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Preparation method of antibacterial peptide gene and encoding peptide and prokaryotic expression thereof Pomfret brandt

A prokaryotic expression and antimicrobial peptide technology, which is applied in the field of aquatic animal immunity, can solve the problems of strong bacterial drug resistance and crisis ecological security, and achieve the effect of easy degradation and drug resistance

Active Publication Date: 2019-03-29
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the disease prevention and treatment of pomfret carp still mainly relies on antibiotics, which leads to increasingly strong bacterial resistance and endangers ecological security.

Method used

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  • Preparation method of antibacterial peptide gene and encoding peptide and prokaryotic expression thereof Pomfret brandt
  • Preparation method of antibacterial peptide gene and encoding peptide and prokaryotic expression thereof Pomfret brandt
  • Preparation method of antibacterial peptide gene and encoding peptide and prokaryotic expression thereof Pomfret brandt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 The preparation method of the antimicrobial peptide gene and its coded peptide and prokaryotic expression of Bradley's pomfret comprises the following steps:

[0039] 1) Cloning and purification of the TroLEAP-2 fragment of the antimicrobial peptide gene of the pomfret pomfret

[0040] ①Extract liver RNA of Pompano brucei, reverse transcribe to obtain the cDNA library of Pomfret brucei, and use LEAP-2F1 / R1 primers to amplify to obtain the full-length sequence of the antimicrobial peptide gene TroLEAP-2 of Pomfret brucei without signal peptide, the specific sequence See SEQ NO.1.

[0041] Among them, the upstream primer LEAP-2F1:

[0042] 5'-AATTGATATCGCCACCATGGGTCCACTGGCCTCTC-3'

[0043] Downstream primer LEAP-2R1:

[0044] 5'-CTAAATTGATATCATAGTTTACGGGCTCTGAGG-3'

[0045] ②Gel recovery kit was used to purify the product, and the purified TroLEAP-2 fragment of the antimicrobial peptide gene of Pomfret pomfret without signal peptide was obtained.

[0046]③ ...

Embodiment 2

[0063] Example 2: In vitro antibacterial activity experiment of recombinant pompano antibacterial peptide

[0064] (1) Colony counting method

[0065] ①Dilute the three pathogenic bacteria grown to the logarithmic phase of Edwardsiella tarda, Vibrio harveii and Streptococcus agalactiae to 1×10 with LB medium. 5 CFU / mL.

[0066] ② Dilute the recombinant Bradley pomfret antimicrobial peptide (rTroLEAP-2 for short) and the prokaryotic expression empty carrier protein rTrx with PBS to 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, in addition, 100 μL PBS as a negative control.

[0067] ③ 100 μL of diluted pathogenic bacteria and 100 μL of proteins diluted to different concentrations were incubated at 28°C for 3 hours.

[0068] ④ After incubation, dilute the incubation solution 100 times, and take 100 μL for coating. Incubate overnight at 28°C.

[0069] ⑤Record the number of single colonies on each plate, the calculation formula is:

[0070] Bacteria...

Embodiment 3

[0078] Example 3: In vivo antibacterial activity experiment of recombinant pompano antimicrobial peptide rTroLEAP-2

[0079] (1) The purified recombinant TroLEAP-2 was resuspended in PBS to a final concentration of 200 μg / mL, and then mixed with an equal amount of aluminum and compound adjuvant. PBS was also mixed with the same amount of aluminum and compound adjuvant as a control group.

[0080] (2) 100 pomfrets weighing about 10 g were randomly divided into two groups, injected with 100 μl rTroLEAP-2 + aluminum and compound adjuvant and aluminum and compound adjuvant respectively, and cultured in a recirculating aquaculture system.

[0081] (3) Eight weeks after immunization, Edwardsiella lentus and Streptococcus agalactiae were used to challenge the virus respectively, and the cumulative mortality of pomfret in each group was observed and recorded. The relative protective effect RPS calculation formula is as follows:

[0082] RPS=(1-% cumulative mortality of sample group / ...

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Abstract

The invention relates to a preparation method of antibacterial peptide gene and encoding peptide and prokaryotic expression thereof in Pomfret brandt and belongs to the technical field of aquatic animal immunity, wherein the gene is shown in SEQ NO.1, the amino acid sequence of the antimicrobial peptide of Pomfret scad encoded by the gene is shown in SEQ No.2. The method also provides the prokaryotic expression of the antimicrobial peptide TroLEAP-2 of pomfret scad and preparation method, the vector is Escherichia coli DH5alpha competent cells, the strain used for recombination is Rosetta (DE3). The antibacterial peptide TroLEAP-2 of pomfret scad obtained by the method has obvious inhibitory effect on Eduard tardy, Vibrio harveyi and Streptococcus lactis at the final concentration of 30 mug / ml. In addition, compared with traditional antibiotics, antimicrobial peptide TroLEAP-2 is easy to degrade and bacteria are not easy to produce drug resistance.

Description

technical field [0001] The invention belongs to the technical field of aquatic animal immunization, and in particular relates to an antibacterial peptide gene of pompano brucei and its coded peptide and a prokaryotic expression preparation method. Background technique [0002] Antimicrobial peptides are a class of small molecule polypeptides discovered in recent years with antimicrobial activity, widely present in bacteria, fungi, plants, insects, crustaceans, fish, amphibians, birds and mammals. Antimicrobial peptides have the characteristics of wide killing range, strong lethality, safety and no side effects, especially their strong inhibitory effect on drug-resistant bacteria, so they can replace antibiotics to a certain extent and become a hot research topic today. [0003] Fish antimicrobial peptides are an important part of the non-specific immune system of fish. When fish are injured or infected by pathogenic microorganisms, they can quickly produce specific types of ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C07K14/46C12N1/21C12N15/70A61K38/17A61P31/04C12R1/19
CPCA61P31/04C12N15/70C07K14/461A61K38/00
Inventor 孙云周永灿雷阳刘春胜曹贞洁王世锋郭伟良
Owner HAINAN UNIVERSITY
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