Application of King Cobra Antibacterial Peptide OH-CATH30 in Treating Enteritis of Fish

Abstract

The present invention relates to the application of the ophiophagus hannah antimicrobial peptide OH-CATH30 in the treatment of fish enteritis, belonging to the technical field of biomedicine. The molecular weight of the ophiophagus hannah antimicrobial peptide OH-CATH30 is 4693.71 Da, and its isoelectric point is 12.08. Its amino acid sequence is KRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPFHHHHHH; the application of the ophiophagus hannah antimicrobial peptide OH-CATH30 in the treatment of fish enteritis. The effect of the present invention is that by mixing and stirring the ophiophagus hannah antimicrobial peptide OH-CATH30 with fish feed and feeding it to silver cod suffering from enteritis, the enteritis of silver cod can be effectively cured.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of the king cobra antimicrobial peptide OH-CATH30 in the treatment of fish enteritis. Background Art

[0002] Fish enteritis is a bacterial enteritis caused by infection with Aeromonas punctata f. intestinalis, and it is one of the most common fish diseases. It can affect fish from fry to adult fish, especially feeding fish such as grass carp and crucian carp are most susceptible. This disease starts to prevail when the water temperature is above 18°C, and the peak prevalence is at 25°C - 30°C. It often co-occurs with bacterial gill rot disease, red skin disease, etc., and is one of the more serious diseases in cultured fish.

[0003] Oxytetracycline is basically used to treat fish enteritis. Oxytetracycline is a broad-spectrum antibiotic. However, with the use of antibiotics, especially the abuse of antibiotics, the bacterial resistance to antibiotics has gradually increased, and the use of antibiotics has greatly contributed to the increase in antibiotic-resistant pathogens.

[0004] The king cobra antimicrobial peptide OH-CATH30 consists of 34 amino acid sequences. This patent takes the king cobra antimicrobial peptide OH-CATH30 as the research object and studies its application in the treatment of fish enteritis. Summary of the Invention

[0005] Another object of the present invention is to provide the application of the king cobra antimicrobial peptide OH-CATH30 in the treatment of fish enteritis, which can effectively cure fish enteritis, regulate the immune level of fish, and improve the feed conversion efficiency.

[0006] The present invention is achieved by the following technical solution: A king cobra antimicrobial peptide OH-CATH30, the king cobra antimicrobial peptide OH-CATH30 consists of 34 amino acid sequences; its molecular weight is 4693.71 Da, and the isoelectric point is 12.08. The amino acid sequence of the king cobra antimicrobial peptide OH-CATH30 is KRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPFHHHHHH.

[0007] Further, the king cobra antimicrobial peptide OH-CATH30 is obtained by the method of Pichia pastoris fermentation.

[0008] The application of the king cobra antimicrobial peptide OH-CATH30 of the present invention in the treatment of fish enteritis.

[0009] Advantages of the present invention: The genetically engineered expression product of the king cobra antimicrobial peptide OH-CATH30 was prepared, its antibacterial activity was identified, and an experiment for treating enteritis was conducted. The experiment showed that the king cobra antimicrobial peptide OH-CATH30 was mixed and stirred with fish feed and fed to silver cod suffering from enteritis, which could effectively cure the enteritis of silver cod. Brief Description of the Drawings

[0010] Figure 1 Picture of the control group of silver cod;

[0011] Figure 2 Picture of the experimental group of silver cod;

[0012] Figure 3 Picture of the control group of the intestinal epithelial tissue of silver cod;

[0013] Figure 4 Picture of the experimental group of the intestinal epithelial tissue of silver cod. Detailed Embodiment

[0014] 1. The king cobra antimicrobial peptide OH-CATH30 is from the venom gland of the king cobra (Ophiophagus hannah).

[0015] 2. Vector and host cell: The pPIC9K plasmid and the Pichia pastoris GS115 strain were both purchased from the Miaoling Plasmid Platform.

[0016] 3. The bacterial strain was purchased from the China General Microbiological Culture Collection Center.

[0017] Unless otherwise specified, the reagents used in the following examples of the present invention are all analytical pure reagents and can be purchased from conventional commercial channels.

[0018] The following is a further description of the present invention, and the protection scope of the present invention is not limited to the following:

[0019] Example 1: Preparation of the king cobra antimicrobial peptide OH-CATH30

[0020] S01. Construction of the king cobra antimicrobial peptide OH-CATH30 expression vector pPIC9K-OH-CATH30, the specific operation is as follows: PCR amplify the king cobra antimicrobial peptide OH-CATH30 gene to obtain the target gene fragment of the antimicrobial peptide OH-CATH30, and ligate the OH-CATH30 gene fragment with the pPIC9K vector to obtain the pPIC9K-OH-CATH30 recombinant vector;

[0021] S02. Transform the recombinant vector obtained in step S01 into the host cell Pichia pastoris GS115, and induce the expression of the host cell to obtain the expression product;

[0022] S03. The expression product obtained in step S02 is separated and purified. The specific operation is as follows: First, the expression product is centrifuged, the supernatant is collected, after dialysis, and finally purified by affinity chromatography to obtain the recombinant polypeptide OH-CATH30.

[0023] Example 2: Preparation of King Cobra Antibacterial Peptide OH-CATH30 as a Fish Feed Additive

[0024] (1). Select 30 kg of Zhongyang brand #3 silver cod feed on the market;

[0025] (2). Add the King Cobra Antibacterial Peptide OH-CATH30 polypeptide sample obtained by the method in Example 1 to the feed at a ratio of 0.5 mg / kg, mix evenly to obtain the feed containing the King Cobra Antibacterial Peptide OH-CATH30 polypeptide.

[0026] Example 3: Experiment on the Effect of King Cobra Antibacterial Peptide OH-CATH30 as a Fish Feed Additive in Treating Silver Cod Enteritis

[0027] (1). Select 50 silver cod with equivalent body weights, and the body weight of each fish is 200 g ± 10 g;

[0028] (2). Conduct a challenge experiment on the silver cod. The challenge experiment uses the intraperitoneal injection method, injecting 0.1 mL of Aeromonas punctata f. intestinalis at a concentration of 1.0×10 8 cfu / ml. Under the condition of a water temperature of 25°C and a daily feeding rate of 3%, feed is given 3 times a day. Observe the silver cod with a significantly decreased feeding situation, which are the silver cod suffering from enteritis.

[0029] (3). Set up an experimental group and a control group;

[0030] Experimental group: Select 10 silver cod suffering from enteritis, and feed the silver cod in the experimental group with the feed containing the King Cobra Antibacterial Peptide OH-CATH30 polypeptide mixed evenly in Example 2. Feed with reduced food intake. Under the condition of a water temperature of 25°C, the daily feeding rate is reduced to 1% to ensure that the medicated bait can be completely eaten by the diseased fish. Feed 3 times a day for 10 consecutive days.

[0031] Control group: Select 10 silver cod suffering from enteritis, and feed the silver cod with the same kind of ordinary fish feed without adding OH-CATH30. The daily feeding rate and the number of feedings per day are the same as those in the experimental group, and also feed for 10 consecutive days.

[0032] (4). After 10 days, dissect the silver cod in the experimental group and the control group.

[0033] As Figure 1As shown, it was found that: among the 10 silver cod in the control group, there was red swelling and flatulence in the intestines, while after 10 days of continuous feeding of the feed containing ophiophagus hannah antimicrobial peptide OH-CATH30 to the 10 silver cod in the experimental group, the intestines returned to normal, no red swelling and flatulence in the intestines were seen, and the intestines were filled with food.

[0034] (5)Intestinal sections of the silver cod in the experimental group and the control group were respectively made to observe the condition of the intestinal epithelial tissue. As Figure 2 shown, the intestinal epithelial tissue of the 10 silver cod in the control group disintegrated, while the intestinal epithelial tissue structure of the silver cod in the experimental group was intact.

[0035] The silver cod feeding experiment shows that feeding ophiophagus hannah antimicrobial peptide OH-CATH30 mixed in the feed to the silver cod suffering from enteritis can effectively cure the enteritis of the silver cod and restore the integrity of the intestinal epithelial tissue structure of the silver cod. Sequence Listing <110> Jiangsu Kangjun Biotechnology Co., Ltd. <120> Application of Ophiophagus Hannah Antimicrobial Peptide OH-CATH30 in the Treatment of Fish Enteritis <130> 20220411 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 105 <212> DNA <213> Ophiophagus hannah <400> 1 aagagattca agaaattttt caagaagctg aagaacagcg tgaagaaacg tgccaagaaa 60 ttcttcaaga agccgagggt catcggagtc tccatcccct tctaa 105 <210> 2 <211> 40 <212> PRT <213> Ophiophagus hannah <400> 2 Lys Arg Phe Lys Lys Phe Phe Lys Lys Leu Lys Asn Ser Val Lys Lys 1 5 10 15 Arg Ala Lys Lys Phe Phe Lys Lys Pro Arg Val Ile Gly Val Ser Ile 20 25 30 Pro Phe His His His His His His 35 40

Claims

1. Application of king cobra antimicrobial peptide OH-CATH30, characterized in that, The application of the king cobra antimicrobial peptide OH-CATH30 in the preparation of a drug for treating the enteritis of silver cod caused by Aeromonas punctata f. intestinalis. The king cobra antimicrobial peptide OH-CATH30 is obtained by the method of fermentation with Pichia pastoris, and its amino acid sequence is KRFKKFFKKLKNSVKKRAKKFFKKPRVIGVSIPFHHHHHH.

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