Phage for controlling vibrio infection of prawns

Abstract

The invention provides a bacteriophage for controlling vibrio infection of prawns, and belongs to the technical field of veterinarians. The recombinant tail chain protein rTC is designed and the expression gene of the recombinant tail chain protein rTC is synthesized by comparing and structurally analyzing the amino acid sequence of the tail chain protein of the vibrio source bacteriophage. The rTC gene is electrically transferred into vibrio parahaemolyticus separated from diseased prawns, PHxp1 bacteriophage separated from seawater is used for infecting host bacteria, after the rTC gene and the PHxp1 bacteriophage are recombined, the collected bacteriophages are screened by vibrio alginolyticus to obtain two bacteriophages PHxp1-1 and PHxp1-2 capable of splitting the vibrio alginolyticus, and the vibrio harveyi is used for screening to obtain one bacteriophage PHxp1-3 capable of splitting the vibrio harveyi. The host spectrums of the three recombinant bacteriophages are widened. When the recombinant tail chain protein bacteriophage is applied to a prawn farm, death and growth stagnation caused by vibrio infection of prawns are greatly reduced, and culture benefits are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary medicine, and in particular relates to a bacteriophage for controlling Vibrio infection in shrimp. Background Art

[0002] Aquaculture is one of my country's pillar food industries, meeting over one-third of the nation's animal protein needs. Shrimp farming, with key species including Penaeus vannamei and Penaeus monodon, is a major global aquaculture sector. While modern, high-density, intensive shrimp farming practices have significantly increased production, they have also led to increasingly frequent bacterial disease outbreaks. Vibrio diseases, in particular, pose a significant threat to the shrimp aquaculture industry. Vibrio disease, a serious aquatic disease caused by bacteria of the genus Vibrio, primarily affects important aquaculture species such as Penaeus vannamei and Penaeus monodon. With its rapid spread and high mortality rate, the disease poses a major threat to the global shrimp aquaculture industry. Key pathogenic Vibrio species include Vibrio harveyi, which causes red body disease and hepatopancreatic necrosis; Vibrio parahaemolyticus, which causes acute hepatopancreatic necrosis and white stool syndrome; Vibrio alginolyticus, which causes white muscle opacity, black gill disease, and ulcers; and Vibrio cholerae, which causes intestinal inflammation and hepatopancreatic atrophy.

[0003] In shrimp farming, antibiotics are commonly used to prevent and control vibriosis, but the effectiveness of treatment is suboptimal. In recent years, the types and levels of bacterial resistance to antibiotics have increased significantly, while antibiotic development has lagged far behind the emergence of this resistance. This has led to a post-antibiotic era for the prevention and control of bacterial diseases, presenting significant challenges. Furthermore, the widespread use of antibiotics has led to the prevalence of drug-resistant bacteria, necessitating the urgent need for safer and more effective treatments for vibriosis. Phage therapy uses virulent bacteriophages with a lytic cycle to target and lyse pathogens, thereby preventing or treating disease. It offers high specificity, safety, non-toxicity, and environmental friendliness, enabling efficient and precise targeted killing of pathogens in aquaculture environments. It provides an innovative and environmentally friendly treatment option for combating various bacterial diseases faced by the aquaculture industry. Currently, phage therapy has been proven to be effective in preventing and controlling some Vibrio diseases in aquaculture. However, researchers seeking to introduce phages into practical applications have acknowledged that their narrow host spectrum often hinders their effectiveness and limits their scope of application. The future of bacteriophages as antimicrobial agents hinges on overcoming their specific limitations in recognizing and lysing host bacteria. Numerous pathogenic Vibrio species are present in shrimp farming, and there is an urgent need for agents that can infect and kill multiple Vibrio species to address the challenge of mixed infections in shrimp farming. Summary of the Invention

[0004] The invention aims to provide a bacteriophage for controlling Vibrio infection in shrimp, belonging to the technical field of veterinary medicine.

[0005] To achieve the above object, the present invention provides the following technical solutions: First, the present invention provides a bacteriophage for controlling Vibrio infection in shrimp, wherein the bacteriophage is integrated with a recombinant tail chain protein gene, and the nucleotide sequence of the recombinant tail chain protein gene is SEQ ID NO.2.

[0006] Furthermore, the amino acid sequence of the recombinant tail chain protein is SEQ ID NO.1.

[0007] Furthermore, the bacteriophage can lyse some Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio harveyi.

[0008] Furthermore, the method for preparing the bacteriophage comprises the following steps: (1) Cultivate the host bacteria of PHxp1 phage, Vibrio parahaemolyticus, to the logarithmic growth phase; (2) The recombinant tail chain protein rTC gene fragment was electroporated into Vibrio parahaemolyticus; (3) The electroporated V. parahaemolyticus was revived at 37°C and 1000 rpm for 1 hour; (4) Add 5 μL of wild-type phage PHxp1 (10 7 PFU / mL), incubated at 37°C for 8 h; (5) Collect the culture supernatant by centrifugation, filter with a 0.22 μm filter membrane, take 100 μL of the filtrate and mix it with 100 μL of logarithmic phase (OD600≈0.8) Vibrio alginolyticus or Vibrio harveyi, inoculate the double-layer plate, pick a single phage plaque, and purify it to obtain pure phage.

[0009] Secondly, the present invention provides a phage recombinant tail chain protein for broadening the host spectrum of Vibrio-derived phage, the amino acid sequence of the recombinant tail chain protein is SEQ ID NO.1.

[0010] Furthermore, the nucleotide sequence of the expression gene of the recombinant tail chain protein is SEQ ID NO.2.

[0011] Beneficial effects: The present invention compares and structurally analyzes the amino acid sequences of the tail chain proteins of Vibrio phage, Vibrio alginolyticus phage, and Vibrio harveyi phage to design a recombinant tail chain protein rTC and synthesize its expression gene. The rTC gene is electroporated into Vibrio parahaemolyticus isolated from diseased shrimp, and the host bacteria are infected with PHxp1 phage isolated from seawater. The phages collected after the rTC gene and PHxp1 phage are recombined with Vibrio alginolyticus to obtain two phages, PHxp1-1 and PHxp1-2, that can lyse Vibrio alginolyticus. Vibrio harveyi is screened to obtain one phage, PHxp1-3, that can lyse Vibrio harveyi. The host populations of the three recombinant phages have been broadened. The recombinant tail chain protein phage is used in shrimp farms to effectively control Vibrio infection in shrimp. The survival rate of the control group was only 36%, while that of the wild-type phage PHxp1 group was 73%, that of the recombinant tail chain protein phage PHxp1-1 group was 85%, that of the PHxp1-3 group was 86%, and that of the PHxp1-2 group was the highest, at 93%. The average weight of surviving shrimp in the control group was only 8.78 grams, while that of the wild-type phage PHxp1 group was 11.08 grams. The average weight of the recombinant tail chain protein phage PHxp1-1 group was 13.36 grams, that of the PHxp1-3 group was 13.17 grams, and that of the PHxp1-2 group was the highest, at 13.84 grams. Recombinant tail chain protein phage can effectively control shrimp mortality and growth stagnation caused by Vibrio infection, significantly improving aquaculture efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 : The effect of bacteriophage on the survival rate of shrimp; Figure 2 : Effects of bacteriophages on the average body weight of shrimp. DETAILED DESCRIPTION

[0013] Example 1: Isolation and identification of Vibrio Shrimp exhibiting symptoms of red body disease, hepatopancreatic necrosis, white feces syndrome, muscle turbidity, black gill disease, and surface ulcers were collected from shrimp farms in different regions of Guangdong, Guangxi, and Fujian provinces. The affected shrimp were dissected, and the hepatopancreas and intestinal tissues were collected and placed in a sterile 1.5 mL centrifuge tube containing 1 mL of PBS. The tissues were ground using a tissue grinder for 5 minutes. The grinding solution was centrifuged at 2000 rpm for 2 minutes at room temperature, and the supernatant was streaked onto TCBS agar plates. The streaked plates were incubated in a 37°C incubator for 16 hours. Suspected colonies were then streaked again. This was repeated three times until the colonies were completely consistent in morphology, indicating that they were pure strains. Genomic DNA of the isolated strains was used as a template for PCR amplification using conventional 16S primers and then sequenced. Sequencing results were then analyzed by blast analysis.

[0014] As a result, 13 strains of Vibrio were successfully isolated from the diseased shrimp samples, including 6 strains of Vibrio parahaemolyticus, which were named VP1, VP2, VP3, VP4, VP5, and VP6; 4 strains of Vibrio alginolyticus, which were named VA1, VA2, VA3, and VA4; and 3 strains of Vibrio harveyi, which were named VH1, VH2, and VH3.

[0015] Example 2: Isolation and purification of bacteriophage (1) Phage Isolation and Purification: Seawater samples were collected from the Xiapu waters of Fujian Province and filtered through a 0.22 μm filter membrane. 500 μL of the filtrate was mixed with 100 μL of 11 isolated Vibrio strains at the logarithmic phase (OD600 ≈ 0.8), and then mixed with 4 mL of water agar (approximately 55°C) and poured onto LBS solid plates. The plates were incubated at 37°C for 5–8 h. A single phage plaque was picked, diluted with Vibrio culture medium, and the above purification procedure was repeated three times to obtain pure phage.

[0016] (2) Determination of phage host spectrum The host spectrum of phage was determined using the double-layer plate method: 2 mL of logarithmic phase Vibrio liquid was mixed with 6 mL of water agar at about 55°C, and poured onto the LBS solid plate prepared in advance. After the plate solidified, 5 μL of phage liquid (10 7 PFU / mL), incubate at 37℃ for 5-8h. If phage plaques appear, the strain can be lysed; otherwise, it cannot be lysed.

[0017] A bacteriophage, designated PHxp1, was isolated from seawater. PHxp1 forms clear, transparent plaques on double-layer plates, approximately 1 mm in diameter and devoid of halos. The host spectrum of PHxp1 was determined using a double-layer plate assay. Table 1 shows that, based on 13 Vibrio strains isolated from diseased shrimp in our laboratory, PHxp1 phage lysed four strains of V. parahaemolyticus and one strain of V. harveyi, but failed to lyse V. alginolyticus. This suggests that PHxp1 primarily infects V. parahaemolyticus and has a narrow host spectrum.

[0018] Table 1 Phage PHxp1 can lyse host bacteria

[0019] Example 3: Phage tail protein design The amino acid sequences of tail chaperonins of Vibrio parahaemolyticus phage, Vibrio alginolyticus phage, and Vibrio harveyi phage were retrieved from GenBank. After alignment and structural analysis, the amino acid sequence of the recombinant tail chaperonin (rTC) was designed based on the conserved region of the V. parahaemolyticus phage tail chaperonin YP_007111887.1 sequence. The amino acid mutations of the V. alginolyticus phage tail chaperonin were introduced to create the recombinant tail chaperonin (rTC) with the amino acid sequence shown as SEQ ID NO. 1. The nucleotide sequence of the recombinant rTC expression gene was optimized based on the codon preference of V. parahaemolyticus and is shown as SEQ ID NO. 2. EcoRV restriction sites were added at both ends of the gene. The gene was synthesized and cloned into the pUC57 vector at the request of Sangon Biotech (Shanghai) Co., Ltd., resulting in the synthetic plasmid pUC57-rTC.

[0020] Example 4: Construction of recombinant tail chain protein phage (1) Take 100 μL of pUC57-rTC plasmid and digest it with EcoRV at 37℃ for 2 hours. Run electrophoresis on agarose gel and cut out the approximately 400 bp band. Use an agarose gel recovery kit to recover the rTC gene fragment.

[0021] (2) Inoculate VP1 Vibrio parahaemolyticus, the host bacteriophage of PHxp1, into a 50 mL centrifuge tube containing 5 mL of NB liquid medium. Incubate overnight at 37°C and 1000 rpm.

[0022] (3) Transfer 50 μL of the overnight culture to a 50 mL centrifuge tube containing 5 mL of NB liquid medium and culture at 37°C and 1000 rpm for about 2 hours until the logarithmic growth phase.

[0023] (4) Use a pipette to draw 1.5 ml of bacterial solution and centrifuge at 4000 rpm for 5 minutes. Discard the supernatant and add 1 ml of ultrapure water to resuspend the bacterial cells at the bottom of the tube.

[0024] (5) Repeat step 5 once.

[0025] (6) Centrifuge at 4000 rpm for 5 minutes, discard the supernatant, retain about 100 μL of water, add 20 μL of rTC gene fragment, pipette to mix, transfer to a 1 mm electroporation cup, and electroporate once at 1350 V.

[0026] (7) Add 1 mL of NB liquid medium to the electroporation cup, resuspend the bacteria in the electroporation cup, and transfer them to a 50 mL centrifuge tube. Resuscitate at 37°C and 1000 rpm for 1 hour.

[0027] (8) Add 5 μL of phage PHxp1 (10 7 PFU / mL), and incubated at 37°C for 8 h.

[0028] (9) Centrifuge at 10,000 rpm for 10 minutes, collect the supernatant, filter through a 0.22 μm filter, and mix 100 μL of the filtrate with 100 μL of 4 isolates of Vibrio alginolyticus and 3 isolates of Vibrio harveyi at the logarithmic phase (OD600 ≈ 0.8). Mix with 4 mL of water agar (approximately 55°C) and pour onto a LBS solid plate. Incubate in a 37°C incubator for 5–8 hours. Pick a single plaque, dilute with Vibrio culture medium, and repeat the above purification process three times to obtain pure phage.

[0029] (10) Use the double-layer plate method to determine the host spectrum of phages: take 2 mL of logarithmic phase Vibrio liquid and mix it with 6 mL of water agar at about 55°C, pour it onto the LBS solid plate prepared in advance, and add 5 μL of phage liquid (10 7 PFU / mL), incubate at 37℃ for 5-8h. If phage plaques appear, the strain can be lysed; otherwise, it cannot be lysed.

[0030] Vibrio parahaemolyticus VP1 electroporated with the rTC gene fragment was infected with PHxp1 phage. The phages collected after the recombination of the rTC gene and PHxp1 phage were screened with Vibrio alginolyticus to obtain two phages that can lyse Vibrio alginolyticus and named them PHxp1-1 and PHxp1-2. A phage that can lyse Vibrio harveyi, PHxp1-3, was obtained by screening with Vibrio harveyi.

[0031] The host spectrum of phages PHxp1-1, PHxp1-2, and PHxp1-3 was determined using a double-layer plate assay. Table 2 shows that phage PHxp1-1 can lyse 5 strains of V. parahaemolyticus, 4 strains of V. alginolyticus, and 1 strain of V. harveyi. Table 3 shows that phage PHxp1-2 can lyse 5 strains of V. parahaemolyticus, 4 strains of V. alginolyticus, and 2 strains of V. harveyi. Table 4 shows that phage PHxp1-3 can lyse 5 strains of V. parahaemolyticus, 2 strains of V. alginolyticus, and 3 strains of V. harveyi.

[0032] It can be seen that integrating the recombinant tail chain protein rTC into conventional Vibrio phage can broaden the host spectrum of the phage, enhance its applicability, and better control the risk of Vibrio infection in shrimp.

[0033] Table 2 Phage PHxp1-1 can lyse host bacteria

[0034] Table 3 Phage PHxp1-2 can lyse host bacteria

[0035] Table 4 Phage PHxp1-3 can lyse host bacteria

[0036] Example 5: Application effect of recombinant tail chain protein phage in shrimp farms (1) In a shrimp farm where Vibrio infection recently occurred, five aquariums were set up and water was pumped in from the same diseased waters. Phage PHxp1 was added to aquarium No. 1, PHxp1-1 was added to aquarium No. 2, PHxp1-2 was added to aquarium No. 3, PHxp1-3 was added to aquarium No. 4, and no phage was added to aquarium No. 5, which served as the control group. The amount of phage added to each experimental group was 1×10 6 PFU / L.

[0037] (2) 500 whiteleg shrimps of the same health and weight (1.05±0.05) g were randomly divided into 5 groups, and each group was placed in an aquarium. The shrimps in each group were fed the same feed and maintained under the same conditions. The water of each group was changed once every week, and 1×10 6 The experimental period was 70 days, and the survival rate and average body weight of each group were calculated at the end of the experiment.

[0038] After one week of feeding, the shrimp in the control group developed obvious symptoms such as body ulcers, white muscle turbidity, hepatopancreatic necrosis, and continued to die. The nucleic acid test of the dead shrimp was positive for Vibrio parahaemolyticus and Vibrio alginolyticus. The number of deaths in the groups with phage added to the water was significantly lower than that in the control group. Figure 1 It can be seen that by the end of the experiment, the survival rate of the control group was only 36%, the survival rate of the wild-type phage PHxp1 group was 73%, the survival rate of the recombinant tail chain protein phage PHxp1-1 group was 85%, the survival rate of the PHxp1-3 group was 86%, and the survival rate of the PHxp1-2 group was the highest at 93%. Figure 2 As can be seen, by the end of the experiment, the average weight of surviving shrimp in the control group was only 8.78 grams, while the wild-type phage PHxp1 group averaged 11.08 grams. The recombinant tail chain protein phage PHxp1-1 group averaged 13.36 grams, the PHxp1-3 group averaged 13.17 grams, and the PHxp1-2 group had the highest average weight of 13.84 grams. This shows that the recombinant tail chain protein phage can effectively control shrimp mortality and growth stagnation caused by Vibrio infection, significantly improving aquaculture efficiency.

Claims

1. A bacteriophage for controlling Vibrio infection in shrimp, characterized in that: The phage is integrated with a recombinant tail chain protein gene, and the nucleotide sequence of the recombinant tail chain protein gene is SEQ ID NO.

2.

2. The bacteriophage according to claim 1, characterized in that The amino acid sequence of the recombinant tail chain protein is SEQ ID NO.

1.

3. The bacteriophage according to claim 1, characterized in that The bacteriophage can lyse some Vibrio parahaemolyticus, Vibrio alginolyticus and Vibrio harveyi.

4. The bacteriophage according to claim 1, characterized in that The method for preparing the bacteriophage comprises the following steps: (1) Cultivate the host bacteria of PHxp1 phage, Vibrio parahaemolyticus, to the logarithmic growth phase; (2) The recombinant tail chain protein rTC gene fragment was electroporated into Vibrio parahaemolyticus; (3) The electroporated V. parahaemolyticus was revived at 37°C and 1000 rpm for 1 hour; (4) Add 5 μL of wild-type phage PHxp1 (10 7 PFU / mL), incubated at 37°C for 8 h; (5) Collect the culture supernatant by centrifugation, filter with a 0.22 μm filter membrane, take 100 μL of the filtrate and mix it with 100 μL of logarithmic phase (OD600≈0.8) Vibrio alginolyticus or Vibrio harveyi, inoculate the double-layer plate, pick a single phage plaque, and purify it to obtain pure phage.

5. A phage recombinant tail chain protein for broadening the host spectrum of Vibrio-derived phage, characterized in that: The amino acid sequence of the recombinant tail chain protein is SEQ ID NO.

1.

6. The phage recombinant tail chain protein according to claim 5, characterized in that The nucleotide sequence of the expression gene of the recombinant tail chain protein is SEQ ID NO.2.

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