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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Problems solved by technology

At present, the disease prevention and treatment of pomfret carp still mainly relies on antibio

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

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Example Embodiment

[0038] Example 1 The preparation method of the antimicrobial peptide gene of Brycephalus pomfret and the peptide encoded and prokaryotic expression thereof includes the following steps:

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

[0040] ①Extract RNA from the liver of Brandt pomfret, reverse transcription to obtain the cDNA library of Brandt's pomfret, and amplify with LEAP-2F1 / R1 primers to obtain the full-length sequence and specific sequence of the antimicrobial peptide gene TroLEAP-2 of the brandt pomfret. See SEQ NO.1.

[0041] The upstream primer LEAP-2F1:

[0042] 5’-AATTGATATCGCCACCATGGGTCCACTGGCCTCTC-3’

[0043] Downstream primer LEAP-2R1:

[0044] 5’-CTAAATTGATATCATAGTTTACGGGCTCTGAGG-3’

[0045] ②The product was purified by using a gel recovery kit to obtain a purified fragment of the antimicrobial peptide gene TroLEAP-2 of Brucet's trevally with the signal peptide removed.

[0046] ③ The purified Bromp...

Example Embodiment

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

[0064] (1) Colony counting method

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

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

[0067] ③100μL of the diluted pathogen and 100μL of protein diluted to different concentrations were incubated at 28℃ for 3h.

[0068] ④ At the end of the 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:

[0070] Bacterial survival rate (%) = number of surviving bacteri...

Example Embodiment

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

[0079] (1) Resuspend the purified recombinant Brandt pomfret antibacterial peptide protein TroLEAP-2 in PBS to a final concentration of 200 μg / mL, and then mix with the same 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 Brandt pomfrets weighing about 10 g were randomly divided into 2 groups, and injected with 100 μl rTroLEAP-2+ aluminum and compound adjuvants and aluminum and compound adjuvants respectively, and were cultured in a circulating aquaculture system.

[0081] (3) Eight weeks after immunization, Edwards tarda and Streptococcus agalactiae were used to challenge the poison. Observe and record the cumulative mortality of the pompano in each group. The relative protection effect RPS calculation formula is as follows:

[0082] RPS=(1-% cumulative dea...

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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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