Recombinant protein of crd of long oyster c-type lectin cgclec-tm2 and application

Abstract

The application belongs to the technical field of molecular biology, and particularly relates to a long oyster C-type lectin CgCLec-TM2 functional domain CRD recombinant protein and application. The amino acid sequence of the long oyster C-type lectin CgCLec-TM2 functional domain CRD recombinant protein is shown as SEQ ID NO. 1. The rCRD obtained by the application has binding capacity to various pathogen-associated molecular patterns (PAMPs), can bind and agglutinate various microorganisms, has a growth inhibiting effect on gram-negative bacteria Vibrio splendidus and Escherichia coli, and can be used for the development of bacteriostatic feed additives and new-type immune enhancers.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a recombinant protein of the CgCLec-TM2 functional domain of the oyster C-type lectin and its applications. Background Technology

[0002] C-type lectins are defined as a class of lectins that contain a sugar recognition domain (CRD) and are expressed as Ca2+. 2+ C-lectins are proteins that rely on specific forms to recognize and bind to carbohydrates. However, with the discovery of more C-lectins, their ligand recognition has expanded beyond carbohydrates to include proteins, lipids, inorganic molecules, and non-carbohydrate ligands such as ice crystals. C-lectins are widely found in mammals, invertebrates, plants, and even microorganisms, and possess various biological activities such as cell agglutination, antibacterial activity, and antiviral activity.

[0003] C-type lectins have wide applications in clinical medicine. They can be used as molecular probes to detect the distribution of glycosyl receptors on the surface and inside tumor cells, and to study tumor differentiation and variation, as well as tumor diagnosis, treatment and prognosis. C-type lectins can also be used as immune adjuvants and in cell typing and identification of microorganisms. In addition, C-type lectins can bind to fluorescein, biotin, enzymes, etc. without affecting their biological activity, and can be widely used in immunocytochemical research. Compared with higher animals, the types and functions of C-type lectins in invertebrates are more diverse. Studies have shown that C-type lectins not only participate in the recognition and binding of invading pathogens as pattern recognition molecules in the innate immune response of invertebrates, but also play an important role as effector molecules in the process of clearing pathogenic microorganisms. For example, (1) recognition and binding of PAMPs and pathogenic microorganisms: the C-type lectin CD94-1 of glass sea squirt has LPS binding activity, and the C-type lectin DL-1 of fruit fly can bind to Escherichia coli and Erwinia chrysogenum. (2) Promotes phagocytosis: C-type lectin MCL-4 in Manila clams can increase the phagocytic rate of hemolymphocytes. (3) Agglutination: C-type lectin in Litopenaeus vannamei can agglutinate erythrocytes in various animals such as rat hemolymphocytes and rabbit hemolymphocytes. C-type lectin CLec-3 in Pacific oyster can agglutinate Gram-negative bacteria, Gram-positive bacteria and fungi. (4) Antibacterial activity: C-type lectin CLec-3 in Pacific oyster shows antibacterial activity against Escherichia coli and Staphylococcus aureus.

[0004] The Pacific oyster is a bivalve molluskaloid inhabiting the intertidal zone and shallow rocky seabeds. It is widely distributed and highly adaptable. To adapt to the complex and ever-changing marine environment, the Pacific oyster has developed a sophisticated immune defense system over a long period of evolution, including immune molecules with specific biological activities. The Pacific oyster genome shows a significant expansion of C-type lectin molecules, with 266 proteins containing CRDs identified, far more than in fruit flies (34) and humans (81). Compared to vertebrates, the structure and function of these CRDs in the Pacific oyster are diverse. First, the number of CRDs in Pacific oyster C-type lectins varies considerably, with one type containing as many as 12 CRDs. Second, the functions of Pacific oyster C-type lectins are also more diverse, exhibiting different microbial recognition and binding abilities. For example, CgCLec-4 and CgCLec-5, which contain only one CRD, can bind to several polysaccharides such as LPS, PGN, GLU, and MAN. They can also bind to Staphylococcus aureus, Escherichia coli, Vibrio anguillarum, and Yersinia lipolyticis. In addition, CgCLec-4 exhibits antimicrobial activity against bacteria and fungi. However, the antimicrobial activity of CgCLec-5 is not obvious. Therefore, unlike vertebrate C-type lectins, their accurate functions cannot be predicted solely by analyzing their sequence and structure. Specific experiments are required for verification. Summary of the Invention

[0005] The purpose of this invention is to provide a recombinant protein of the CgCLec-TM2 functional domain of oyster C-type lectin and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A recombinant protein with the CgCLec-TM2 functional domain CRD of oyster C-type lectin, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] A method for preparing a recombinant CRD protein with the CgCLec-TM2 functional domain of a type C lectin.

[0009] a. The CRD coding region fragment of the CgCLec-TM2 molecular functional domain of the Pacific oyster was amplified by PCR using primers P1 and P2. 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.

[0010] b. The PCR amplification product and pET-30a vector were digested with KpnI and EcoRI, ligated with T4 ligase, transformed, and sequenced to identify the recombinant.

[0011] c. The recombinant was transformed into Escherichia coli Transetta(DE3) expression strain to achieve prokaryotic in vitro recombinant expression. Subsequently, the protein was purified and renatured to obtain a recombinant protein with the amino acid sequence in SEQ ID NO.1.

[0012] A primer for preparing recombinant protein CgCLec-TM2 domain CRD of oyster C-type lectin, P1: (5'-CGGGGTACCCCGTGTAGCGAAGGTTGGACCTTAA-3'); P2: (5'-CCGGAATTCTTCACACACAGACATTAAA-3').

[0013] Application of a recombinant protein with the CgCLec-TM2 functional domain CRD of oyster C-type lectin, specifically in the preparation of antibacterial agents, immune enhancers, or feed additives.

[0014] To elaborate further,

[0015] The recombinant protein with the CgCLec-TM2 domain of the oyster C-type lectin can bind to a variety of pathogen-associated molecular patterns (PAMPs);

[0016] The recombinant protein with the CRD functional domain of the oyster C-type lectin (CgCLec-TM2) exhibits binding activity with a variety of microorganisms.

[0017] The recombinant protein with the CRD functional domain of the oyster C-type lectin (CgCLec-TM2) exhibits agglutination activity against a variety of microorganisms.

[0018] The recombinant protein with the CRD domain of the oyster C-type lectin (CgCLec-TM2) has an inhibitory effect on the growth of Gram-negative bacteria.

[0019] Advantages of this invention:

[0020] This invention utilizes in vitro recombinant expression technology to obtain a recombinant CRD protein with the functional domain of oyster C-type lectin (CgCLec-TM2). The obtained CRD recombinant protein exhibits biological activities involving the binding of poly(I:C), PGN, mannose, and LPS. It also demonstrates binding and agglutination activity against Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Vibrio splendidus, Bacillus subtilis, and Pichia pastoris, and inhibits the growth of Gram-negative bacteria Vibrio splendidus and Escherichia coli. The recombinant CRD protein with the functional domain of oyster C-type lectin (CgCLec-TM2) obtained in this invention has application value in the preparation of antibacterial drugs, novel immunomodulators, and feed additives. Attached Figure Description

[0021] Figure 1 The image shows the purification process of recombinant protein CgCLec-TM2 functional domain CRD from oyster C-type lectin, as provided in this embodiment of the invention.

[0022] Figure 2 The graph shows the detection effect of the binding activity of the recombinant protein CRD domain of oyster C-type lectin (CgCLec-TM2) with poly(I:C), PGN, mannose and LPS provided in the embodiments of the present invention.

[0023] Figure 3 This is a graph showing the effect of detecting the binding activity of the CRD functional domain of oyster C-type lectin (CgCLec-TM2) with microorganisms, provided in an embodiment of the present invention.

[0024] Figure 4 This is a graph showing the effect of the functional domain CRD of the oyster C-type lectin (CgCLec-TM2) on the detection of microbial agglutination activity provided in the embodiments of the present invention.

[0025] Figure 5 This is a graph showing the detection effect of the CRD functional domain of oyster C-type lectin (CgCLec-TM2) inhibiting microbial growth, provided in an embodiment of the present invention. Detailed implementation method:

[0026] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0027] The rCRD obtained in this invention has the ability to bind to multiple pathogen-associated molecular patterns (PAMPs), and can bind and agglutinate various microorganisms. It has an inhibitory effect on the growth of Gram-negative bacteria Vibrio splenita and Escherichia coli, and can be used in the development of antibacterial feed additives and novel immune enhancers. Experimental Example 1: In vitro prokaryotic expression and purification of the CRD functional domain of the oyster C-type lectin CgCLec-TM2 of this invention, including the following steps:

[0028] 1. Construction of recombinant vectors

[0029] The recombinant vector used in this invention is Novagen's pET-30a prokaryotic expression vector. Using PCR technology, primers P1 and P2, with KpnI and EcoRI restriction sites added to their 5' ends respectively, amplified the CRD functional domain coding region of the oyster C-type lectin CgCLec-TM2 molecule.

[0030] The reaction conditions were as follows: first, pre-denaturation at 94℃ for 5 min, followed by the following cycles: denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 2 min, for a total of 35 cycles, and a final extension at 72℃ for 10 min. The amplified fragment was purified and recovered, and ligated into the pMD19-T vector. Positive clones were screened after transformation, and plasmids were extracted. The plasmid was double-digested with KpnI and EcoRI, and the target fragment was purified and recovered using a gel purification kit (Dalian Takara Bio Inc.). The recovered target fragment was ligated into the expression vector pET-30a, which had been digested with KpnI and EcoRI, to complete the vector construction.

[0031] 2. Expression of CgCLec-TM2 functional domain CRD recombinant protein

[0032] The constructed recombinant vector was transformed and expressed in *Escherichia coli* Transetta(DE3), and positive single clones were screened and inoculated into 200 mL of LB liquid medium and cultured at 220 rpm and 37 °C until OD500. 600 =0.4-0.8. Add IPTG to achieve a final concentration of 1 mmol / L and continue culturing for 4 h. Centrifuge at 5000 rpm for 10 min at 4 °C, collect the bacterial cells, and freeze at -20 °C for later use. Centrifuge 1 mL of bacterial culture, discard the supernatant, add 80 μL of water and 20 μL of 5× loading buffer, boil at 99 °C for 10 min, centrifuge briefly, and detect the expression product by SDS-PAGE.

[0033] 3. Purification and refolding of CgCLec-TM2 domain CRD recombinant protein

[0034] In this invention, the recombinant protein was purified using a nickel agarose gel FF column. The denatured recombinant protein was obtained and then renatured by dialyzing with dialysis buffer.

[0035] The specific steps are as follows:

[0036] The following are the steps for purifying and separating His-tagged recombinant proteins in the denatured state using nickel agarose gel column:

[0037] (1) Nickel agarose gel FF packing (1.6×20cm), column bed volume is 10mL;

[0038] (2) Equilibrate 2 to 5 bed volumes with buffer I (50 mmol / L Tris-HCl, 0.5 mol / L NaCl, pH 7.2) at a flow rate of 2 mL / min;

[0039] (3) Cells induced by IPTG expression were resuspended in buffer I, sonicated at 150W for 30 min, centrifuged at 12000 rpm and 4℃ for 30 min, and the supernatant was filtered through a 0.45 μm filter membrane and passed through a column at a flow rate of 1 mL / min.

[0040] (4) Wash with buffer I for 2-5 bed volumes at a flow rate of 1 mL / min;

[0041] (5) Wash with buffer I containing 50 mmol / L imidazole for 2-5 column volumes at a flow rate of 2 mL / min;

[0042] (6) Elute the target protein with imidazole buffer I containing 400 mmol / L and collect it;

[0043] (7) Detect the expression of the fusion protein using SDS-PAGE;

[0044] (8) Wash with pure water for 5 column volumes, then wash with 20% ethanol for 3 column volumes at a flow rate of 2 mL / min. Store the column at 4°C. Purify the recombinant protein in the denatured state by dialysis in the refolding buffer to remove urea, allowing the protein to refold correctly and restore its correct conformation. The denatured and purified product was refolded 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 a gradient of decreasing urea. The urea concentration was gradually changed from 6 M to 4 M, 3 M, 2 M, 1 M, and 0 M. No glycerol was added during the last dialysis until no urea was added. Dialysis was performed at 4°C for 12 h each time to obtain the CRD functional domain of the recombinant oyster CgCLec-TM2 as shown in SEQ ID NO. 1.

[0045] SEQ ID NO.1

[0046] CSEGWTLNDGSCYWLNKDFLSWDEAEEACRKRSATLVEITNGNENMFL

[0047] EEFAISYLIWVGVKVIESNTRINTCSQTPSSFGLTYTREFGKCIESITIFDGT

[0048] GEPFPFFVNRDCNDRLMSVCE

[0049] Length: 122 amino acids

[0050] Type: Amino acid

[0051] Chain type: single chain

[0052] Characteristics: Molecular weight is 13.94 kDa, isoelectric point is 4.43, and it has a conserved CRD functional domain.

[0053] Source: Oyster

[0054] Experimental results:

[0055] SDS-PAGE results showed that the molecular weight of the purified oyster CgCLec-TM2 domain CRD recombinant protein was 19.8 kDa, consistent with the predicted molecular weight. Figure 1 ).

[0056] Primers are

[0057] P1: (5'-CGGGGTACCCCGTGTAGCGAAGGTTGGACCTTAA-3');

[0058] P2: (5'-CCGGAATTCTTCACACACAGACATTAAA-3').

[0059] Experimental Example 2: Detection of the binding activity of the CgCLec-TM2 functional domain CRD recombinant protein of the present invention with PAMPs

[0060] Different PAMPs (LSP, PGN, MAN, and poly(I:C)) were diluted to 100 μg / mL using coating buffer (50 mM Na2CO3-NaHCO3 buffer, pH 9.6), and 100 μL was added to each well of a 96-well microplate. The plates were incubated overnight at 4°C. To avoid nonspecific binding, 200 μL of 3% BSA was added, and the plates were blocked at 37°C for 1 h. Afterward, 100 μL of recombinant protein rCRD, serially diluted 2-fold with TBS, was added to each well, followed by the addition of Ca2+ to a final concentration of 10 mM. 2+Incubate at 18°C ​​for 3 hours. Add 100 μL of mouse anti-His-taq monoclonal antibody diluted with 3% BSA (1:1000) and incubate at 37°C for 1 hour. Add 100 μL of HRP-labeled goat anti-mouse IgG (1:3000) and incubate at 37°C for 1 hour. Wash three times with TBST between each incubation step, 5 min each time. After thorough washing, add 100 μL of TMB to each well, incubate in the dark for 30 min, then add 50 μL of 2MH2SO4 to stop the color development. Read and record the values ​​at 450 nm using a microplate reader. Set up a negative control group for rTrx protein and a blank control group for TBS. Each sample is tested in triplicate, and the average value is taken from the three measurements. For data analysis, wells with P / N = experimental group (P) - control (B) / negative control group (N) - blank control (B) > 2.1 are considered positive (see Figure 2 ).

[0061] Experimental results showed that the recombinant protein rCRD in Ca 2+ At a concentration of 10 mM, the P / N ratios of each PAMP gradually increased with increasing protein concentration, indicating a gradually enhanced ability to bind sugars. Among these, rCRD showed stronger binding ability to PGN and poly(I:C). This experiment demonstrates that the recombinant protein rCRD binds to calcium... 2+ In the presence of these proteins, LSP, PGN, MAN, and poly(I:C) are recognized and bound. Example 3: Detection of the binding activity of the CgCLec-TM2 functional domain CRD recombinant protein of this invention with microorganisms.

[0062] The binding activity of the recombinant protein to three Gram-negative bacteria (Vibrio splendens, Vibrio aureus, and Escherichia coli), three Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteus), and one fungus (Pichia pastoris) was detected using Western blotting. The strains used were sourced as follows: Vibrio splendens (V. splendens JZ6) from Beijing Microbial Culture Collection Center, Escherichia coli (E. coli) from TransGen Biotech, Vibrio anguillarum (V. anguillarum), Staphylococcus aureus (S. aureus), Micrococcus luteus (M. luteus) from Beijing Microbial Culture Collection Center, Bacillus subtilis (B. subtilis) from Beijing Microbial Culture Collection Center, and Pichia pastoris (P. pastoris GS115) from Invitrogen. The specific procedures are as follows:

[0063] (1) The above seven microorganisms were cultured overnight. The culture methods were as follows: Micrococcus luteus and Escherichia coli were cultured on LB medium at 37°C for 20 h; Staphylococcus aureus and Bacillus subtilis were cultured on LB medium at 28°C for 20 h; Vibrio splenium and Vibrio anguillarum were cultured on 2216E medium at 28°C for 20 h; and Pichia pastoris was cultured on YPD medium at 28°C for 20 h.

[0064] (2) Collect bacterial cells by centrifugation, resuspend in TBS buffer, and adjust the concentration of each bacterial cell to 1×10⁻⁶. 8 CFU / mL;

[0065] (3) Take 100 μL of the microbial suspension and mix it with an equal volume of the oyster C-type lectin rCRD (0.25 mg / mL) obtained in the present invention, and add Ca to a final concentration of 10 mM. 2+ Or not add Ca 2+ Under the conditions specified, incubate by rotation at room temperature for 30 minutes;

[0066] (4) Centrifuge at 10,000 rpm for 2 min to collect bacterial cells, and wash the bacterial cells four times with TBS buffer;

[0067] (5) After washing, collect the bacterial cells and resuspend them in 40 μL of sterile water;

[0068] (6) Add 10 μL of 5× protein electrophoresis buffer, heat at 99℃ for 10 min, and separate the protein samples by SDS-PAGE electrophoresis;

[0069] (7) After electrophoresis, remove the gel and cut out NC membranes and filter paper of the same size and immerse them together in the electrophoresis buffer for 10 minutes.

[0070] (8) Place the filter paper, NC membrane, gel and filter paper into the electroporation apparatus in order from top to bottom. Set the corresponding current according to the size of the gel block area and transfer the membrane for 20 minutes.

[0071] (9) Remove the NC membrane and wash it three times with TBS buffer, 5 min each time;

[0072] (10) Wash three times with TBST buffer, 5 min each time;

[0073] (11) Place the NC membrane in 5% skim milk powder (dissolved in TBST) and seal at room temperature for 2 hours;

[0074] (12) Remove the NC membrane and wash it three times with TBST buffer, 5 min each time;

[0075] (13) Immerse the NC membrane in a His-tagged monoclonal antibody (purchased from Shanghai Sangon Biotech) solution (5% skim milk powder, TBST buffer) diluted according to the ratio and incubate at room temperature for 1 h;

[0076] (14) Remove the NC membrane and wash it three times with TBST buffer, 5 min each time;

[0077] (15) Place the NC membrane in a solution of goat anti-mouse HRP secondary antibody (purchased from Shanghai Sangon Biotech) diluted according to the ratio (5% skim milk powder TBST buffer) and incubate at room temperature for 1 h;

[0078] (16) Remove the NC membrane and wash it three times with TBST buffer for 10 min each time;

[0079] (17) ECL method development, imager records Western blotting results, such as Figure 3 As shown.

[0080] The results showed that the CgCLec-TM2 functional domain CRD recombinant protein of the present invention was in Ca 2+ The presence of these bacteria increases their binding affinity to Gram-negative bacteria (Vibrio anguillarum, Escherichia coli, and Vibrio splenium), Gram-positive bacteria (Bacillus subtilis), and the fungus Pichia pastoris. It also increases their binding affinity to Staphylococcus aureus and Micrococcus luteus. Figure 3 A), negative control group rTrx( Figure 3 C) and without Ca 2+ Positive control group ( Figure 3 No obvious bands were found in B).

[0081] Experimental Example 4: Detection of the microbial agglutination activity of the CgCLec-TM2 functional domain CRD recombinant protein of the present invention from oyster *Crassostrea gigas*.

[0082] The agglutination activity of the CgCLec-TM2 functional domain CRD recombinant protein from the oyster *Crassostrea gigas* against Gram-negative bacteria (*Vibrio splenium*, *Escherichia coli*), Gram-positive bacteria (*Micrococcus luteus*, *Staphylococcus aureus*), and fungi (*Pichia pastoris*) was observed using fluorescence microscopy. The strains were referenced in the above examples. The experimental procedures are as follows:

[0083] 1. Fluorescently labeled microorganisms

[0084] (1) The above 5 microorganisms were cultured overnight, and the culture method was the same as in Experiment 3(1);

[0085] (2) Collect the bacterial cells by centrifugation, and then fix them with formaldehyde for 10 min;

[0086] (3) After washing three times with 0.1M NaHCO3, resuspend in NaHCO3 (0.1mol / L, pH 9.0) solution containing 1mg / mL fluorescein isothiocyanate (FITC), and incubate overnight in the dark at 4℃.

[0087] (4) Clean with TBS three times and adjust the microbial concentration to 1×10⁻⁶. 8 CFU / mL, store at 4℃ for later use.

[0088] 2. Aggregation activity detection

[0089] (1) Take 10 μL of fluorescently labeled microorganisms and an equal volume of CRD recombinant protein (0.5 mg / mL) and incubate at 25℃ for 20 min (rTrx is the negative control and TBS is the blank control);

[0090] (2) Take 10 μL of the above incubation mixture, spread it on a glass slide, observe and photograph it using a fluorescence microscope. The results are as follows: Figure 4 As shown.

[0091] Experimental results:

[0092] The results showed that the recombinant CgCLec-TM2 domain CRD protein of the oyster in this embodiment of the invention had strong agglutination activity against Gram-negative bacteria, Gram-positive bacteria, and fungi. Figure 4 ).

[0093] Experimental Example 5: Detection of microbial growth inhibition by recombinant protein with CgCLec-TM2 functional domain of Ostrea gigas in this invention.

[0094] The antibacterial activity of the recombinant protein rCRD from Pacific oyster was tested against Gram-negative bacteria Vibrio splenti and Escherichia coli, and then detected using an enzyme-linked immunosorbent assay (ELISA) reader (Tecan Infinite M1000 PRO).

[0095] The strains were sourced as described above.

[0096] The specific steps are as follows:

[0097] (1) The experimental bacteria were prepared with a TBS-free solution to a concentration of approximately 4 × 10⁻⁶. 4 Bacterial suspension with cell / mL;

[0098] (2) Add 50 μL of rCRD (final concentration of 0.2 mg / mL) to a 96-well microplate, and then add 50 μL of test bacterial solution to each well. Also set up negative control wells (rTRX) and TBS blank control wells, with a total volume of 100 μL per well.

[0099] (3) After mixing the protein samples with the bacteria to be detected, the samples were placed in an ELISA reader and incubated at the appropriate temperature for 15 hours. The OD of each well was measured and recorded every hour. 600 .

[0100] (4) Statistical analysis was performed using SPSS 6.0 software. Significant differences (p<0.05) were marked with *, and significant differences (p<0.01) were marked with **. Growth curves for each detected bacteria were plotted for comparison. Results are as follows: Figure 5 As shown. The results show that the rCRD of the long oyster in the embodiment of the present invention is effective against Vibrio splenida (…). Figure 5 A) and Escherichia coli ( Figure 5 The growth of B) has a certain inhibitory effect.

Claims

1. A C-type lectin from the long oyster Cg CLec-TM2 domain CRD recombinant protein, characterized by: Oyster C-type lectin Cg The amino acid sequence of the CLec-TM2 functional domain CRD recombinant protein is shown in SEQ ID NO.

1.

2. A C-type lectin as described in claim 1 Cg A method for preparing CLec-TM2 functional domain CRD recombinant protein, characterized in that... Follow these steps in sequence: a. Using primers P1 and P2 on the long oyster. Cg The CLec-TM2 molecular functional domain CRD coding region fragment was amplified by PCR. The DNA sequence of primer P1 was 5'-CGGGGTACCCCGTGTAGCGAAGGTTGGACCTTAA-3', and the DNA sequence of primer P2 was 5'-CCGGAATTCTTCACACACAGACATTAAA-3'. b. The PCR amplification products were mixed with the pET-30a vector and... Kpn I and EcoR After digestion with enzyme I, the recombinants were ligated using T4 ligase, transformed, and sequenced to identify the recombinants. c. The recombinant was transformed into Escherichia coli Transetta (DE3) expression strain to achieve prokaryotic in vitro recombinant expression. Subsequently, the protein was purified and renatured to obtain a recombinant protein with the amino acid sequence in SEQ ID NO.

1.

3. A method for preparing C-type lectin from oysters. Cg Primer pairs for CLec-TM2 functional domain CRD recombinant proteins, characterized by: Primer pairs are P1: 5'-CGGGGTACCCCGTGTAGCGAAGGTTGGACCTTAA-3'; P2: 5'-CCGGAATTCTTCACACACAGACATTAAA-3'.

4. A C-type lectin of the long oyster as described in claim 1 Cg The application of CLec-TM2 functional domain CRD recombinant proteins is characterized by: The C-type lectin of the long oyster as described in claim 1 Cg Application of CLec-TM2 domain CRD recombinant protein in the preparation of antibacterial agents; The bacteria in question are either Vibrio brilliance or Escherichia coli.

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