CN114306574B - Koi protein for resisting pathogenic bacteria infection and application thereof - Google Patents

Abstract

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

Description

Koi protein for resisting pathogenic bacteria infection and application thereof

Technical Field

The application relates to a koi protein for resisting pathogenic bacteria infection and application thereof in the field of biotechnology.

Background

The fancy carp is called a swimming artwork and is one of the highest ornamental fish varieties in the world. Unlike eating carp, the fancy carp undergoes multiple sorting in different growth stages, and the operations of net pulling, catching and the like can cause stress reaction of the fancy carp, so that immunity is reduced, various pathogenic microorganisms are easy to breed, and diseases are frequent. Many diseases of koi are caused by bacteria and parasites, and there are few diseases caused by viruses. The industry and koi lovers mainly use antibiotics and bactericides for treatment, but pathogenic bacteria have poor treatment effect due to drug resistance of the antibiotics, and in addition, the use of the antibiotics can pollute water bodies and influence the physical health of fishes and human beings. Therefore, the principle of preventing and treating ornamental fish is particularly important, and a new idea is provided for healthy cultivation and disease prevention and control of the fancy carp based on immune epidemic prevention product development for improving the immune disease resistance level of the fancy carp.

Disclosure of Invention

The technical problem to be solved by the application is how to improve the immunity of fish and how to resist infection of pathogenic bacteria.

In order to solve the technical problems, the application firstly provides application of protein for resisting pathogenic bacteria infection in preparing a product with any one of the following functions:

d1 Improving immunity of fish;

d2 Treating and/or preventing diseases caused by pathogenic bacterial infection of fish;

d3 Inhibiting pathogenic bacteria from growing in the fish body;

d4 Removing pathogenic bacteria in the fish body;

d5 Against infestation of fish by pathogenic bacteria;

d6 Protection of fish infected with pathogenic bacteria;

d7 Improving the survival rate of fish infected by pathogenic bacteria;

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

a1 A protein having an amino acid sequence of SEQ ID NO. 1;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in SEQ ID NO.1 in the sequence table and has the same function;

a3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate purification of the protein of A1), a tag as shown in the following table may be attached to the amino-terminus or the carboxyl-terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence Listing.

Table: tag sequence

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

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

The protein in A2) can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.

The gene encoding the protein in A2) above can be obtained by deleting one or more amino acid residues in the DNA sequence shown in SEQ ID NO.2, and/or performing missense mutation of one or more base pairs, and/or ligating the coding sequences of the tags shown in the above table at the 5 'and/or 3' ends thereof. Wherein the DNA molecule shown in SEQ ID NO.2 encodes the protein shown in SEQ ID NO.1.

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

d1 Improving immunity of fish;

d2 Treating and/or preventing diseases caused by pathogenic bacterial infection of fish;

d3 Inhibiting pathogenic bacteria from growing in the fish body;

d4 Removing pathogenic bacteria in the fish body;

d5 Against infestation of fish by pathogenic bacteria;

d6 Protection of fish infected with pathogenic bacteria;

d7 Improving the survival rate of fish infected by pathogenic bacteria;

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

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A cell line containing the nucleic acid molecule of B1) or a cell line containing the expression cassette of B2).

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

b11 A cDNA molecule or a DNA molecule of SEQ ID NO.2 in the sequence table;

b12 A DNA molecule shown in SEQ ID NO.2 of the sequence table;

b13 A cDNA molecule or genomic DNA molecule having 75% or more identity to the nucleotide sequence defined in b 11) or b 12) and encoding PBRRP;

b14 Under stringent conditions with the nucleotide sequence defined in b 11) or b 12) or b 13), and a cDNA molecule or genomic DNA molecule encoding 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 application can be easily mutated by a person skilled in the art using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the isolated PBRRP protein of the application are derived from the nucleotide sequence of the application and are equivalent to the sequence of the application as long as they encode the PBRRP protein and function as 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 with the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the present application. 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 evaluate 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 NaPO ₄ Hybridization with 1mM EDTA in a mixed solution at 50℃2×SSC, rinsing in 0.1% sds; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; the method can also be as follows: hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; the method can also be as follows: hybridization and washing of membranes were performed at 65℃in 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above applications, the expression cassette (PBRRP gene expression cassette) comprising a nucleic acid molecule encoding a PBRRP protein as described in B2) refers to DNA capable of expressing the PBRRP protein in a host cell, which DNA may include not only a promoter for initiating transcription of the PBRRP gene but also a terminator for terminating 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 applications, the vector may be a plasmid, cosmid, phage or viral vector. The plasmid may specifically be pcdna3.1.

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

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

In the above applications, the cell line does not include propagation material.

PBRRP or said biological material also fall within the scope of protection of this aspect.

In the present application, the immunity may be immunity of fish to pathogenic bacteria.

The pathogenic bacteria may be Aeromonas (Aeromonas) bacteria.

Further, the Aeromonas (Aeromonas) bacterium may be Aeromonas veronii (Aeromonas veronii) or Aeromonas hydrophila (Aeromonas hydrophila). In one embodiment of the present application, the Aeromonas verrucosa (Aeromonas veronii) is Aeromonas verrucosa (Aeromonas veronii) CGMCC No.1.927 (China general microbiological culture collection center (CGMCC, strain number: 1.927). In one embodiment of the present application, the Aeromonas hydrophila (Aeromonas hydrophila) is Aeromonas hydrophila (Aeromonas hydrophila) NX830 (national aquatic animal pathogen base, accession number: BYK 20130805).

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

e1, a cyprinid;

e2, a carp species;

e3, carp;

e4, koi (Cyprinus carpio koi).

In the application, the fish body can be in the fish spleen.

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

Drawings

FIG. 1 shows the expression profile of the 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 and kidney PBRRP gene after infection of aeromonas veronii. PBS was used as a control group and A.v as an challenge group. * Indicating that the difference reached a significant level p < 0.05.

FIG. 3 shows expression changes of the spleen PBRRP gene of koi after infection with Aeromonas veronii. PBS was used as a control group and A.v as an challenge group. * Indicating that the difference reached a significant level p < 0.05.

FIG. 4 shows expression changes of the liver PBRRP gene of koi after infection with Aeromonas veronii. PBS was used as a control group and A.v as an challenge group. * Indicating that the difference reached a significant level p < 0.05.

FIG. 5 spleen bacterial infection colony count of koi. pcdna3.1 is control group and PBRRP is test group.

FIG. 6. Protective effect of koi PBRRP on koi against pathogenic infection. pcdna3.1 is control group and PBRRP is test group.

FIG. 7 shows the variation of the expression of the immune-related genes of the head and kidney tissues of koi after injection of PBRRP and control plasmid. pcdna3.1 is control group and PBRRP is test group. * Indicating that the difference reached a significant level p < 0.05.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate 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 a strain number: 1.927, the date of preservation was 1975, 9 and 9. The strain websites are as follows:

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

example 1,

Discovery of PBRRP Gene

The inventor finds a gene whose expression is obviously up-regulated (about 2 times) through transcriptome sequencing of spleen of the fancy carp after infection of aeromonas verrucosa, which indicates that the gene is likely to be related to immunity of the fancy carp, and the gene is marked as PBRRP gene. In the koi, the coding sequence of the PBRRP gene is shown as SEQ ID NO.2, and has 441 nucleotides, the amino acid sequence of the coding protein is shown as SEQ ID NO.1, and the koi contains no signal peptide.

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

ATGGGATTCTGGACCATCTCTGTGTCTCTCTGTCTGCTCTTTGTTATGAATGCATCAGGAGCCTGTCGGTTTGGGTGGTCTCGATATGGACATGAATGCTTCAAGGTTTTTACCAATCCATTGTCCTGGGGTGATGCAGAGGTGACGTGCTTGAACAGTGGTGGGAACCTTGCCTCTGTGCACAGTAAACAGCAGTATGATTTCTTAAAGAGCCTGATCTCAAGTTCACAATCATACTGGATAGGAGGCTATGATGCTGTTTCAGAGGGAAAGTGGTTCTGGAGTGATGGGTCCCAAATGAATTACAGACTTTGGAACCCTGGAGAGCCCAACAACCTACAGAGGGCTGAGCACTGCATTCAGATGAACTATGGAGCTGCAGGAAATTGGAATGACCAAAAATGTACAGACAAGATACCATTTGTGTGTGTCATTTCTTGA。

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

MGFWTISVSLCLLFVMNASGACRFGWSRYGHECFKVFTNPLSWGDAEVTCLNSGGNLASVHSKQQYDFLKSLISSSQSYWIGGYDAVSEGKWFWSDGSQMNYRLWNPGEPNNLQRAEHCIQMNYGAAGNWNDQKCTDKIPFVCVIS。

tissue expression profile of koi PBRRP

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

The specific operation method comprises the following steps: 12 tissues of healthy koi (body weight about 20 g) were randomly selected, respectively: gill, eye, head and kidney, spleen, kidney, heart, muscle, skin, liver, blood, brain and intestine. Total RNA from tissue was extracted with RNAiso Plus (Takara, 9109) and reverse transcribed into cDNA using a reverse transcription kit (Takara, RR 047A).

Primers for fluorescent quantitative PCR were designed based on the cDNA sequence of PBRRP, the primer sequences were as follows:

qNattF：5’-AACAGTGGTGGGAACCTTGC-3’；

qNattR：5’-GGACCCATCACTCCAGAACC-3’；

the primer sequences of the 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 PBRRP in the 12 tissues by using an ABI7500 real-time fluorescence quantitative PCR instrument. The reaction conditions are as follows: 95 ℃ for 30s;95℃for 5s,59.6℃for 30s,72℃for 30s,40 cycles.

The results showed that the PBRRP gene was expressed in all of the above 12 tissues, among which the expression level was highest in the head kidney, followed by kidney, gill, spleen and skin, as shown in FIG. 1. The head kidney and spleen are the main immune organs of fish, and the skin and gill are the main mucosal immune organs, which shows that the gene can participate in the koi mucosal immune reaction and play a role in the fish body immune reaction.

Expression change of koi PBRRP Gene after infection with Aeromonas veronii

Taking out Aeromonas verrucosa (Aeromonas veronii) CGMCC No.1.927 from ultra-low temperature refrigerator, streaking on LB plate, culturing in 28 deg.C incubator, resuscitating strain, picking single colony, expanding culture in LB liquid medium, centrifuging to collect thallus, re-suspending with PBS, and adjusting to 5×10 ⁶ CFU/mL, obtaining the Aeromonas veronii suspension.

60 healthy fancy carp (about 20 g) are randomly selected, and the average of the healthy fancy carp is divided into 2 groups of 3 parallel cylinders, namely a virus attack group and a control group. The virus-attacking group is injected with 100 mu L of aeromonas veronii suspension per tail koi abdominal cavity, and the control group is injected with 100 mu LPBS per tail koi abdominal cavity. Before infection (0 h) and 6h,12h,24h,48h,96h and 7d after infection, 6 tail koi is randomly selected from a virus attack group and a control group respectively, spleen, liver and head and kidney tissues are collected, quickly frozen in liquid nitrogen and stored at-80 ℃ for RNA extraction, and in order to reduce individual errors, every 2 fishes are mixed. And detecting the relative expression quantity of the PBRRP gene of the koi by adopting a real-time fluorescence quantitative PCR method.

The results (figure 2) show that the PBRRP gene of the koi appears to be significantly up-regulated in the tissue expression of the head and kidney and then is down-regulated 6 hours after the infection of the aeromonas veronii; the expression level of spleen PBRRP gene is obviously increased (P is less than 0.05) 48h after the infection of aeromonas veronii (figure 3) until 7d after the infection, and the expression level of the gene is higher than that of a control group; in the liver, the PBRRP gene was significantly increased only at 96h after pathogen infection, and then decreased to the control level (fig. 4). The result shows that the PBRRP gene of the koi responds to the infection of aeromonas veronii and can play a non-specific immunity role in the koi body.

Construction of koi PBRRP eukaryotic expression plasmid

According to the sequencing result, the mRNA sequence of the koi PBRRP gene designs an amplification primer F (SEQ ID NO.3: CC) respectively carrying HindIII and XhoI restriction enzyme sitesAAGCTTGGGATGGGATTCTGGACCATCTCTGTG) and R (SEQ ID NO.4: CCCTCGAGTCAGTGGTGGTGGTGGTGGTGAGAAATGACACACAC). And (3) performing PCR amplification by taking the spleen cDNA of the koi (Cyprinus carpio koi) as a template and taking F and R as primers to obtain a DNA fragment containing the PBRRP coding sequence.

The eukaryotic expression vector pCDNA3.1 (Invitrogen) was digested with restriction enzymes HindIII and XhoI and then subjected to gel recovery to obtain a vector backbone; the DNA fragment containing the PBRRP coding sequence was digested with restriction enzymes HindIII and XhoI, the resulting fragment was ligated to the vector backbone, and the resulting recombinant vector with the correct sequence was designated pCDNA3.1-PBRRP.

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

Application of koi PBRRP eukaryotic expression plasmid

1. Effect of koi PBRRP on eliminating koi pathogenic bacteria-Aeromonas veronii

(1) Plasmid injection: the 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 koi (about 20 g) were randomly divided into 2 groups of 4 tails each, two groups being control and test groups, respectively. Each fish of the test group was injected with 100 μl pcdna3.1-PBRRP plasmid injection, respectively, and each fish of the control group was injected with 100 μl control plasmid injection, respectively.

(2) Preparation of pathogenic bacteria suspension: culturing Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 to OD600 of 0.6-0.8 in LB medium, centrifuging (8000 g,2 min), pouring the supernatant, suspending thallus with PBS, and adjusting final concentration to 5×10 ⁶ CFU/mL, namely the aeromonas veronii suspension.

(3) Attack of toxic infection: at 72h after plasmid injection in step (1), 100. Mu.L of the Aeromonas veronii suspension of (2) above 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 rod, 100. Mu.L of spleen homogenate was spread on LB plates, 2 plates were spread on each sample, the average value was taken, colonies were counted after the plates were incubated at 28℃for 24 hours, and statistical analysis was performed. As shown in Table 1 and FIG. 5, the number of colonies of the spleens of the test group koi (5 spleens/mg spleens) was significantly lower (P < 0.05) than that of the control group koi spleens (44 spleens/mg spleens).

Table 1, spleen bacterial infection colony count of koi (individual/mg spleen)

2. Protective effect of koi PBRRP on koi against pathogenic bacteria-Aeromonas veronii infection

(1) Plasmid injection: the 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 koi (about 20 g) were randomly divided into 2 groups of 8 tails each, two groups being control and test groups, respectively. Each fish of the test group was injected with 100 μl pcdna3.1-PBRRP plasmid injection, respectively, and each fish of the control group was injected with 100 μl control plasmid injection, respectively.

(2) Preparation of pathogenic bacteria suspension: culturing Aeromonas veronii (Aeromonas veronii) CGMCC No.1.927 to OD600 of 0.6-0.8 in LB medium, centrifuging (8000 g,2 min), pouring the supernatant, suspending thallus with PBS, and adjusting final concentration to 5×10 ⁷ CFU/mL, namely the aeromonas veronii suspension.

(3) 72h after plasmid injection in step (1), 100. Mu.L of the Aeromonas veronii suspension in (2) above was injected into each fish of the control group and the test group. And (5) observing and counting the death condition of each group of koi in 7d, finding out dead fish in time, and drawing a survival curve by using Graphpad.

The results (fig. 6) show that the survival rate for 7 days of the test group is (100%) which is significantly higher than the survival rate for 7 days of the control group (62.5%). The PBRRP can enhance the infection of the koi against pathogenic bacteria.

3. Effect of koi PBRRP on eliminating koi pathogenic bacteria-aeromonas hydrophila

(1) Plasmid injection: the 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 mug/mL in PBS buffer solution to obtain the control plasmid injection. The 6-tail koi (about 30 g) is randomly divided into 2 groups of 3 tails each, and the two groups are a control group and a test group respectively. Each fish of the test group was intramuscular injected with 100 μl of plasmid injection (injected at the rich muscle site in front of the dorsal fin root) and each fish of the control group was intramuscular injected with 100 μl of control plasmid injection.

(2) Preparation of pathogenic bacteria suspension: the LB medium was used to culture Aeromonas hydrophila (Aeromonas hydrophila, A.h) NX830 (national aquatic animal pathogen reservoir, accession number: BYK 20130805) to an OD600 of 0.6-0.8, centrifuged (8000 g,2 min), the supernatant was poured, and the cells were suspended in PBS buffer to a final concentration of 1X 10 ⁸ CFU/mL is the aeromonas hydrophila suspension.

(3) Attack of toxic infection: at 72h after plasmid injection in step (1), 100 μl (2) of aeromonas hydrophila suspension was injected per fish of control and test groups. 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 rod, 50. Mu.L of spleen homogenate was spread on LB plates, 2 plates were spread on each sample, the average value was taken, colonies were counted after incubation of the plates at 28℃for 24 hours, and statistical analysis was performed. As a result, as shown in Table 2, the number of colonies of the spleens of the test group koi (18 spleens/mg) was significantly lower (P < 0.05) than that of the spleens of the control group koi (81 spleens/mg).

TABLE 2 fancy carp (30 g) spleen colony count (individual/mg spleen)

4. And (3) performing expression analysis on the head and kidney tissue immunity related genes after injecting the koi PBRRP eukaryotic expression plasmid, and diluting the pCDNA3.1-PBRRP to 200 mug/mL in PBS buffer solution, namely the pCDNA3.1-PBRRP plasmid injection. The blank plasmid pCDNA3.1 is diluted to 200 mug/mL in PBS buffer solution to obtain the control plasmid injection. The 20 koi (about 30 g) were randomly divided into 2 groups of 10 tails each, two groups being control group and test group respectively. Each fish of the test group was intramuscular injected with 100 μl of plasmid injection (injected at the rich muscle site in front of the dorsal fin root) and each fish of the control group was intramuscular injected with 100 μl of control plasmid injection. At 72h after injection, 6 fish were randomly selected for each group, the immune tissue head and kidney were collected, total RNA was extracted, and reverse transcribed into cDNA. In order to reduce individual errors, 2 fish are mixed, and the relative expression quantity of mRNA of several immune related genes is detected by adopting a real-time fluorescence quantitative PCR method.

The results showed (as shown in FIG. 7), that the pro-inflammatory factor IL-6, TNF- α expression (9.96-fold and 2.47-fold, respectively) in koi head and kidney tissues, major histocompatibility complex MHCII expression (1.81-fold compared to the control plasmid), and complement system C8 expression (56.15-fold compared to the control plasmid) could be rapidly up-regulated 72h after injection of the PBRRP eukaryotic expression plasmid. The result shows that PBRRP can promote the reaction of the head nephritis, induce the immune processes such as antigen presentation and the like participated by MHCII, and activate the complement system to play a role in immunoregulation.

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

Sequence listing

<110> Beijing aquatic science research institute (national fresh water fishery engineering research center)

<120> a protein of koi against pathogenic bacteria infection and use thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 146

<212> PRT

<213> fancy carp (Cyprinus carpio haematopterus)

<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 haematopterus)

<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 (8)

1. The application of protein for resisting pathogenic bacteria infection in preparing products with any one of the following functions:

d1 Improving immunity of fish;

d2 Treating and/or preventing diseases caused by pathogenic bacterial infection of fish;

d3 Inhibiting pathogenic bacteria from growing in the fish body;

d4 Removing pathogenic bacteria in the fish body;

d5 Against infestation of fish by pathogenic bacteria;

d6 Protection of fish infected with pathogenic bacteria;

d7 Improving the survival rate of fish infected by pathogenic bacteria;

the protein for resisting pathogenic bacteria infection is the following A1) or A2):

a1 A protein having an amino acid sequence of SEQ ID NO. 1;

a2 A fusion protein obtained by connecting a label to the N end or/and the C end of A1);

the pathogenic bacteria is aeromonas spAeromonas) Bacteria;

the fish is koi carpCyprinus carpio koi）。

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

3. Use according to claim 1 or 2, characterized in that: the aeromonas spAeromonas) The bacteria are aeromonas veroniiAeromonas veronii) Or aeromonas hydrophilaAeromonas hydrophila）。

4. Use of a biomaterial associated with a protein resistant to pathogenic bacterial infection as defined in claim 1 for the preparation of a product having any one of the following functions:

d1 Improving immunity of fish;

d2 Treating and/or preventing diseases caused by pathogenic bacterial infection of fish;

d3 Inhibiting pathogenic bacteria from growing in the fish body;

d4 Removing pathogenic bacteria in the fish body;

d5 Against infestation of fish by pathogenic bacteria;

d6 Protection of fish infected with pathogenic bacteria;

d7 Improving the survival rate of fish infected by pathogenic bacteria;

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

b1 A nucleic acid molecule encoding a protein of claim 1 against infection by a pathogenic bacterium;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A cell line containing the nucleic acid molecule of B1) or a cell line containing the expression cassette of B2);

the pathogenic bacteria is aeromonas spAeromonas) Bacteria;

the fish is koi carpCyprinus carpio koi）。

5. The use according to claim 4, characterized in that: b1 The nucleic acid molecule is b 11) or b 12) as follows:

b11 A cDNA molecule or a DNA molecule of SEQ ID NO.2 in the sequence table;

b12 A DNA molecule shown in SEQ ID NO.2 of the sequence Listing.

6. Use according to claim 4 or 5, characterized in that: the immunity is the immunity of fish to pathogenic bacteria.

7. Use according to claim 4 or 5, characterized in that: the aeromonas spAeromonas) The bacteria are aeromonas veroniiAeromonas veronii) Or aeromonas hydrophilaAeromonas hydrophila）。

8. A protein as claimed in claim 1 or a biological material as claimed in claim 4 or 5 against pathogenic bacteria.