Preparation and application of tilapia streptococcus antigen protein and segmented subunit nano-carrier vaccine of tilapia streptococcus antigen protein

Abstract

The invention discloses a tilapia streptococcus antigen protein and preparation and application of a segmented subunit nano-carrier vaccine of the tilapia streptococcus antigen protein, and belongs to the technical field of biological medicine. The invention discloses a preparation method of BNC-rEno (B-rE) and a segmented nano-carrier soaking vaccine (B-rE1 / B-rE2 / B-rE3) of the BNC-rEno (B-rE). The invention further discloses an immune protection effect of the subunit nano vaccine on tilapia mossambica to resist streptococcus infection. According to the Eno segmented subunit nano vaccine (B-rE3) disclosed by the invention, the serum antibody level of tilapia is remarkably improved, the death rate of tilapia infected with streptococcus agalactiae and streptococcus iniae is remarkably reduced, and the Eno segmented subunit nano vaccine has a relatively good immune protection effect on tilapia against streptococcus infection, can be used as a potential candidate vaccine for tilapia against streptococcus infection, and can be used for preparing the tilapia vaccine for preventing streptococcus infection. A new thought and an effective choice are provided for immune prevention and control of tilapia streptococcosis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the preparation and application of a tilapia streptococcus antigen protein and a segmented subunit nanocarrier vaccine thereof. Background Art

[0002] Tilapia occupies an important position in global aquaculture and is the second largest fish species after carps. However, high-density farming makes tilapia susceptible to different pathogens, especially large-scale outbreaks of bacterial diseases, which is one of the main limiting factors threatening tilapia production. Streptococcus agalactiae and Streptococcus iniae are the main pathogens of bacterial diseases in tilapia. The huge mortality rate caused by this bacterial infection is as high as 80%, which has a serious impact on the development of tilapia farming.

[0003] At present, the use of vaccines for aquaculture fish is the most ideal means of disease prevention and control. It can not only effectively control and prevent fish diseases, reduce the economic losses of the industry, but also reduce the health hazards caused by the large-scale use of chemical drugs. In recent years, extensive research has been conducted on vaccines for fish streptococcal disease. Among them, subunit vaccines with the characteristics of single immunogen and good safety have become the preferred solution for the development of aquatic vaccines, which can provide better immune protection for fish. Compared with other immunization methods, immersion immunization is easy to use in large quantities, does not require excessive labor costs, and can effectively activate the mucosal immunity of fish, thereby inducing protective immune responses. Studies have shown that nanomaterial-based delivery systems can overcome the technical limitations of applying immersion vaccines to fish by enhancing antigen absorption, allowing vaccines to adhere to mucosal surfaces, and promoting the delivery of specific antigens to target immune cells, thereby potentially improving the efficacy of vaccines and significantly increasing protection against pathogen infection. Bacterial nanocellulose (BNC) is a viable candidate for aquaculture vaccine carrier systems.

[0004] Enolase (Eno) exists as a plasminogen receptor on the surface of Streptococcus, enabling bacteria to destroy the fibrinolytic activity of plasminogen to facilitate tissue invasion. Based on comparative genomic analysis and multiple genomic screening, Eno has been suggested as a potential vaccine candidate. There are currently no reports on how to construct an enolase subunit nanocarrier vaccine. Summary of the invention

[0005] The purpose of the present invention is to provide a tilapia streptococcal antigen protein and its segmented subunit nanocarrier vaccine preparation and application, so as to solve the problems existing in the above-mentioned prior art. In order to solve the dilemma of the tilapia breeding industry and promote the healthy development of the aquaculture industry, the present invention aims to provide a method for preparing a tilapia streptococcal subunit nanocarrier vaccine, dividing the surface antigen Eno of Streptococcus agalactiae into three segments (E1 / E2 / E3), constructing and expressing recombinant proteins rE / rE1 / rE2 / rE3. Then, a subunit nanocarrier vaccine is prepared by combining bacterial nanocellulose (BNC), which can provide better immune protection for tilapia against streptococcal infection.

[0006] To achieve the above object, the present invention provides the following solutions: One of the technical solutions of the present invention is a segmented protein rE3 of the Streptococcus agalactiae antigen protein Eno, and its amino acid sequence is shown in SEQ ID NO.3.

[0007] The second technical solution of the present invention is a DNA molecule encoding the segmented protein rE3, and its nucleotide sequence is shown in SEQ ID NO.4.

[0008] The third technical solution of the present invention is a recombinant expression vector, which includes the DNA molecule.

[0009] A fourth technical solution of the present invention is a recombinant strain, comprising the recombinant expression vector.

[0010] A fifth technical solution of the present invention is the use of the segmented protein rE3 in the preparation of a vaccine for preventing tilapia streptococcal disease.

[0011] The sixth technical solution of the present invention is a subunit vaccine for preventing tilapia streptococcal disease, wherein the subunit vaccine comprises the segmented protein rE3 and a nanocellulose carrier.

[0012] The seventh technical solution of the present invention is a method for preparing the subunit vaccine, comprising the following steps: mixing the segmented protein rE3 and the cellulose nanosuspension for reaction, collecting the precipitate by centrifugation after the reaction, and dissolving the precipitate to prepare the nanocarrier vaccine.

[0013] Based on the above technical solution, the present invention has the following technical effects: First, based on a large number of analyses and experiments, the present invention divides the surface antigen Eno of Streptococcus agalactiae into three segments (E1 / E2 / E3) on average, and searches for a protein fragment (rE3) with good immunogenicity and cross-protection properties through injection immunization. The nanocarrier vaccine (B-rE3) prepared based on this is used to immunize tilapia with Streptococcus agalactiae and Streptococcus dolphinus bacterial fluids 4 weeks later, and the relative immune protection rates are 73.33% and 70.00%, respectively.

[0014] Second, the constructed bacterial nanocellulose-loaded vaccine system can achieve the goal of large-scale, simple and rapid seedling immunization through immersion immunization, greatly improve the effect of immersion immunization, and realize the industrialization of commercial fishery vaccines represented by tilapia streptococcal vaccine. It will also lay a theoretical foundation for the research and application of nano-loaded genetic engineering vaccines for other aquatic animals and provide new research ideas, which is of great significance to the sustainable development of fisheries and the safe production of aquatic products.

[0015] Based on a large number of analyses and experiments, the present invention divides the surface antigen Eno of Streptococcus agalactiae into three segments (E1 / E2 / E3) on average, obtains recombinant proteins rE / rE1 / rE2 / rE3 through prokaryotic expression, and then performs injection immunization to find a protein fragment (rE3) with good immunogenicity and cross-protection performance, which is then connected to bacterial nanocellulose to prepare a tilapia streptococcal subunit nanocarrier vaccine (B-rE3). Studies have shown that the vaccine is safe and effective, and can produce a good protective effect after immunizing tilapia. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 The identification results of the recombinant expression plasmids. A represents the recombinant expression plasmid pET28a-E, B represents the recombinant expression plasmid pET28a-E1, C represents the recombinant expression plasmid pET28a-E2, and D represents the recombinant expression plasmid pET28a-E3; M1: DL2000 Plus DNA Marker; 1: plasmid PCR identification; 2: double enzyme digestion identification; 3: plasmid single enzyme digestion; M2: DL15000 DNAMarker.

[0018] Figure 2This is the SDS-PAGE detection of recombinant proteins. A represents recombinant protein rE, B represents recombinant protein rE1, C represents recombinant protein rE2, and D represents recombinant protein rE3; M1: protein marker; 1: E. coli BL21 (pET28a / 28a) supernatant; 2: E. coli BL21 (pET28a / 28a) precipitate; 3: uninduced BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3) supernatant; 1: supernatant; 2: precipitate of uninduced BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3); 3: supernatant of induced BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3); 4: precipitate of uninduced BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3); 5: supernatant of induced BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3).

[0019] Figure 3 Western blot detection of purified recombinant protein. A represents recombinant protein rE, B represents recombinant protein rE1, C represents recombinant protein rE2, and D represents recombinant protein rE3; lane M is protein marker; lane 1 is purified recombinant protein.

[0020] Figure 4 Detection of serum antibody levels in different groups of tilapia immunized by injection. Different letters indicate significant differences (p < 0.05), the same below.

[0021] Figure 5 The survival rate and relative protection rate of tilapia in different immune groups after infection with Streptococcus agalactiae.

[0022] Figure 6 Detection of serum antibody levels in different groups of tilapia immunized by immersion.

[0023] Figure 7 The survival rate and relative protection rate of tilapia in different immune groups after pathogen infection. Among them, A represents Streptococcus agalactiae and B represents Streptococcus iniae. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0030] The embodiment of the present invention provides a segmented protein rE3 of the Streptococcus agalactiae antigen protein Eno, and the amino acid sequence thereof is shown in SEQ ID NO.3.

[0031] The embodiment of the present invention also provides a DNA molecule encoding the segmented protein rE3, and its nucleotide sequence is shown in SEQ ID NO.4.

[0032] An embodiment of the present invention further provides a recombinant expression vector, which includes the DNA molecule.

[0033] The embodiment of the present invention also provides a recombinant strain, including the recombinant expression vector.

[0034] The embodiment of the present invention also provides the use of the segmented protein rE3 in preparing a vaccine for preventing tilapia streptococcal disease.

[0035] In some specific embodiments, the tilapia streptococcal disease comprises streptococcal disease caused by Streptococcus agalactiae and / or Streptococcus iniae.

[0036] The embodiment of the present invention also provides a subunit vaccine for preventing tilapia streptococcal disease, wherein the subunit vaccine comprises the segmented protein rE3 and a nanocellulose carrier.

[0037] The embodiment of the present invention also provides a method for preparing the subunit vaccine, comprising the following steps: mixing the segmented protein rE3 and the cellulose nanosuspension for reaction, collecting the precipitate by centrifugation after the reaction, and dissolving the precipitate to prepare the nanocarrier vaccine.

[0038] In some specific embodiments, the cellulose nanosuspension comprises BNC, EDAC and NHS; the mass ratio of BNC, EDAC and NHS is 50:2:3; and the BNC is carboxylated BNC.

[0039] In some specific embodiments, the mass ratio of the segmented protein rE3 to the BNC in the cellulose nanosuspension is 1:1.

[0040] The present invention prepares Eno and its segmented recombinant proteins rE / rE1 / rE2 / rE3 through molecular biology and chemical connection techniques, adopts BNC delivery vector, constructs subunit nanocarrier vaccine through chemical connection techniques, and immunizes tilapia by soaking. The present invention finds that the constructed Eno segmented recombinant protein rE3 has good immunogenicity to Streptococcus agalactiae, and the nanocarrier vaccine B-rE3 can significantly improve the serum antibody level and non-specific immune indicators such as lysozyme and total antioxidant capacity of tilapia, significantly reduce the mortality of tilapia infected with Streptococcus agalactiae and Streptococcus iniae, and provide a good immune protection effect for tilapia against streptococcal infection.

[0041] The embodiment of the present invention further provides a Streptococcus agalactiae antigen protein Eno and its segments (E / E1 / E2 / E3), wherein the amino acid sequence of the recombinant protein rE / rE1 / rE2 / rE3 is shown in SEQ ID NO.1.

[0042] The embodiment of the present invention also provides a nucleic acid sequence encoding the recombinant protein rE / rE1 / rE2 / rE3, and the nucleotide sequence is shown in SEQ ID NO.2.

[0043] The present invention also provides a method for preparing the recombinant protein rE / rE1 / rE2 / rE3, the method comprising the following steps: Step 1, amplification of E / E1 / E2 / E3 target genes; Step 2, construction of recombinant Escherichia coli BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3) expressing rE / rE1 / rE2 / rE3 proteins; Step 3, induced expression of recombinant Escherichia coli BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3); Step 4: Centrifuge the bacterial solution after induced expression, collect the bacterial precipitate, resuspend the precipitate with PBS, freeze-thaw the resuspended bacterial solution three times in a row, and then perform ultrasonic disruption in an ice bath. Centrifuge the disrupted bacterial solution at 4°C, 12000 rpm, collect the bacterial protein, and dissolve the bacterial protein with 8 M urea to obtain a protein solution.

[0044] Step 5, purification of recombinant protein: Use nickel Ni2+ chelate affinity chromatography column to purify the protein, and dialyze the purified protein. After the dialysis, collect the dialyzed protein solution and store it at 4°C for future use.

[0045] The embodiments of the present invention also provide the use of the recombinant proteins rE / rE1 / rE2 / rE3 in preparing a vaccine for preventing tilapia streptococcal disease.

[0046] Preferably, the vaccine is a subunit vaccine, and the subunit vaccine is prepared from recombinant proteins rE / rE1 / rE2 / rE3 and bacterial nanocellulose carriers.

[0047] Preferably, the method for preparing the subunit vaccine comprises the following steps: Step 1, preparation and purification of recombinant protein rE / rE1 / rE2 / rE3; Step 2, preparation of cellulose nanocarrier vaccine: recombinant protein rE / rE1 / rE2 / rE3 is mixed with cellulose nanosuspension for reaction, and the precipitate is collected by centrifugation after the reaction, and the precipitate is dissolved to prepare the nanocarrier vaccine, wherein the concentration of the target recombinant protein in the vaccine is 20 mg / mL.

[0048] Preferably, the step one is: dividing the surface antigen Eno of Streptococcus agalactiae into three segments (E1 / E2 / E3) on average, constructing a recombinant Escherichia coli BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3) expressing the recombinant protein rE / rE1 / rE2 / rE3; fermenting, expressing and purifying the recombinant Escherichia coli BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3) to prepare rE / rE1 / rE2 / rE3 proteins.

[0049] Preferably, the bacterial nanocellulose delivery system in step 2 is prepared by the following steps: (1) Carboxylation of bacterial cellulose: Mix wet bacterial cellulose with TEMPO, sodium bromide and sodium hypochlorite in proportion, stir at room temperature for 12 h (control the pH at around 10.5), and add an appropriate amount of anhydrous ethanol to terminate the reaction.

[0050] (2) Preparation of carboxylated bacterial nanocellulose BNC: The above reaction solution was filtered with distilled water until pH ≤ 8, 70% concentrated sulfuric acid was prepared and mixed with the filtered cellulose, heated and stirred at 50°C for 2 h to prepare carboxylated nanocellulose of relatively uniform size (nano size mainly concentrated between 200-300 nm), and distilled water was added to terminate the reaction. Dialysis (pH close to neutral) and freeze-dried.

[0051] (3) Mix BNC, EDAC and NHS in appropriate proportions and stir at room temperature for 4 h in MES buffer (pH = 6.0). Add an appropriate amount of mercaptoethanol to terminate the reaction.

[0052] (4) Resuspend an equal amount of rSSF protein and BNC in phosphate buffer (pH = 7.2), then mix them in a ratio of protein resuspension solution: BNC reaction solution = 1:1 (V / V) and stir at room temperature for 4 h.

[0053] (5) The reaction product was dialyzed using a dialysis bag with a cutoff of 10,000 for 48 h and freeze-dried using a freeze dryer to obtain a solid powder. Example 1

[0054] The full-length amino acid sequence of the Streptococcus agalactiae antigen protein Eno is shown in SEQ ID NO.1: MSIITDVYAREVLDSRGNPTLEVEVYTESGAFGRGMVPSGASTGEHEAVELRDGDKSRYGGLGTQKAVDNVNNVIAEAIIGYDVRDQQAIDRAMIALDGTPNKGKLGANAILGVSIAVARAAADYLEVPLYSYLGGFNTKVLPTPMMNIINGGSHSDAPIAFQEFMIMPVGAPTFKEALRWGAEVFHALKKILKERGLETAVGDEGGFAPKFEGTEDGVETILKAIEAAGYEAGENGIMIGFDCASSEFYDAERKVYDYGKFEGEGGAVRTAAEQIDYLEELVNKYPIITIEDGMDENDWDGWKALTERLGGRVQLVGDDFFVTNTDYLARGIKEEAANSILIKVNQIGTLTETFEAIEMAKEAGYTAVVSHRSGETEDSTIADIAVATNAGQIKTGSLSRTDRIAKYNQLLRIEDQLGEVAQYKGIKSFYNLKK; The nucleotide sequence of its encoding gene Eno is shown in SEQ ID NO.2:

[0055] To further determine the dominant antigenic region of the Eno protein, the Eno gene was equally divided into three segments, namely E1 / E2 / E3 (the nucleotide sequences of which are shown in SEQ ID NO.3-5), and recombinant expression plasmids and recombinant proteins were prepared for each of them.

[0056] SEQ ID NO.3: ATGTCAATTATTACTGATGTTTACGCACGCGAAGTCCTTGACTCACGCGGTAACCCAACACTTGAAGTAGAAGTTTATACAGAATCAGGTGCTTTCGGACGTGGTATGGTTCCTTCAGGAGCTTCTACTGGTGAACACGAAGCAGTTGAACTTCGTGACGGCGACAAGTCTCGTTACGGTGGTCTTGGAACACAAAAAGCTGTTGACAACGTAAACAA CGTTATTGCAGAAGCAATCATTGGTTATGATGTACGCGACCAACAAGCTATCGACCGTGCTATGATCGCTCTTGACGGAACTCCTAATAAAGGTAAATTAGGTGCAAATGCTATTCTTGGTGTTTCTATTGCCGTAGCACGTGCTGCAGCTGATTACCTTGAAGTACCACTTTACAGCTACCTTGGTGGATTCAACACTAAAGTTCTTCCAACTCCA; SEQ ID NO.4: ATGATGAACATCATCAACGGTGGTTCTCACTCAGACGCTCCAATCGCATTCCAAGAATTCATGATTATGCCAGTTGGTGCACCAACTTTCAAAGAAGCACTTCGTTGGGGTGCTGAAGTATTCCATGCTCTTAAGAAAATCCTTAAAGAACGTGGTCTTGAAACAGCTGTTGGTGATGAAGGTGGATTTGCACCTAAATTTGAAGGTACTGAAGATGGTGTAGAAACTATTCTTAAAGCTATTGAAGCAGCTGGTTACGAAGCTGGCGAAAATGGAATCATGATTGGTTTTGACTGTGCTTCATCAGAGTTCTACGATGCAGAACGTAAAGTATACGACTACGGTAAATTCGAAGGTGAAGGTGGAGCAGTTCGTACTGCTGCAGAACAAATCGATTACCTTGAAGAATTGGTTAACAAATACCCAATCATCACT; SEQ ID NO.5: ATTGAAGATGGTATGGATGAAAACGACTGGGATGGTTGGAAAGCTCTTACAGAACGCCTTGGTGGACGTGTACAATTAGTTGGTGACGATTTCTTCGTTACAAATACTGATTACTTAGCACGTGGTATCAAAGAAGAAGCTGCTAACTCAATCCTTATCAAAGTTAACCAAATCGGTACTTTGACAGAAACTTTCGAAGCCATTGAAATGGCTAAAGAAGCAGGTTACACTGCGGTTGTATCACACCGTTCAGGTGAAACTGAAGATTCAACAATCGCTGATATCGCAGTTGCAACTAACGCTGGTCAAATTAAAACAGGTTCATTATCACGTACTGACCGTATTGCTAAATACAACCAATTACTTCGTATCGAAGACCAACTTGGTGAAGTTGCTCAATACAAAGGTATCAAATCATTCTACAACTTAAAAAAATAG。

[0057] 1 Construction and identification of recombinant Escherichia coli BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3) 1.1 Construction of recombinant E. coli BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3) expressing rE / rE1 / rE2 / rE3 proteins The E / E1 / E2 / E3 fragments were obtained by PCR cloning, and the target fragments were recovered using a gel recovery kit and connected to the pMD-19T vector. The ligation product was transformed into E. coli DH5α competent cells, and the positive bacteria were selected for expansion culture and plasmids were extracted. The correctly sequenced pMD19T-E / pMD19T-E1 / pMD19T-E2 / pMD19T-E3 plasmids were double-digested with pET-28a or pET-28a prokaryotic expression vectors, and connected using T4 DNA ligase to obtain the prokaryotic expression plasmids pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3. The plasmids were transformed into E. coli In BL21 (DE3), spread on LB solid medium containing antibiotics (ampicillin or kanamycin), and invert and culture at 37°C overnight. Then pick a single colony for colony PCR detection. Inoculate the colony with positive colony PCR into LB culture medium containing antibiotics and culture it at 37°C and 180 rpm overnight. Extract the plasmid according to the instructions of the plasmid extraction kit, perform PCR and single and double enzyme digestion identification, and perform agarose gel electrophoresis on the product at 120V for 30 min. The results are as follows Figure 1 As shown, the PCR results and single and double enzyme digestion results of the recombinant expression plasmids pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3 all showed target bands consistent with the expected size, indicating that the recombinant expression plasmids were successfully constructed.

[0058] 1.2 Inducible expression of recombinant E. coli BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3) Use an inoculation loop to pick up a small amount of the preserved bacterial solution, streak it onto LB solid medium, incubate it upside down at 37°C overnight, pick a single colony and inoculate it into LB medium, incubate it at 37°C and 180 rpm overnight as seed solution, then inoculate it into LB liquid medium at 1% (V / V), and add antibiotics to a final concentration of 50 μg / mL. Incubate at 37°C for 5-7 h until the bacterial solution OD 600 When the value was 1.1-1.3, IPTG was added to a final concentration of 0.001 mol / L and induced at 37°C for 6 h.

[0059] 1.3 Bacterial liquid treatment and ultrasonic disruption The fermentation product was centrifuged at 10,000 rpm at room temperature, the bacteria were collected, washed three times with PBS (0.01 mol / L, pH = 7.2), the collected bacteria were resuspended at a ratio of 1:10 between wet bacteria and PBS solution, and ultrasonically disrupted in an ice bath. The disrupted bacterial solution was centrifuged at 4°C and 10,000 rpm, the bacterial protein was collected, and dissolved with 8 M urea to obtain a protein solution.

[0060] 1.4 Protein purification Dissolve each gram of protein precipitate with 10 mL of dissolving solution (5 mmol / L imidazole, 0.5 mmol / L sodium chloride, 8 M urea, 20 mmol / L Tirs-HCl, pH 7.9), shake and dissolve at 200 rpm at room temperature for 2 h, centrifuge at 10,000 rpm for 30 min at 4°C, and collect the supernatant. Use equilibrium buffer (5 mmol / L imidazole, 0.5 mmol / L sodium chloride, 8 M urea, 20 mmol / L Tirs-HCl, pH 7.9) to fully balance the metals (Ni 2＋ ) chelate affinity chromatography column, load the sample according to 2 column volumes, balance with equilibrium buffer, and then elute with elution buffer (0.5 mol / L imidazole, 0.5 mmol / L sodium chloride, 8 M urea, 20mmol / L Tirs-HCl, pH7.9) to collect the protein. Place 200 mL of the purified protein in a SnakeSkin™ dialysis bag (10K MWCO) and immerse it directly and completely in 10 L of refolding solution (150 mM NaCl, 2.5 mM KCl, 10 mM Na2HPO4, 2mM KH2PO4, 1% Tween-20, 10 mM β-cyclodextrin, 1 mL-cysteine, 3 mM reduced and 1 mM oxidized glutathione, pH7.9) at 4°C for 12 h. During this period, the solution was changed every 6 h. After the dialysis, the dialyzed protein solution was collected and stored at 4°C for later use.

[0061] 1.5 Detection of recombinant proteins 1.5.1 SDS-PAGE: Add 5× gel loading buffer to the supernatant and resuspended precipitate after disruption, and directly add 5× gel loading buffer to the supernatant without ultrasonic disruption. Boil for 10 min, and detect by 12% SDS-PAGE electrophoresis. Then perform Coomassie Brilliant Blue staining and decolorize with decolorizing solution until the bands are clear. At the same time, set up BL21 (pET28a-E / pET28a-E1 / pET28a-E2 / pET28a-E3) culture without IPTG induction as a control. The results are shown in Figure 2As shown, the four recombinant proteins rE / rE1 / rE2 / rE3 were expressed in both the supernatant (lane 5) and the precipitate (lane 6), but they were mainly expressed in the precipitate in the form of inclusion bodies.

[0062] 1.5.2 Western-blot identification: First, perform SDS-PAGE electrophoresis on the recombinant protein in the same way as above. After the electrophoresis, take a piece of film and place it on the transfer instrument, and transfer the membrane at 200 mA for 1 hour. Place the PVDF membrane loaded with the protein in a plastic washing box, add 5% skim milk to block at room temperature for 2 hours, pour out the skim milk, wash 3 times with TBST, add the primary antibody mouse anti-6×His monoclonal antibody (1:2000 dilution), slowly shake at room temperature for 2 hours, pour out the primary antibody, wash 3 times with TBST, add the horseradish peroxidase (HRP) labeled goat anti-mouse IgG secondary antibody (1:3000 dilution), slowly shake at room temperature for 2 hours, pour out the secondary antibody, wash 3 times with TBST, and place the membrane in a plate in a dark room. Use the chemiluminescence (ECL) kit to develop the color and obtain the expected size band (rE is about 45 kDa, rE1 / rE2 / rE3 are all about 18 kDa). The results are as follows Figure 3 As shown, the four recombinant proteins rE / rE1 / rE2 / rE3 can react with mouse anti-6×His antibody and show positive target bands, indicating that the recombinant proteins are successfully expressed. Example 2

[0063] 2 Evaluation of the immune effect of Eno segmented vaccine 2.1 ELISA test of serum antibody levels 250 healthy tilapia weighing 4.0 ± 0.5 g were divided into 5 groups (50 / group), namely PBS, rE, rE1, rE2 and rE3. The vaccine group was injected intraperitoneally at a concentration of 10 μg / g (50 μL / tail), and the control group was injected with an equal amount of PBS buffer. Three fish were randomly selected from each group at the 1st, 2nd, 3rd and 4th week of immunization to collect tail vein blood. The collected blood was left to stand at room temperature for 2 h and placed in a 4°C refrigerator overnight. The next day, the blood was centrifuged at 5000 g in a low-temperature refrigerated centrifuge for 15 min, and the upper serum was taken and stored at -20°C.

[0064] Serum antibody levels were determined by indirect ELISA: serum was diluted to an appropriate concentration with coating diluent, added to each well of a 96-well ELISA plate at 100 μL, and each sample was repeated three times. The plate was placed at 4°C for overnight coating. After the coating was completed, the liquid in the well was discarded; 5% calf serum was added and the plate was blocked at 37°C for 1 h. After the blocking was completed, the plate was washed three times with TBST, each time for 3 min; the antigen protein rE / rE1 / rE2 / rE3 was diluted to an appropriate concentration, added to the ELISA reaction well, 100 μL per well, and incubated at 37°C for 1 h. Then wash three times with TBST, 3 min each time; dilute mouse anti-6×His monoclonal antibody (1:2000), add 100 μL per well to the enzyme-labeled reaction wells, incubate at 37°C for 1 h, and then wash three times with TBST, 3 min each time; dilute horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (1:3000), add 100 μL per well to the enzyme-labeled reaction wells, incubate at 37°C for 1 h, and then wash three times with TBST, 3 min each time; add 100 μL of TMB colorimetric solution to each well and place it at 37°C away from light for 5-10 min; add 50 μL of stop solution to each well to terminate the reaction, and measure the absorbance at 450 nm within 20 min.

[0065] The results showed that the serum antibody levels of each vaccine group reached the highest level at the 4th week. The antibody levels of the rE3 group at the 3rd and 4th weeks after immunization were significantly higher (p < 0.05) than those of the other groups. Figure 4 shown.

[0066] 2.2 Detection of immune protection rate after artificial bacterial infection After 4 weeks of observation, the immunized tilapia were inoculated with Streptococcus agalactiae (bacterial content was 1×10 8 Each group was challenged with bacterial solution (100 μL / tail) by intraperitoneal injection. The fish were observed for 14 consecutive days after challenge and the mortality of tilapia was recorded.

[0067] The results show that ( Figure 5 ), 4 weeks after injection of tilapia, the relative survival rates (RPS) of tilapia against Streptococcus agalactiae by Eno full-length protein vaccine and three segmented vaccines rE1 / rE2 / rE3 were 31.03%, 32.11%, 12.72% and 38.58%, respectively, indicating that the segmented vaccine rE3 had the best immune effect. It was speculated that it was the dominant antigenic region of Eno and was selected for the next experiment.

[0068] The amino acid sequence of rE3 is shown in SEQ ID NO.6: MELVNKYPIITIEDGMDENDWDGWKALTERLGGRVQLVGDDFFVTNTDYLARGIKEEAANSILIKVNQIGTLTETFEAIEMAKEAGYTAVVSHRSGETEDSTIADIAVATNAGQIKTGSLSRTDRIAKYNQLLRIEDQLGEVAQYKGIKSFYNLKK; The nucleotide sequence encoding rE3 is shown in SEQ ID NO.7: ATGGAATTGGTTAACAAATACCCAATCATCACTATTGAAGATGGTATGGATGAAAACGACTGGGATGGTTGGAAAGCTCTTACAGAACGCCTTGGTGGACGTGTACAATTAGTTGGTGACGATTTCTTCGTTACAAATACTGATTACTTAGCACGTGGTATCAAAGAAGAAGCTGCTAACTCAATCCTTATCAAAGTTAACCAAATCGGTACTTTGACAGAAACTTTCGAAGCCAT TGAAATGGCTAAAGAAGCAGGTTACACTGCGGTTGTATCACACCGTTCAGGTGAAACTGAAGATTCAACAATCGCTGATATCGCAGTTGGTCAAATTAAAACAGGTTCATTATCACGTACTGACCGTATTGCTAAATACAACCAATTACTTCGTATCGAAGACCAACTTGGTGAAGTTGTCAATACAAAGGTATCAAATCATTCTACAACTTAAAAAAATAA. Example 3

[0069] 3 Preparation of Nanocarrier Vaccines 3.1 Preparation of bacterial nanocellulose delivery system (1) Carboxylation of bacterial cellulose: 400 mL of bacterial cellulose solution (concentration of 1 mg / mL, Chengdu Starding Biotechnology Co., Ltd.) was mixed with 60 mg of TEMPO, 400 mg of sodium bromide and 10 mL of sodium hypochlorite, stirred at room temperature for 12 h (control the pH at around 10.5), and an appropriate amount of anhydrous ethanol was added to terminate the reaction.

[0070] (2) Preparation of carboxylated bacterial nanocellulose BNC: The above reaction solution was filtered with distilled water to a pH of ≤ 8, and 70% concentrated sulfuric acid was prepared and mixed with the filtered cellulose. The ratio of concentrated sulfuric acid to cellulose was 1:2. The mixture was heated and stirred at 50°C for 2 h to prepare carboxylated nanocellulose of relatively uniform size (nano size was mainly concentrated between 200-300 nm). Distilled water was added to terminate the reaction. The mixture was dialyzed (pH was close to neutral) and freeze-dried.

[0071] (3) Mix BNC, EDAC, and NHS in a mass ratio of 50:2:3 and stir in MES buffer (pH = 6.0) at room temperature for 4 h. Add an appropriate amount of mercaptoethanol to terminate the reaction.

[0072] 3.2 Preparation of subunit nanocarrier vaccines (1) Resuspend the purified rE / rE1 / rE2 / rE3 protein with an equal mass to BNC in phosphate buffer (pH = 7.2), then mix them in a ratio of protein resuspension: BNC reaction solution = 1:1 (V / V) and stir at room temperature for 4 h.

[0073] (2) The reaction product was dialyzed for 48 h using a dialysis bag with a cutoff of 10,000, and the product was freeze-dried using a freeze dryer to obtain a solid powder; (3) Add 20 mg of solid precipitate to 1 mL of PBS to make a protein suspension and store it at 4°C until use. Example 4

[0074] 4 Evaluation of the immune effect of subunit nanocarrier vaccines 4.1 ELISA test of serum antibody levels 450 healthy tilapia were collected and divided into 6 groups (75 per group), namely PBS, B, rE, rE3, B-rE and B-rE3. The fish were immersed in water containing vaccine solution (protein content was 20 mg / L), and the control group was replaced with saline instead of vaccine. After 8 hours of immersion immunization, they were transferred to normal aquaculture water. Blood was collected from each group of tilapia at 1, 2, 3 and 4 weeks, serum was separated and stored, and serum antibody levels were determined according to 2.1 as described above.

[0075] The results showed that the serum antibody levels of each vaccine group reached the highest level in the third week after immunization. The antibody levels of the B-rE3 group were significantly higher than those of the other groups at each week after immunization (p < 0.05). Figure 6 shown.

[0076] 4.2 Detection of immune protection rate after artificial bacterial infection After 4 weeks of observation, the immunized tilapia were inoculated with Streptococcus agalactiae (bacterial content was 1×10 8CFU / mL) and Streptococcus iniae (bacterial content was 1×10 9 Each group was challenged with bacterial solution (100 μL / tail) by intraperitoneal injection. The fish were observed for 14 consecutive days after challenge and the mortality of tilapia was recorded.

[0077] The results showed that after 4 weeks of immersion immunization of tilapia, the relative protection rates of subunit nanocarrier vaccine B-rE3 against Streptococcus agalactiae and Streptococcus iniae infection were 73.33% and 70.00%, respectively. Figure 7 shown.

[0078] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A segmented protein rE3 of the antigenic protein Eno of Streptococcus agalactiae, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

3.

2. A DNA molecule encoding the segmented protein rE3 according to claim 1, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

4.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the DNA molecule according to claim 2.

4. A recombinant strain, characterized in that: Comprising the recombinant expression vector according to claim 3.

5. Use of the segmented protein rE3 as claimed in claim 1 in preparing a vaccine for preventing tilapia streptococcal disease.

6. The vaccine according to claim 5, characterized in that The streptococcal disease of tilapia includes streptococcal disease caused by Streptococcus agalactiae and / or Streptococcus iniae.

7. A subunit vaccine for preventing tilapia streptococcal disease, characterized in that: The subunit vaccine comprises the segmented protein rE3 described in claim 1 and a nanocellulose carrier.

8. The method for preparing a subunit vaccine according to claim 7, characterized in that: The method comprises the following steps: mixing the segmented protein rE3 and the cellulose nano suspension for reaction, collecting the precipitate by centrifugation after the reaction, and dissolving the precipitate to prepare the nano carrier vaccine.

9. The preparation method according to claim 8, characterized in that: The cellulose nanosuspension comprises BNC, EDAC and NHS; the mass ratio of the BNC, EDAC and NHS is 50:2:3; and the BNC is carboxylated BNC.

10. The preparation method according to claim 8, characterized in that: The mass ratio of the segmented protein rE3 to the BNC in the cellulose nanosuspension is 1:1.

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