Recombinant protein rCgDM9CP-7 containing DM9 domain from Crassostrea gigas and its application

Abstract

The present invention belongs to the technical field of molecular biology and relates to a recombinant protein rCgDM9CP-7 from the Pacific oyster Crassostrea gigas containing a DM9 domain and its application. The amino acid sequence of the recombinant protein rCgDM9CP-7 from the Pacific oyster Crassostrea gigas containing a DM9 domain is shown in SEQ ID NO.1; an amino acid sequence having a homology of greater than or equal to 90% thereto. The application of the recombinant protein in preparing an agglutination preparation for Vibrio splendidus, Escherichia coli, Staphylococcus aureus or Bacillus subtilis to inhibit the growth of the strains.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and relates to a recombinant protein rCgDM9CP-7 containing a DM9 domain from the Pacific oyster (Crassostrea gigas) and its application. Background Art

[0002] DM9 is a novel pattern recognition domain initially discovered in Drosophila. Subsequently, a toxin containing a DM9 domain was found in fish. The N-terminus of this toxin contains a DM9 domain, and the C-terminus contains a bacterial toxin (ETX / MTX2) domain, which was named "Natterin". It can exert toxic effects on human cells, leading to cell necrosis, edema, and local swelling. In recent years, more and more proteins containing the DM9 domain (DM9-domain containing protein, DM9CP) have been discovered in arthropods and mollusks. For example, when the liver fluke parasitizes on the liver surface, the expression level of DM9 increases significantly, triggering an immune response in the body. The expression levels of DM9CPs in the lateral lobes of the salivary glands of Anopheles gambiae increase with the increase in the number of sporozoites infected after the invasion of Plasmodium falciparum. Studies have found that the Pacific oyster CgDM9CP-1 can recognize microbial and pathogen-associated molecular patterns (Pathogen-Associated Molecular Patterns, PAMPs) and has relatively strong binding activity to mannose. At the same time, it has been reported that CgDM9CP-1 has the ability to recognize and bind to strains, and CgDM9CP-2 can bind to D-Mannose, LPS, and PGN.

[0003] However, so far, there have been no reports on the recombinant protein containing the DM9 domain from the Pacific oyster as a preparation for inhibiting specific strains and its activity in recognizing specific PAMPs. Summary of the Invention

[0004] The present invention is to solve the above problems existing in the prior art and provides a recombinant protein rCgDM9CP-7 containing a DM9 domain from the Pacific oyster and its application.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] A recombinant protein rCgDM9CP-7 containing a DM9 domain from the Pacific oyster, the amino acid sequence of the recombinant protein rCgDM9CP-7 containing a DM9 domain from the Pacific oyster is as shown in SEQ ID NO.1, or an amino acid sequence with a homology of greater than or equal to 90% thereto.

[0007] A preparation method of a recombinant protein rCgDM9CP-7 containing a DM9 domain from the Pacific oyster, which is carried out in the following steps in sequence:

[0008] a. The coding region fragment of the CgDM9CP-7 gene of the oyster Crassostrea gigas was amplified by PCR using primers P1 and P2.

[0009] b. The PCR amplification product and the pET30a vector were digested with BamH I and Xhol I and then ligated with T4 ligase, transformed, and the recombinant was identified by sequencing.

[0010] c. The recombinant was transferred into the Escherichia coli Transetta(DE3) expression strain for induction culture, and then purified and refolded to obtain the recombinant protein rCgDM9CP-7 with the amino acid sequence in Sequence Listing SEQ ID NO.1.

[0011] The primer P1 is CGCGGATCCATGAAAAAGAAGGTAAAAAT;

[0012] The primer P2 is ACGCGTCGACCTACTTGATCTTACAGAGGA.

[0013] Use of the recombinant protein rCgDM9CP-7 containing the DM9 domain of the oyster Crassostrea gigas, the use of the recombinant protein rCgDM9CP-7 in recognizing interferon inducer (Poly(I:c)).

[0014] Use of the recombinant protein rCgDM9CP-7 containing the DM9 domain of the oyster Crassostrea gigas, the use of the recombinant protein rCgDM9CP-7 in preparing an agglutination preparation of Vibrio splendidus, Escherichia coli, Staphylococcus aureus or Bacillus subtilis to inhibit the growth of the strain.

[0015] Advantages of the present invention:

[0016] The recombinant protein rCgDM9CP-7 of the Pacific oyster (Crassostrea gigas) containing the DM9 domain in the present invention was cloned from a cDNA library of the Pacific oyster. rCgDM9CP-7 has the activity of binding to MAN, LPS, PGN and Poly(I:c). rCgDM9CP-7 can bind to the fungi Y. lipolytica, P. pastoris, the Gram-positive bacteria S. aureus, M. luteus and the Gram-negative bacteria E. coli, V. splendidus. rCgDM9CP-7 can significantly inhibit the growth of V. splendidus, E. coli and S. aureus, mediate the agglutination of V. splendidus, E. coli and B. subtilis and cause wrinkles on the cell surface. As an effective pattern recognition receptor, it has application value in the preparation of antibacterial drugs, new immune preparations and feed additives. Compared with CgDM9CP-2, CgDM9CP-7 can also bind Poly(I:c) in addition to binding to MAN, LPS and PGN. And it has stronger antibacterial ability against E. coli, V. splendidus and S. aureus. At the same time, CgDM9CP-7 can promote the agglutination of V. splendidus, E. coli and B. subtilis and cause wrinkles on the surface of B. subtilis. Brief Description of the Drawings

[0017] Figure 1 It is the detection effect diagram of the activity of the recombinant protein rCgDM9CP-7 of the Pacific oyster containing the DM9 domain binding bacteria in the embodiment of the present invention.

[0018] Figure 2 It is the detection effect diagram of the activity of the recombinant protein rCgDM9CP-7 of the Pacific oyster containing the DM9 domain binding PAMP in the embodiment of the present invention.

[0019] Figures 3 - 5 It is the detection diagram of the growth of V. splendidus, E. coli and S. aureus after incubation with the recombinant protein rCgDM9CP-7 of the Pacific oyster containing the DM9 domain in the embodiment of the present invention.

[0020] Figure 6 It is the electron microscope observation result of the morphological structure of V. splendidus, E. coli and B. subtilis after incubation with the recombinant protein rCgDM9CP-7 of the Pacific oyster containing the DM9 domain in the embodiment of the present invention. Detailed Embodiments

[0021] The following further illustrates the detailed embodiments of the present invention with reference to examples. It should be noted that the detailed embodiments described here are only for explaining and interpreting the present invention and are not limited to the present invention.

[0022] Example 1

[0023] The preparation method of the recombinant protein CgDM9CP-7 containing the DM9 domain from Crassostrea gigas is carried out in the following steps in sequence:

[0024] 1. Construction of the recombinant vector

[0025] In the embodiment of the present invention, the recombinant vector used is the prokaryotic expression vector pET-30a(+) of Novagen. By PCR technology, primers P1 and P2 with BamH I and Xhol I restriction enzyme cleavage sites added to the 5' ends respectively are used to amplify the coding region of Crassostrea gigas CgDM9CP-7 mRNA;

[0026] The primer P1 is CGCGGATCCATGAAAAAGAAGGTAAAAAT;

[0027] The primer P2 is ACGCGTCGACCTACTTGATCTTACAGAGGA.

[0028] The PCR reaction conditions are as follows: First, pre-denature at 94°C for 5 min, and then enter the following cycle: denature at 94°C for 30 s, anneal at 57°C for 30 s, extend at 72°C for 1 min, and perform 30 cycles in total. Finally, extend at 72°C for 10 min; the amplified fragment is purified and recovered by agarose gel electrophoresis, ligated with the pMD19-T vector; after transformation, positive clones are screened, plasmids are extracted, and the plasmids are double-digested with BamH I and Sal I; the target fragment is recovered and ligated with the expression vector pET-30a(+) digested with BamH I and Sal I to complete the construction of the recombinant plasmid.

[0029] 2. Expression of the recombinant protein rCgDM9CP-7

[0030] The constructed recombinant plasmid is transformed into the expression Escherichia coli Transetta(DE3). Single colonies are picked and inoculated into 400 mL of LB liquid medium, cultured at 220 rpm and 37°C until OD 600 = 0.4 - 0.8; IPTG (final concentration 1 mmol / L) is added, and the culture is continued for 12 h. Then, centrifuge at 4°C and 12,000×g for 5 min to collect the bacterial cells, and store them at -80°C for later use; at the same time, take 1 mL of the bacterial liquid, centrifuge, discard the supernatant, add 80 μL of water and 20 μL of 5× protein loading buffer, boil at 99°C for 10 min, centrifuge slightly, and detect the expression product by SDS-PAGE.

[0031] 3. Purification and renaturation of the recombinant protein rCgDM9CP-7

[0032] The expressed product was purified using a nickel agarose gel FF column to obtain a denatured recombinant protein, which was renatured by dialysis with a dialysis buffer. The specific operation steps are as follows:

[0033] (1) Pack the nickel agarose gel FF column, 1.6×20 cm, and the column bed volume is 10 mL;

[0034] (2) Equilibrate with Buffer I (50 mmol / L Tris-HCl buffer, pH 9.0, 50 mmol / L NaCl, 8 mol / L urea) for 2 - 5 bed volumes at a flow rate of 2 mL / min;

[0035] (3) Take the IPTG-induced expression cells, resuspend them with Buffer I, sonicated at 150 W for 30 min, centrifuge at 12,000×g for 30 min at 4°C. After filtering the supernatant with a 0.45 μm filter membrane, load it onto the column at a flow rate of 1 mL / min;

[0036] (4) Wash with Buffer I for another 2 - 5 bed volumes at a flow rate of 2 mL / min;

[0037] (5) Wash with Buffer I containing 50 mmol / L imidazole for another 2 - 5 column bed volumes at a flow rate of 2 mL / min;

[0038] (6) Elute the target protein with Buffer I containing 400 mmol / L imidazole and collect it;

[0039] (7) Detect the expression of the fusion protein by SDS-PAGE;

[0040] (8) Wash with pure water for 5 column bed volumes, then wash with 20% ethanol for 3 column bed volumes at a flow rate of 2 mL / min. The column is stored at 4°C. To purify the recombinant protein in the denatured state, urea needs to be removed by dialysis in the renaturation buffer to allow the protein to refold correctly and regain the correct conformation. The denatured purified product is renatured by dialysis with 2 mM reduced glutathione, 0.4 mM oxidized glutathione, 1 mM EDTA, 50 mM Tris-HCl, 100 mM NaCl, 10% glycerol, 1% glycine and gradually decreasing urea. The urea concentration starts from 6 M and is gradually replaced to 4 M, 3 M, 2 M, 1 M, 0 M. Glycerol is not added in the last dialysis when there is no urea dialysis buffer. Each dialysis is carried out at 4°C for 12 h, and the recombinant protein rCgDM9CP-7 containing the DM9 domain of Crassostrea gigas is obtained. The amino acid sequence of the recombinant protein rCgDM9CP-7 containing the DM9 domain of Crassostrea gigas is shown in SEQ ID NO.1.

[0041] SEQ ID NO.1:

[0042] MKKKVKMAVWVTTTGCHIPEHAIRAGYEADGRPLFIARASMEGTLTPGKCGFHLPGAHIPYGCKENIAHQYEVLVHPNNAQGFYDWQRAADGNVPEHALKTDTDTYVGRAYFSGSLVPCKIATSSPHMCAYMGYGGKEHNTKDYEVLCKIK-

[0043] Length: 152 amino acids

[0044] Type: Amino acid

[0045] Chain type: Single chain

[0046] Characteristics: The molecular weight is 16.67 kDa, the isoelectric point is 8.49, and it has two DM9 domains.

[0047] Experimental Example 2: Detection of the binding activity of the recombinant protein rCgDM9CP-7 containing the DM9 domain from the Pacific oyster Crassostrea gigas to bacteria

[0048] Based on the Western blotting method, the binding activities of the recombinant protein rCgDM9CP-7 to two Gram-negative bacteria (Vibrio splendidus and Escherichia coli), two Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus), and two fungi (Yarrowia lipolytica and Pichia pastoris) were detected. The sources of the bacterial strains used are as follows: Vibrio splendidus JZ6 was purchased from the Beijing Microbial Culture Collection Center, Escherichia coli was purchased from TransGen Biotech Co., Ltd., Staphylococcus aureus was purchased from the Beijing Microbial Culture Collection Center, Micrococcus luteus was purchased from the Beijing Microbial Culture Collection Center, Yarrowia lipolytica was purchased from the Beijing Microbial Culture Collection Center, and Pichia pastoris GS115 was purchased from Invitrogen.

[0049] The specific operations are as follows:

[0050] (1) The above 7 microorganisms were cultured overnight. The culture methods are as follows: Micrococcus luteus and Escherichia coli were cultured in LB medium at 37 °C for 20 h, Staphylococcus aureus was cultured in LB medium at 28 °C for 20 h, Vibrio splendidus and Yarrowia lipolytica were cultured in 2216E medium at 28 °C for 20 h, and Pichia pastoris was cultured in YPD medium at 28 °C for 20 h;

[0051] (2) Centrifuge the obtained bacterial solutions above to collect the bacterial cells, resuspend them with TBS buffer, and adjust the bacterial concentration to 1×10 8 CFU / mL;

[0052] (3) Pipette 100 μL of each microbial suspension and mix it with an equal volume of the recombinant protein rCgDM9CP-7 prepared in the above examples, and incubate with rotation at room temperature for 30 min;

[0053] (4) Centrifuge at 10,000×g for 2 min to collect the bacterial cells, and wash the bacterial cells four times with TBS buffer;

[0054] (5) After washing, collect the bacterial cells and resuspend them with 40 μL of sterile water;

[0055] (6) Add 10 μL of 5× protein electrophoresis buffer, heat at 99 °C for 10 min, and separate the protein samples by SDS-PAGE electrophoresis;

[0056] (7) After electrophoresis, remove the gel, cut out NC membranes and filter papers of the same size, and immerse them in the electrotransfer buffer and let stand for 10 min;

[0057] (8) Place the filter paper, NC membrane, gel, and filter paper into the electrotransfer apparatus in order from top to bottom, set the corresponding current according to the area of the gel block, and transfer the membrane for 25 min;

[0058] (9) Take out the NC membrane and wash it three times with TBS buffer, 5 min each time;

[0059] (10) Wash it three times with buffer TBST, 5 min each time;

[0060] (11) Place the NC membrane into 5% skim milk powder (dissolved in TBST) and block it at room temperature for 2 h;

[0061] (12) Take out the NC membrane and wash it three times with TBST buffer, 5 min each time;

[0062] (13) Immerse the NC membrane in the His-tag monoclonal antibody (purchased from Sangon Biotech, Shanghai) solution diluted in proportion (1:2000 (v / v)) (5% skim milk powder, TBST buffer) and incubate at room temperature for 1 h;

[0063] (14) Take out the NC membrane and wash it three times with TBST buffer, 5 min each time.

[0064] (15) Place the NC membrane into the goat anti-mouse HRP secondary antibody (purchased from Sangon Biotech, Shanghai) solution diluted in proportion (1:2000 (v / v)) (5% skim milk powder, TBST buffer) and incubate at room temperature for 1 h;

[0065] (16) Take out the NC membrane and wash it three times with TBST buffer for 10 min each time;

[0066] (17) Develop using the ECL method and record the Western blotting results with an imager, as Figure 1 shown.

[0067] The results showed that the recombinant protein rCgDM9CP-7 containing the DM9 domain from the Pacific oyster (Crassostrea gigas) in the examples of the present invention had different degrees of binding activity to Gram-negative bacteria, Gram-positive bacteria and fungi, and its binding activities to Vibrio splendidus, Escherichia coli, Staphylococcus aureus and Micrococcus luteus were higher than those to Pichia pastoris and Yarrowia lipolytica. There were no obvious bands in the rTrx negative control group.

[0068] Experimental Example 3: Detection of the sugar-binding activity of the recombinant protein rCgDM9CP-7 containing the DM9 domain from the Pacific oyster (Crassostrea gigas) of the present invention

[0069] The binding of the recombinant protein rCgDM9CP-7 containing the DM9 domain from the Pacific oyster (Crassostrea gigas) of the present invention to various PAMPs was detected by enzyme-linked immunosorbent assay (ELISA). The four sugars MAN, LPS, PGN and Poly(I:c) used were all purchased from Sigma.

[0070] The specific operation steps are as follows:

[0071] (1) Dissolve the four sugars MAN, LPS, PGN and Poly(I:c) with the coating solution (pH 7.6) prepared from Na2CO3 (15 mmol / L) and NaHCO3 (35 mmol / L) respectively and adjust the concentration to 125 μg / mL. Then add 100 μL to each well of the enzyme-linked immunosorbent assay (ELISA) plate and incubate overnight at 4 °C;

[0072] (2) Discard the coating liquid and wash 4 times with TBS-T for 4 min each time;

[0073] (3) After washing, add 250 μL of 3% BSA to the wells and incubate at 37 °C in a constant temperature incubator for 1 h;

[0074] (4) After blocking, repeat step 2;

[0075] (5) Add 100 μL of recombinant protein rCgDM9CP-7 at different concentrations (15.625, 31.25, 62.5, 125, 250 and 500 μg / mL) to each well (add rTrx protein to the negative control and add TBS to the control wells), and incubate at room temperature for 2 h;

[0076] (6) Repeat step (4);

[0077] (7) Add 100 μL of His-tagged primary antibody to each well at a ratio of 1:1,000 (v / v), and incubate at 37 °C for 1 h;

[0078] (8) Same as step (2);

[0079] (9) Replace the antibody in step (7) with HRP-conjugated secondary antibody and incubate in a 37 °C incubator for 1 h;

[0080] (10) Wash each well 5 times with TBST, 3 min each time;

[0081] (11) After developing the color with TMB chromogenic solution for about 30 min, add 45.65 μL of stop solution (concentration: 2 M HCl) to stop the color development, and then measure the OD 595 value, and the results are as Figure 2 shown.

[0082] The results showed that the recombinant protein rCgDM9CP-7 of the present invention has binding activity with MAN, LPS, PGN, and Poly(I:c), and the recombinant protein rCgDM9CP-7 has the strongest affinity with MAN and LPS (P / N = 7.45; 6.44), followed by the affinity with PGN (P / N = 5.10) and the affinity with Poly(I:c) (P / N = 4.21). And the affinity of the recombinant protein rCgDM9CP-7 with MAN, LPS, PGN, and Poly(I:c) is enhanced with the increase of the concentration of the recombinant protein rCgDM9CP-7. The negative control rTrX has no binding activity with the above four sugars.

[0083] Experimental Example 4: Detection of the activity of the recombinant protein rCgDM9CP-7 containing the DM9 domain of the present invention in inhibiting bacterial growth (growth curve method)

[0084] After incubating rCgDM9CP-7 by the growth curve method, the growth of the Gram-negative bacteria V. splendidus, E. coli and the Gram-positive bacteria S. aureus was detected.

[0085] The sources of the above-mentioned bacterial strains are as above.

[0086] The specific operations are as follows:

[0087] (1) The experimental bacteria were prepared into a bacterial suspension with a concentration of about 4×10 4 cells / mL with TBS solution;

[0088] (2) Take 100 μL of the recombinant protein rCgDM9CP-7 and mix it with 100 μL of the bacterial suspension, and incubate at room temperature for 2 h;

[0089] (3) After incubation, centrifuge at 10,000×g for 2 min to collect the bacterial cells, and wash them 3 times with buffer TBS;

[0090] (4) Add 20 μL of the test bacterial solution and 200 μL of 2216E or LB culture medium into a 96-well microplate. At the same time, set up negative control wells (rTRX) as controls, with a total volume of 220 μL per well;

[0091] (5) Incubate at an appropriate temperature in a microplate reader for 10 - 12 h until reaching the plateau phase, and read the OD 600 value every half hour;

[0092] (6) Plot the growth curves of each detected bacterium for comparison. The results are as Figures 3 - 5 shown.

[0093] The results showed that the recombinant protein rCgDM9CP-7 containing the DM9 domain from the Pacific oyster in the examples of the present invention significantly inhibited the growth of Vibrio splendidus, Escherichia coli, and Staphylococcus aureus at 11 h and 2.5 h compared with the control rTrx protein.

[0094] Experimental Example 5: Electron microscopy detection of the bacterial agglutination activity of the recombinant protein rCgDM9CP-7 containing the DM9 domain from the Pacific oyster of the present invention

[0095] Some proteins can bind to the cell wall of bacteria, causing agglutination and damage to varying degrees. The Gram-negative bacteria V. splendidus, E. coli, and the Gram-positive bacterium B. subtilis were detected by electron microscopy observation.

[0096] Bacillus subtilis was purchased from the Beijing Center for Microbial Culture Collection and cultured in LB medium at 28 °C for 20 h. The sources of the other bacterial strains were as above; then the experiment was carried out, and the following operations were performed using Bacillus subtilis as an example.

[0097] The specific operations are as follows:

[0098] (1) Bacillus subtilis was cultured in LB medium at 28 °C for 20 h. Subsequently, the experimental bacterial solution was prepared into a bacterial suspension with a concentration of about 4×10 4 cells / mL with TBS solution;

[0099] (2) Take 3 mL of the recombinant protein rCgDM9CP-7 and mix it with 3 mL of the bacterial suspension. At the same time, set up a negative control (rTRX) and incubate at room temperature for 3 h;

[0100] (3) After incubation, centrifuge at 5,000×g for 2 min to collect the bacterial cells, and wash them 3 times with buffer TBS;

[0101] (4) After discarding the supernatant, the precipitated bacterial sludge was fixed with 2.5% glutaraldehyde for 12 h;

[0102] (5) After fixation, it was washed 3 times with phosphate buffer at 5,000×g;

[0103] (6) Subsequently, it was soaked in 1% osmium tetroxide for 5 h and washed 3 times with buffer at 5,000×g;

[0104] (7) Dehydration with gradient ethanol, once each for 30%, 50%, 70%, 85%, 95% and twice for 100% ethanol, 20 min each time;

[0105] (8) Replacement with isoamyl acetate twice, 20 min each time;

[0106] (9) Fold the qualitative filter paper in half to form a small paper bag, staple one end firmly, forming a small pocket. Drop the centrifugally concentrated bacterial solution into the small paper bag, seal it with staples, and immediately put it into the sample chamber of the critical point dryer for CO2 critical point drying;

[0107] (10) After drying, cut open the filter paper bag, pour the dried powdery pure bacteria into a petri dish. Stick one side of the carbon conductive tape on a 1 / 4 cover glass, gently press the other side upside down on the bacterial powder, and after turning it over, gently scrape and flatten the bacteria with forceps. After ion sputtering with gold, perform scanning electron microscopy observation. The results are as Figure 6 shown.

[0108] The results showed that compared with the control rTrx protein, the recombinant protein rCgDM9CP-7 containing the DM9 domain in the oyster Crassostrea gigas in the embodiment of the present invention showed obvious agglutination effects on Vibrio splendidus and Escherichia coli, and the surface of the bacteria was rough and viscous, while obvious wrinkling phenomena appeared on the surface of Bacillus subtilis.

Claims

1. A recombinant protein rDM9CP-7 containing the DM9 domain from the Pacific oyster (Crassostrea gigas), characterized in that: C Recombinant protein r containing DM9 domain from Crassostrea gigas C DM9CP-7, its amino acid sequence is shown in SEQ ID NO.

1. ​ 2. A preparation method of the recombinant protein rDM9CP-7 of Crassostrea gigas containing the DM9 domain as described in claim 1, characterized in that C Follow the steps below: ​ a. Use primers P1 and P2 to perform PCR amplification on the coding region fragment of the DM9CP-7 gene of Crassostrea gigas C and obtain the PCR amplification product of the coding region fragment of the DM9CP-7 gene b. The PCR amplification product and the pET30a vector are digested with Bam HI and Xhol I, and then ligated with T4 ligase, transformed, and the recombinants are identified by sequencing; c. Transfer the recombinant into Escherichia coli Transetta (DE3) expression strain for induced culture, and then purify and refold it to obtain the recombinant protein r with the amino acid sequence in Sequence Listing SEQ ID NO.1 C DM9CP-7; The primer P1 is CGCGGATCCATGAAAAAGAAGGTAAAAAT; Primer P2 is ACGCGTCGACCTACTTGATCTTACAGAGGA.

3. Use of the recombinant protein r containing the DM9 domain in the oyster gigas as described in claim 1 C for DM9CP-7, characterized in that: The application of the recombinant protein r C DM9CP-7 in identifying interferon inducers.

4. Use of the recombinant protein r C containing the DM9 domain in Crassostrea gigas as claimed in claim 1, DM9CP-7, characterized in that: The recombinant protein r C Application of DM9CP-7 in preparing an agglutination preparation of Vibrio splendidus, Escherichia coli or Bacillus subtilis to inhibit the growth of strains.

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