A bacteriophage for controlling infection by vibrio penaeicida

Abstract

The application provides a bacteriophage for controlling Vibrio infection, and belongs to the field of veterinary technology. The application designs a recombinant tail chain protein rTC and synthesizes an expression gene of the recombinant tail chain protein rTC through amino acid sequence alignment and structure analysis of tail chain proteins of Vibrio phages. The rTC gene is electroporated into Vibrio parahaemolyticus isolated from diseased prawns, and the host bacteria are infected with PHxp1 bacteriophage isolated from seawater; after the rTC gene is recombined with the PHxp1 bacteriophage, the collected bacteriophage is screened with Vibrio alginolyticus to obtain two bacteriophages PHxp1-1 and PHxp1-2 capable of lysing Vibrio alginolyticus; the bacteriophage is screened with Vibrio harveyi to obtain one bacteriophage PHxp1-3 capable of lysing Vibrio harveyi. The host spectrum of the three recombinant bacteriophages is widened. The recombinant tail chain protein bacteriophage greatly reduces the death and growth stagnation of prawns caused by Vibrio infection in prawn farms, and improves the breeding benefit.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of veterinary technology, and particularly relates to a bacteriophage for controlling Vibrio infection of shrimp. BACKGROUND

[0002] Aquaculture is one of the pillar food industries in China, which meets more than 1 / 3 of the demand for animal protein of people, and shrimp culture is an important industry in global aquaculture, including Penaeus vannamei and Marsupenaeus japonicus. However, the modern high-density intensive shrimp culture mode leads to more frequent bacterial disease outbreaks while greatly improving the yield. Vibrio disease has a huge impact on the shrimp culture industry. Shrimp vibrio disease is a serious aquatic disease caused by Vibrio bacteria, which mainly affects important breeding varieties such as Penaeus vannamei and Marsupenaeus japonicus. The disease spreads quickly and has a high mortality rate, and is one of the main threats to global shrimp culture. The main pathogenic Vibrio species include Vibrio harveyi causing shrimp red body disease, hepatopancreas necrosis, Vibrio parahaemolyticus causing acute hepatopancreas necrosis disease and white stool syndrome, Vibrio alginolyticus causing muscle opacity, black gill disease and body surface ulcer, and Vibrio cholera causing intestinal inflammation and hepatopancreas atrophy.

[0003] In the process of shrimp culture, antibiotics are usually used to prevent and treat Vibrio disease, but the treatment effect is not ideal. In recent years, more and more bacterial antibiotic-resistant species have emerged, and the degree of drug resistance has become stronger, while the development of antibiotics lags far behind the emergence of bacterial drug resistance, which makes the prevention and control of bacterial diseases enter the post-antibiotic era, facing severe challenges. Moreover, the use of a large amount of antibiotics has led to the spread of drug-resistant bacteria, and there is an urgent need for safer and more effective treatment methods for Vibrio disease. Phage therapy is a means of using virulent phages with lytic cycles to target and lyse pathogenic bacteria, thereby preventing or treating diseases. It has strong specificity, is safe and non-toxic, and will not pollute the environment, and can efficiently and accurately target and kill pathogenic bacteria in aquaculture environments, providing an innovative and environmentally friendly treatment solution for various bacterial diseases faced by the aquaculture industry. Currently, phage therapy has been proven to be effective in preventing and treating diseases caused by some Vibrio in aquaculture. However, researchers who attempt to introduce phages into practical applications are well aware that the narrow host spectrum of phages often affects their application effect and limits their application range. The application prospect of phages as antibacterial agents depends on breaking through the specificity restrictions of host bacteria recognition and lysis. There are many pathogenic Vibrio species in shrimp culture, and it is urgent to develop phages that can infect and kill multiple Vibrio species to solve the problem of mixed infection of multiple Vibrio species in shrimp culture. SUMMARY

[0004] The present application belongs to the field of veterinary technology, and particularly relates to a bacteriophage for controlling Vibrio infection of shrimp.

[0005] To achieve the above object, the present application provides the following technical scheme:

[0006] Firstly, the present invention provides a bacteriophage for controlling Vibrio infection in shrimp, wherein the bacteriophage integrates a recombinant tail chain protein gene, the nucleotide sequence of which is SEQ ID NO.2.

[0007] Furthermore, the amino acid sequence of the recombinant tail chain protein is SEQ ID NO.1.

[0008] Furthermore, the bacteriophage is capable of lysing some Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio harveyi.

[0009] Furthermore, the method for preparing the bacteriophage includes the following steps:

[0010] (1) Culture Vibrio parahaemolyticus, the host bacteriophage of PHxp1, to the logarithmic growth phase;

[0011] (2) The recombinant tail chain protein rTC gene fragment was electroporated into Vibrio parahaemolyticus;

[0012] (3) Vibrio parahaemolyticus after electroporation was revived for 1 hour at 37°C and 1000 rpm;

[0013] (4) Add 5 μL of wild-type phage PHxp1 (10 7 PFU / mL), incubate at 37℃ for 8 hours;

[0014] (5) Centrifuge to collect culture supernatant, filter with 0.22 μm filter membrane, take 100 μL of filtrate and mix with 100 μL of Vibrio alginolyticus or Vibrio harveyi in the logarithmic phase (OD600≈0.8), inoculate with double-layer plate, pick out a single phage plaque, and purify to obtain pure phage.

[0015] Secondly, the present invention provides a recombinant tail chain protein of bacteriophage for broadening the host spectrum of Vibrio-derived bacteriophages, wherein the amino acid sequence of the recombinant tail chain protein is SEQ ID NO.1.

[0016] Furthermore, the nucleotide sequence of the gene expressing the recombinant tail chain protein is SEQ ID NO.2.

[0017] Beneficial Effects: This invention, through comparison and structural analysis of the amino acid sequences of the tail chain proteins of Vibrio phage, Vibrio alginolyticus phage, and Vibrio harveyi phage, designed a recombinant tail chain protein rTC and synthesized its expression gene. The rTC gene was electrotransferred into Vibrio parahaemolyticus isolated from diseased shrimp, and the host bacterium was infected with PHxp1 phage isolated from seawater. Phages collected after recombining the rTC gene with PHxp1 phage were screened using Vibrio alginolyticus to obtain two phages capable of lysing Vibrio alginolyticus, PHxp1-1 and PHxp1-2, and screened using Vibrio harveyi to obtain one phage capable of lysing Vibrio harveyi, PHxp1-3. The host spectrum of the three recombinant phages was broadened. The application of the recombinant tail chain protein phage in shrimp farms effectively controlled Vibrio infection in shrimp. The survival rate of the control group was only 36%, while 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 highest survival rate of the PHxp1-2 group was 93%. The average weight of the surviving shrimp in the control group was only 8.78 grams, the average weight 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, the average weight of the PHxp1-3 group was 13.17 grams, and the highest average weight of the PHxp1-2 group was 13.84 grams. Recombinant tail chain protein phage can effectively control shrimp mortality and growth retardation caused by Vibrio infection, significantly improving aquaculture efficiency. Attached Figure Description

[0018] Figure 1 The effect of bacteriophages on shrimp survival rate;

[0019] Figure 2 The effect of bacteriophages on the average weight of shrimp. Detailed Implementation

[0020] Example 1: Isolation and Identification of Vibrio

[0021] Shrimp exhibiting symptoms such as red body disease, hepatopancreatic necrosis, white feces syndrome, muscle opacity, black gill disease, and body surface ulcers were collected from different shrimp farms in Guangdong, Guangxi, and Fujian provinces. Diseased shrimp were dissected, and hepatopancreatic and intestinal tissues were collected in sterile 1.5 mL centrifuge tubes containing 1 mL PBS and homogenized for 5 min using a tissue homogenizer. The homogenate was centrifuged at 2000 rpm for 2 min at room temperature, and the supernatant was streaked onto TCBS agar plates. After incubating the streaked plates at 37℃ for 16 h, suspected colonies were picked and streaked again. This streaking process was repeated three times until the colony morphology was completely consistent, indicating a pure culture colony. Using the genomic DNA of the isolated strain as a template, PCR amplification was performed using standard 16S primers, and the sequencing results were analyzed by BLAST.

[0022] As a result, 13 strains of Vibrio were successfully isolated from diseased shrimp samples. Among them, 6 strains of Vibrio parahaemolyticus were named VP1, VP2, VP3, VP4, VP5, and VP6, respectively; 4 strains of Vibrio alginolyticus were named VA1, VA2, VA3, and VA4, respectively; and 3 strains of Vibrio harveyi were named VH1, VH2, and VH3, respectively.

[0023] Example 2: Isolation and purification of bacteriophages

[0024] (1) Phage isolation and purification: Seawater samples were collected from the Xiapu sea area 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 in the logarithmic phase (OD600≈0.8), and then mixed with 4 mL of water agar (about 55℃) and poured into LBS solid plates. The plates were incubated at 37℃ for 5-8 h. Single phage plaques were picked, diluted with Vibrio culture medium, and the above operation was repeated 3 times to obtain pure phages.

[0025] (2) Phage host spectrum determination: The host spectrum of phages was determined using the double-layer plate method: 2 mL of logarithmic Vibrio bacterial suspension was mixed with 6 mL of water agar at approximately 55°C, and poured onto a pre-prepared LBS solid plate. After the plate solidified, 5 μL of phage solution (10 μL / mL) was added. 7 (PFU / mL), incubate at 37℃ for 5-8 hours. If phage plaques appear, the strain can be lysed; otherwise, it cannot be lysed.

[0026] A bacteriophage strain was isolated from seawater and named PHxp1. Phage PHxp1 formed clear, transparent plaques approximately 1 mm in diameter on double-layer plates, without any halo. The host spectrum of PHxp1 bacteriophage was determined using the double-layer plate method. Table 1 shows that, based on 13 Vibrio strains isolated from diseased shrimp in our laboratory, PHxp1 bacteriophage could lyse 4 strains of Vibrio parahaemolyticus and 1 strain of Vibrio harveyi, but could not lyse Vibrio alginolyticus, indicating that PHxp1 bacteriophage mainly infects Vibrio parahaemolyticus and has a narrow host spectrum.

[0027] Table 1. Bacteriophage PHxp1 can lyse host bacteria

[0028]

[0029] Example 3: Design of phage tail chain proteins

[0030] The amino acid sequences of tail chaperonin from Vibrio parahaemolyticus phage, Vibrio alginolyticus phage, and Vibrio harveyi phage were retrieved from GenBank. After alignment and structural analysis, based on the conserved region of the Vibrio parahaemolyticus phage tail chaperonin YP_007111887.1 sequence, an amino acid mutation of the Vibrio alginolyticus phage tail chaperonin was introduced, and the amino acid sequence of the recombinant tail chaperonin rTC was designed as SEQ ID NO.1. The nucleotide sequence of the recombinant tail chaperonin rTC expression gene was optimized according to the codon bias of Vibrio parahaemolyticus as SEQ ID NO.2, and EcoRV restriction sites were added to both ends of the gene. The gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd. and cloned into the pUC57 vector, and the synthesized plasmid was pUC57-rTC.

[0031] Example 4: Construction of recombinant tail chain protein phage

[0032] (1) Take 100 μL of pUC57-rTC plasmid and digest it with EcoRV at 37℃ for 2 hours. Perform agarose gel electrophoresis to cut out a band of about 400 bp. Recover the rTC gene fragment using an agarose gel recovery kit.

[0033] (2) Inoculate the host bacteriophage of PHxp1, Vibrio parahaemolyticus VP1, into a 50 mL centrifuge tube containing 5 mL of NB liquid medium. Incubate overnight at 37°C and 1000 rpm.

[0034] (3) Transfer 50 μL of overnight culture bacteria to a 50 mL centrifuge tube containing 5 mL of NB liquid medium and incubate at 37 °C and 1000 rpm for about 2 hours until the logarithmic growth phase.

[0035] (4) Use a pipette to draw 1.5 ml of bacterial solution, centrifuge at 4000 rpm for 5 minutes, discard the supernatant, and add 1 mL of ultrapure water to resuspend the bacterial solution at the bottom of the tube.

[0036] (5) Repeat step 5 once.

[0037] (6) Centrifuge at 4000 rpm for 5 minutes, discard the supernatant, retain about 100 μL of water, add 20 μL of rTC gene fragment, mix by pipetting, transfer to a 1 mm electric transfer cup, and electrolyze once at 1350 V.

[0038] (7) Add 1 mL of NB liquid culture medium to the electroporation cup, resuspend the bacteria in the electroporation cup, and transfer it to a 50 mL centrifuge tube. Recover the bacteria at 37°C and 1000 rpm for 1 hour.

[0039] (8) Add 5 μL of phage PHxp1 (10 7 PFU / mL), incubate at 37℃ for 8 hours.

[0040] (9) Centrifuge at 10,000 rpm for 10 minutes, collect the supernatant, filter through a 0.22 μm filter membrane, take 100 μL of the filtrate and mix it with 100 μL of 4 strains of Vibrio alginolyticus and 3 strains of Vibrio harveyi isolated in the logarithmic phase (OD600≈0.8), mix with 4 mL of water agar (about 55℃) and pour into LBS solid plates. Incubate at 37℃ for 5-8 h, pick a single phage plaque, dilute with Vibrio culture medium and repeat the above operation 3 times to obtain pure phage.

[0041] (10) Determining the host spectrum of bacteriophages using the double-layer plate method: Mix 2 mL of logarithmic Vibrio bacterial suspension with 6 mL of water agar at approximately 55°C, pour the mixture onto a pre-prepared LBS solid plate, and after the plate solidifies, add 5 μL of bacteriophage solution (10 μL). 7 (PFU / mL), incubate at 37℃ for 5-8 hours. If phage plaques appear, the strain can be lysed; otherwise, it cannot be lysed.

[0042] Vibrio parahaemolyticus VP1, whose rTC gene fragment was electroporated, was infected with PHxp1 phage. After recombination of the rTC gene with PHxp1 phage, the collected phages were screened with Vibrio alginolyticus to obtain two phages that could lyse Vibrio alginolyticus, which were named PHxp1-1 and PHxp1-2. One phage that could lyse Vibrio harveyi was obtained by screening with Vibrio harveyi, PHxp1-3.

[0043] The host spectrum of phages PHxp1-1, PHxp1-2, and PHxp1-3 was determined using the double-layer plate method. Table 2 shows that phage PHxp1-1 could lyse 5 strains of Vibrio parahaemolyticus, 4 strains of Vibrio alginolyticus, and 1 strain of Vibrio harveyi. Table 3 shows that phage PHxp1-2 could lyse 5 strains of Vibrio parahaemolyticus, 4 strains of Vibrio alginolyticus, and 2 strains of Vibrio harveyi. Table 4 shows that phage PHxp1-3 could lyse 5 strains of Vibrio parahaemolyticus, 2 strains of Vibrio alginolyticus, and 3 strains of Vibrio harveyi.

[0044] It is evident that integrating recombinant tail chain protein rTC into conventional Vibrio phages can broaden the host spectrum of phages, enhance their applicability, and better control the risk of Vibrio infection in shrimp.

[0045] Table 2. Bacteriophage PHxp1-1 can lyse host bacteria

[0046]

[0047] Table 3. Bacteriophage PHxp1-2 can lyse host bacteria

[0048]

[0049] Table 4. Bacteriophage PHxp1-3 can lyse host bacteria

[0050]

[0051] Example 5: Application effect of recombinant tail chain protein phage in shrimp farms

[0052] (1) In a shrimp farm where vibriosis had recently occurred, five aquariums were set up, and water was pumped in from the same diseased area using a water pump. Aquarium 1 was given bacteriophage PHxp1, aquarium 2 was given bacteriophage PHxp1-1, aquarium 3 was given bacteriophage PHxp1-2, aquarium 4 was given bacteriophage PHxp1-3, and aquarium 5 was given no bacteriophage and served as the control group. The amount of bacteriophage added to each experimental group was 1×10⁻⁶. 6 PFU / L.

[0053] (2) Five hundred healthy whiteleg shrimp with a weight of (1.05±0.05) g were randomly divided into five groups, each group was placed in an aquarium. All groups were fed the same feed and managed under the same conditions. Water was changed once a week for each group, and 1×10⁻⁶ shrimp feed was added to each experimental group. 6 The phage concentration was PFU / L. The experimental period was 70 days. At the end of the experiment, the viability and average body weight of each group were recorded.

[0054] After one week of rearing, shrimp in the control group gradually developed obvious symptoms such as body surface ulcers, muscle opacity, and hepatopancreatic necrosis, and continued to die. Nucleic acid testing of the dead shrimp showed positive results for Vibrio parahaemolyticus and Vibrio alginolyticus. The number of cases and deaths in the groups where bacteriophages were added to the water was significantly lower than 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 wild-type phage PHxp1 was 73%, the survival rate of recombinant tail chain protein phage PHxp1-1 was 85%, the survival rate of PHxp1-3 was 86%, and the survival rate of PHxp1-2 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 average weight 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, the average weight of the PHxp1-3 group was 13.17 grams, and the highest average weight of the PHxp1-2 group was 13.84 grams. This demonstrates that the recombinant tail chain protein phage can effectively control shrimp mortality and growth retardation caused by Vibrio infection, significantly improving aquaculture efficiency.

Claims

1. A bacteriophage for controlling Vibrio infection in shrimp, characterized in that, The phage integrates a recombinant tail protein gene, the nucleotide sequence of which 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. A recombinant tail chain protein of bacteriophage for broadening the host spectrum of Vibrio-derived bacteriophages, characterized in that, The amino acid sequence of the recombinant tail chain protein is SEQ ID NO.

1.

4. The phage recombinant tail chain protein according to claim 3, characterized in that, The nucleotide sequence of the gene expressing the recombinant tail chain protein is SEQ ID NO.2.

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