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A kind of antibacterial protein and isolated nucleic acid, antibacterial drug and application

Active Publication Date: 2021-11-23
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Edwardsiella catfish disease has caused serious economic losses to the aquaculture industry in my country, there are still relatively few basic applied researches on Edwardsiella catfish in China

Method used

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  • A kind of antibacterial protein and isolated nucleic acid, antibacterial drug and application
  • A kind of antibacterial protein and isolated nucleic acid, antibacterial drug and application
  • A kind of antibacterial protein and isolated nucleic acid, antibacterial drug and application

Examples

Experimental program
Comparison scheme
Effect test

no. 1 example

[0042] Constructing the expression vector containing the antibacterial protein nucleic acid comprises the following steps.

[0043] 1. Primer Design

[0044] RIP2 in wild zebrafish infected with E. silguris and in RIP2 - / - The differentially expressed histone H2A nucleic acid was screened in the transcriptome library of the knockout homozygous zebrafish, and a pair of specific primers were designed on this basis. The specific primers include upstream primers and downstream primers.

[0045] Wherein, the base sequence of the upstream primer is:

[0046] 5'-GTC AAGCTT AAGACCAAGATGAGCGGAA-3' (as shown in SEQ ID NO.25)

[0047] Wherein, the underlined AAGCTT is the restriction site of Hind III.

[0048] The base sequence of the above-mentioned downstream primers is:

[0049] 5'-GAT GGTACC TTGCCTTTGGCAGCCTTCTC-3' (as shown in SEQ ID NO.26)

[0050] Wherein, the underlined place GGTACC is the enzyme cleavage site of KPN I.

[0051] 2. PCR amplification

[0052] Using PCR...

no. 2 example

[0061] To verify the expression of histone H2A-V2 and histone H2A-V8 in fish somatic cells.

[0062] In this experimental example, the expression of histone H2A-V2 and histone H2A-V8 in fish somatic cells was examined by Western blot (Western blotting). The specific operation of Western blot is as follows:

[0063] 1. Collect the target protein

[0064] The histone H2A-V2 and histone H2A-V8 plasmids constructed in the first example were transfected into fish EPC cells, and the target protein was extracted after 48 hours.

[0065] 2. Electrophoresis

[0066] Prepare a 12% SDS-PAGE gel, add 20 μl of the target protein, and conduct electrophoresis at 80v and then at 120v.

[0067] 3. Transfer film

[0068] Transfer the target protein to PVDF membrane, 30v constant pressure transfer membrane for 90 minutes, 5% skimmed milk powder (TBST preparation) to block for one hour, then add mouse anti-monoclonal FLAG tag antibody (Sigma company, 1:5000 dilution) and incubate at 4°C Over...

no. 3 example

[0073] Validation of expression of histone H2A-V2 and histone H2A-V8 in fish infected with E. caturis.

[0074] In this experimental example, qRT-PCR was used to test the recombinant expression of histone H2A-V2 and histone H2A-V8 plasmids in fish. The specific operations are as follows:

[0075] First, the plasmids of histone H2A-V2 and histone H2A-V8 provided in the first embodiment were microinjected into zebrafish embryos by microinjection. Then, on the fourth day after fertilization, after the zebrafish larvae were fully hatched, the plasmid groups injected with the empty plasmid group, histone H2A-V2 and histone H2A-V8 were divided into groups for soaking infection, and the infection concentration was 2 ×10 8 pfu (plaque forming unit) / ml. 3 replicates per group, 30 fish per replicate. At 24h and 48h after infection, 10 juvenile fish were taken from each group, washed with PBS, and then homogenized with Trizol. Then, the RNA of the 3 groups of samples was extracted an...

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Abstract

The invention provides an antibacterial protein, isolated nucleic acid, antibacterial drug and application, and relates to the field of biotechnology. The histone H2A provided by the invention includes one or more of the histones described in SEQ ID NO.1-12 , which can inhibit bacterial infection efficiently, safely and conveniently, and has the function of immune regulation; it can be used in the fields of antibacterial drugs, immune enhancers and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibacterial protein, isolated nucleic acid, antibacterial drug and application. Background technique [0002] Histones are important components of nucleosomes. According to their molecular weight and amino acid composition, histones can be divided into H1, H2A, H2B, H3 and H4. Among the five histones, H2A is the least conserved in evolution. At present, many histones H2A and H3 are found. The difference of these histone H2A mainly lies in the length and sequence of the C-terminal tail, which plays an important role in the morphological structure and stability of chromosomes. In recent years, antimicrobial peptides derived from histones have become a research hotspot. These histone-derived antimicrobial peptides are degraded and secreted by histones in organisms under the action of proteases. [0003] Edwardsiella ictaluri is the pathogen that causes catfish intestinal sepsis....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/46C12N15/12C12N15/63A61K38/16A61P31/04A61P37/04
Inventor 昌鸣先武小曼谢海侠李楠聂品
Owner INST OF AQUATIC LIFE ACAD SINICA
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