Vibrio harveyi recombinant outer membrane protein OmpK and application thereof

Abstract

The invention discloses a vibrio harveyi recombinant outer membrane protein OmpK and application thereof.The recombinant outer membrane protein OmpK has high immunogenicity and can effectively stimulate immune response of fishes, induce generation of specific serum antibodies aiming at vibrio harveyi and remarkably improve the activity of catalase, superoxide dismutase, acid (alkali) phosphatase and lysozyme. The vaccine is an efficient and safe vaccine for preventing or treating vibrio harveyi infection of fishes, and solves the problems of unstable protection effect, easy secondary infection, strong virulence and the like of the existing inactivated vaccine and attenuated vaccine. The recombinant protein and the biological material can be used for preparing products for preventing or treating diseases caused by Vibrio harveyi infection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant outer membrane protein OmpK of Vibrio harveyi and an application thereof. Background Art

[0002] Vibrio harveyi, also known as Vibrio harveyi, is a common pathogen in the ocean. It was first reported in 1936 and is one of the most important diseases of vibriosis. Fish infected with Vibrio harveyi have severe liver lesions, varying degrees of damage to the renal tubules and renal parenchyma, damage to the hematopoietic system leading to loss or decline of function, skin ulceration and bleeding, and pathogens secrete a large amount of hemolytic toxins. At present, the treatment of bacterial diseases is still mainly based on antibiotics. The bacterial resistance caused by long-term use of antibiotics and the problem of drug residues in aquatic products cannot be ignored. Immune prevention and control is to improve the body's resistance to diseases by stimulating the body's own immune defense system, which can largely avoid the problems of bacterial resistance and drug residues, and has a good disease prevention effect. Therefore, the research and development and application of fish vaccines have received widespread attention.

[0003] At present, vaccines are mainly divided into traditional vaccines and new vaccines. Traditional vaccines include live vaccines, inactivated vaccines and subunit vaccines; new vaccines include genetically engineered subunit vaccines, anti-idiotypic vaccines, synthetic peptide vaccines, live vector vaccines, gene-deleted attenuated live vaccines, genetically modified plant vaccines and nucleic acid vaccines. Attenuated vaccines have good immune effects, but they are inconvenient to store and transport, and there may also be problems with virulence reversion. DNA vaccines cannot be used in the short term due to problems such as high technical difficulty, unstable antigen expression, low protection rate and high immunization cost. Subunit vaccines and genetically engineered vaccines have received widespread attention due to their low toxicity and good immune effects.

[0004] Genetically engineered subunit vaccines, also known as recombinant subunit vaccines, refer to vaccines made by using recombinant DNA technology to construct prokaryotic expression vectors, express antigen genes, and purify recombinant expression products. It has the characteristics of high yield, high purity, and good stability. At present, subunit vaccines are mainly recombinant prokaryotically expressed pathogen surface proteins. Pathogen surface proteins are antigens that can be recognized by the host immune system, can induce the host's immune response, produce specific immune antibodies, and prevent the appearance of pathogen infection symptoms.

[0005] Outer membrane proteins (OMPs) are core components of bacterial proteins. They are present in Neisseria gonorrhoeae, Haemophilus influenzae, Treponema pallidum and other pathogens and are highly conserved. They are common surface antigens and vaccine candidates. Outer membrane proteins have been proposed as candidate vaccine molecules as early as the twentieth century, and the Omp85 protein family has been shown to be essential for the assembly of the bacterial outer membrane, assembling β-barrel protein substrates into the outer membrane. In addition, bacteria can secrete a variety of substrates, such as small molecules and proteins toxic to the host, through several secretion systems. These secretion systems are also mainly composed of OMPs, and their adhesion function has been well demonstrated in several fish pathogens (such as Aeromonas and Vibrio). Bacterial adhesion to host cells mediated by Omps can stimulate the host's immune response, because the host's immune defense system can easily recognize the outer membrane components as foreign substances, thereby inducing the upregulation of proinflammatory cytokines. These findings indicate that OMPs play an important role in both bacterial invasion and the stimulation of host immune responses. At the same time, OMPs also carry pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), which can be recognized by pathogen recognition receptors (PRRs) on host cells (such as monocytes, macrophages, neutrophils and dendritic cells), which are involved in antigen uptake, processing and presentation to adaptive immune system cells to induce long-term protective immunity. In summary, OMPs can serve as ideal candidate vaccines for intracellular or extracellular bacteria prevalent in aquaculture. However, there is currently no recombinant subunit vaccine for the outer membrane protein of Harveyella on the market. Summary of the invention

[0006] Based on this, the present invention provides a recombinant outer membrane protein OmpK of Vibrio harveyi and its application, which solves the problems in the above-mentioned background technology.

[0007] One of the technical solutions adopted by the present invention to solve the technical problem is: a recombinant protein, wherein the recombinant protein is the recombinant outer membrane protein OmpK of Vibrio harveyi, and the amino acid sequence thereof is shown in SEQ ID NO:1.

[0008] The second technical solution adopted by the present invention to solve the technical problem is: providing the above-mentioned recombinant protein-related biological material, which is any one of the following A1) to A8):

[0009] A1) a nucleic acid molecule encoding the above recombinant protein;

[0010] A2) an expression cassette containing the nucleic acid molecule described in A1);

[0011] A3) a recombinant vector containing the nucleic acid molecule described in A1),

[0012] A4) a recombinant vector containing the expression cassette described in A2);

[0013] A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

[0014] A6) a recombinant microorganism containing the expression cassette described in A2);

[0015] A7) a recombinant microorganism containing the recombinant vector described in A3);

[0016] A8) A recombinant microorganism containing the recombinant vector described in A4).

[0017] Preferably, the vector comprises an Escherichia coli, Bacillus subtilis, lactic acid bacteria or yeast expression vector.

[0018] Preferably, the recombinant microorganism comprises an engineered bacterium.

[0019] Preferably, the nucleic acid molecule sequence is shown as SEQ ID No.2.

[0020] The third technical solution adopted by the present invention to solve its technical problem is: to provide the use of the above-mentioned recombinant protein in the preparation of a product for preventing or treating Vibrio harveyi infection.

[0021] The third technical solution adopted by the present invention to solve its technical problem is: to provide the use of the above-mentioned biological material in the preparation of a product for preventing or treating Vibrio harveyi infection.

[0022] The present invention has the following beneficial effects:

[0023] The present invention performs genome sequencing and outer membrane protein library analysis on Vibrio harveyi isolated from diseased grouper in Fujian Province, selects OmpK gene from it, and constructs recombinant outer membrane protein OmpK, which has a molecular weight of 56KDa, has strong immunogenicity, can effectively stimulate the immune response of fish, OmpK can induce the production of specific serum antibodies against Vibrio harveyi, and can significantly improve the activities of catalase, superoxide dismutase, acid (base) phosphatase and lysozyme. It is an efficient and safe method for preventing or treating Vibrio harveyi infection in fish, and solves the problems of unstable protective effect, easy to cause secondary infection, and strong toxicity in current inactivated seedlings and attenuated seedlings. The recombinant protein and biomaterial of the present invention can be used to prepare products for preventing or treating diseases caused by Vibrio harveyi infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the sequencing depth distribution diagram of the M01 strain.

[0025] The horizontal axis represents the length of reads (bp); the vertical axis on the left represents the number of reads, corresponding to the green bar graph; the vertical axis on the right represents the total number of bases (Mb) contained in reads greater than the corresponding length, corresponding to the blue curve; the red dotted line represents the N50 length of reads.

[0026] Figure 2 This is the circle map of the genome of strain M01.

[0027] The outermost circle indicates the genome size, with each scale being 5 kb;

[0028] The second and third circles are genes on the positive and negative strands of the genome, respectively, and different colors represent different COG functional classifications;

[0029] The fourth circle is a repeating sequence;

[0030] The fifth circle is tRNA and rRNA, blue is tRNA, purple is rRNA;

[0031] The sixth circle is the GC content. The light yellow part indicates that the GC content of this region is higher than the average GC content of the genome. The higher the peak value, the greater the difference from the average GC content. The blue part indicates that the GC content of this region is lower than the average GC content of the genome.

[0032] The innermost circle is GC-skew, dark gray represents areas with greater G content than C, and red represents areas with greater C content than G.

[0033] Among them, A in the figure is the partial genome sequence Scaffold1 spliced ​​from short DNA fragments generated by genome sequencing;

[0034] B is the partial genome sequence Scaffold 2, which is spliced ​​from short DNA fragments generated by genome sequencing;

[0035] C is the partial genome sequence Scaffold 3, which is spliced ​​from short DNA fragments generated by genome sequencing;

[0036] D is the partial genome sequence Scaffold 4, which is spliced ​​from short DNA fragments generated by genome sequencing;

[0037] E is the partial genome sequence Scaffold 5, which is spliced ​​from short DNA fragments generated by genome sequencing;

[0038] F is the partial genome sequence Scaffold 6, which is spliced ​​from short DNA fragments generated by genome sequencing.

[0039] Figure 3 This is the ORF-TMH screening result of M01 strain.

[0040] Figure 4 This is the ORF-SCL screening result of M01 strain.

[0041] Figure 5 This is the result of the “double library” screening of the M01 strain.

[0042] Figure 6 It is the amplified product of the recombinant outer membrane protein OmpK gene of Vibrio harveyi.

[0043] M is the DNA molecular weight marker;

[0044] 1 is the OmpK gene amplification band.

[0045] Figure 7 The results of SDS-PAGE identification of recombinant outer membrane protein OmpK of Vibrio harveyi. A is the expression and purification diagram of OmpK protein,

[0046] M in A: protein marker; 1: OmpK precipitate after crushing; 2: OmpK supernatant after crushing; 3: washing solution; 4-8: OmpK elution

[0047] B is Western-blot detection of OmpK immunogenicity,

[0048] M in B: protein marker; OmpK: OmpK specifically binds to rabbit anti-grouper IgM antibody

[0049] Figure 8 Survival rate of grouper after intraperitoneal injection of Vibrio harveyi.

[0050] Fig. 9 The survival rate after intraperitoneal injection of Vibrio harveyi after OmpK immunization.

[0051] Fig.10 For histopathological analysis of grouper.

[0052] Fig.11 The serum antibody titer was determined by indirect ELISA method.

[0053] Fig.12 These are the results of the activity measurements of A lysozyme, B superoxide dismutase, C alkaline phosphatase, D acid phosphatase, and E catalase in serum.

[0054] **, * indicate that there are statistically significant differences between the immune group and the GST protein control group (P<0.01, P<0.05)

[0055] Fig.13 The qRT-PCR method was used to detect the expression of head and kidney immune-related genes.

[0056] Among them, A is IL1β; B is IL10; C is TLR3; D is CD4; E is complement C3; F is MHCII; G is the gene expression level of IgM.

[0057] **, * indicate that there are statistically significant differences between the immune group and the GST protein control group (P<0.01, P<0.05)

[0058] Fig.14 The qRT-PCR method was used to detect the expression of immune-related genes in the spleen.

[0059] Among them, A is IL1β; B is IL10; C is TLR3; D is CD4; E is complement C3; F is MHCII; G is the gene expression level of IgM.

[0060] **, * indicate that there are statistically significant differences between the immune group and the GST protein control group (P<0.01, P<0.05)

[0061] Fig.15 The qRT-PCR method was used to detect the expression of liver immune-related genes.

[0062] **, * indicate that there are statistically significant differences between the immune group and the GST protein control group (P<0.01, P<0.05)

[0063] Among them, A is IL1β; B is IL10; C is TLR3; D is CD4; E is complement C3; F is MHCII; G is the gene expression level of IgM.

[0064] **, * indicate that there are statistically significant differences between the immune group and the GST protein control group (P<0.01, P<0.05)

[0065] Fig.16 The expression of intestinal immunity-related genes was detected by qRT-PCR.

[0066] Among them, A is IL1β; B is IL10; C is TLR3; D is CD4; E is complement C3; F is MHCII; G is the gene expression level of IgM.

[0067] **, * indicate that there are statistically significant differences between the immune group and the GST protein control group (P<0.01, P<0.05) DETAILED DESCRIPTION

[0068] In order to better understand the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, those skilled in the art understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0069] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0070] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0071] Example 1 Construction of recombinant outer membrane protein OmpK of Vibrio harveyi

[0072] 1. DNA extraction and whole genome sequencing analysis of Vibrio harveyi

[0073] The bacterial strain M-01 was provided by the Fujian Fisheries Research Institute and isolated from diseased grouper in the waters of Fujian Province. The bacterial liquid was streaked onto TCBS medium by the plate streaking method and then placed in a 28°C incubator overnight until a single colony grew. The single colony was picked and cultured in 5 ml of TSB liquid and cultured at 28°C and 200 rpm. The successfully grown strain was centrifuged and DNA was extracted from the precipitate using the Tiangen Bacterial DNA Extraction Kit and commissioned to Beijing Biomarker Biotechnology Co., Ltd. for whole genome sequencing. Genome analysis Prodigal, Infernal, and tRNAscan-SE were used to predict and annotate CDS, rRNA, and tRNA, respectively. RPS BLAST was used for homology group (COG) analysis. Antibiotic resistance genes and virulence factor encoding genes were identified by BLAST retrieval of the Comprehensive Antibiotic Resistance Database (CARD) and the Virulence Factor Database (VFDB) of Pathogenic Bacteria. The Circos v0.66 software was used to draw the genome circle map.

[0074] The GC content and average depth of read coverage of the assembled sequence were counted to analyze the GC bias and repetitive sequences of the genome. The analysis results are as follows Figure 1 As shown, the genome content distribution of strain M-01 tends to be consistent, the GC content is concentrated in the range of 40-50%, and there is no obvious horizontal block distribution in the figure, indicating that this data sample is not contaminated by other species.

[0075] Using the genome information obtained through assembly and prediction, such as tRNA, rRNA, repeat sequences, GC content, and gene function information, the Circos v0.66 software was used to draw genome circulograms (e.g. Figure 2 Scaffold 3 does not have rRNA, and Scaffold 5 does not have tRNA. The genome of Vibrio harveyi M-01 strain contains 2 chromosomes and 4 plasmids, of which 2 plasmids are newly discovered plasmids ( Figure 2 C and E in Figure 3), the lengths of the newly discovered plasmids were 163436 bp and 20244 bp, respectively.

[0076] In summary, the bacterial genome size was 505598104 bp, the genome GC content was 44.76%, and it encoded a total of 5325 genes. A total of 801 virulence genes were predicted, of which 502 virulence genes were on Scaffold2 and 299 virulence genes were on Scaffold1. No virulence genes were found on the newly discovered plasmid.

[0077] 2. Construction of the outer membrane protein library of Vibrio harveyi

[0078] TMHMM and Phobius were used to comprehensively analyze the proteins with signal peptides to identify sequences without transmembrane helical structures. Then, the lipoprotein and β-barrel transmembrane protein ORFs were identified using the online websites LipoP and PRED-TMBB to form the ORF-TMH library. At the same time, the ORFs with signal peptides were analyzed by PSORTb and Cell-Ploc to form the ORF-SCL library. The Vibrio harveyi outer membrane protein gene library was constructed by comprehensive double library screening. The successfully constructed outer membrane protein library was compared with the published Vibrio harveyi Omp gene by BLAST, and the sequences with higher similarity were selected and cloned using primers designed by Primer5.0.

[0079] like Figure 3 As shown in the figure, 612 ORFs with signal peptides were comprehensively predicted and analyzed by TMHMM and Phobius software, and 563 protein sequences without transmembrane helices were obtained. Lipoproteins and β-barrel transmembrane proteins were identified by LipoP and PRED-TMBB. Finally, 136 ORFs were screened to form the ORF-TMH library. Figure 4 As shown in the figure, 27 of the 612 ORFs with signal peptides were analyzed by PSORTb and 23 by Cell-Ploc, and a total of 29 ORFs were included in the ORF-SCL library. Figure 5 ) showed that there were 17 proteins that appeared in both ORF-TMH and ORF-SCL of the outer membrane protein library, and OmpK protein was screened for subsequent study.

[0080] 3. Construction of recombinant plasmid

[0081] The specific primers of OmpK gene sequence were designed by Primer5.0, and the restriction sites BamHI and SmaⅠ (italic part) were inserted into the upstream and downstream primers respectively; the primer sequences are shown in Table 1, which were synthesized by Shanghai Shenggong Biological Co., Ltd. PCR amplification was performed using the synthesized primers and the extracted bacterial DNA as templates, and the amplification parameters were as follows: 95℃ (denaturation), 60, 58, 56℃ (annealing), 1min30s (extension), and the reaction was carried out for 40 consecutive cycles. The two restriction sites of SmaⅠ and BamHI were introduced into the PGEX-4T-2 vector by double restriction digestion technology, and the PCR product was connected to the double-digested vector at 16℃ overnight. SolutionA ligase was used in the connection process to obtain the recombinant plasmid. The recombinant plasmid was transferred into E. coli DH5α, and the bacterial solution was spread on the ampicillin fixed medium and placed in a 37°C constant temperature incubator for culture. After 16 hours, a single colony was grown and picked up. A sterilized toothpick was used to pick up a single colony for bacterial inspection. The bacterial solution that successfully tested was sent for testing. After successful comparison, an equal volume of 50% glycerol was added for seed preservation. At the same time, the recombinant plasmid PGEX-4T-2-Omp was double-digested with BamHⅠ and SmaⅠ for verification. After verification, the recombinant plasmid was transformed into the host bacteria BL21 (DE3) competent state as an engineering strain.

[0082] Table 1 Primers for amplification of outer membrane protein gene of Vibrio harveyi

[0083]

[0084]

[0085] Using the genomic DNA of Vibrio harveyi as a template, PCR amplification was performed to obtain the 30KD outer membrane protein gene. The results showed that the size of the 30KD outer membrane protein gene was 780bp, which was consistent with the expected size ( Figure 6 The sequencing results showed that the coding sequence of the Vibrio harveyi outer membrane protein gene was correct (its nucleotide sequence is shown in SEQ ID NO: 2), indicating that the gene was amplified correctly.

[0086] IV. Expression and purification of recombinant proteins

[0087] The recombinant strain identified correctly by sequencing was inoculated into LB liquid medium containing 80 μg / mL ampicillin and cultured at 37°C with shaking at 180 rpm until OD 600 nm 0.6~0.8, add IPTG to a final concentration of 0.5mmol / L, induce at 16℃ for 6h, and set up positive clones without IPTG induction as controls. Ultrasonication in an ice bath, collect supernatant and precipitate after centrifugation, and perform SDS-PAGE electrophoresis to detect the expression of the target protein.

[0088] Protein purification was performed using the Bio-Tech GST-tagged protein purification kit. The purification steps are as follows: Add lysis buffer at a ratio of 4 ml per gram of wet weight of bacterial pellet to fully resuspend the bacteria. Add an appropriate amount of protease inhibitor cocktail (PMSF) to the lysis buffer. Add lysozyme to a final concentration of 1 mg / ml and mix well, place in an ice-water bath or on ice for 30 minutes. Ultrasonicate and lyse bacteria on ice. Ultrasonic power 200-300 W, ultrasonic treatment for 10 seconds each time, 10 seconds interval each time, for a total of 6 ultrasonic treatments. Centrifuge at 4°C 10,000g for 20-30 minutes, collect the supernatant of the bacterial lysate and place in an ice-water bath or on ice. 20 μl of the supernatant can be taken for subsequent detection. Take 1ml of the mixed BeyoGold GST-tag Purification Resin, centrifuge at 4℃ (1000g×10s), discard the storage solution, add 0.5ml lysis buffer to the gel to resuspend and balance the gel, centrifuge at 4℃ (1000g×10s), discard the liquid, repeat the balance 1-2 times, and discard the liquid. Add about 4ml of bacterial lysate supernatant to it, and shake slowly on a side-swing shaker or horizontal shaker at 4℃ for 60min. Load the mixture of lysate and BeyoGold GST-tag Purification Resin into the empty column tube of the affinity chromatography column provided in this kit. Open the lid at the bottom of the purification column, let the liquid in the column flow out under the action of gravity, and collect about 20 microliters of flow-through for subsequent analysis. Wash the column 5 times, adding 0.5-1ml lysis buffer each time, and collect about 20 microliters of the liquid that flows through the column each time for subsequent analysis and detection. Elute the target protein 6-10 times, using 0.5ml elution buffer each time. The eluted solution was collected into different centrifuge tubes. The concentration of the eluted protein was detected by ELISA at 595 nm and then the purification effect was detected by SDS-PAGE.

[0089] The recombinant protein was ultrasonically broken and analyzed by SDS-PAGE. Figure 7 A) in the figure shows that the target protein OmpK was found in both the supernatant and the precipitate, indicating that the protein was expressed in the form of soluble protein and inclusion body. At the same time, after His-tag affinity chromatography, the expression product showed a single band with a purity of more than 90%. Western-blot detection results ( Figure 7 B) in the figure shows that the antigen-antibody binding of the Vibrio harveyi specific antiserum shows the presence of a strong immunoreactive band at 56KD. These experimental results show that the prepared protein is an outer membrane protein of Vibrio harveyi and has strong immunogenicity.

[0090] The amino acid sequence of OmpK is shown in SEQ ID NO: 1:

[0091] SEQ ID NO: 1

[0092] The nucleotide sequence of the OmpK-encoding gene is shown in SEQ ID NO: 2 as follows: MRKSLLALSLLAATSAPVMAADYSDGDIHKNDYKWMQFNLMGAFDELPGESSHDYLEMEFGGRSGIFDLYGYVDVFNLTSDPGSDKAGAEKIFMKFAPRMSLDALTGKDLSFGPVQELYVSTLMEWGGASQVNTQKVGLGSDVMVPWFGKVGLNLYGTYDSNRKDWNGFQISTNWFKPFYFFENGSFISYQGYIDYQFGMKDEYTTASSGGAMFNGIYWHSDRFAVGYGLKGYKDIYGIKDTDAFKSTGFGHYLAVTYKF

[0093] SEQ ID NO: 2

[0094] ATGCGTAAATCACTTTTAGCTCTTAGCCTTCTAGCGGCTACTTCAGCTCCTGTAATGGCTGCTGACTACTCAGACGGCGACATCCACAAGAACGATTACAAGTGGATGCAATTCAACCTAATGGGTGCATTTGACGAACTTCCAGGTGAGTCTTCACACGACTACCTAGAAATGGAATTTGGCGGTCGCTCTGGT ATCTTCGACCTATACGGTTACGTTGACGTATTCAACCTAACTTCAGATCCAGGCAGCGACAAAGCTGGTGCAGAGAAGATCTTCATGAAGTTCGCACCACGTATGTCTCTAGACGCACTAACTGGTAAAGACCTATCTTTCGGTCCAGTTCAAGAGCTTTACGTATCTACTCTTATGGAGTGGGGTGGCGCTTCTC AAGTTAACACTCAAAAAGTAGGTCTTGGTTCTGACGTAATGGTTCCTTGGTTCGGTAAAGTAGGTCTTAACCTATACGGTACTTACGATTCAAACCGAAAGACTGGAACGGTTTCCAAATCTCGACTAACTGGTTCAAACCATTCTACTTCTTCGAGAACGGTTCATTCATTTCTTACCAAGGTTACATCGATTA CCAATTCGGTATGAAAGATGAATACACAACTGCTAGCAGTGGCGGCGCAATGTTCAACGGTATCTACTGGCACTCTGACCGCTTCGCAGTGGGTTACGGTCTAAAAGGTTACAAAGACATCTACGGTATCAAAGACACTGACGCATTCAAGTCTACTGGCTTTGGTCACTACCTAGCTGTAACTTACAAGTTCTAA

[0095] Example 2 Evaluation of the effect of recombinant outer membrane protein OmpK of Vibrio harveyi

[0096] 1. Preparation of rabbit anti-grouper specific antibodies and immunoblot analysis

[0097] Preparation of inactivated bacteria: Streak the bacteria stored at -80 °C onto TCBS medium, pick a single colony and culture it in TSB liquid medium to expand the culture. Adjust the bacterial solution to a concentration of 1×10 9CFU / ml, and formaldehyde was added at a final concentration of 0.2%. The bacterial solution was incubated at 180 rpm and 28°C for 24 hours. After inactivation, the formaldehyde inactivation effect of Vibrio harveyi was confirmed by inoculating it in a tryptic soy agar plate and incubating it in a 28°C incubator overnight.

[0098] Vibrio harveyi inactivated by 0.2% formaldehyde was injected into grouper, and a second immunization was performed 14 days later. Blood was collected from the caudal vertebrae 28 days later, and the serum was separated by centrifugation at 3000 rpm for 10 minutes after standing at 4°C overnight and then stored in a -80°C refrigerator.

[0099] The grouper IgM antibody was purified using GE HiTrap ProteinA HP1ml prepacked column. The purification steps were as follows: the thawed serum was taken out of the -80 refrigerator and centrifuged at 12000rpm for 10min to remove impurities and precipitation, diluted with an equal amount of PBS, filtered through a 0.22μm PES filter and placed in an ice box. The purification instrument used was an AKTA pure 25 protein purifier, and 20% anhydrous ethanol, distilled water, PBS, glycine-hydrochloric acid buffer (PH=2.8), a 10ml syringe, etc. were required.

[0100] The serum was loaded onto a 1ml pre-loaded column and incubated on a shaker at 10°C for 150min. Unbound proteins were then eluted with 10cv of PBS at a flow rate of 0.75ml / min, and 2ml was repeated for incubation for 150min. Unbound proteins were eluted with 20cvPBS, and bound proteins were finally eluted with glycine-HCl buffer, and the eluate was collected in steps of 200μl. To prevent the protein from being in an acidic environment for a long time, the pH was adjusted to 7.4-8.0 with Tris-HCL neutralizing solution. The protein concentration was determined with a Bradford kit and then freeze-dried for storage. Detection of grouper IgM purification effect: SDS-PAGE, the purified antibody was immunized with rabbits to prepare rabbit anti-grouper IgM antiserum.

[0101] The prepared antibodies were screened from the recombinant outer membrane proteins by Western-bot technology to select Omp with strong immunogenicity. After the protein was separated by 10% SDSPAGE separation gel, it was transferred to PVDF membrane by semi-dry method and blocked with PBST containing 5% skim milk powder at 4℃ for 2h; Vibrio harveyi antiserum (1:2000 dilution) was added and incubated overnight; washed once with PBST and twice with PBS, each time for 10min, and the prepared rabbit anti-grouper IgM antibody (1:5000 dilution) was added and reacted at room temperature for 2h; then incubated with goat anti-rabbit HRP at room temperature for 1h30min, and the membrane was washed 3 times (same as above); ECL color developer was added for color development and photography.

[0102] 2. Determination of the half-lethal concentration (LD50) of Vibrio harveyi to grouper

[0103] The healthy groupers were randomly divided into 5 groups, 18 in each group, and the injection volume was 100 μL. The concentration gradient was 2×10 4 CFU / mL, 2×10 5 CFU / mL, 2×10 6 CFU / mL, 2×10 7 CFU / mL and a control group. The mortality of fish was observed and recorded within 14 days.

[0104] The mortality of grouper caused by intraperitoneal injection of different concentrations of Vibrio harveyi within 14 days was statistically analyzed (as shown in Table 2). The results showed that in the control group, no deaths were recorded, that is, the survival rate was 100%. However, when infected with 2.0×10 7 cfu / mL of Vibrio harveyi resulted in acute mortality, resulting in a 0% survival rate for grouper. In contrast, a concentration of 2.0×10 6 , 2.0×10 5 and 2.0×10 4 The survival rates after infection with cfu / mL Vibrio harveyi were 43%, 52% and 75% respectively.

[0105] When the highest mortality rate reached 100% and the lowest mortality rate was ≠ 0%, the modified Koch method was used to treat 2×10 4 , 2.0×10 5 , 2.0×10 6 , 2.0×10 7 CFU / mL calculation of the median lethal concentration of Vibrio harveyi at four concentrations. Calculated according to the median lethal concentration formula: logLD 50 =Xk-i(Σp-(3-Pm-Pn) / 4)=7.30103-1×(2.17-(3-1-0.17) / 4)=5.59, and we get: LD 50 =10 5.59 =3.89×10 5 CFU / mL.

[0106] Table 2. Statistical analysis of mortality caused by Vibrio infection

[0107]

[0108] 3. Immunity and toxicity of Vibrio harveyi Omp to grouper

[0109] Preparation of subunit vaccine: Mix recombinant proteins of known concentrations and GST protein (control) samples with PBS to ensure that the concentration of each recombinant protein is 200μg / mL. Groupers with an average body weight of 30g±g were divided into 4 groups, including GST-tagged protein, Omp1, Omp2, and Omp3. Each group was intraperitoneally injected with 200μg / mL of GST-tagged protein and three outer membrane proteins. The initially purified Omp was diluted to 200μg / mL with PBS and then immunized with grouper. The control group was injected with the same concentration of GST-tagged protein. The second immunization dose on 14 days was 100μg / tail, and the immunization method was intraperitoneal injection. Blood and tissue samples were collected at 7, 14, 21, and 28 days. After 28 days, 20×LD 50 The mortality rate within 14 days of the challenge with Vibrio harveyi was counted. The fish that died during the challenge were sterilely dissected and the bacteria were isolated and cultured to determine whether the death was caused by the challenge strain.

[0110] Grouper in the immunized group and the GST control group were intraperitoneally injected with cultured Vibrio harveyi ( Fig. 9 ) to observe their survival rate. The GST protein immunization group began to die on the second day after the attack, and only 10% of the groupers survived after the eighth day. A small number of groupers in the two outer membrane protein immunization groups began to die from the fourth day, and maintained a survival rate of more than 50% after the ninth day. These results revealed that the recombinant outer membrane protein effectively stimulated the immune response of groupers.

[0111] Histopathological sections after challenge ( Fig.10 ), the results showed that compared with normal grouper tissue, the liver of the GST protein group after infection with bacteria had serious lesions and structural disorder. Edema and atrophy of hepatocytes led to widening of the interstitium and congestion of the hepatic veins. Compared with the control group, the liver necrosis and round vacuoles in the OmpK group were less.

[0112] 4. Serum antibody titer and enzyme activity detection

[0113] In the 1st to 4th week after immunization, 6 fish were randomly selected from each group, anesthetized with MS-222, and blood was collected from the tail vein. Serum was collected by centrifugation. The antibody titer level was detected by indirect ELISA. The serum was diluted in series and incubated with 1:5000 diluted rabbit anti-grouper IgM serum and 1:5000 diluted HRP-labeled goat anti-rabbit IgG at 37°C, and TMB substrate was added for color development, and the reading was read at 450nm.

[0114] The results of ELISA ( Fig.11 ) showed that OmpK can induce the production of specific serum antibodies against Vibrio harveyi. The level of specific antibodies in the serum of grouper vaccinated with OmpK vaccine began to increase significantly from the third week after vaccination and reached a peak in the fourth week.

[0115] Within 28 days after inoculation, the activities of catalase, superoxide dismutase, acid (alkaline) phosphatase and lysozyme in the sera of the immunized group were significantly higher than those in the GST protein control group ( Fig.12 ).

[0116] 5. Real-time fluorescence quantitative (RT-qPCR) detection of changes in expression of important immune-related genes in grouper

[0117] To evaluate the immune response of grouper after inoculation with recombinant protein vaccine, the expression levels of 7 immune-related genes in the liver, spleen, head kidney and intestine of grouper were detected at 7d, 14d, 21d and 28d after inoculation. Six fish were randomly selected from each group in the first to fourth weeks after immunization. After anesthesia, the head kidney, spleen and liver were removed with RNAse-free scissors and forceps and placed in a sterile EP tube filled with RNA protection solution and stored at -80℃. 30-50mg of frozen tissue was taken, and total RNA was extracted using RNA small-scale extraction kit (Tiangen). After the RNA concentration and purity were detected by NanoDrop2000, it was reverse transcribed into cDNA and stored at -20℃ for later use. RT-qPCR was used to detect the expression of interleukin-1β (IL-1β), interleukin 10 (IL-10), toll-like receptor 3 (TLR3), cluster of differentiation 4 (CD4), major histocompatibility complex II (MHCⅡ) and immunoglobulin M (IgM) genes in the liver, spleen and head kidney.

[0118] The total number of primers used is shown in Table 3. β-actin gene was used as internal control in all RT-qPCR experiments. The reaction conditions were: 95°C for 5 min; 95°C for 30 s, 60°C for 30 s and 72°C for 30 s, 40 cycles. After normalization to β-actin, the expression level of immune genes was calculated by 2-ΔΔCt method. All samples were performed in triplicate.

[0119] Table 3 Quantitative primers for grouper immune-related genes

[0120] Target gene Primer sequences 5′real-β-actin 5′-TTGTGCGTGACATCAAGGAG-3′ (such as SEQ ID NO: 5) 3′real-β-actin 5′-TCATTGCCGATGGTGATGAC-3′ (such as SEQ ID NO: 6) 5′real-IL1β 5′-AAGGAGGAAACTGTGAACGC-3′ (such as SEQ ID NO: 7) 3′real-IL1β 5′-TGTGCAGGTTGCTTTTCAGG-3′ (such as SEQ ID NO: 8) 5′real-IL10 5′-ACCATTGCTGTCGATTCGTG-3′ (such as SEQ ID NO: 9) 3′real-IL10 5′-TCCAAGTCGTCGTTTGCTTC-3′ (such as SEQ ID NO: 10) 5′real-TLR3 5′-AGGCCCCAAGTGTTCAAAAC-3′ (such as SEQ ID NO: 11) 3′real-TLR3 5′-TGTCACGAACCACAACATGC-3′ (such as SEQ ID NO: 12) 5′real-CD4 5′-ATCGTTGTGTTCGAGTCAGC-3′ (such as SEQ ID NO: 13) 3′real-CD4 5′-AAATCAACGCTGCTGGAAG-3′ (such as SEQ ID NO: 14) 5′real-complement C3 5′-ACAAACTGGACCGCGAAATC-3′ (such as SEQ ID NO: 15) 3′real-complement C3 5′-TTCAAATGCCTTGGCCTTGG-3′ (such as SEQ ID NO: 16) 5′real-MHCⅡ 5′-GTGGGTTGCTTTGAAACGG-3′ (such as SEQ ID NO: 17) 3′real-MHCⅡ 5′-TGGGCACAGTGTAAACAACC-3′ (such as SEQ ID NO: 18) 5′real-IgM 5′-TACAGCCTCTGGATTAGACATTAG-3′ (such as SEQ ID NO: 19) 3′real-IgM 5′-CTGCTGTCTGCTGTTGTCTGTGGAG-3′ (such as SEQ ID NO: 20)

[0121] qRT-PCR was used to analyze the expression profile of immune-related genes. Fig.13 , Fig.14 , Fig.15 , Fig.16 As shown in the figure, OmpK can significantly induce the expression of IL-1, IL10, TLR3, CD4, complement C3, MHCII and IgM in the head kidney, spleen, liver and intestine of grouper. The expression levels of each gene have similar trends, which are significantly increased from 7 to 21 days and significantly decreased to the lowest value at 28 days. In the head kidney group, the expression levels of the above genes after OmpK injection were higher than those in the OmpW group, and the levels of IL1 in the head kidney and spleen tissues reached a peak at 7 days.

[0122] In summary, the present invention successfully constructed a recombinant outer membrane protein OmpK of Vibrio harveyi, which has strong immunogenicity, can effectively stimulate the immune response of fish, induce the production of specific serum antibodies against Vibrio harveyi, and significantly improve the activity of catalase, superoxide dismutase, acid (base) phosphatase and lysozyme. It is an efficient and safe method for preventing or treating Vibrio harveyi infection in fish, and solves the problems of unstable protective effect, easy to cause secondary infection, and strong virulence existing in current inactivated and attenuated vaccines. The recombinant protein and biomaterial of the present invention can be used to prepare products for preventing or treating diseases caused by Vibrio harveyi infection.

[0123] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A recombinant protein, characterized in that The recombinant protein is the recombinant outer membrane protein OmpK of Vibrio harveyi, and its amino acid sequence is shown in SEQ ID NO:

1.

2. The biological material related to the recombinant protein according to claim 1 is any one of the following A1) to A8): A1) a nucleic acid molecule encoding the recombinant protein according to claim 1; A2) an expression cassette containing the nucleic acid molecule described in A1); A3) a recombinant vector containing the nucleic acid molecule described in A1), A4) a recombinant vector containing the expression cassette described in A2); A5) a recombinant microorganism containing the nucleic acid molecule described in A1); A6) a recombinant microorganism containing the expression cassette described in A2); A7) a recombinant microorganism containing the recombinant vector described in A3); A8) A recombinant microorganism containing the recombinant vector described in A4).

3. The biomaterial according to claim 2, characterized in that: The vector includes an expression vector of Escherichia coli, Bacillus subtilis, lactic acid bacteria or yeast.

4. The biomaterial according to claim 2, characterized in that: The recombinant microorganisms include engineered bacteria.

5. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule sequence is shown as SEQ ID No.

2.

6. Use of the recombinant protein according to claim 1 in the preparation of a product for preventing or treating Vibrio harveyi infection.

7. Use of the biological material according to claim 2 in preparing a product for preventing or treating Vibrio harveyi infection.

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