Recombinant protein rCgDM9CP-8 containing DM9 domain from Crassostrea gigas and its applications

Abstract

The present invention belongs to the technical field of molecular biology and relates to a recombinant protein rCgDM9CP-8 from the Pacific oyster (Crassostrea gigas) containing a DM9 domain and its application. The amino acid sequence is as shown in SEQ ID NO.1, or an amino acid sequence with a homology of greater than or equal to 90% thereto. For the specific preparation, the coding region fragment of the CgDM9CP-8 gene from the Pacific oyster was amplified by PCR using primers P1 and P2. The PCR amplification product and the pET30a vector were digested with BamH I and Sal I and then ligated by T4 ligase, followed by transformation and sequencing to identify the recombinant. 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-8 with the amino acid sequence in SEQ ID NO.1 in the sequence listing; the application of the recombinant protein rCgDM9CP-8 in recognizing an interferon inducer (Poly(I:c)).

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and relates to a recombinant protein rCgDM9CP-8 containing a DM9 domain from the Pacific oyster (Crassostrea gigas) and its application. Background Art

[0002] The Pacific oyster (Crassostrea gigas) is an important mariculture shellfish in China. The Pacific oyster lacks an adaptive immune system and mainly relies on the innate immune system to resist the invasion of external pathogenic microorganisms. The DM9 domain-containing proteins (CgDM9CPs) from the Pacific oyster are a new type of pattern recognition molecules identified in recent years. They can specifically recognize microorganisms and pathogen-associated molecular patterns (PAMPs) as pattern recognition receptors and participate in the processes of microbial phagocytosis, killing, and clearance. Aspartic acid and lysine in the DM9 domain play a key role in the specific binding of CgDM9CPs to MAN. Previous studies have found that CgDM9CP-2 can only bind to MAN, D-Mannose, LPS, and PGN. The CgDM9CP-1 protein only has recognition and binding activity for some strains.

[0003] However, so far, there have been no reports on the use of the DM9 domain from the Pacific oyster as a drug against Staphylococcus aureus, Bacillus subtilis, Vibrio splendidus, and Escherichia coli. 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-8 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:

[0006] A recombinant protein rCgDM9CP-8 containing a DM9 domain from the Pacific oyster, the amino acid sequence of the recombinant protein rCgDM9CP-8 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 method for preparing a recombinant protein rCgDM9CP-8 containing a DM9 domain from the Pacific oyster, which is carried out in the following steps in sequence:

[0008] a. PCR amplify the coding region fragment of the CgDM9CP-8 gene from the Pacific oyster using primers P1 and P2;

[0009] b. Digest the PCR amplification product and the pET30a vector with BamH I and Sal I, and then ligate them with T4 ligase, transform, and sequence and identify the recombinant.

[0010] 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 rCgDM9CP-8 with the amino acid sequence in Sequence Listing SEQ ID NO.1.

[0011] The P1 primer is CGCGGATCCATGGCAGTGTGGGTTACAACGA;

[0012] The P2 primer is ACGCGTCGACCTACTTAATCTTACAGAGGACT.

[0013] Application of the recombinant protein rCgDM9CP-8 containing DM9 domain from Crassostrea gigas, the application of the recombinant protein rCgDM9CP-8 in damaging Staphylococcus aureus, Bacillus subtilis, Vibrio splendidus or Escherichia coli to form wrinkles, thereby inhibiting the growth of the strains.

[0014] Application of the recombinant protein rCgDM9CP-8 containing DM9 domain from Crassostrea gigas, the application of the recombinant protein rCgDM9CP-8 in recognizing interferon inducer (Poly(I:c)).

[0015] Advantages of the present invention:

[0016] The recombinant protein rCgDM9CP-8 containing DM9 domain from Crassostrea gigas of the present invention is cloned from the cDNA library of Crassostrea gigas, can bind to various PAMPs such as MAN, PGN, LPS and Poly(I:c), and has relatively strong binding activity with MAN and PGN. Moreover, it also has broad-spectrum binding activity against fungi Y. lipolytica, P. pastoris, Gram-positive bacteria S. aureus, M. luteus and Gram-negative bacteria E. coli, V. splendidus. rCgDM9CP-8 can participate in mediating phenomena such as surface perforation of V. splendidus and E. coli and wrinkling of the cell surface of B. subtilis, and at the same time significantly inhibit the growth of V. splendidus, E. coli and S. aureus. Description of the drawings

[0017] Figure 1 It is the detection effect diagram of the activity of the recombinant protein rCgDM9CP-8 containing DM9 domain from Crassostrea gigas binding bacteria in the example of the present invention.

[0018] Figure 2 It is the detection effect diagram of the activity of the recombinant protein rCgDM9CP-8 containing DM9 domain from Crassostrea gigas binding PAMPs in the example of the present invention.

[0019] Figures 3 - 5 This is the activity detection effect diagram of the recombinant protein rCgDM9CP-8 containing the DM9 domain from the Pacific oyster Crassostrea gigas in inhibiting the growth of Vibrio splendidus, Escherichia coli, and Staphylococcus aureus in the embodiments of the present invention.

[0020] Figure 6 This is the electron microscope observation result of the morphological structure of Vibrio splendidus, Escherichia coli, and Bacillus subtilis after incubation with the recombinant protein rCgDM9CP-8 containing the DM9 domain from the Pacific oyster Crassostrea gigas in the embodiments of the present invention. Specific Embodiments

[0021] The following further describes the specific embodiments of the present invention in combination with examples. It should be noted that the specific embodiments described herein are only for explaining and interpreting the present invention and are not limited to the present invention.

[0022] Experimental Example 1 Preparation method of the recombinant protein CgDM9CP-8 containing the DM9 domain from the Pacific oyster Crassostrea gigas in the present invention is carried out in the following steps in sequence:

[0023] 1. Construction of recombinant vector

[0024] In the embodiments of the present invention, the recombinant vector used is the prokaryotic expression vector pET-30a(+) from Novagen. By PCR technology, primers P1 and P2 with BamH I and Xhol I restriction enzyme cleavage sites added at the 5' ends respectively are used to amplify the coding region of the Pacific oyster CgDM9CP-8 mRNA. The DNA sequence of primer P1 is shown in SEQ ID NO.2, and the DNA sequence of primer P2 is shown in SEQ ID NO.3.

[0025] The P1 primer is CGCGGATCCATGGCAGTGTGGGTTACAACGA;

[0026] The P2 primer is ACGCGTCGACCTACTTAATCTTACAGAGGACT.

[0027] 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; The recovered product is 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.

[0028] 2. Expression of recombinant protein rCgDM9CP-8

[0029] The constructed recombinant plasmid was transformed into Escherichia coli Transetta(DE3) for expression. Single colonies were picked and inoculated into 400 mL of LB liquid medium, and cultured at 37 °C with a rotation speed of 220 rpm until the OD 600 = 0.4 - 0.8; IPTG with a final concentration of 1 mmol / L was added, and the culture continued for 12 h. Then, the cells were centrifuged at 4 °C and 12,000×g for 5 min to collect the bacterial cells, which were stored at -80 °C for later use. At the same time, 1 mL of the bacterial liquid was centrifuged, the supernatant was discarded, 80 μL of water and 20 μL of 5× protein loading buffer were added, and the mixture was boiled at 99 °C for 10 min, slightly centrifuged, and the expression product was detected by SDS-PAGE.

[0030] 3. Purification and refolding of recombinant protein rCgDM9CP-8

[0031] The expression product was purified using a nickel-agarose gel FF column to obtain the denatured recombinant protein, which was refolded by dialysis with a dialysis buffer. The specific operation steps are as follows:

[0032] (1) Pack the nickel-agarose gel FF column, with a size of 1.6×20 cm and a column bed volume of 10 mL;

[0033] (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 column bed volumes at a flow rate of 2 mL / min;

[0034] (3) Take the cells induced by IPTG, resuspend them with Buffer I, disrupt them by ultrasonic wave at 150 W for 30 min, centrifuge at 12,000×g and 4 °C for 30 min, filter the supernatant through a 0.45 μm filter membrane, and then pass it through the column at a flow rate of 1 mL / min;

[0035] (4) Wash with Buffer I for another 2 - 5 column bed volumes at a flow rate of 2 mL / min;

[0036] (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;

[0037] (6) Elute the target protein with Buffer I containing 400 mmol / L imidazole and collect it;

[0038] (7) Detect the expression of the fusion protein by SDS-PAGE;

[0039] (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 in a 4°C environment. To purify the recombinant protein in a denatured state, urea needs to be removed by dialysis in a refolding buffer to allow the protein to refold correctly and regain its correct conformation. The denatured purified product is dialyzed and refolded 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 concentrations of urea. The initial urea concentration is 6 M and is gradually replaced with 4 M, 3 M, 2 M, 1 M, 0 M. Glycerol is not added in the last dialysis when using a urea-free dialysis buffer. Each dialysis is carried out at 4°C for 12 h, and the recombinant protein rCgDM9CP-8 containing the DM9 domain from the Pacific oyster Crassostrea gigas is obtained. The amino acid sequence of the obtained rCgDM9CP-8 is shown in SEQ ID NO.1.

[0040] SEQ ID NO.1

[0041] MAVWVTTTGCHIPEHAIRAGYEADGRPLFIARASMEGTLTPGKCGFHLPGAHIPYGCKENIAHQYEVLVHPNNTQGFYDWRRACLGKVPEDALKTDTDTYVGRAYFSGSLVPCKIATSYPHMCAYMGYGGKEYNAIDYEVLCKIK-

[0042] Length: 152 amino acids

[0043] Type: Amino acid

[0044] Chain type: Single chain

[0045] Characteristics: Molecular weight is 16.67 kDa, isoelectric point is 8.49, contains two DM9 domains.

[0046] Experimental Example 2: Detection of the binding activity of the recombinant protein rCgDM9CP-8 containing the DM9 domain from the Pacific oyster Crassostrea gigas of the present invention to bacteria

[0047] The binding activity of the recombinant protein rCgDM9CP-8 against 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) was detected by Western blotting. The sources of the strains used are as follows: Vibrio splendidus JZ6 was isolated and preserved in the laboratory in the previous stage, Escherichia coli was purchased from Beijing TransGen Biotech Co., Ltd., Staphylococcus aureus was purchased from the Beijing Center for Preservation of Microbial Strains, Micrococcus luteus was purchased from the Beijing Center for Preservation of Microbial Strains, Yarrowia lipolytica was purchased from the Beijing Center for Preservation of Microbial Strains, and Pichia pastoris GS115 was purchased from Invitrogen.

[0048] The specific operations are as follows:

[0049] (1) The above 7 microorganisms were cultured overnight, and 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;

[0050] (2) The cells of the above-obtained bacterial solutions were collected by centrifugation respectively, resuspended with TBS buffer, and the bacterial concentrations were adjusted to 1×10 8 CFU / mL;

[0051] (3) 100 μL of each microbial suspension was respectively mixed with an equal volume of the recombinant protein rCgDM9CP-8 prepared in the above example, and incubated with rotation at room temperature for 30 min;

[0052] (4) The cells were collected by centrifugation at 10,000×g for 2 min, and the cells were washed 4 times with TBS buffer;

[0053] (5) After washing, the cells were collected and resuspended with 40 μL of sterile water;

[0054] (6) 10 μL of 5× protein electrophoresis buffer was added, and the mixture was heated at 99 °C for 10 min, and the protein samples were separated by SDS-PAGE electrophoresis;

[0055] (7) After electrophoresis, the gel was removed, and the same-sized NC membrane and filter paper were cut and immersed in the electrotransfer buffer and allowed to stand for 10 min;

[0056] (8) Place the filter paper, NC membrane, gel, and filter paper into the electrotransfer apparatus in sequence from top to bottom. Set the corresponding current according to the size of the gel block area, and transfer the membrane for 25 minutes.

[0057] (9) Take out the NC membrane and wash it 3 times with TBS buffer for 5 minutes each time.

[0058] (10) Wash it 3 times with TBST buffer for 5 minutes each time.

[0059] (11) Place the NC membrane into 5% skim milk powder (dissolved in TBST) and block it at room temperature for 2 hours.

[0060] (12) Take out the NC membrane and wash it 3 times with TBST buffer for 5 minutes each time.

[0061] (13) Immerse the NC membrane into the His-tag monoclonal antibody (purchased from Sangon Biotech, Shanghai) solution diluted in proportion (1:2000) (5% skim milk powder, TBST buffer), and incubate it at room temperature for 1 hour.

[0062] (14) Take out the NC membrane and wash it 3 times with TBST buffer for 5 minutes each time.

[0063] (15) Place the NC membrane into the goat anti-mouse HRP secondary antibody (purchased from Sangon Biotech, Shanghai) solution diluted in proportion (1:2000) (5% skim milk powder, TBST buffer), and incubate it at room temperature for 1 hour.

[0064] (16) Take out the NC membrane and wash it 3 times with TBST buffer for 10 minutes each time.

[0065] (17) Develop by ECL method, and record the Western blotting results with an imager, as Figure 1 shown.

[0066] The results showed that the rCgDM9CP-8 of the embodiment of the present invention had different degrees of binding activities to Gram-negative bacteria, Gram-positive bacteria, and fungi, and the binding activities to Vibrio splendidus, Escherichia coli, and Staphylococcus aureus were higher than those to Micrococcus luteus, Pichia pastoris, and Yarrowia lipolytica. There were no obvious bands in the rTrx negative control group.

[0067] Experimental Example 3: Detection of the activity of the recombinant protein rCgDM9CP-8 containing the DM9 domain from the Pacific oyster Crassostrea gigas of the present invention in binding different PAMPs

[0068] The binding of the recombinant protein rCgDM9CP-8 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 used MAN, LPS, PGN, and Poly(I:c) were all purchased from Sigma.

[0069] The specific operation steps are as follows:

[0070] (1) Prepare a coating solution with a pH of 7.6 by mixing Na2CO3 and NaHCO3 at concentrations of 15 mmol / L and 35 mmol / L, respectively. Dissolve MAN, LPS, PGN, and Poly(I:c) and adjust their concentrations 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;

[0071] (2) Discard the coating liquid and wash 4 times with TBS-T, 4 minutes each time;

[0072] (3) After washing, add 250 μL of 3% BSA to each well and incubate in a constant temperature incubator at 37 °C for 1 hour for blocking;

[0073] (4) After blocking, repeat step 2;

[0074] (5) Add 100 μL of recombinant protein rCgDM9CP-8 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 wells and add TBS to the control wells), and incubate at room temperature for 2 hours;

[0075] (6) Repeat step (4);

[0076] (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 hour;

[0077] (8) Same as step (2);

[0078] (9) Replace the antibody in step (7) with HRP-conjugated secondary antibody and incubate in a constant temperature incubator at 37 °C for 1 hour;

[0079] (10) Wash each well 5 times with TBST, 3 minutes each time;

[0080] (11) Use TMB chromogenic solution for color development for about 30 minutes, then add 45.65 μL of stop solution (2 M HCl) to terminate the color development, and then measure the OD 595 value, and the results are as Figure 2 shown.

[0081] The results showed that the recombinant protein rCgDM9CP-8 of the embodiments of the present invention had binding activities with MAN, LPS, PGN, and Poly(I:c), and the recombinant protein rCgDM9CP-8 had the strongest affinities for MAN and PGN (P / N = 10.73; 8.12), followed by LPS (P / N = 6.18) and Poly(I:c) (P / N = 5.71). Moreover, the affinities of rCgDM9CP-8 for MAN, LPS, PGN, and Poly(I:c) increased with their increase. The negative control rTrX had no binding activity with the above PAMPs.

[0082] Experimental Example 4: Detection of the activity of the recombinant protein rCgDM9CP-8 containing the DM9 domain from the Pacific oyster Crassostrea gigas of the present invention in inhibiting bacterial growth (growth curve method)

[0083] Some proteins can bind to the cell wall of bacteria and destroy the components of the cell wall, and can inhibit the growth of bacteria by affecting the formation of the cell wall. The growth curve method was used in the present invention to detect the growth of Gram-negative bacteria V. splendidus, E. coli and Gram-positive bacteria S. aureus.

[0084] The sources of the above-mentioned bacterial strains were as described above.

[0085] The specific operations were as follows:

[0086] (1) The experimental bacteria were prepared into a bacterial suspension with a concentration of about 4×10 4 cell / mL with TBS solution;

[0087] (2) 100 μL of the recombinant protein rCgDM9CP-8 was mixed with 100 μL of the bacterial suspension and incubated at room temperature for 2 h;

[0088] (3) After incubation, the bacteria were collected by centrifugation at 10,000×g for 2 min, and washed 3 times with the buffer TBS;

[0089] (4) 20 μL of the test bacterial solution and 200 μL of 2216E or LB culture medium were added to a 96-well microplate, and at the same time, a negative control well (rTRX) was set as a control well, and the volume of each well was 220 μL in total;

[0090] (5) It was placed in a microplate reader and incubated at an appropriate temperature for 10 - 12 h until the plateau phase was reached, and the OD 600 value was read every half hour;

[0091] (6) The growth curves of each detected bacterium were plotted and compared. The results were as Figures 3 - 5 shown.

[0092] The results showed that after incubation with the recombinant protein rCgDM9CP-8 containing the DM9 domain from the Pacific oyster (Crassostrea gigas) of the present invention for 14.5 h and 2.5 h, the OD of Vibrio splendidus, Escherichia coli and Staphylococcus aureus in the treatment group was 600 significantly lower than that in the control group of rTrx protein, indicating that rCgDM9CP-8 has the activity of inhibiting the growth of the above three bacteria.

[0093] Experimental Example 4: Detection of the activity of the recombinant protein rCgDM9CP-8 containing the DM9 domain from the Pacific oyster (Crassostrea gigas) of the present invention in agglutinating bacteria (electron microscopy)

[0094] Some proteins can bind to the cell wall of bacteria, causing varying degrees of agglutination and damage. The morphological structures of the Gram-negative bacteria V. splendidus and E. coli and the Gram-positive bacterium B. subtilis after incubation with rCgDM9CP-8 were detected by electron microscopy.

[0095] 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.

[0096] The specific operations were as follows:

[0097] (1) Bacillus subtilis was cultured in LB medium at 28 °C for 20 h, and then the experimental bacterial solution was prepared into a bacterial suspension with a concentration of about 4×10 4 cell / mL with TBS solution;

[0098] (2) 3 mL of the recombinant protein rCgDM9CP-8 was mixed with 3 mL of the bacterial suspension, and a negative control (rTRX) was set and incubated at room temperature for 3 h;

[0099] (3) After incubation, the cells were centrifuged at 5,000×g for 2 min to collect the bacterial cells, and washed 3 times with buffer TBS;

[0100] (4) After discarding the supernatant, the precipitated bacterial sludge was fixed with 2.5% glutaraldehyde for 12 h;

[0101] (5) After fixation, it was washed 3 times with phosphate buffer at 5,000×g;

[0102] (6) Subsequently, it was soaked in 1% osmium tetroxide for 5 h and washed 3 times with buffer at 5,000×g;

[0103] (7) Gradient dehydration with ethanol, once each with 30%, 50%, 70%, 85%, 95% and twice with 100% ethanol, 20 min each time;

[0104] (8) Replacement with isoamyl acetate twice, 20 min each time;

[0105] (9)Fold qualitative filter paper in half to form a small paper packet, staple one end firmly to form a small pocket. After dropping the centrifugally concentrated bacterial liquid into the small paper packet and sealing it with staples, immediately place it in the sample chamber of a critical point dryer for CO2 critical point drying;

[0106] (10) After drying, cut open the filter paper packet and pour the dried powdery pure bacteria into a petri dish. Stick one side of a carbon conductive tape to a 1 / 4 coverslip, and gently press the other side against the bacterial powder in an inverted manner. After turning it right side up, gently scrape and flatten the bacteria with forceps. After ion sputtering with gold, perform scanning electron microscopy observation.

[0107] The results showed that, compared with the rTrx protein group of the control group, in the treatment group with the recombinant protein rCgDM9CP-8 containing the DM9 domain from the Pacific oyster in the examples of the present invention, the surface of Vibrio splendidus was rough and obvious holes appeared; the surface of the original Escherichia coli bacteria was damaged with wrinkles and perforations; while the surface of Bacillus subtilis showed obvious wrinkles and the content overflowed, presenting an overall shriveled state.

Claims

1. A recombinant protein r of Crassostrea gigas containing the DM9 domain Cg DM9CP-8, characterized in that: Recombinant protein r Cg The amino acid sequence of DM9CP-8 is shown in SEQ ID NO.

1.

2. A recombinant protein r of the Pacific oyster containing the DM9 domain as described in claim 1 Cg A method for preparing DM9CP-8, characterized in that It is carried out successively according to the following steps: a. Perform PCR amplification on the coding region fragment of the DM9CP-8 gene of Crassostrea gigas using primers P1 and P2 Cg ; b. The PCR amplification product and the pET30a vector are digested with Bam HI and Sal I, and then ligated by T4 ligase, transformed, and the recombinant is 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 Cg DM9CP-8; The P1 primer is CGCGGATCCATGGCAGTGTGGGTTACAACGA; The P2 primer is ACGCGTCGACCTACTTAATCTTACAGAGGACT.

3. Use of the recombinant protein r of Crassostrea gigas containing the DM9 domain as described in claim 1 Cg DM9CP-8, characterized in that: The recombinant protein r Cg Application of DM9CP-8 in damaging and forming wrinkles on Bacillus subtilis, Vibrio splendidus or Escherichia coli, thereby inhibiting the growth of the strains.

4. Use of the recombinant protein r of Crassostrea gigas containing the DM9 domain as described in claim 1 Cg DM9CP-8, characterized in that: The recombinant protein r Cg Application of DM9CP-8 in recognizing interferon inducers.

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