epitope antigen amino acid sequence described in claim 1 into a nucleotide sequence, synthesizing the Vibrio outer membrane protein A gene and cloning it into a pET-28a(+) cloning vector, then enzyme-cutting and connecting it to the pET-28a(+) expression vector, transforming the plasmid containing the Vibrio outer membrane protein A gene into a protein expression competent cell BL21(DE3) to screen out positive clones, inoculating the positive clones into an Escherichia coli liquid culture medium and culturing them at 37°C and 120r / min until the bacterial solution OD 600 When the pH value is 0.4-0.6, IPTG with a final concentration of 0.5 mM is added for induction at 37°C for 5 hours; after the induction, the mixture is centrifuged at a speed of 10000 r / min for 5 minutes, the Escherichia coli liquid culture medium is discarded, the bacteria are resuspended with a 10-fold buffer solution, and then crushed at a low temperature under a pressure of 55-65 MPa using a nano high-pressure homogenizer, and centrifuged at 4°C and 10000 r / min for 20 minutes. After the supernatant is poured into a nickel column for 1-2 hours, the impurities are washed with 70 mM imidazole until the protein peak drops to a stable state, and then 500 mM imidazole is replaced to collect the eluate to obtain the Vibrio outer membrane protein A multi-epitope recombinant antigen, wherein the buffer solution formula is 25 mM Tris, 150 mM NaCl, pH=8.

0.

4. A use of the Vibrio outer membrane protein A multi-epitope recombinant antigen obtained by the preparation method of claim 2 in the preparation of multi-epitope antibodies, characterized in that The steps of the method for preparing multi-epitope antibodies are as follows: the Vibrio outer membrane protein A multi-epitope recombinant antigen is mixed with Freund's complete adjuvant for the first immunization, and then mixed with Freund's incomplete adjuvant for three subsequent immunizations, and mice are immunized by intraperitoneal injection. Blood is collected after 4 immunizations to obtain immune mouse serum, i.e., multi-epitope antibodies.

5. A method for detecting Vibrio in a sample liquid using the multi-epitope antibody prepared by the preparation method of claim 4, characterized in that Using multi-epitope antibodies as marker proteins, micro-thermophoresis technology is used to detect Vibrio in the sample liquid to be tested. The specific steps are as follows: (1) Mix equal volumes of Lysine RED-NHS II dye and NHS labeling buffer, and mix them evenly with a pipette to obtain a dye solution with a final concentration of 300 μM; (2) After mixing a multi-epitope antibody with a concentration of 10 μM and a dye solution with a concentration of 300 μM at a volume ratio of 9:1, gently pipette to mix, and place in a dark place at room temperature for 30 minutes to obtain a dye-antibody protein labeling reaction solution; (3) Transfer 100 μL of the dye-antibody protein labeling reaction solution to the resin in the center of the balanced protein purification column. After the liquid completely enters the resin, add 550 μL of binding buffer to the column. After elution, add another 450 μL of binding buffer to the column, and collect the eluate to obtain the calibrated antibody protein. (4) The calibrated antibody protein is added to the sample solution containing Vibrio to be tested, and the red light detection program in the micro-thermophoresis instrument and the MO-K022 capillary are used to detect at a fluorescence intensity of 400-800 A.U. The type of Vibrio in the sample to be tested is determined based on the affinity between the antibody and the antigen.

6. The method for detecting Vibrio in a sample liquid using a multi-epitope antibody according to claim 5, characterized in that: The binding buffer described in step (3) is a 0.01 M phosphate buffer with a pH of 7.

4.

7. The method for detecting Vibrio in a sample liquid using a multi-epitope antibody according to claim 5, characterized in that: In step (4), when the affinity signal-to-noise ratio displayed on the microthermophoresis instrument is 9.7, the sample liquid to be tested contains Vibrio alginolyticus, when the affinity signal-to-noise ratio is 7.2, the sample liquid to be tested contains Vibrio splendens, and when the affinity signal-to-noise ratio is 5.3, the sample liquid to be tested contains Vibrio parahaemolyticus.

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