Recombinant serum amyloid a of crassostrea gigas, preparation method and application thereof

Abstract

This invention discloses a recombinant serum amyloid A (r) protein from the Pacific oyster. Cg SAA), preparation method and application, r Cg The SAA amino acid sequence is shown in SEQ ID NO. 1; the preparation method is to use primers P1 and P2 to... Cg The SAA coding region fragment was amplified by PCR, and the DNA sequences of primers P1 and P2 are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively. The PCR amplification product was then mixed with the pET-30a vector via... Bam HI and Xhol After digestion with enzyme I, ligation was performed using T4 ligase, followed by transformation to construct recombinants; the recombinants were then transformed into *E. coli*. Transetta In the (DE3) expression strain, the expression strain was then cultured and IPTG was added to induce expression, followed by purification and refolding to obtain r. Cg SAA protein. The prepared r Cg SAA inhibits the growth of Vibrio splenti and Vibrio anguillarum under non-acidic conditions, and can also kill Vibrio splenti. It can be used to prepare drugs against Vibrio splenti and Vibrio anguillarum, and can be applied directly, effectively and safely to aquaculture.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and particularly relates to a recombinant oyster serum amyloid protein A, its preparation method, and its application. Background Technology

[0002] The aquaculture industry faces a serious threat from diseases caused by Vibrio bacteria, with Vibrio splenti and Vibrio anguillarum being particularly prominent in their pathogenicity. Vibrio splenti, a halophilic bacillus, can directly damage host tissues by secreting virulence factors such as metalloproteinases and hemolysins, leading to tissue ulceration, organ necrosis, and even septicemia. Furthermore, its intracellular infection mechanism helps it evade the host's immune defenses, posing a serious threat to many high-value marine aquaculture species such as sea cucumbers, oysters, and scallops. Vibrio anguillarum invades the host through the skin or gills using flagella and adhesins. It adapts to osmotic pressure changes via its outer membrane proteins and obtains nutrients through its siderophore system to support its reproduction. Its secreted toxins can cause hemorrhagic septicemia, and its host range covers more than 50 species of fish, including salmon, trout, and sea bass. In summary, these two pathogens are highly pathogenic and can easily cause large-scale mortality or quality decline in farmed organisms, resulting in significant economic losses for aquaculture farmers.

[0003] Serum amyloid A (SAA) is a key acute-phase protein (APP) that, in addition to serving as an important biomarker for early disease diagnosis, plays multiple immunological functions in the host's defense against infection. Current research has confirmed that SAA responds rapidly in the early stages of infection, with its plasma concentration significantly increasing within hours and rapidly amplifying inflammatory signals. At the level of immune recognition and recruitment, SAA can migrate to the site of infection via chemotaxis of neutrophils and monocytes / macrophages. In the immune clearance mechanism, SAA can bind to the surface of pathogens, thereby enhancing the recognition and phagocytosis efficiency of phagocytes. Furthermore, SAA also has immunomodulatory functions, activating innate immune cells, promoting the release of pro-inflammatory factors, and participating in the regulation of adaptive immune responses. SAA not only binds to bacterial membrane phospholipids through its hydrophobic N-terminal region but also exerts a direct bactericidal effect by disrupting bacterial cell membranes under acidic conditions (pH 4–6), such as directly killing Staphylococcus aureus and Escherichia coli. However, to date, there have been no reports of SAA having an inhibitory or killing effect on important pathogenic bacteria (Vibrio splenti and Vibrio anguillarum) in aquaculture under non-acidic conditions. Summary of the Invention

[0004] The present invention aims to solve the above-mentioned technical problems existing in the prior art, and provides a recombinant oyster serum amyloid protein A, its preparation method and its application.

[0005] The technical solution of the present invention is: a recombinant serum amyloid protein A from oyster, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0006] A method for preparing the above-mentioned recombinant oyster serum amyloid A is carried out according to the following steps: a. Using primers P1 and P2 on the Pacific oyster. Cg The SAA coding region fragment was amplified by PCR. 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. b. Mix the PCR amplification products with... p ET-30a(+) carrier via Bam HI and Xhol After digestion with enzyme I, the recombinant was ligated using T4 ligase, transformed, and sequenced to obtain the correct recombinant. c. Transform the recombinant into Escherichia coli. Transetta The (DE3) expression strain was cultured and IPTG was added to induce expression. Then, it was purified and refolded to obtain recombinant oyster serum amyloid protein A.

[0007] The application of the above-mentioned recombinant oyster serum amyloid A in the preparation of drugs that inhibit Vibrio splenti and Vibrio anguillarum.

[0008] The application of the above-mentioned recombinant oyster serum amyloid A in the preparation of a drug to kill Vibrio splenti.

[0009] This invention uses specific primer pairs Cg The SAA coding region fragment was amplified by PCR, and the PCR amplification product was then mixed with the pET-30a vector. Bam HI and Xhol After digestion with enzyme I, ligation was performed using T4 ligase, followed by transformation to construct recombinants; the recombinants were then transformed into *E. coli*. Transetta (DE3) expression strains were cultured and induced to express by IPTG. After purification and refolding, recombinant oyster serum amyloid A (r) was obtained. Cg SAA). The r prepared in this invention Cg SAA can inhibit the growth of Vibrio splendidus and Vibrio anguillarum under non-acidic conditions and can directly kill Vibrio splendidus. It can be used to prepare anti-Vibrio splendidus and anti-Vibrio anguillarum drugs, which can be applied directly, effectively and safely to aquaculture. Attached Figure Description

[0010] Figure 1 This is a graph showing the activity detection effect of recombinant oyster serum amyloid protein A binding to bacteria in an embodiment of the present invention.

[0011] Figure 2 This is a graph showing the detection effect of Vibrio splendidus growth after co-incubation of recombinant serum amyloid A from oysters with Vibrio splendidus in an embodiment of the present invention.

[0012] Figure 3 This is a graph showing the detection effect of the growth of Vibrio anguillarum after co-incubation of recombinant oyster serum amyloid A with Vibrio anguillarum in an embodiment of the present invention.

[0013] Figure 4 The results are obtained by electron microscopy observation of the morphology and structure of Vibrio splendidus after incubation of recombinant oyster serum amyloid A with Vibrio splendidus in an embodiment of the present invention. Detailed Implementation

[0014] The method for preparing recombinant oyster serum amyloid A according to the present invention comprises the following steps: 1. Construction of recombinant plasmids The recombinant vector used in this embodiment of the invention is from Novagen. p ET-30a(+) prokaryotic expression vector. Using PCR technology, the 5' ends were laced with... Bam HI and Xhol I. Restriction enzyme sites primers P1 and P2, for the oyster cDNA library. Cg The SAAmRNA coding region was amplified.

[0015] The DNA sequence for primer P1 is TACTCAGGATCCTATGACTTCGCCAGGCAGGCT; The DNA sequence for primer P2 is TACTCACTCGAGATCGGGTCTGTATCGATTCGG.

[0016] The PCR 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 54℃ for 30 s, extension at 72℃ for 2 min, for a total of 30 cycles, and a final extension at 72℃ for 10 min; the amplified fragments were purified and recovered by agarose gel electrophoresis, and then... p MD19-T vector ligation; positive clones were screened after transformation, plasmids were extracted, and used... Bam HI and Xhol I. The plasmid was double-digested with enzymes; the target fragment and the enzyme were recovered. Bam HI and Xhol Expression vector digested with enzyme I p ET-30a (+) ligation completes the construction of the recombinant plasmid.

[0017] 2. Recombinant Pacific oyster serum amyloid A (r Cg SAA) expression The constructed recombinant plasmid was transformed and expressed in *E. coli* Transetta (DE3). Single colonies were picked and inoculated into 200 mL LB liquid medium and cultured at 220 rpm and 37°C until OD500.600 =0.4-0.8; Add IPTG (final concentration 1 mmol / L), continue culturing for 4 h, centrifuge at 10,000 rpm for 5 min at 4℃, collect the bacterial cells, and freeze at -80℃ for later use; at the same time, take 1 mL of bacterial culture, centrifuge, discard the supernatant, add 80 μL of water and 20 μL of 5× protein loading buffer, boil at 99℃ for 10 min, centrifuge briefly, and detect the expression product by SDS-PAGE.

[0018] 3. Recombinant Pacific oyster serum amyloid A (r Cg SAA purification and refolding The expression product was purified using a nickel agarose gel FF column to obtain the denatured recombinant protein, which was then refolded by dialyzing with dialysis buffer. The specific operational steps are as follows: (1) Nickel agarose gel FF packing, 1.6×20 cm, column bed volume is 10 mL; (2) Equilibrate 2-5 column volumes with buffer I (50 mmol / L Tris-HCl buffer, pH 9.0, 50 mmol / L NaCl, 8 mol / L urea) at a flow rate of 2 mL / min; (3) Take the cells that have been induced to express by IPTG, resuspend them in buffer I, sonicate them at 150 W for 30 min, centrifuge at 12,000 rpm and 4℃ for 30 min, filter the supernatant through a 0.45 μm filter membrane, and pass it through a column at a flow rate of 1 mL / min. (4) Wash with buffer I for 2-5 column volumes at a flow rate of 2 mL / min; (5) Wash with 2-5 column volumes of imidazole buffer I containing 50 mmol / L at a flow rate of 2 mL / min; (6) Elute the target protein with imidazole buffer I containing 400 mmol / L and collect the protein further; (7) The protein expression and purification status were detected by SDS-PAGE; (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℃. Purification of the recombinant protein requires dialysis in a refolding buffer to remove urea, allowing the protein to refold correctly and restore its proper 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 concentrations. The urea concentration was initially 6 M, gradually replaced with 4 M, 3 M, 2 M, 1 M, and 0 M. No glycerol was added during the final dialysis until no urea remained in the dialysate. Dialysis was performed at 4℃ for 12 h each time to obtain recombinant oyster serum amyloid A (r Cg SAA), the obtained r Cg The amino acid sequence of SAA is shown below: MRVFAVLSVLFIGLVLTEQTLAQGFLDRIRDRIRSGYDFARQAGQGTRDMYRAYSDMREANWRNSDQYFHARGNHDAASRGPGGRWAARVISNAREGYQSGLSGQGPEDTARDQEANRWGRNGGDPNRYRPDGLPDRY.

[0019] Length: 138 amino acids Type: Amino acid Chain type: single chain Characteristics: Molecular weight is 15.66 kDa, and isoelectric point is 9.84.

[0020] Experiment 1: The recombinant serum amyloid A (r) protein of the present invention from the oyster *Crassostrea gigas* Cg SAA (Sterile Acid) binding activity assay Detection of recombinant protein r based on Western blotting method Cg SAA and four types of Gram-negative bacteria (Vibrio anguillarum) Vibrio anguillarum Vibrio splendens ( Vibrio splendidus ), Escherichia coli ( Escherichia coli ) and alginolytic Vibrio ( Vibrio alginolyticus ), three Gram-positive bacteria (Micrococcus luteus ( Micrococcus luteus Staphylococcus aureus ( Staphylococcus aureus ) and Bacillus subtilis ( Bacillus subtilis ( ) Binding activity. The bacterial strains used in this experiment were obtained from the following sources: Vibrio brilliance, Vibrio alginolyticus, Vibrio anguillarum, Staphylococcus aureus, Micrococcus luteus, and Bacillus subtilis were all purchased from Beijing Microbial Culture Collection Center, and Escherichia coli was purchased from Beijing TransGen Biotech Co., Ltd.

[0021] The specific steps are as follows: (1) The above 7 microorganisms were cultured overnight. The culture method was as follows: Vibrio anguillarum, Vibrio splenium, and Vibrio alginolyticus were cultured in 2216E medium at 28°C for 20 h; Escherichia coli, Micrococcus luteus, Staphylococcus aureus, and Bacillus subtilis were cultured in LB liquid medium at 37°C for 20 h. (2) Collect each bacterial cell by centrifugation, resuspend in TBS buffer at pH 7.2-7.6, and adjust the concentration of each cell to 1×10⁻⁶. 8 CFU / mL; (3) Take 100 μL of each microbial suspension and mix it with an equal volume of the recombinant protein r prepared in the above examples. Cg SAA mixture, rTRX with bacterial suspension as negative control, and incubated at room temperature for 3 hours by rotation. (4) Collect bacterial cells by centrifugation at 4,000 rpm for 5 min, and wash the bacterial cells four times with TBS buffer; (5) After washing, collect the bacterial cells and resuspend them in 40 μL of sterile water; (6) Add 10 μL of 5× protein electrophoresis buffer, heat at 99℃ for 10 min, and separate the protein samples by SDS-PAGE electrophoresis; (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 min. (8) Place the filter paper, NC membrane, gel and filter paper into the electroporation apparatus in the order from top to bottom, set the corresponding current according to the size of the gel block, and transfer the membrane for 25 minutes; (9) Remove the NC membrane and wash it three times with TBS buffer, 5 min each time; (10) Wash three times with TBST buffer, 5 min each time; (11) Place the NC membrane in 5% skim milk powder (dissolved in TBST) and seal at room temperature for 2 h; (12) Remove the NC membrane and wash it three times with TBST buffer, 5 min each time; (13) Immerse the NC membrane in a His-tagged monoclonal antibody (purchased from Shanghai Sangon Biotech) solution diluted (1:1000) (5% skim milk powder, TBST buffer) and incubate at 4°C for 16 h; (14) Remove the NC membrane and wash it three times with TBST buffer, 5 min each time; (15) Place the NC membrane in a solution of goat anti-mouse HRP secondary antibody (purchased from Shanghai Sangon Biotech) diluted in proportion (1:1000) (5% skim milk powder TBST buffer) and incubate at room temperature for 1 h; (16) Remove the NC membrane and wash it three times with TBST buffer, 5 min each time; (17) ECL method development, imager recording of western blotting results, such as Figure 1 As shown.

[0022] The results show that the embodiment r of the present invention Cg SAA showed binding activity only with Vibrio anguillarum and Vibrio splenium, while no obvious bands were observed in the other 5 bacteria and the rTrx negative control group.

[0023] Experiment 2: The recombinant serum amyloid A (r) protein of the present invention from the oyster serum of the present invention Cg SAA (Sterile Antimicrobial Activity) activity against bacterial growth (growth curve method) detection The growth curve method was used to analyze r. Cg After co-incubating SAA with Vibrio splenium or Vibrio anguillarum, the growth of Vibrio splenium and Vibrio anguillarum was detected. The source of the strains is as described in Experiment 1.

[0024] The specific steps are as follows: (1) The experimental bacteria were prepared with TBS solution at pH 7.2-7.6 to a concentration of approximately 4×10⁻⁶. 4 Bacterial suspension with cell / mL; (2) Add 50 μL r Cg After mixing SAA and bacterial culture at a 1:1 ratio, the mixture was incubated for 3 h. 50 μL of PBS with bacterial culture was used as a blank control, and 50 μL of rTRX with bacterial culture was used as a negative control. (3) Take 20 μL of each bacterial culture to be tested and mix it into a 96-well plate. Add 200 μL of the corresponding culture medium and incubate in a microplate reader at the appropriate temperature for 12-16 h until the plateau phase is reached. Read the OD every half hour. 600 Numerical value; (4) Plot the growth curves of each detected bacteria for comparison. The results are as follows: Figure 2 , Figure 3 As shown.

[0025] Figure 2 , Figure 3 The results show that black (rTRX) bacteria grow normally, while the growth of red (rSAA) bacteria is significantly inhibited. These results demonstrate the effectiveness of the embodiments of the present invention. Cg SAA has a significant inhibitory effect on the growth of both Vibrio splendidus and Vibrio anguillarum.

[0026] Experiment 3: Recombinant oyster serum amyloid A (r) of this invention Cg Electron microscopy results of SAA on surface damage of Vibrio splendidus The strains were obtained as described in Experiment 1.

[0027] The specific steps are as follows: (1) Vibrio splenium was prepared with TBS solution at pH 7.2-7.6 to a concentration of approximately 4 × 10⁻⁶. 4 Bacterial suspension with cell / mL; (2) Take 100 μL of the microbial suspension and mix it with an equal volume of the recombinant oyster serum amyloid A (r) obtained in the invention example. Cg The mixture of SAA and negative control (rTRX) was incubated at room temperature by rotation for 3 h. (3) After incubation for 3 h, the cells were collected by centrifugation at 4,000 rpm for 5 min and washed four times with TBS buffer. (4) Fix the bacterial cells with 2.5% glutaraldehyde solution and incubate overnight at 4°C; (5) After centrifugation, the sample was washed three times with TBS solution to remove residual glutaraldehyde. Dehydration was then performed using a gradient of ethanol: 30%, 50%, 70%, 80%, 90%, and 100% ethanol were used sequentially, each for 15 min. After each treatment, the supernatant was discarded by centrifugation, and the sample was then replaced with the next concentration of ethanol. After dehydration, the sample was evenly spread onto a glass slide and dried at room temperature. Finally, the slide was sputtered with gold and observed under a scanning electron microscope. The results are as follows: Figure 4 As shown.

[0028] Figure 4 The image shows that the Vibrio splendidus in the negative control group (top row) is morphologically intact, exhibiting a typical rod or arc shape, smooth surface, and clear structure; its magnified image shows an intact bacterial cell membrane with no obvious damage or leakage of contents. The bottom row shows... Cg In the SAA group, *Vibrio splendidus* exhibited significant morphological abnormalities, with multiple depressions, wrinkles, and ruptures (indicated by red arrows). Partial cell membrane integrity was disrupted, showing signs of content leakage or cell collapse. These results indicate that the recombinant *Crassostrea gigas* serum amyloid A (r) protein in this embodiment of the invention... Cg Compared with the control group rTRX protein, SAA showed a puncture-killing effect on Vibrio brilliance.

Claims

1. A recombinant serum amyloid protein A from oyster shell, characterized in that: The amino acid sequence characteristics are shown in SEQ ID NO.

1.

2. A method for preparing recombinant oyster serum amyloid A as described in claim 1, characterized in that... Follow these steps in sequence: a. Using primers P1 and P2 on the Pacific oyster. Cg The SAA coding region fragment was amplified by PCR. 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. b. Mix the PCR amplification products with... p ET-30a(+) carrier via Bam HI and Xhol After digestion with enzyme I, the recombinant was ligated using T4 ligase, transformed, and sequenced to obtain the correct recombinant. c. Transform the recombinant into Escherichia coli. Transetta The expression strain was cultured and induced to express by IPTG, then purified and refolded to obtain recombinant oyster serum amyloid A.

3. The use of recombinant oyster serum amyloid A as described in claim 1 in the preparation of drugs that inhibit Vibrio splendidus and Vibrio anguillarum.

4. The use of recombinant oyster serum amyloid A as described in claim 1 in the preparation of a drug to kill Vibrio splenti.

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