CN114306574A - Koi protein capable of resisting pathogenic bacteria infection and application thereof - Google Patents

Abstract

The invention discloses a koi protein for resisting pathogenic bacteria infection and application thereof. The invention discloses a koi protein for resisting pathogenic bacteria infection, which is a protein with an amino acid sequence of SEQ ID NO.1. Experiments prove that the immunoprotein and the coding gene thereof can eliminate pathogenic bacteria in fish bodies and improve the survival rate: after the plasmid expressing the immune protein is injected into the body, the pathogenic bacteria in the fish body infected with the pathogenic bacteria are obviously lower than the control injected with the plasmid not expressing PBRRP, and the survival rate of the former is obviously higher than that of the latter. The immunoprotein and the coding gene thereof can be used for improving the immunity of the fish and further can be used for treating and preventing the pathogenic bacteria infection of the fish.

Description

Koi protein capable of resisting pathogenic bacteria infection and application thereof

Technical Field

The invention relates to the field of biotechnology, and discloses koi protein capable of resisting pathogenic bacteria infection and application thereof.

Background

The fancy carp is called as a swimming artwork and is one of the top ornamental fish varieties in the world. Different from edible carps, the fancy carps are subjected to multiple sorting in different growth stages, the operations of pulling a net, fishing and the like can cause the stress reaction of the fancy carps, so that the immunity is reduced, various pathogenic microorganisms are easy to breed, and the diseases are frequent. The diseases of koi are mostly caused by bacteria and parasites, and there are also a few diseases caused by viruses. The antibiotic and the bactericide are mainly used by amateurs and fancy carp fans for treatment, but the treatment effect is poor due to the drug resistance of pathogenic bacteria to the antibiotic, and in addition, the use of the antibiotic causes pollution to water bodies and influences the health of fishes and human beings. Therefore, the principle of 'prevention over treatment' for ornamental fishes is particularly important, and a new idea is provided for healthy breeding and disease prevention and control of fancy carps based on development of immune epidemic prevention products for improving the immune disease resistance level of the fancy carps.

Disclosure of Invention

The invention aims to solve the technical problems of improving the immunity of fish and resisting the infection of pathogenic bacteria.

In order to solve the technical problems, the invention firstly provides the application of resisting pathogenic bacteria infection protein in preparing any functional product as follows:

D1) improving the immunity of the fish;

D2) treating and/or preventing disease in fish caused by infection with pathogenic bacteria;

D3) inhibiting the growth of pathogenic bacteria in the fish body;

D4) pathogenic bacteria in the fish body are eliminated;

D5) combating infestation of fish by pathogenic bacteria;

D6) protecting fish from pathogenic bacteria infection;

D7) the survival rate of fish infected by pathogenic bacteria is improved;

the protein for resisting pathogenic bacteria infection is derived from koi (Cyprinus carpio koi) and is named as PBRRP (PBRRP), wherein the PBRRP is A1), A2) or A3):

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown by SEQ ID NO.1 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate the purification of the protein of A1), the amino terminus or the carboxy terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence Listing may be attached with the tags shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein A2) is a protein having identity of 75% or more than 75% with the amino acid sequence of the protein shown in SEQ ID NO.1 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The protein of A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of A2) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in SEQ ID NO.2, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching a coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in SEQ ID NO.2 codes the protein shown in SEQ ID NO.1.

The invention also provides any one of the following applications of the biological material related to the PBRRP:

D1) improving the immunity of the fish;

D2) treating and/or preventing disease in fish caused by infection with pathogenic bacteria;

D3) inhibiting the growth of pathogenic bacteria in the fish body;

D4) pathogenic bacteria in the fish body are eliminated;

D5) combating infestation of fish by pathogenic bacteria;

D6) protecting fish from pathogenic bacteria infection;

D7) the survival rate of fish infected by pathogenic bacteria is improved;

the biomaterial is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding a PBRRP;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a cell line comprising B1) the nucleic acid molecule or a cell line comprising B2) the expression cassette.

In the above application, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) as follows:

b11) the coding sequence is cDNA molecule or DNA molecule of SEQ ID NO.2 in the sequence table;

b12) DNA molecule shown as SEQ ID NO.2 in the sequence table;

b13) a cDNA molecule or a genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding a PBRRP;

b14) hybridizes with the nucleotide sequence defined by b11) or b12) or b13) under strict conditions and encodes a cDNA molecule or a genomic DNA molecule of the PBRRP.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the PBRRP protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 75% or more identity to the nucleotide sequence of the PBRRP protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode the PBRRP protein and have the function of the PBRRP protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID NO.1. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

The above-mentioned identity of 75% or more may be 80%, 85%, 90% or 95% or more.

In the above applications, the expression cassette containing a nucleic acid molecule encoding a PBRRP protein (PBRRP gene expression cassette) according to B2) refers to a DNA capable of expressing the PBRRP protein in a host cell, and the DNA may include not only a promoter for initiating the transcription of the PBRRP gene but also a terminator for terminating the transcription of the PBRRP gene. Further, the expression cassette may also include an enhancer sequence.

The existing expression vector can be used for constructing a recombinant vector containing the PBRRP gene expression cassette.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid may be specifically pcdna3.1.

B3) The recombinant vector can be pCDNA3.1-PBRRP. The pCDNA3.1-PBRRP is a recombinant vector obtained by replacing a DNA fragment between HindIII and XhoI recognition sequences of pCDNA3.1 with a DNA fragment shown by SEQ ID NO.2 in a sequence table. pCDNA3.1-PBRRP can express the fusion protein of PBRRP shown in SEQ ID NO.1 and 6 × His tag.

In the above application, the microorganism may be yeast, bacteria, algae or fungi.

In the above application, the cell line does not comprise propagation material.

PBRRP or the biological material also belongs to the protection scope of the present aspect.

In the invention, the immunity can be the immunity of the fish to pathogenic bacteria.

The pathogenic bacteria may be bacteria of the genus Aeromonas (Aeromonas).

Further, the bacterium of the genus Aeromonas (Aeromonas) may be Aeromonas veronii (Aeromonas veronii) or Aeromonas hydrophila (Aeromonas hydrophila). In one embodiment of the present invention, Aeromonas veronii (Aeromonas veronii) is Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 (China general microbiological culture Collection center (CGMCC, Strain No. 1.927.) in one embodiment of the present invention, Aeromonas hydrophila (Aeromonas hydrophila) NX830 (national aquatic animal pathogen Bank, Collection No. BYK 20130805).

The fish may be E1, E2, E3 or E4:

e1, cyprinid;

e2, carp;

e3, carp;

e4, Koi (Cyprinus carpio koi).

In the present invention, the fish body may be in the spleen of a fish.

Experiments prove that the PBRRP and the coding gene thereof can eliminate pathogenic bacteria in fish bodies and improve the survival rate: after the plasmid expressing the PBRRP is injected into the body, pathogenic bacteria in the fish body infected with the pathogenic bacteria are obviously lower than a control injected with the plasmid not expressing the PBRRP, and the survival rate of the former is obviously higher than that of the latter. The PBRRP and the coding gene thereof can be used for improving the immunity of the fish and further can be used for treating and preventing the pathogenic bacteria infection of the fish.

Drawings

FIG. 1 expression distribution of PBRRP gene in Koi tissue. There was no significant difference in gene expression between tissues labeled with the same letter, and there was a significant difference in gene expression between tissues labeled with different letters.

FIG. 2 shows the expression change of the Koi head kidney PBRRP gene after infection of Aeromonas veronii. PBS was used as control group, A.v was used as challenge group. Indicates that the difference reaches a significant level p < 0.05.

FIG. 3 shows the expression change of fancy carp spleen PBRRP gene after infection of Aeromonas veronii. PBS was used as control group, A.v was used as challenge group. Indicates that the difference reaches a significant level p < 0.05.

FIG. 4 shows the expression change of the Koi liver PBRRP gene after infection of Aeromonas veronii. PBS was used as control group, A.v was used as challenge group. Indicates that the difference reaches a significant level p < 0.05.

FIG. 5 spleen bacterial infection colony count of koi. pCDNA3.1 is a control group, and PBRRP is a test group.

FIG. 6 shows the protective effect of Koi PBRRP against pathogenic bacteria infection. pCDNA3.1 is a control group, and PBRRP is a test group.

FIG. 7 shows the variation of the expression of the koi head and kidney tissue immune related genes after PBRRP and control plasmid injection. pCDNA3.1 is a control group, and PBRRP is a test group. Indicates that the difference reaches a significant level p < 0.05.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 in the following examples is a China general microbiological culture Collection center (CGMCC) strain with the strain number: 1.927, the preservation date is 9/1975. The website of the strain is as follows:

http://www.cgmcc.net/directory/detailcgmccid＝1.927&number＝1.927&genus＝&species＝&yiming＝&page＝1。

examples 1,

Discovery of PBRRP Gene

The inventor discovers a gene with obviously up-regulated expression (about 2 times) by performing transcriptome sequencing on the spleen of the fancy carp with the disease after the infection of the aeromonas veronii, indicates that the gene is probably related to the immunity of the fancy carp, and records the gene as a PBRRP gene. In koi, the coding sequence of the PBRRP gene is shown as SEQ ID NO.2, and has 441 nucleotides in total, the amino acid sequence of the coding protein is shown as SEQ ID NO.1, and the coding protein does not contain a signal peptide.

The nucleotide sequence of ORF of PBRRP gene (SEQ ID NO.2) is as follows:

ATGGGATTCTGGACCATCTCTGTGTCTCTCTGTCTGCTCTTTGTTATGAATGCATCAGGAGCCTGTCGGTTTGGGTGGTCTCGATATGGACATGAATGCTTCAAGGTTTTTACCAATCCATTGTCCTGGGGTGATGCAGAGGTGACGTGCTTGAACAGTGGTGGGAACCTTGCCTCTGTGCACAGTAAACAGCAGTATGATTTCTTAAAGAGCCTGATCTCAAGTTCACAATCATACTGGATAGGAGGCTATGATGCTGTTTCAGAGGGAAAGTGGTTCTGGAGTGATGGGTCCCAAATGAATTACAGACTTTGGAACCCTGGAGAGCCCAACAACCTACAGAGGGCTGAGCACTGCATTCAGATGAACTATGGAGCTGCAGGAAATTGGAATGACCAAAAATGTACAGACAAGATACCATTTGTGTGTGTCATTTCTTGA。

the PBRRP amino acid sequence (SEQ ID NO.1) is as follows:

MGFWTISVSLCLLFVMNASGACRFGWSRYGHECFKVFTNPLSWGDAEVTCLNSGGNLASVHSKQQYDFLKSLISSSQSYWIGGYDAVSEGKWFWSDGSQMNYRLWNPGEPNNLQRAEHCIQMNYGAAGNWNDQKCTDKIPFVCVIS。

II, tissue expression distribution of fancy carp PBRRP

And detecting the relative expression quantity of the PBRRP gene in different tissues in the healthy koi body by adopting a real-time fluorescent quantitative PCR method, and researching the tissue distribution of the PBRRP mRNA.

The specific operation method comprises the following steps: randomly selecting 12 tissues of healthy koi (with the weight of about 20g), wherein the tissues are as follows: gill, eye, head kidney, spleen, kidney, heart, muscle, skin, liver, blood, brain, and intestine. Total tissue RNA was extracted using RNAioso Plus (Takara 9109) and reverse transcribed to cDNA using reverse transcription kit (Takara RR 047A).

The primers for the fluorescent quantitative PCR were designed based on the cDNA sequence of PBRRP and the primer sequences were as follows:

qNattF：5’-AACAGTGGTGGGAACCTTGC-3’；

qNattR：5’-GGACCCATCACTCCAGAACC-3’；

the primer sequences of the internal reference gene 40S ribosomal protein S11 gene are as follows:

qS11F：5’-CCGTGGGTGACATCGTTACA-3’；

qS11R：5’-TCAGGACATTGAACCTCACTGTCT-3’。

and detecting the relative expression quantity of the PBRRP in the 12 tissues by adopting an ABI7500 real-time fluorescence quantitative PCR instrument. The reaction conditions are as follows: at 95 ℃ for 30 s; 95 ℃ for 5s, 59.6 ℃ for 30s, 72 ℃ for 30s, 40 cycles.

The results showed (as shown in FIG. 1) that the PBRRP gene was expressed in all of the 12 tissues, with the highest expression level in the head and kidney, followed by kidney, gill, spleen and skin. The head kidney and spleen are the most main immune organs of fish, and the skin and gill are the main mucosal immune organs, which indicates that the gene is possibly involved in the mucosal immune response of koi and plays a role in the immune response of fish bodies.

Expression change of fancy carp PBRRP gene after infection of aeromonas veronii

Taking Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 out of an ultralow temperature refrigerator, streaking on an LB plate, culturing in an incubator at 28 ℃, recovering strains, selecting a single colony to perform amplification culture in an LB liquid culture medium, centrifugally collecting the thallus, resuspending the thallus by PBS, and adjusting the thallus to 5 x 10⁶CFU/mL to obtain suspension of Aeromonas veronii.

Randomly selecting 60 healthy fancy carps (about 20g), and randomly and averagely dividing into 2 groups, a toxicity counteracting group and a control group, wherein each group comprises 3 parallel cylinders. 100 mu L of Aeromonas veronii suspension is injected into the abdominal cavity of each fancy carp in the toxicity counteracting group, and 100 mu L of LPBS is injected into the abdominal cavity of each fancy carp in the control group. Randomly selecting 6 koi from the challenge group and the control group respectively before (0h) infection and 6h,12h,24h,48h,96h and 7d after infection, collecting spleen, liver and head kidney tissues, placing in liquid nitrogen for quick freezing, storing at-80 ℃ for RNA extraction, and mixing every 2 fish samples to reduce individual errors. And detecting the relative expression quantity of the fancy carp PBRRP gene by adopting a real-time fluorescent quantitative PCR method.

The results (fig. 2) show that the fancy carp PBRRP gene is significantly up-regulated and then reduced in the head and kidney tissue expression 6h after the Aeromonas veronii infection; the expression level of the spleen PBRRP gene is obviously increased (P is less than 0.05) 48h after the Aeromonas veronii infection (figure 3) until 7d after the infection, the expression level of the gene is higher than that of a control group; in the liver, the PBRRP gene was significantly elevated at 96h after pathogen infection and then reduced to control levels (fig. 4). The result shows that the fancy carp PBRRP gene responds to the Aeromonas veronii infection and may play a non-specific immune role in the fancy carp body.

Construction of koi PBRRP eukaryotic expression plasmid

Designing amplification primers F respectively carrying HindIII and XhoI enzyme cutting sites according to the mRNA sequence of the koi PBRRP gene in the sequencing result (SEQ ID NO.3: CC)AAGCTTGGGATGGGATTCTGGACCATCTCTGTG) and R (SEQ ID NO.4: CC)CTCGAGTCAGTGGTGGTGGTGGTGGTGAGAAATGACACACAC). Taking spleen cDNA of koi (Cyprinus carpio koi) as a template, and taking F and R as primers to carry out PCR amplification, thus obtaining a DNA fragment containing the PBRRP coding sequence.

The eukaryotic expression vector pCDNA3.1(Invitrogen) is digested by restriction enzymes HindIII and XhoI and then gel is recovered to obtain a vector framework; the DNA fragment containing the coding sequence of PBRRP is cut by restriction enzymes HindIII and XhoI, the obtained fragment is connected with a vector framework, and the obtained recombinant vector with correct sequence is marked as pCDNA3.1-PBRRP.

pCDNA3.1-PBRRP is a recombinant vector obtained by replacing a DNA fragment between HindIII and XhoI recognition sequences of pCDNA3.1 with a DNA fragment shown by SEQ ID NO.2 in a sequence table. pCDNA3.1-PBRRP can express a fusion protein formed by PBRRP shown in SEQ ID NO.1 and 6 XHis tag.

Application of Koi PBRRP eukaryotic expression plasmid

1. Effect of PBRRP of fancy carp on eliminating pathogenic bacteria-aeromonas veronii

(1) Plasmid injection: pCDNA3.1-PBRRP was diluted to 200. mu.g/mL in PBS, i.e., pCDNA3.1-PBRRP plasmid injection. The blank plasmid pCDNA3.1 was diluted to 200. mu.g/mL in PBS, the control plasmid injection. 8 brocade carps (about 20g) are randomly divided into 2 groups, each group has 4 carps, and the two groups are respectively a control group and a test group. Each fish of the test group was injected with 100. mu.L of pCDNA3.1-PBRRP plasmid injection, and each fish of the control group was injected with 100. mu.L of control plasmid injection.

(2) Preparation of pathogen suspension: culturing Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 in LB medium to OD600 of 0.6-0.8, centrifuging (8000g, 2min), pouring supernatant, suspending the thallus in PBS, adjusting to final concentration of 5 × 10⁶CFU/mL, namely the Aeromonas veronii suspension.

(3) Infection with offensive toxin: at 72h after the plasmid injection of step (1), 100. mu.L of Aeromonas veronii suspension of the above (2) was injected into each fish of the control group and the test group. At 24h post-infection, koi was anesthetized with MS-222, dissected, spleen tissue removed, and weighed. Sterilized PBS was added at 10. mu.L/mg, ground with a sterile grinding bar, 100. mu.L of spleen homogenate was spread on LB plates, 2 plates were applied per sample to obtain an average, and colony counting was performed after the plates were incubated for 24 hours at 28 ℃ in an incubator, and statistical analysis was performed. As a result, as shown in Table 1 and FIG. 5, the number of colonies in the spleen of koi in the test group (5 colonies/mg spleen) was significantly lower (P < 0.05) than that in the spleen of koi in the control group (44 colonies/mg spleen).

TABLE 1 spleen bacterial infection colony counts for fancy carp (counts/mg spleen)

2. Protective effect of fancy carp PBRRP on resistance of fancy carp to pathogenic bacterium-aeromonas veronii infection

(1) Plasmid injection: pCDNA3.1-PBRRP was diluted to 200. mu.g/mL in PBS, i.e., pCDNA3.1-PBRRP plasmid injection. The blank plasmid pCDNA3.1 was diluted to 200. mu.g/mL in PBS, the control plasmid injection. 16 fancy carps (about 20g) are randomly divided into 2 groups, each group has 8 tails, and the two groups are respectively a control group and a test group. Each fish of the test group was injected with 100. mu.L of pCDNA3.1-PBRRP plasmid injection, and each fish of the control group was injected with 100. mu.L of control plasmid injection.

(2) Preparation of pathogen suspension: culturing Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 in LB medium to OD600 of 0.6-0.8, centrifuging (8000g, 2min), pouring supernatant, suspending the thallus in PBS, adjusting to final concentration of 5 × 10⁷CFU/mL, namely the Aeromonas veronii suspension.

(3) At 72 hours after the plasmid injection in step (1), 100. mu.L of Aeromonas veronii suspension of (2) above was injected into each fish of the control group and the test group. And observing and counting the death conditions of all groups of fancy carps within 7d, fishing out the dead carps in time, and drawing a survival curve by using Graphpad.

The results (fig. 6) show that the survival rate of the test group for 7 days was (100%), which was significantly higher than that of the control group for 7 days (62.5%). The PBRRP can enhance the resistance of the fancy carp to the infection of pathogenic bacteria.

3. Effect of fancy carp PBRRP in eliminating pathogenic bacteria-aeromonas hydrophila of fancy carp

(1) Plasmid injection: pCDNA3.1-PBRRP was diluted to 200. mu.g/mL in PBS buffer, i.e., pCDNA3.1-PBRRP plasmid injection. The blank plasmid pCDNA3.1 is diluted to 200 mu g/mL in PBS buffer solution, and then the control plasmid injection is obtained. 6 brocade carps (about 30g) are randomly divided into 2 groups, each group has 3 carps, and the two groups are respectively a control group and a test group. Each fish in the test group was injected intramuscularly with 100. mu.L of plasmid injection (injected into the muscle rich area in front of dorsal fin root), and each fish in the control group was injected with 100. mu.L of control plasmid injection.

(2) Preparation of pathogen suspension: culturing Aeromonas hydrophila (Aeromonas hydrophila, A.h) NX830 (national aquatic animal pathogen library, preservation number: BYK20130805) in LB culture medium to OD600 of 0.6-0.8, centrifuging (8000g, 2min), pouring supernatant, suspending the thallus in PBS buffer solution, adjusting to final concentration of 1 × 10⁸CFU/mL, namely the aeromonas hydrophila suspension.

(3) Infection with offensive toxin: at 72h after the plasmid injection in step (1), 100. mu.L of (2) suspension of Aeromonas hydrophila was injected into each fish of the control group and the test group. At 96h post-infection, koi was anesthetized with MS-222, dissected, spleen tissue removed, and weighed. Sterilized PBS buffer was added at 10. mu.L/mg, ground with a sterile grinding bar, 50. mu.L of spleen homogenate was spread on LB plates, 2 plates were applied per sample to obtain an average, and colony counting was performed after the plates were incubated for 24 hours at 28 ℃ in an incubator, and statistical analysis was performed. As a result, as shown in Table 2, the number of colonies in the spleen of koi in the test group (18 colonies/mg spleen) was significantly lower (P < 0.05) than that in the spleen of koi in the control group (81 colonies/mg spleen).

TABLE 2 spleen colony counts (counts/mg spleen) for fancy carp (30g)

4. After injecting Koi PBRRP eukaryotic expression plasmid, the expression analysis of head and kidney tissue immune related gene is realized by diluting pCDNA3.1-PBRRP in PBS buffer solution to 200 mug/mL, namely pCDNA3.1-PBRRP plasmid injection. The blank plasmid pCDNA3.1 is diluted to 200 mu g/mL in PBS buffer solution, and then the control plasmid injection is obtained. 20 brocade carps (about 30g) are randomly divided into 2 groups, 10 carps in each group, and the two groups are respectively a control group and a test group. Each fish in the test group was injected intramuscularly with 100. mu.L of plasmid injection (injected into the muscle rich area in front of dorsal fin root), and each fish in the control group was injected with 100. mu.L of control plasmid injection. At 72h after injection, 6 fishes in each group were randomly selected, immune tissue head and kidney were collected, total RNA was extracted, and reverse transcription was performed to cDNA. In order to reduce individual errors, 2 fishes are mixed, and the relative expression quantity of mRNA of several immune related genes is detected by a real-time fluorescence quantitative PCR method.

The results show (as shown in fig. 7), that after 72h of PBRRP eukaryotic expression plasmid injection, proinflammatory factor IL-6, TNF-alpha expression (9.96 times and 2.47 times of control plasmid group, respectively), mhc ii expression of major histocompatibility complex (1.81 times of control plasmid group), and expression of complement system C8 (56.15 times of control plasmid group) of koi head and kidney tissues can be rapidly up-regulated. The results suggest that PBRRP can promote the inflammatory reaction of head and kidney, induce immune processes such as antigen presentation in which MHCII participates, and activate the complement system to play a role in immune regulation.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

<110> research institute of Water science in Beijing city (national research center for freshwater fishery engineering)

<120> koi protein for resisting pathogenic bacteria infection and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 146

<212> PRT

<213> fancy carp (Cyprinus carpio haemattopterus)

<400> 1

Met Gly Phe Trp Thr Ile Ser Val Ser Leu Cys Leu Leu Phe Val Met

1 5 10 15

Asn Ala Ser Gly Ala Cys Arg Phe Gly Trp Ser Arg Tyr Gly His Glu

20 25 30

Cys Phe Lys Val Phe Thr Asn Pro Leu Ser Trp Gly Asp Ala Glu Val

35 40 45

Thr Cys Leu Asn Ser Gly Gly Asn Leu Ala Ser Val His Ser Lys Gln

50 55 60

Gln Tyr Asp Phe Leu Lys Ser Leu Ile Ser Ser Ser Gln Ser Tyr Trp

65 70 75 80

Ile Gly Gly Tyr Asp Ala Val Ser Glu Gly Lys Trp Phe Trp Ser Asp

85 90 95

Gly Ser Gln Met Asn Tyr Arg Leu Trp Asn Pro Gly Glu Pro Asn Asn

100 105 110

Leu Gln Arg Ala Glu His Cys Ile Gln Met Asn Tyr Gly Ala Ala Gly

115 120 125

Asn Trp Asn Asp Gln Lys Cys Thr Asp Lys Ile Pro Phe Val Cys Val

130 135 140

Ile Ser

145

<210> 2

<211> 441

<212> DNA

<213> fancy carp (Cyprinus carpio haemattopterus)

<400> 2

atgggattct ggaccatctc tgtgtctctc tgtctgctct ttgttatgaa tgcatcagga 60

gcctgtcggt ttgggtggtc tcgatatgga catgaatgct tcaaggtttt taccaatcca 120

ttgtcctggg gtgatgcaga ggtgacgtgc ttgaacagtg gtgggaacct tgcctctgtg 180

cacagtaaac agcagtatga tttcttaaag agcctgatct caagttcaca atcatactgg 240

ataggaggct atgatgctgt ttcagaggga aagtggttct ggagtgatgg gtcccaaatg 300

aattacagac tttggaaccc tggagagccc aacaacctac agagggctga gcactgcatt 360

cagatgaact atggagctgc aggaaattgg aatgaccaaa aatgtacaga caagatacca 420

tttgtgtgtg tcatttcttg a 441

<210> 3

<211> 35

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 3

ccaagcttgg gatgggattc tggaccatct ctgtg 35

<210> 4

<211> 44

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 4

ccctcgagtc agtggtggtg gtggtggtga gaaatgacac acac 44

Claims (10)

1. The application of resisting pathogenic bacteria infection protein in preparing any functional product as follows:

D1) improving the immunity of the fish;

D2) treating and/or preventing disease in fish caused by infection with pathogenic bacteria;

D3) inhibiting the growth of pathogenic bacteria in the fish body;

D4) pathogenic bacteria in the fish body are eliminated;

D5) combating infestation of fish by pathogenic bacteria;

D6) protecting fish from pathogenic bacteria infection;

D7) the survival rate of fish infected by pathogenic bacteria is improved;

the protein for resisting pathogenic bacteria infection is A1), A2) or A3) as follows:

A1) a protein having an amino acid sequence of SEQ ID No. 1;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown by SEQ ID NO.1 in the sequence table and has the same function;

A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2).

2. Use according to claim 1, characterized in that: the immunity is immunity of the fish to pathogenic bacteria.

3. Use according to claim 1 or 2, characterized in that: the pathogenic bacteria are Aeromonas (Aeromonas) bacteria;

further, the bacterium belonging to the genus Aeromonas (Aeromonas) is Aeromonas veronii (Aeromonas veronii) or Aeromonas hydrophila (Aeromonas hydrophylla).

4. Use according to any one of claims 1 to 3, characterized in that: the fish is the following E1, E2, E3 or E4:

e1, cyprinid;

e2, carp;

e3, carp;

e4, Koi (Cyprinus carpio koi).

5. Use of a biomaterial as claimed in claim 1 in association with a protein conferring resistance to pathogenic infection as claimed in any one of the following:

D1) improving the immunity of the fish;

D2) treating and/or preventing disease in fish caused by infection with pathogenic bacteria;

D3) inhibiting the growth of pathogenic bacteria in the fish body;

D4) pathogenic bacteria in the fish body are eliminated;

D5) combating infestation of fish by pathogenic bacteria;

D6) protecting fish from pathogenic bacteria infection;

D7) the survival rate of fish infected by pathogenic bacteria is improved;

the biomaterial is any one of the following B1) to B5):

B1) a nucleic acid molecule encoding a protein according to claim 1 that is resistant to infection by a pathogenic bacterium;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a cell line comprising B1) the nucleic acid molecule or a cell line comprising B2) the expression cassette.

6. Use according to claim 5, characterized in that: B1) the nucleic acid molecule is b11) or b12) or b13) or b14) as follows:

b11) the coding sequence is cDNA molecule or DNA molecule of SEQ ID NO.2 in the sequence table;

b12) DNA molecule shown as SEQ ID NO.2 in the sequence table;

b13) a cDNA molecule or genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) and encoding a protein according to claim 1 which is resistant to infection by pathogenic bacteria;

b14) a cDNA molecule or a genomic DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) or b13) and encodes a protein resistant to infection by pathogenic bacteria according to claim 1.

7. Use according to claim 5 or 6, characterized in that: the immunity is immunity of the fish to pathogenic bacteria.

8. Use according to any one of claims 5 to 7, characterized in that: the pathogenic bacteria are Aeromonas (Aeromonas) bacteria;

further, the bacterium belonging to the genus Aeromonas (Aeromonas) is Aeromonas veronii (Aeromonas veronii) or Aeromonas hydrophila (Aeromonas hydrophylla).

9. Use according to any one of claims 5 to 8, characterized in that: the fish is the following E1, E2, E3 or E4:

e1, cyprinid;

e2, carp;

e3, carp;

e4, Koi (Cyprinus carpio koi).

10. A protein according to claim 1 or a biomaterial according to claim 5 or 6 for combating pathogenic bacterial infection.

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