A pathogenic Vibrio Pir B virulence protein binding peptide P2 and its application

Abstract

The application discloses a pathogenic Vibrio Pir B virulence protein binding peptide P2 and application thereof. The small-molecule binding peptide P2 has high affinity with the Pir B virulence protein and has an amino acid sequence of LGSPLLTYGRPQ. The application screens the binding peptide with high affinity with the pathogenic Vibrio Pir B virulence protein from a random peptide library through phage display technology, and the binding peptide is chemically synthesized. After injection of the binding peptide, the survival rate of shrimps after infection with Vibrio can be effectively improved, and the shrimps have a remarkable protection effect in shrimp culture. The binding peptide can efficiently bind to the pathogenic Vibrio Pir B virulence protein, and effectively reduce the virulence of the Pir B protein. In addition, the method can effectively reduce the use of antibiotics in healthy shrimp culture, reduce the generation of drug-resistant strains, and has a good application prospect in the prevention and treatment of shrimp AHPND.

Description

Technical fields:

[0001] This invention belongs to the field of biochemistry and molecular biology, specifically a pathogenic Vibrio Pir B virulence protein-binding peptide P2 and its application in the preparation of a drug for the prevention and treatment of acute hepatopancreatic necrosis disease (AHPND) in shrimp. Background technology:

[0002] Litopenaeus vannamei, commonly known as the Pacific white shrimp or Litopenaeus vannamei, is native to the tropical eastern Pacific Ocean and is the world's and my country's most produced farmed shrimp species. According to the "2021 China Fisheries Yearbook," my country's annual production of Litopenaeus vannamei in 2020 exceeded 1.86 million tons, playing a significant role in meeting the people's demand for high-quality protein food.

[0003] With the continuous expansion of the Litopenaeus vannamei farming industry and the increasing diversification of farming environments and methods, outbreaks of diseases in Litopenaeus vannamei are becoming more frequent. Vibrio infection is the most common and damaging bacterial disease in shrimp farming. Acute hepatopancreatic necrosis disease (AHPND) in Litopenaeus vannamei was formerly known as "early death syndrome" (EMS). According to estimates by the Global Aquaculture Federation, AHPND causes economic losses of up to $1 billion annually; however, there are still no effective control measures to curb its outbreaks.

[0004] The virulence protein Pir AB secreted by *Vibrio parahaemolyticus*, a pathogenic bacterium in *Litopenaeus vannamei* (AHPND), specifically binds to receptor proteins on the hepatopancreatic cells of *Litopenaeus vannamei*, causing cell detachment and subsequently inducing acute necrosis of the hepatopancreatic tissue, ultimately leading to shrimp death. Analysis of the protein structure of Pir AB revealed that it contains a pore-binding domain I and a receptor-binding domain II. Designing a Pir AB-binding peptide to block its binding to the host's receptor protein can alleviate the toxicity of Pir AB to hepatopancreatic target cells, thereby reducing mortality after infection with pathogenic *Vibrio parahaemolyticus*. Summary of the Invention:

[0005] The purpose of this invention is to provide a pathogenic Vibrio Pir B virulence protein binding peptide P2, which can specifically bind to the pathogenic Vibrio Pir B virulence protein, reduce the mortality rate of shrimp infected with Vibrio, and can be applied to the prevention and treatment of acute hepatopancreatic necrosis disease (AHPND) in shrimp.

[0006] Based on the reported gene sequence of the pathogenic Vibrio virulence protein Pir B, the inventors exogenously expressed the Pir B virulence protein in prokaryotes. The expressed protein was then purified, denatured, and renatured to obtain an active Pir B virulence protein. Using the purified Pir B virulence protein as a target, phage display technology was employed to screen a small molecule binding peptide P2 with high affinity for Pir B virulence protein from a random 12-peptide library. The amino acid sequence of P2 is: LGSPLLTYGRPQ. The P2 binding peptide of this invention is structurally novel and has not been reported in any previous literature.

[0007] Therefore, the first objective of this invention is to provide a small molecule binding peptide P2 with high affinity for Pir B virulence protein, characterized in that the amino acid sequence is LGSPLLTYGRPQ.

[0008] A second objective of this invention is to provide a gene encoding a small molecule binding peptide P2.

[0009] A third objective of this invention is to provide the application of the above-mentioned small molecule binding peptide P2 or its encoding gene in the preparation of drugs for inhibiting Vibrio.

[0010] Preferably, the Vibrio refers to Vibrio species that carry genes encoding the Pir B virulence protein, including but not limited to Vibrio species such as Vibrio halys and Vibrio parahaemolyticus.

[0011] The binding peptide P2 of the present invention can be obtained by chemical synthesis or genetic engineering recombination expression.

[0012] Preferably, the small molecule binding peptide can be a vector expressing the small molecule binding peptide P2, an engineered bacterial strain or cell line containing the vector.

[0013] Preferably, the application of small molecule binding peptide P2 or its encoding gene in the preparation of drugs for the prevention and treatment of acute hepatopancreatic necrosis in shrimp.

[0014] The fourth objective of this invention is to provide a drug for the prevention and treatment of Vibrio or acute hepatopancreatic necrosis in shrimp, which contains a small molecule binding peptide P2, a vector expressing the small molecule binding peptide P2, and an engineered strain or cell line containing the vector as active ingredients.

[0015] This invention utilizes phage display technology to screen a binding peptide with high affinity for the Pir B virulence protein of pathogenic Vibrio from a random peptide library. This binding peptide is then chemically synthesized. Injection of this peptide effectively improves the survival rate of shrimp infected with Vibrio, demonstrating a significant protective effect on shrimp farming. This binding peptide can efficiently bind to the Pir B virulence protein of pathogenic Vibrio, effectively reducing the virulence of the Pir B protein. Furthermore, this method can effectively reduce the use of antibiotics in healthy shrimp farming, decreasing the emergence of drug-resistant strains, and shows great promise for the prevention and control of AHPND in shrimp. Attached image description:

[0016] Figure 1 This is an SDS-PAGE electrophoresis image of the purified Pir B virulence protein;

[0017] Figure 2 It is a screening for peptides that randomly bind to Pir B virulence proteins with high affinity;

[0018] Figure 3 The study investigated the effect of peptide P2 on the mortality rate of Litopenaeus vannamei after infection with pathogenic Vibrio. Detailed implementation method:

[0019] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described are only for illustration and explanation of the present invention and are not limited to the present invention.

[0020] Example 1

[0021] Prokaryotic expression and purification of Pir B virulence protein of pathogenic Vibrio

[0022] The amino acid sequence of Pir B virulence protein is shown in SEQ ID No. 1, and the encoded nucleotide sequence is shown in SEQ ID No. 2.

[0023] The correctly sequenced Pir B virulence protein sequence (nucleotide sequence as shown in SEQ ID NO.2) was cloned into the pet28a plasmid and transformed into *E. coli* strain BL21 for exogenous prokaryotic expression. After sonication and SDS-PAGE analysis, the target protein was found to exist primarily in inclusion body form. The inclusion bodies were then purified, renatured, and affinity purified to obtain the finally active Pir B virulence protein. SDS-PAGE analysis showed that the renatured Pir B virulence protein had a molecular weight of approximately 64 kDa and a purity greater than 80%. (See appendix) Figure 1 )

[0024] Example 2

[0025] Screening for Pir B virulence protein-binding peptide P2 of pathogenic Vibrio

[0026] Based on the prepared Pir B virulence protein, Pir B virulence protein binding peptides were selected from a phage 12-peptide random peptide library using an ELISA plate method. The experimental steps were as follows: Pir B virulence protein was coated onto an ELISA plate and incubated overnight at 4°C to a coating concentration of 2 μg / mL; the plate was then incubated at 37°C for 1 hour with 4% PBSM to block the Pir B protein; phage was diluted 1:1 in 4% PBSM, and 100 μL was added to each well, and the plate was incubated at 37°C for 1 hour; the plate was washed three times with TBST and PBS; Anti-M13-HRP antibody was diluted 1:2000 in 4% PBSM and incubated at 37°C for 40 minutes; the plate was then washed three times with TBST and PBS; 100 μL of TMB chromogenic buffer was added to each well, and the plate was incubated for 15 minutes; finally, the absorbance of each well was read at 450 nm using a microplate reader. The above process was followed for three rounds of panning. In the second and third rounds, six additional washes were performed each time. After three rounds of enrichment, the infected bacterial solutions were collected by picking monoclonal phage supernatants for ELISA titer detection. A total of 190 phage monoclonal infections were selected. The Pir B virulence protein concentration was reduced (1 μg / mL) to raise the screening standard. Further screening of the selected positive clones resulted in 11 positive clones with high affinity for the target protein and no recognition of irrelevant proteins. (Appendix) Figure 2 )

[0027] The binding peptide P2, which has a high affinity for Pir B virulence protein, was selected and sequenced. Its amino acid sequence is: LGSPLLTYGRPQ.

[0028] Example 3

[0029] The effect of Pir B virulence protein-binding peptide P2 on the survival rate of shrimp infected with pathogenic Vibrio virulence.

[0030] To verify the function of the Pir B virulence protein binding peptide P2 obtained during the pathogenic Vibrio infection process, healthy Litopenaeus vannamei shrimp (approximately 5g / shrimp) were temporarily housed in culture tanks for functional verification of the binding peptide.

[0031] The artificially synthesized binding peptide P2 (LGSPLLTYGRPQ) was administered via intramuscular injection in vitro. Its function was verified by observing its effect on mortality in Litopenaeus vannamei infected with pathogenic Vibrio. The experiment was divided into two groups (n=30): the experimental group received the selected binding peptide, and the control group received an equal volume of PBS. The synthesized binding peptide P2 was dissolved in PBS, the concentration was adjusted, and Litopenaeus vannamei was injected intramuscularly at a dose of 10 μg / g bwt, with an injection volume of 20 μL. Twenty-four hours after injection, pathogenic Vibrio parahaemolyticus (a pathogenic strain of Vibrio parahaemolyticus carrying the Pir B virulence protein gene) was injected intramuscularly again at a dose of 2 × 10⁻⁶. 6 CFU / g bwt, injection volume 20 μL. To exclude individuals that died from two injections, mortality was recorded for each group of shrimp starting 6 hours after Vibrio injection, for a total of 168 hours. Compared to the negative control group (injected with PBS, survival rate 3.45%), injection of the short peptide P2, which has a high affinity for Pir B virulence protein, significantly improved the survival rate of shrimp infected with Vibrio (survival rate 40%) (see Appendix). Figure 3 ).

[0032] The amino acid sequence of SEQ ID No. 1 - Pir B virulence protein

[0033] MTNEYVVTMSSLTEFNPNNARKSYLFDNYEVDPNYAFKAMVSFGLSNIPYAGGFLSTLWNIFWPNTPNEPDIENIWEQLRDRIQDLVDESIIDAINGILDSKIKETRDKIQDINETIENFGYAAAKDDYIGLVTHYLIGLEENFKRELDGDEWLGYAILPLLATTVSLQITYMACGLDYKDE FGFTDSDVHKLTRNIDKLYDDVSSYITELAAWADNDSYNNANQDNVYDEVMGARSWCTVHGFEHMLIWQKIKELKKVDVFVHSNLISYSPAVGFPSGNFNYIATGTEDEIPQPLKPNMFGERRNRIVKIESWNSIEIHYYNRVGRLKLTYENGEVVELGKAHKYDEHYQSIELNGAYIKYVD VIANGPEAIDRIVFHFSDDRTFVVGENSGKPSVRLQLEGHFICGMLADQEGSDKVAAFSVAYELFHPDEFGTEK

[0034] The nucleotide sequence of the gene encoding the virulence protein Pir B (SEQ ID No. 2)

[0035] ATGACTAACGAATACGTTGTAACAATGTCATCTTTGACGGAATTTAACCCTAACAATGCTCGTAAAAGTTATTTATTTGATAACTATGAAGTTGATCCTAACTATGCTTTCAAAGCAATGGTTTCATTTGGTCTTTCAAATATTCCTTACGCGGGTGGTTTTTTATCAACGTTATGGAATATCTTTTGGCCAAATACGCCAAATGAGCCAGATATTGAAAACATTTGGGAACAATTACGTGACAGAATCCAAGATTTAGTAGATGAATCGATTATAGATGCCATCAATGGAATATTGGATAGCAAAATCAAAGAGACACGCGATAAAATTCAAGACATTAATGAGACTATCGAAAACTTCGGTTATGCTCGGCAAAAGATGATTACATTGGTTTAGTTACTCATTACTTGATTGGACTTGAAGAGAACTTTAAGCGCGAGCTAGACGGTGATGAATGGCTTGGTTATGCGATATTGCCTCTATTAGCAACAACTGT AAGTCTTCAAATTACTTACATGGCTTGTGGTCTGGATTATAAGGATGAATTCGGTTTCACCGATTCTGATGTGCATAAGCTAACACGTAATATTGATAAGCTTTATGATGATGTATCGTCTTACATTACAGAACTCGCTGCGTGGGCTGATAACGACTCTTACAATAATGCAAACCAAGATAACGTGTATGATGAAGTGATGGGTGCTCGTAGTTGGTGTACGGTTCACGGCTTTGAACATATGCTTATTTGGCAAAAAATCAAAGAGTTGAAAAAAGTTGATGTGTTTGTTCACAGTAATTTAATTTCATATTCACCTGCTGTTGGTTTTCCTAGTGGTAATTTCAACTATATTGCTACAGGTACGGAAGATGAAATACCTCAACCATTAAAACCAAATATGTTTGGGGAACGTCGAAATCGTATTGTAAAAATTGAATCATGGAACAGTATTGAAATACATTATTACAATCGCGTAGGTCGACTTAAACTAACTTATGAAAATGGGGAAGTGGTAGAACTAGGCAAGGCTCATAAATATGACGAGCATTACCAATCTATTGAGTTAAACGGCGCTTACATTAAAATATGTTGATGTTATTGCCAATGGACCTGAAGCAATTGATCGAATCGTATTTCATTTTTCAGATGATCGAACATTTGTTGTTGGTGAAAACTCAGGCAAGCCAAGTGTGCGTTTGCAACTGGAAGGTCATTTTATTTGTGGCATGCTTGCGGATCAAGAAGGTTCTGACAAAGTTGCCGCGTTTAGCGTGGCTTATGAATTGTTTCATCCCGATGAATTTGGTACAGAAAAGTAG

Claims

1. A small molecule binding peptide P2 with high affinity for Pir B virulence protein, characterized in that, The amino acid sequence of the small molecule binding peptide P2 is LGSPLLTYGRPQ, and the amino acid sequence of the Pir B virulence protein is shown in SEQ ID No.

1.

2. A gene encoding the small molecule binding peptide P2 as described in claim 1.

3. The use of the small molecule binding peptide P2 of claim 1, the vector expressing the small molecule binding peptide P2, the engineered strain or cell line containing the vector, or the gene encoding the small molecule binding peptide P2 in the preparation of a drug for preventing acute hepatopancreatic necrosis caused by Vibrio parahaemolyticus infection in shrimp, wherein the Vibrio parahaemolyticus carries the Pir B virulence protein, and the amino acid sequence of the Pir B virulence protein is shown in SEQ ID No.

1.

4. The application according to claim 3, characterized in that, The small molecule binding peptide P2 is obtained through chemical synthesis or genetic engineering recombinant expression.

5. A drug for preventing acute hepatopancreatic necrosis caused by Vibrio parahaemolyticus infection in shrimp, characterized in that, The Vibrio parahaemolyticus contains the small molecule binding peptide P2 as described in claim 1 or a vector expressing the small molecule binding peptide P2, and carries the Pir B virulence protein, the amino acid sequence of which is shown in SEQ ID No. 1.

Images