CN107540735B - Antibacterial protein, isolated nucleic acid, antibacterial drug and application - Google Patents
Antibacterial protein, isolated nucleic acid, antibacterial drug and application Download PDFInfo
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Abstract
The invention provides an antibacterial protein, a separated nucleic acid, an antibacterial drug and application, and relates to the technical field of biology, wherein the histone H2A provided by the invention comprises one or more of the histone described by SEQ ID NO. 1-12, can efficiently, safely and conveniently inhibit bacterial infection, and has the function of immunoregulation; it can be used in the fields of antibacterial agents, immunopotentiators and the like.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an antibacterial protein, a separated nucleic acid, an antibacterial drug and application.
Background
Histones are important components of nucleosomes and are classified into H1, H2A, H2B, H3 and H4, depending on molecular weight and amino acid composition. Of these five classes of histones, H2A is the least evolutionarily conserved class. More histones H2A and H3 were found, and these histones H2A differ mainly in the length and sequence of the C-terminal tail, which plays an important role in the morphological structure and stability of the chromosome. In recent years, antibacterial peptides derived from histones have been the focus of research. These histone-derived antibacterial peptides are degraded and secreted by histone in the organism under the action of protease.
Edwardsiella cati (Edwardsiella ictaluri) is the causative agent of intestinal sepsis in cats. The catfish Edwardsiella mainly infects catfishes, and is the most important bacterial pathogen in channel catheterus (Ictalurus punctatus) culture. The breeding of clarias leather is also harmed by the pathogen. Other important cultured fishes of the order catfishes, such as southern catfish and leiocassis longirostris, are also attacked by the pathogen. Although Edwardsiellosis catfish causes serious economic loss to aquaculture industry in China, the basic application research on Edwardsiella catfish in China is less. Therefore, there is a strong need for new immunosuppressive drugs that are effective in controlling edwardsiella cati.
Disclosure of Invention
The first purpose of the invention is to provide an antibacterial protein which is used as an antibacterial peptide and has the functions of not only having antibacterial effect, but also having immunoregulation.
The second purpose of the invention is to provide a nucleic acid for coding the antibacterial protein, which can code the antibacterial protein, has antibacterial effect and also has the function of immunoregulation.
The third objective of the invention is to provide an expression vector, which comprises the nucleic acid.
It is a fourth object of the present invention to provide a host cell comprising the above-described nucleic acid.
The fifth object of the present invention is to provide an antibacterial agent comprising the above nucleic acid, which has an inhibitory effect on bacteria.
The sixth purpose of the invention is to provide the application of the antibacterial protein in the preparation of antibacterial drugs.
The seventh purpose of the invention is to provide the application of the antibacterial protein in preparing an immunopotentiator.
The invention is realized by the following steps:
an antibacterial protein comprises one or more of histone H2A-V1, histone H2A-V2, histone H2A-V3, histone H2A-V4, histone H2A-V5, histone H2A-V6, histone H2A-V7, histone H2A-V8, histone H2A-V9, histone H2A-V10, histone H2A-V11 and histone H2A-V12. Wherein, the amino acid sequence of the histones H2A-V1 is shown as SEQ ID NO.1, the amino acid sequence of the histones H2A-V2 is shown as SEQ ID NO.2, the amino acid sequence of the histones H2A-V3 is shown as SEQ ID NO.3, the amino acid sequence of the histones H2A-V4 is shown as SEQ ID NO.4, the amino acid sequence of the histones H2A-V5 is shown as SEQ ID NO.5, the amino acid sequence of the histones H2A-V6 is shown as SEQ ID NO.6, the amino acid sequence of the histones H2A-V7 is shown as SEQ ID NO.7, the amino acid sequence of the histones H2A-V8 is shown as SEQ ID NO.8, the amino acid sequence of the histones H2A-V9 is shown as SEQ ID NO.9, the amino acid sequence of the histones H2A-V10 is shown as SEQ ID NO.10, the amino acid sequence of the histones H2-V A-V8653 is shown as SEQ ID NO. 8611, and the amino acid sequence of the histones H2-V8653 is shown as SEQ ID NO. 8653.
The beneficial effects of the invention include:
the antibacterial protein provided by the invention can effectively, safely and conveniently inhibit bacterial infection, such as the infection of Edwardsiella cati, and has an immunoregulation function. The nucleic acid for coding the antibacterial protein, the expression vector containing the nucleic acid and the host cell provided by the invention can be used for expressing the target protein in the host cell by connecting the nucleic acid of the antibacterial protein with the expression vector, so that the target protein can be obtained rapidly in a large amount. In addition, the active ingredients of the antibacterial drug containing the antibacterial protein and the application thereof provided by the invention comprise one or more of the antibacterial protein or derivatives thereof, so that the popularization and the application of the antibacterial drug are facilitated.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is an electrophoretogram of twelve histone H2A expression plasmids constructed according to a first embodiment of the present invention;
FIG. 2 is a graph showing the results of Western blot experiments on the expression of histones H2A-V2 and histones H2A-V8 in fish somatic cells, according to a second embodiment of the present invention;
FIG. 3 is a graph showing the results of qRT-PCR for expression of histones H2A-V2 and histone H2A-V8 in bacterial-infected fish somatic cells according to the third embodiment of the present invention;
FIG. 4 is a graph showing the results of the inhibition of bacterial proliferation in fish by histones H2A-V2 and histone H2A-V8 provided in the fourth embodiment of the present invention;
FIG. 5 is a graph showing the results of qRT-PCR of histone H2A-V8 in fish in cooperation with RIP2 to enhance the nucleic acid expression of histone H2A in accordance with a fifth embodiment of the present invention;
FIG. 6 is a graph showing the results of qRT-PCR of histone H2A-V8 in fish in cooperation with RIP2 to enhance the expression of RIP2 nucleic acid in fish according to a fifth embodiment of the present invention;
FIG. 7 is a graph showing the results of qRT-PCR of histone H2A-V8 in fish with RIP2 to enhance mhc2a nucleic acid expression in cooperation with fish in accordance with a fifth embodiment of the present invention;
FIG. 8 is a graph showing the results of qRT-PCR in fish with cooperative enhancement of mhc2dab nucleic acid expression by RIP2 of histone H2A-V8 provided in the fifth embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The histone H2A provided by the embodiment of the invention, the nucleic acid, the expression vector, the host cell, the antibacterial drug and the application thereof, and the application of the histone H2A in the immunopotentiator are specifically explained below.
In eukaryotes, DNA and Histones (Histones) are combined to form chromatin, and nucleosomes are the basic structural units of chromosomal organization that contain a core histone octamer structure. The core histone octamer is composed of four histones H2A, H2B, H3 and H4, each histone is formed of two molecules, and DNA molecules of about 200 base pairs are wrapped around the outside of the core histone octamer to form one nucleosome unit. In chromatin fibers, the interaction of the connecting DNA with histone H1 links the nucleosome core components with the chromatin fibers, further folding to form chromatin.
There are different variants of histone H2A, and these also belong to histones, and H2A exists in nature (including animals and parts of plants), such as h2a.z, MacroH2A, h2a.bbd, h2a.x, etc., and these can form normal nucleosomes. Among the core histones, the H2A family is most abundant in species, and the diversity of H2A reflects the instability between H2A and H2B dimers and nucleosomes and DNA. The differences in H2A are mainly due to the length and sequence of the C-terminal (C-terminal end of DNA) tail, which plays an important role in the morphological structure and stability of chromosomes.
In recent years, antibacterial peptides derived from histones have been the focus of research. These histone-derived antibacterial peptides are degraded and secreted by histone in the organism under the action of protease. All antimicrobial peptides derived from histone H2A belong to the class of cationic antimicrobial peptides. In vitro experiments show that they have broad-spectrum antibacterial activity.
Edwardsiella cati (Edwardsiella ictaluri) is the causative agent of intestinal sepsis in cats. The catfish Edwardsiella mainly infects catfishes, and is the most important bacterial pathogen in channel catheterus (Ictalurus punctatus) culture. The breeding of clarias leather is also harmed by the pathogen. Other important cultured fishes of the order catfishes, such as southern catfish and leiocassis longirostris, are also attacked by the pathogen. Although the catedwardsiellosis causes serious economic loss to the aquaculture industry in China. Therefore, there is a strong need for new immunosuppressive drugs that are effective in controlling edwardsiella cati.
In teleost fish, histone H2A has a high homology. The identity of the histone H2A of the zebra fish (carp fish) and the histone H2A of other fishes such as channel catfish (catfish) is over 95 percent.
In one aspect, the invention provides an antimicrobial protein comprising one or more of histone H2A-V1, histone H2A-V2, histone H2A-V3, histone H2A-V4, histone H2A-V5, histone H2A-V6, histone H2A-V7, histone H2A-V8, histone H2A-V9, histone H2A-V10, histone H2A-V11, and histone H2A-V12. The antibacterial protein has the function of inhibiting bacterial proliferation, such as inhibiting the growth of the catfish Edwardsiella, can be applied to antibacterial drugs, also has the function of regulating immunity, and can be applied to immunopotentiators.
Furthermore, the amino acid sequence of the histones H2A-V1 is shown as SEQ ID NO.1, the amino acid sequence of the histones H2A-V2 is shown as SEQ ID NO.2, the amino acid sequence of the histones H2A-V3 is shown as SEQ ID NO.3, the amino acid sequence of the histones H2A-V4 is shown as SEQ ID NO.4, the amino acid sequence of the histones H2A-V5 is shown as SEQ ID NO.5, the amino acid sequence of the histones H2A-V6 is shown as SEQ ID NO.6, the amino acid sequence of the histones H2A-V7 is shown as SEQ ID NO.7, the amino acid sequence of the histones H2H 84-V8 is shown as SEQ ID NO.8, the amino acid sequence of the histones H2A-V9 is shown as SEQ ID NO.9, the amino acid sequence of the histones H462-V465 is shown as SEQ ID NO. 3748-V8, the amino acid sequence of the histones H5811 is shown as SEQ ID NO.24, the amino acid sequence of the histone H2A-V12 is shown as SEQ ID NO. 12.
In another aspect, the invention also provides an isolated nucleic acid encoding the above-mentioned antimicrobial protein, which comprises one or more of the nucleic acid sequences shown in SEQ ID NO. 13-24.
Furthermore, the nucleic acid sequence coding for histone H2A-V1 is shown in SEQ ID NO.13, the nucleic acid sequence coding for histone H2A-V2 is shown in SEQ ID NO.14, the nucleic acid sequence coding for histone H2A-V3 is shown in SEQ ID NO.15, the nucleic acid sequence coding for histone H2A-V4 is shown in SEQ ID NO.16, the nucleic acid sequence coding for histone H2A-V5 is shown in SEQ ID NO.17, the nucleic acid sequence coding for histone H2A-V6 is shown in SEQ ID NO.18, the nucleic acid sequence coding for histone H2A-V7 is shown in SEQ ID NO.19, the nucleic acid sequence coding for histone H2 84-V8 is shown in SEQ ID NO.20, the nucleic acid sequence coding for histone H2A-V9 is shown in SEQ ID NO.21, the nucleic acid sequence coding for histone H462-V A-V395 is shown in SEQ ID NO. 3723, the nucleic acid sequence coding for histone H2-V4623 is shown in SEQ ID NO. A, the nucleic acid sequence for coding the histone H2A-V12 is shown as SEQ ID NO. 24.
In another aspect, the present invention also provides an expression vector comprising the above-described nucleic acid.
Furthermore, the expression vector adopts p3 XFLAG CMV plasmid, wherein CMV is a promoter, FLAG is a label, and Hind III and KPN I endonucleases are adopted as restriction enzymes.
In another aspect, the invention also provides a host cell comprising the histone H2A described above, and in the examples of the invention, Escherichia coli TOP10 is used, and the host cell is transformed with an expression vector containing the histone H2A nucleic acid described above.
In another aspect, the invention also provides an antibacterial drug. The antibacterial medicine contains the antibacterial protein.
In another aspect, the invention provides the use of the histone H2A in the preparation of an immunopotentiator.
The features and properties of the present invention are described in further detail below with reference to examples.
First embodiment
Constructing an expression vector comprising an antimicrobial protein nucleic acid comprising the following steps.
1. Primer design
RIP2 in wild zebrafish infected with Edwardsiella catus and in wild Edwardsiella catus-/-Knocking out a transcriptome library of homozygous zebra fish, screening out a histone H2A nucleic acid which is differentially expressed, and designing a pair of specific primers on the basis. The specific primer comprises an upstream primer and a downstream primer.
Wherein, the base sequence of the upstream primer is as follows:
5’-GTCAAGCTTAAGACCAAGATGAGCGGAA-3' (e.g. as inShown as SEQ ID NO. 25)
Among them, the underlined AAGCTT is the enzyme cleavage site of Hind III.
The base sequence of the downstream primer is as follows:
5’-GATGGTACCTTGCCTTTGGCAGCCTTCTC-3' (shown in SEQ ID NO. 26)
Wherein, the underlined GGTACC is the cleavage site of KPN I.
PCR amplification
By adopting a PCR technology, wild zebra fish infected by the Edwardsiella cati is taken as template DNA, heated to about 94 ℃ for a certain time, the temperature is reduced to about 55 ℃, the template DNA becomes single-stranded, the primers (shown as SEQ ID NO.25 and SEQ ID NO. 26) are paired and combined with the template DNA single-stranded to form a DNA template and primer combination, the combination takes dNTP as a reaction raw material and a target sequence as a template under the action of Taq enzyme, and the process is repeatedly circulated according to the base pairing and semi-conservative replication principle to amplify the open reading frame of the zebra fish histone H2A. Then the amplified fragment is cut by endonuclease Hind III and KPN I and then connected to the p3 XFLAG-CMV-14 expression vector which is cut by the same double enzyme.
3. Sequencing
And transforming the constructed expression vector into a host cell, and proliferating and expressing to obtain the target nucleic acid. The recombinant plasmid containing the target nucleic acid is adhered to the surface of the host cell, the target nucleic acid is promoted to be absorbed by the host cell through short-time heat shock treatment at 42 ℃, then the next generation is cultured in a non-selective culture medium (without antibiotics), the antibiotic nucleic acid carried on the expression vector is expressed, and the successfully transformed host cell can grow in the culture medium containing the antibiotics to form a colony.
In this example, the host cell used was E.coli TOP 10. Because the propagation of the escherichia coli is fast, only 20-30 minutes are needed for the next generation of propagation under proper conditions, and the common plasmid can be propagated in the escherichia coli to hundreds of copies. Therefore, the objective plasmid can be amplified greatly in a short time by culturing E.coli which has been transformed.
Thirty clones were then selected from the colonies and sequenced to find twelve histone H2A variant nucleic acid sequences with sequence differences, which were identified by sequencing as correct and sequence-different recombinant plasmids were named separately, and which encode histone H2A-V1, histone H2A-V2, histone H2A-V3, histone H2A-V4, histone H2A-V5, histone H2A-V6, histone H2A-V7, histone H2A-V8, histone H2A-V9, histone H2A-V10, histone H2A-V11 and histone H2A-V12, respectively.
Wherein the nucleic acid sequence coding for histone H2A-V1 is shown in SEQ ID NO.13, the nucleic acid sequence coding for histone H2A-V2 is shown in SEQ ID NO.14, the nucleic acid sequence coding for histone H2A-V3 is shown in SEQ ID NO.15, the nucleic acid sequence coding for histone H2A-V4 is shown in SEQ ID NO.16, the nucleic acid sequence coding for histone H2A-V5 is shown in SEQ ID NO.17, the nucleic acid sequence coding for histone H2A-V6 is shown in SEQ ID NO.18, the nucleic acid sequence coding for histone H2A-V7 is shown in SEQ ID NO.19, the nucleic acid sequence coding for histone H2A-V8 is shown in SEQ ID NO.20, the nucleic acid sequence coding for histone H2A-V9 is shown in SEQ ID NO.21, the nucleic acid sequence coding for histone H2A-V395 is shown in SEQ ID NO.22, the nucleic acid sequence coding for histone H2A-V A is shown in SEQ ID NO.24, the nucleic acid sequence for coding the histone H2A-V12 is shown as SEQ ID NO. 24.
The recombinant plasmid containing the histone H2A-V1-H2A-V12 is subjected to double enzyme digestion and then subjected to electrophoresis experiments, and the experimental results are shown in the attached drawing 1.
Please refer to fig. 1, which is an electrophoretogram of the twelve histone H2A variant expression plasmids, sequentially from left to right, a plasmid containing histone H2A-V1 gene, a plasmid containing histone H2A-V2 gene, a plasmid containing histone H2A-V3 gene, a plasmid containing histone H2A-V4 gene, a plasmid containing histone H2A-V5 gene, a plasmid containing histone H2A-V6 gene, a plasmid containing histone H2A-V7 gene, a plasmid containing histone H2A-V8 gene, a plasmid containing histone H2A-V9 gene, a plasmid containing histone H2A-V10 gene, a plasmid containing histone H2A-V11 gene, and a plasmid containing histone H2A-V12 gene. As shown in the attached FIG. 1, the plasmids containing twelve histone H2A genes have the same mass, which indicates that the construction of the twelve histone H2A recombinant plasmids is successful.
Second embodiment
The expression of the histones H2A-V2 and the histones H2A-V8 in fish body cells is verified.
In this experimental example, expression of histones H2A-V2 and histones H2A-V8 in fish somatic cells was examined by Western blot (Western blotting). The Western blot is specifically operated as follows:
1. collecting the protein of interest
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 48 hours later.
2. Electrophoresis
A12% SDS-PAGE gel was prepared, and 20. mu.l of the target protein was added, followed by electrophoresis at 80v and then at 120 v.
3. Rotary film
The target protein was transferred to PVDF membrane, 30v stabilized voltage transfer for 90 minutes, blocked with 5% skimmed milk powder (made in TBST) for one hour, then incubated overnight at 4 ℃ with mouse anti-monoclonal FLAG-tag antibody (Sigma Co., 1:5000 dilution), and then washed 3 times for 5 minutes each with TBST. A secondary antibody (ThermoFisher, 1:5000) corresponding to horseradish peroxidase was added thereto, and the mixture was incubated for thirty minutes, followed by washing the membrane 3 times for 5 minutes each with TBST.
4. Detection of
ECL luminescence solution was used and analyzed by gel imaging, and the results of the experiment are shown in FIG. 2.
As shown in the attached figure 2, the negative control has no signal expression, and the plasmids of the histones H2A-V2 and the histones H2A-V8 can be well expressed in the EPC cells of the fishes.
Third embodiment
It was verified that histones H2A-V2 and histone H2A-V8 were expressed in vivo in fish infected with Edwardsiella cati.
In the experimental example, recombinant expression conditions of histone H2A-V2 and histone H2A-V8 plasmids in fish bodies are tested by qRT-PCR, and the specific operation is as follows:
first, the eggs provided in the first embodiment were assembled by microinjectionPlasmids of white H2A-V2 and histone H2A-V8 were microinjected into zebrafish embryos. Then, on the fourth day after fertilization, after the zebra fish juvenile fish were completely hatched, the plasmid fractions injected with the empty plasmid fraction, histone H2A-V2 and histone H2A-V8 were subjected to infection by immersion in groups at an infection concentration of 2X 108pfu (plaque forming units)/ml. Each group had 3 replicates, each replicate 30 fish. At 24h and 48h after infection, 10 young fish were taken from each group, washed with PBS, and then homogenized by adding Trizol. Then, RNA from 3 sets of samples was extracted and used for qRT-PCR after reverse transcription into cDNA.
qRT-PCR, a real-time fluorescent quantitative reverse transcription polymerase chain reaction, consists of three steps, firstly, RNA of a sample is reversely transcribed into cDNA by relying on reverse transcriptase, then the cDNA is amplified by a PCR method, and the amplified product is detected and quantified in real time.
The results of the experiment are shown in table 1 and fig. 3.
TABLE 1
As is clear from Table 1 and FIG. 3, compared with the empty plasmid injection, the expression of the histones H2A-V2 was 2644-and 321-fold respectively at 24H (day 5 after fertilization) and 48H (day 6 after fertilization), and the expression of the histones H2A-V8 was 838-and 425-fold respectively at 24H and 48H after infection. It is known that the plasmids of histone H2A-V2 and histone H2A-V8 can be well expressed in the bodies of fish infected by Edwardsiella cati.
Fourth embodiment
The effect of histone H2A-V2 and histone H2A-V8 on bacterial proliferation was verified.
The effect of the overexpression of histone H2A-V2 and histone H2A-V8 in fish on bacterial proliferation was examined by plate counting method in this example. The specific operation is as follows:
1. sample preparation
The plasmids of the histones H2A-V2 and the histones H2A-V8 provided in the first example were microinjected into zebrafish embryos by microinjection, and on day 4 after embryo fertilization, after the zebrafish larvae had hatched, the plasmid groups injected with empty plasmid group (FLAG), histones H2A-V2(histone H2A-V2) and histones H2A-V8 (histones H2A-V8) were respectively subjected to grouped immersion infection with Edwardsiella cati at an infection concentration of 2X 108pfu/ml. Each group had 3 replicates, each replicate 30 fish.
2. Dilution of
At 24h and 48h after infection, 10 young fish were taken from each group, washed with PBS, and then homogenized with 500. mu.l PBS, the homogenate product from each group was diluted in multiples, the homogenate diluted at 24h at 100X and 1000X was counted on plates, and the homogenate diluted at 48h at 400X and 4000X was counted on plates.
3. Counting
The results of the experiment are shown in table 2 and fig. 4.
TABLE 2
As can be seen from Table 2 and FIG. 4, at 24H after infection, the number of bacteria injected with the empty plasmid control group (FLAG) was 5250000.0000 + -356791.3000, the number of bacteria injected with the histones H2A-V8 was 803333.3000 + -37859.3900, the number of bacteria injected with the histones H2A-V2 was 373333.300 + -388501.4000, and the overexpression of the histones H2A-V2 and the histones H2A-V8 in fish bodies significantly inhibited the proliferation of Edwardsiella catalis.
At 48H after infection, the number of the bacteria in the control group injected with the empty plasmid is 1.620000e +007 +/-445421.2000, the number of the bacteria in the group injected with the histones H2A-V8 is 3106667.0000 +/-1583077.0000, the number of the bacteria in the group injected with the histones H2A-V2 is 4613334.000 +/-612318.0000, and the over-expression of the histones H2A-V2 and the histones H2A-V8 in the fish body obviously inhibits the proliferation of the Edwardsiella catfish.
Therefore, the overexpression of the histones H2A-V2 and the histones H2A-V8 in the fish body obviously inhibits the proliferation of the Edwardsiella cati.
Fifth embodiment
It was verified that histone H2A synergizes with RIP2 to enhance the expression of immune nucleic acids.
RIP2 is a key adaptor protein on NOD1 antibacterial signaling pathway, RIP2 against wild zebrafish infected with Edwardsiella cati and Edwardsiella cati-/-When the homozygous knockout zebra fish is subjected to comparative transcriptome sequencing, analysis shows that the knockout of RIP2 nucleic acid obviously influences the expression of histone family nucleic acid. Knockout of RIP2 gene significantly affected the signaling pathway for MHC antigen presentation, and this example provides validation of the effect of histone H2A on the signaling pathway for MHC antigen presentation in the presence or absence of RIP protein expression.
1. Experimental procedure
FLAG + ptGFP1(FLAG is p3 XFLAG-CMV-14, both FLAG and ptGFP1 are empty plasmids as a control), FLAG + RIP2 groups (RIP2 is connected on a ptGFP1 expression vector), ptGFP1+ histone H2A-V8 groups (histone H2A-V8 is connected on a FLAG expression vector), RIP2+ histone H2A-V8 groups (RIP2 and H2A-V8 are mixed at a ratio of 1: 1) four combinations of plasmids are injected into zebrafish embryos by microscopy, the zebrafish embryos are collected at 24H and 48H after the microinjection, RNA of four groups of samples is extracted by Trizol reagent, and cDNA is extracted for qRT-PCR. Each group of 3 replicates, each replicate 100 or so embryos, and examined for expression of histone H2A, expression of RIP2, expression of MHC2a (MHC class II a chain), and expression of MHC2DAB (DAB nucleic acid on MHC class II molecules).
2. Results of the experiment
The results of the experiment on histone H2A expression are shown in table 3 and fig. 5.
TABLE 3
As shown in Table 3 and FIG. 5, at 24H, the FLAG + RIP2 group was not significantly different from the blank control group, and in the ptGFP1+ histone H2A-V8 group, the expression of histone H2A was increased 64-fold compared with the control group. In the RIP2+ histone H2A-V8 group, expression of histone H2A was increased 1465-fold.
At 48H, expression of histone H2A was increased 63.7-fold in ptGFP1+ histone H2A-V8 group compared to the blank control group. In the RIP2+ histone H2A-V8 group, expression of histone H2A increased 10232.9-fold.
Therefore, the expression of histone H2A can be synergized by histone H2A-V8 and RIP 2.
The results of the experiment with RIP2 expression are shown in Table 4 and FIG. 6.
TABLE 4
As shown in Table 4 and FIG. 6, at 24h, the expression of RIP2 was increased 2249.6-fold in the FLAG + RIP2 group. In the ptGFP1+ histone H2A-V8 group, the expression of RIP2 increased 5.6-fold. In the RIP2+ histone H2A-V8 group, the expression of RIP2 increased 3918.6-fold.
At 48h, expression of RIP2 was increased 2985.6-fold in the FLAG + RIP2 group. In the ptGFP1+ histone H2A-V8 group, the expression of RIP2 increased 9.1-fold. In the RIP2+ histone H2A-V8 group, the expression of RIP2 increased 15054.3-fold. Therefore, histone H2A-V8 and RIP2 can synergize the expression of RIP 2.
The mhc 2-type molecule is composed of two polypeptide chains, namely an a chain and a beta chain, which are non-covalently linked. MHC2a (MHC II a chain) expression as shown in Table 5 and FIG. 7,
TABLE 5
As can be seen from Table 5 and FIG. 7, mhc2a increased expression by 3.7-fold at 24h in the FLAG + RIP2 group. In the ptGFP1+ histone H2A-V8 group, expression of mhc2a was increased by 1.4-fold, and in the RIP2+ histone H2A-V8 group, expression of mhc2a was increased by 3.79-fold.
At 48h, expression of mhc2a was increased 4.1-fold in the FLAG + RIP2 group. In the ptGFP1+ histone H2A-V8 group, expression of mhc2a increased 3.3-fold. In the RIP2+ histone H2A-V8 group, expression of mhc2a increased 37.7-fold. Therefore, histone H2A-V8 and RIP2 can synergize the expression of mhc 2a.
MHC2DAB (DAB nucleic acid on MHC class II molecules) is shown in table 6 and fig. 8. . In zebrafish MHC2dab contains conserved upstream regulatory sequences similar to human MHC2(MHC II).
TABLE 6
As can be seen from Table 6 and FIG. 8, mhc2dab expression was increased 64.9-fold in the FLAG + RIP2 group at 24 h. In the ptGFP1+ histone H2A-V8 group, expression of mhc2dab was increased 1.3 fold. In the RIP2+ histone H2A-V8 group, expression of mhc2dab was increased 100.1 fold.
At 48h, mhc2dab expression was increased 13.0 fold in the FLAG + RIP2 group. In the ptGFP1+ histone H2A-V8 group, expression of mhc2dab was increased 1.3 fold. In the RIP2+ histone H2A-V8 group, mhc2dab expression increased 120.9 fold.
Therefore, histone H2A-V8 and RIP2 can synergize the expression of mhc2 dab.
From the above experimental results, it was found that there was mutual induction between histone H2A-V8 and RIP2, and that there was correlation between histone H2A-V8 and RIP2 in induction of expression of MHC class II nucleic acid.
In conclusion, the antibacterial protein provided by the invention comprises histone H2A-V1 (shown in an amino acid sequence of SEQ ID NO. 1), histone H2A-V2 (shown in an amino acid sequence of SEQ ID NO. 2), histone H2A-V3 (shown in an amino acid sequence of SEQ ID NO. 3), histone H2A-V4 (shown in an amino acid sequence of SEQ ID NO. 4), histone H2A-V5 (shown in an amino acid sequence of SEQ ID NO. 5), histone H2A-V6 (shown in an amino acid sequence of SEQ ID NO. 6), histone H2A-V7 (shown in an amino acid sequence of SEQ ID NO. 7), histone H2A-V8 (shown in an amino acid sequence of SEQ ID NO. 8), histone H2A-V9 (shown in an amino acid sequence of SEQ ID NO. 9), histone H2A-V10 (shown in an amino acid sequence of SEQ ID NO. 10), One or more of histone H2A-V11 (amino acid sequence is shown as SEQ ID NO. 11) and histone H2A-V12 (amino acid sequence is shown as SEQ ID NO. 12). It can effectively, safely and conveniently inhibit bacterial infection and has the function of immunoregulation.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> institute of aquatic organisms of Chinese academy of sciences
<120> an antibacterial protein and isolated nucleic acid, antibacterial drug and application
<130> PA17039351WH
<160> 26
<170> PatentIn version 3.5
<210> 1
<211> 127
<212> PRT
<213> Zebrafish
<400> 1
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Glu
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 2
<211> 127
<212> PRT
<213> Zebrafish
<400> 2
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Ile Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 3
<211> 127
<212> PRT
<213> Zebrafish
<400> 3
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Ile Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 4
<211> 127
<212> PRT
<213> Zebrafish
<400> 4
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 5
<211> 127
<212> PRT
<213> Zebrafish
<400> 5
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 6
<211> 127
<212> PRT
<213> Zebrafish
<400> 6
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 7
<211> 127
<212> PRT
<213> Zebrafish
<400> 7
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Thr Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 8
<211> 127
<212> PRT
<213> Zebrafish
<400> 8
Met Ser Gly Arg Gly Lys Thr Gly Gly Gln Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 9
<211> 127
<212> PRT
<213> Zebrafish
<400> 9
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 10
<211> 127
<212> PRT
<213> Zebrafish
<400> 10
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 11
<211> 127
<212> PRT
<213> Zebrafish
<400> 11
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 12
<211> 127
<212> PRT
<213> Zebrafish
<400> 12
Met Ser Gly Arg Gly Lys Thr Gly Gly Lys Ala Arg Ala Lys Ala Lys
1 5 10 15
Ser Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
20 25 30
Arg Leu Leu Arg Lys Gly Asn Tyr Ala Gln Arg Val Gly Ala Gly Ala
35 40 45
Pro Val Tyr Leu Ala Ala Val Leu Glu Tyr Leu Thr Ala Glu Ile Leu
50 55 60
Glu Leu Ala Gly Asn Ala Ala Arg Asp Asn Lys Lys Thr Arg Ile Ile
65 70 75 80
Pro Arg His Leu Gln Leu Ala Val Arg Asn Asp Glu Glu Leu Asn Lys
85 90 95
Leu Leu Gly Gly Val Thr Ile Ala Gln Gly Gly Val Leu Pro Asn Ile
100 105 110
Gln Ala Val Leu Leu Pro Lys Lys Thr Glu Lys Ala Ala Lys Gly
115 120 125
<210> 13
<211> 392
<212> DNA
<213> Zebrafish
<400> 13
aagaccaaga tgagcggaag aggcaaaacc ggtggaaagg ctagagctaa ggctaagtct 60
cgctcctcta gagcagggct tcagtttccc gttggccgtg ttcacaggct cctccgcaag 120
ggtaactatg ctcagcgggt cggtgctggt gctccagtgt acttggccgc cgtgctcgag 180
tatctgaccg ctgagatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgtatcatcc cccgtcacct gcagttggcg gtgcgcaacg acgaggagct gaacgaactt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 14
<211> 392
<212> DNA
<213> Zebrafish
<400> 14
aagaccaaga tgagcggaag aggcaaaacc ggtggaaagg ctagagctaa ggctaagtct 60
cgctcctcta gagcagggct tcagtttccc gttggccgtg ttcacaggct cctccgcaag 120
ggtaactatg ctcagcgggt cggtgctggt gctccagtgt acttggccgc cgtgctcgag 180
tatctgaccg ctgagatcct ggagttggct ggaaacgccg ctcgggacaa caagaagacc 240
cgtatcatcc cccgtcacct gcagttggcg gtgcgcatcg acgaggagct gaacaaactt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 15
<211> 392
<212> DNA
<213> Zebrafish
<400> 15
aagaccaaga tgagcggaag aggcaaaacc ggtggaaagg ctagagctaa ggctaagtct 60
cgctcctcta gagcagggct tcagtttccc gttggccgtg ttcacaggct cctccgcaag 120
ggtaactatg ctcagcgggt cggtgctggt gctccagtgt acttggccgc cgtgctcgag 180
tatctgaccg ctgagatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgtatcatcc cccgtcatct acagctggcg gtgcgcaacg acgaggagct gaacaaactt 300
ctgggtggag tgatcatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 16
<211> 392
<212> DNA
<213> Zebrafish
<400> 16
aagaccaaga tgagcggaag aggcaaaacc ggcggcaagg ccagagctaa ggctaagtca 60
cgttcttcca gggctggact tcagtttccc gtcggccgtg ttcacaggct gctccgcaag 120
ggtaactatg ctcagcgtgt gggtgccggt gccccggttt acttggccgc tgtgctcgag 180
tatctgaccg ctgagatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgtatcatcc cccgtcacct gcagctggcg gtgcgcaacg acgaggagct gaacaaactt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 17
<211> 392
<212> DNA
<213> Zebrafish
<400> 17
aagaccaaga tgagcggaag aggcaaaacc ggcggcaagg ccagagctaa ggccaagtca 60
cgttcttcca gggctggact gcagtttccc gtcggccgtg tccacaggct gctccgcaag 120
ggtaactatg ctcagcgtgt gggtgccggt gccccggttt acttggccgc tgtgctcgag 180
tatctgaccg ctgagatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgtatcatcc cccgtcacct gcagttggcg gtgcgcaacg acgaggagct gaacaaactt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 18
<211> 392
<212> DNA
<213> Zebrafish
<400> 18
aagaccaaga tgagcggaag aggcaaaacc ggcggcaagg ccagagcaaa ggctaagtca 60
cgttcttcca gggctggact gcagtttccc gtcggccgtg tccacaggct gctccgcaag 120
ggtaactatg ctcagcgtgt gggtgccggt gccccggttt acctggccgc tgtgctcgag 180
tatctgaccg ctgagatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgtatcatcc cccgtcacct gcagctggcg gtgcgcaacg acgaggagct gaacaaactt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 19
<211> 392
<212> DNA
<213> Zebrafish
<400> 19
aagaccaaga tgagcggaag aggcaaaacc ggcggcaagg ccagagctaa ggctaagtca 60
cgttcttcca gggctggact tcagtttccc gtcggccgtg ttcacaggct gctccgcaag 120
ggtaactatg ctcagcgtgt gggtgccggt gccccggttt acttggccgc tgtgctcgag 180
tatctgaccg ctgagatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgtaccatcc cccgtcacct gcagttggcg gtgcgcaacg acgaggagct gaacaaactt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 20
<211> 392
<212> DNA
<213> Zebrafish
<400> 20
aagaccaaga tgagcggaag aggcaaaacc ggcgggcagg ccagagctaa ggctaagtca 60
cgttcttcca gggctggact tcagtttccc gtcggccgtg ttcacaggct gctccgcaag 120
ggtaactatg ctcagcgtgt gggtgccggt gccccggttt acttggccgc tgtgctcgag 180
tatctgaccg ctgagatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgtatcatcc cccgccacct gcagttggcg gtgcgcaacg acgaggagct gaacaaactt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 21
<211> 392
<212> DNA
<213> Zebrafish
<400> 21
aagaccaaga tgagcggaag aggcaaaacc ggaggaaaag cccgcgccaa ggctaagact 60
cgttcttcta gagcagggct tcagtttcca gtcggccgtg ttcacagact tctccgcaag 120
gggaactatg ctcagcgcgt cggtgctgga gctccagtgt atctggccgc tgtgctcgag 180
tatctgaccg ctgagatcct ggagttggct ggaaatgccg ctcgggacaa caagaagacc 240
cgtatcatcc ccagacatct gcagctggcg gtgcgcaacg atgaagagct gaacaaactt 300
ctgggcggtg tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaga ccgagaaggc tgccaaaggc aa 392
<210> 22
<211> 392
<212> DNA
<213> Zebrafish
<400> 22
aagaccaaga tgagcggaag aggcaaaacc ggcggcaagg ccagagctaa ggctaagtca 60
cgttcttcca gggctggact tcagtttccc gtcggccgtg ttcacaggct gctccgcaag 120
ggtaactatg ctcagcgtgt gggtgccggt gccccggttt acttggccgc tgtgctcgag 180
tatctgaccg ctgagatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgtatcatcc cccgccacct gcagttggcg gtgcgcaacg acgaggagct gaacaaactt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 23
<211> 392
<212> DNA
<213> Zebrafish
<400> 23
aagaccaaga tgagcggaag aggcaaaacc ggcggcaagg ccagagctaa ggctaagtca 60
cgttcttcca gggctggact tcagtttccc gtcggccgtg ttcacaggct gctccgcaag 120
ggtaactatg ctcagcgtgt gggtgccggt gccccggttt acttggccgc tgtgctcgag 180
tatctgaccg ctgagatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgtatcatcc cccgtcacct gcagttggcg gtgcgcaacg acgaggagct gaacaaactt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cccaagaaaa ccgagaaggc tgccaaaggc aa 392
<210> 24
<211> 392
<212> DNA
<213> Zebrafish
<400> 24
aagaccaaga tgagcggaag aggcaaaacc ggtggcaagg ctagagcaaa ggcgaagtct 60
cgctcatcca gggctggtct tcagtttccc gttggccgtg ttcacagact gctccgtaag 120
ggtaactatg ctcagcgtgt tggtgccggt gctccagtct acttggccgc tgtgctcgag 180
tatctgaccg ctgaaatcct ggagttggcc ggaaacgccg ctcgggacaa caagaagacc 240
cgcatcatcc cccgtcacct gcagctggcg gtgcgcaacg acgaggagtt gaacaagctt 300
ctgggtggag tgaccatcgc tcagggtggt gtgctgccca acatccaggc cgtgctgctg 360
cctaagaaga ccgagaaggc tgccaaaggc aa 392
<210> 25
<211> 28
<212> DNA
<213> Artificial sequence
<400> 25
gtcaagctta agaccaagat gagcggaa 28
<210> 26
<211> 29
<212> DNA
<213> Artificial sequence
<400> 26
gatggtacct tgcctttggc agccttctc 29
Claims (10)
1. An antibacterial protein, which is characterized by comprising one or more of histone H2A-V2, histone H2A-V8;
the amino acid sequence of the histone H2A-V2 is shown as SEQ ID NO.2, and the amino acid sequence of the histone H2A-V8 is shown as SEQ ID NO. 8.
2. An isolated nucleic acid encoding the antimicrobial protein of claim 1.
3. The nucleic acid according to claim 2, wherein the nucleic acid comprises one or more of the nucleic acid sequences shown as SEQ ID No.14, SEQ ID No. 20.
4. An expression vector comprising the nucleic acid of claim 2 or 3.
5. The expression vector of claim 4, wherein the expression vector is a p3 XFLAG-CMV vector.
6. A host cell comprising the nucleic acid of claim 2 or 3.
7. An antibacterial agent comprising the antibacterial protein according to claim 1 or the nucleic acid according to claim 2.
8. The antibacterial agent according to claim 7, wherein the bacterium is Edwardsiella cati.
9. Use of the antimicrobial protein of claim 1 or the nucleic acid of claim 2 in the preparation of an antimicrobial medicament.
10. Use of the antimicrobial protein of claim 1 or the nucleic acid of claim 2 in the preparation of an immunopotentiator.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101052413A (en) * | 2003-11-05 | 2007-10-10 | C·B·F·莱蒂私人有限公司 | Use of specific histones for the treatment of parasitic diseases |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101052413A (en) * | 2003-11-05 | 2007-10-10 | C·B·F·莱蒂私人有限公司 | Use of specific histones for the treatment of parasitic diseases |
Non-Patent Citations (5)
| Title |
|---|
| In Yup Park等.Parasin I, an antimicrobial peptide derived from histone H2A in the cat¢sh, Parasilurus asotus.《FEBS Letters》.1998,第437卷 * |
| NP_001096634.1;Strausberg,R.L.等;《GenBank》;20130418;全文 * |
| Strausberg,R.L.等.NP_001096634.1.《GenBank》.2013, * |
| 青鱼、草鱼、鲢鱼和鳙鱼组蛋白Ｈ２Ａ Ｎ－端基因克隆及其衍生抗菌肽;武向敏等;《水产科学》;20140515;第33卷(第5期);311-316 * |
| 鱼类组蛋白Ｈ２Ａ衍生抗菌肽研究进展;武向敏等;《水产科学》;20130515;第32卷(第5期);第306页左栏第1-3段 * |
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