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Binding protein of pathogenic vibrio PirB protein and application thereof

A technology of pathogenic Vibrio and protein binding, applied in the field of genetic engineering, can solve the problems of intractable diseases in aquaculture, the existence of drug residues, and the destruction of the micro-ecological balance of aquatic animals, so as to reduce the mortality rate of prawns and have a good application prospect Effect

Active Publication Date: 2022-06-07
INST OF OCEANOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the continuous use of antibiotics, it is easy to produce drug-resistant strains, drug residues, and damage the micro-ecological balance of aquatic animals. On the one hand, aquaculture diseases are becoming more and more difficult to treat; on the other hand, the quality and safety of aquatic products cannot be guaranteed. [8-9]
In addition, disinfectants, water quality protection agents, basic bait organisms, probiotics, Chinese herbal medicines and microecological preparations are used for the prevention and treatment of AHPND [10-15] , but these methods have limited effects and cannot specifically target a specific pathogen to achieve a better control effect

Method used

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  • Binding protein of pathogenic vibrio PirB protein and application thereof
  • Binding protein of pathogenic vibrio PirB protein and application thereof
  • Binding protein of pathogenic vibrio PirB protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] VP AHPND Isolation and identification of candidate interacting protein LvFABP of toxin PirB

[0030] Vibrio parahaemolyticus (VP) infecting this species with acute hepatopancreatic necrosis AHPND ) of the toxin protein PirB as bait to screen the shrimp yeast library. The total RNA was extracted from the hepatopancreas, stomach and intestinal tissues of Penaeus vannamei, followed by mRNA purification and reverse transcription and cDNA synthesis and purification experiments, and then transferred to the pGADT7 vector to construct a yeast two-hybrid library, and the bait gene ( The cDNA clone of pirb) was constructed on the bait vector of yeast two-hybrid, and the self-activation and toxicity detection of the target gene were carried out during the screening of the yeast two-hybrid library; the bait plasmid and the library plasmid were transformed into yeast cells; yeast screening; screening positive results PCR Detecting gel images; screening positive results for all seq...

Embodiment 2

[0037] Example 2 Double-stranded RNA (dsLvFABP) synthesis experiment

[0038]According to the conserved domain of LvFABP gene obtained above, dsLvFABP with a length of about 300 bp was designed, and primers were designed to amplify it. The primers were dsLvFABP-F / R and dsEGFP-F / R. The primers used are shown in Table 1. Using TranscriptAid T7 High Yield (Thermo) transcription kit to synthesize dsRNA in vitro, the steps are as follows:

[0039] (a) dsLvFABP amplification: plasmids (LvFABP and EGFP) as templates. PCR system (50μL): 2×Taq PCRMasterMix (Tiangen) 25μl, Primer F 1ul, Primer R 1ul, DNA template 2ul, ddH 2 O 21ul; PCR reaction conditions: pre-denaturation at 95°C for 5min, denaturation at 95°C for 30s, annealing at 55°C for 30s, extension at 72°C for 1.5min, 30 cycles, incubation at 72°C for 10min, PCR products are recovered by gel for use.

[0040] (b) dsLvFABP transcription: using the purified PCR product as a template, the transcription system is 50 μl: 5×Transcri...

Embodiment 3

[0044] Example 3 Double-stranded RNA (dsLvFABP) interference experiment

[0045] (a) Optimal interference dose experiment of dsLvFABP:

[0046] Using the above-obtained double-stranded RNA (dsLvFABP) as the interfering agent, different interfering doses were set, and dsLvFABP was diluted with NaCl at different concentrations, and the final concentrations were 2 μg / 10 μl, 6 μg / 10 μl and 10 μg / 10 μl, respectively. The experimental group (different doses of dsLvFABP) and two control groups (NaCl group and EGFP group) were set up, with 3 biological replicates. It was injected from the side of the third muscle of the tail of Litopenaeus vannamei. After 48 hours of interference, 3 prawns in each group were used as a sampling unit, and their hepatopancreas, stomach and intestinal tract were respectively placed in 5ml cryopreservation tubes, and immediately placed in liquid Store in nitrogen. After sampling, transfer the samples to a -80°C refrigerator for long-term storage for subse...

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Abstract

The invention relates to genetic engineering, in particular to binding protein (LvFABP) of pathogenic vibrio PirB protein and application of the binding protein in prevention and treatment of AHPND (acute hepatopancreatic necrosis disease) of prawns. The invention discloses a binding protein of pathogenic vibrio PirB protein. The base sequence of the binding protein of the pathogenic vibrio PirB protein is as shown in SEQ ID NO: 1. The invention discloses application of double-stranded RNA (dsLvFABP) of a partial gene sequence of binding protein of pathogenic vibrio PirB protein as shown in SEQ ID NO: 1 in inhibition of pathogenic vibrio infection. The base sequence of the double-stranded RNA (dsLvFABP) of the partial gene sequence of the binding protein of the pathogenic vibrio PirB protein is as shown in SEQ ID NO: 2. After the double-stranded RNA is injected, the death rate of prawns infected by pathogenic vibrios can be effectively reduced, and the double-stranded RNA has a remarkable protection effect on prawn culture. The LvFABP gene and the encoding protein thereof can be used for molecular markers for prawn variety breeding, and the double-stranded RNA or the protein antibody can be used for preventing and treating diseases, so that the LvFABP gene has a good application prospect in prawn AHPND prevention and treatment.

Description

technical field [0001] The invention relates to genetic engineering, in particular to a pathogenic Vibrio PirB protein binding protein (LvFABP) and its application in the prevention and treatment of acute hepatopancreatic necrosis disease (AHPND) in prawns. Background technique [0002] Litopenaeus vannamei is the world's main farmed shrimp species, with aquaculture production of nearly 5 million tons in 2018 [1] . In recent years, due to the increasing market demand for P. vannamei, the shrimp aquaculture industry has developed rapidly, resulting in problems such as high aquaculture density, deterioration of water quality, and frequent occurrence of shrimp diseases. [0003] In recent years, acute hepatopancreatic necrosis disease (AHPND) of shrimp caused by bacteria has become a major threat to shrimp aquaculture in many Asian countries. AHPND is a bacterial contagious disease that mainly infects Litopenaeus vannamei and Penaeus monodon [2] . In the early stage, the di...

Claims

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Application Information

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IPC IPC(8): C07K14/435C12N15/12C12N15/113A61K31/713A61P1/16A61P1/18A61P31/04
CPCC07K14/43509C12N15/113A61K31/713A61P31/04A61P1/16A61P1/18C12N2310/141Y02A50/30
Inventor 谷晓倩刘梅王雷王宝杰蒋克勇
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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