CN113717268B

CN113717268B - Application of koi serum amyloid A5 or encoding gene thereof in regulation and control of koi antipathogenic bacterial infection

Info

Publication number: CN113717268B
Application number: CN202111136494.2A
Authority: CN
Inventors: 王晓雯; 朱华; 刘丽丽; 伍广涛
Original assignee: Beijing Fisheries Research Institute (national Engineering Research Center For Freshwater Fisheries)
Current assignee: Beijing Fisheries Research Institute (national Engineering Research Center For Freshwater Fisheries)
Priority date: 2021-09-27
Filing date: 2021-09-27
Publication date: 2023-07-07
Anticipated expiration: 2041-09-27
Also published as: CN113717268A

Abstract

The invention relates to the technical field of genetic engineering, in particular to application of koi serum amyloid A5 or a coding gene thereof in regulation and control of koi antipathogenic bacterial infection. The invention researches and obtains a differential expression gene, namely a fancy carp Serum amino acid A-5 gene, aiming at the fancy carp infected by pathogenic bacteria, wherein the expression level of the gene in different tissues is obviously changed after the fancy carp is infected by the pathogenic bacteria. The invention over-expresses the fancy carp Serum ammonioid A-5 gene in the healthy fancy carp body, the capability of resisting pathogenic bacteria infection is obviously improved, and the discovery has important significance in the field of resisting pathogenic bacteria infection of the fancy carp.

Description

Application of koi serum amyloid A5 or encoding gene thereof in regulation and control of koi antipathogenic bacterial infection

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of koi serum amyloid A5 or a coding gene thereof in regulation and control of koi antipathogenic bacterial infection.

Background

The koi (Cyprinus carpio haematopterus) is a common ornamental fish, and various diseases caused by pathogenic bacteria are serious problems in the koi cultivation process in the field of koi cultivation. In the prior art, the disease of the fancy carp is avoided by adopting the ways of adjusting the culture environment, daily water quality management, feed management and the like and enhancing the resistance of the fancy carp; or after the occurrence of the disease sign, the water temperature is increased, and the treatment is carried out by means of antibiotics and the like. However, these approaches are only effective against a portion of pathogenic bacteria.

Serum Amyloid A (SAA) is an acute response phase protein secreted by the liver to synthesize and participate in pathogenic defensive responses and immune homeostasis. Human SAA is believed to be a cytokinin that enhances phagocytic activity of phagocytes against gram-negative bacteria and is involved in inflammatory and infectious processes. SAA contains an RGN fibronectin binding domain that can bind cell surface integrins to regulate cell activity, such as to regulate chemotaxis of cells. However, there is little research concerning SAA function in fish.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides application of Serum amyloid A5 (SAA 5) of koi or a coding gene thereof in regulating and controlling the infection of koi with pathogenic bacteria.

In a first aspect, the invention provides application of koi serum amyloid A5 or a coding gene thereof in regulating and controlling koi antipathogenic infection.

The invention further provides application of the koi serum amyloid A5 or the coding gene thereof in cultivation of an antipathogenic koi variety.

Further, the application is to improve the anti-pathogenic bacteria infection capability of the koi by improving the expression level of serum amyloid A5 of the koi.

Further, the pathogenic bacteria include one or more of aeromonas veronii, aeromonas hydrophila, aeromonas wenyujin or aeromonas guinea.

Further, the koi serum amyloid A5 comprises an amino acid sequence as set forth in any one of the following:

i) An amino acid sequence as shown in SEQ ID NO. 1;

ii) amino acid sequences having the same or similar function after addition, substitution, or reduction of one or more amino acid sequences from i).

Further, the koi serum amyloid A5 is encoded by a nucleotide sequence as set forth in any one of the following:

i) A nucleotide sequence as shown in SEQ ID NO. 2;

ii) a nucleotide sequence having the same or similar function after one or more nucleotide sequences are added, replaced or reduced from i).

In a second aspect, the present invention provides a method for modulating the ability of a koi to resist pathogenic bacterial infection comprising:

regulating and controlling the antipathogenic bacteria infection capacity of the koi by regulating and controlling the expression level of serum amyloid A5 of the koi in the genome of the koi;

the koi serum amyloid A5 comprises an amino acid sequence as set forth in any one of the following:

i) An amino acid sequence as shown in SEQ ID NO. 1;

Further, by increasing the expression level of the encoding gene of the serum amyloid A5 of koi in the genome of koi, the ability of koi to resist pathogenic bacterial infection is increased;

the encoding gene of the koi serum amyloid A5 comprises a nucleotide sequence as set forth in any one of the following:

i) A nucleotide sequence as shown in SEQ ID NO. 2;

Further, the method comprises the steps of: constructing the encoding gene of the koi serum amyloid A5 on a vector to obtain an expression plasmid;

transferring the expression plasmid into the koi body for expression.

Further, the vector is one or more of pcDNA3.1, pCMV, pSI, pCGN, pCEP4, pcDNA4 or pCI.

The invention has the following beneficial effects:

according to the invention, a differential expression gene koi Serum amyloid A-5 gene is discovered by researching koi infected by pathogenic bacteria, and can code koi Serum amyloid A5. The invention provides a method for regulating and controlling the resistance of the fancy carp to pathogenic bacteria infection based on the fancy carp Serum ammooid A-5 gene, which obviously improves the resistance of the fancy carp to pathogenic bacteria infection by over-expressing the fancy carp Serum ammooid A-5 gene in the fancy carp body, so that the survival rate of the fancy carp on the 10 th day is improved from 50% to 87.5%, and the method has important significance in the field of resistance of the fancy carp to pathogenic bacteria infection.

Drawings

FIG. 1 is a comparison of the expression levels of the Serum amyoid A-5 gene of example 1 of the present invention in different tissues of healthy koi.

FIG. 2 is a schematic diagram showing the change of the expression level of the fancy carp Serum ammooid A-5 gene in spleen tissue of a fancy carp infected with Aeromonas veronii according to example 2 of the present invention.

FIG. 3 is a schematic diagram showing the change of the expression level of the fancy carp Serum ammooid A-5 gene in the head and kidney tissues of the fancy carp infected with Aeromonas verrucosa, provided by the embodiment 2 of the invention.

FIG. 4 is a schematic diagram showing the change of the expression level of the fancy carp Serum ammooid A-5 gene in the liver tissue of the fancy carp infected with Aeromonas veronii provided by the embodiment 2 of the invention.

Fig. 5 is a graph showing the comparison result of the survival rate of koi in the test group and the control group provided in example 3 of the present invention.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

Example 1 acquisition of the coding Gene for serum amyloid A5 of koi

In order to study the condition of up-regulating the activation of immune related genes of fish body by pathogenic bacteria infected koi, the embodiment carries out transcriptome sequencing on spleen of the pathogenic koi after infection of aeromonas veronii, and finds a differential expression gene with obviously improved expression level (about 189 times) according to sequencing results. Further based on the alignment of homologous sequences and the analysis of protein structure simulation, the present example speculates that the protein encoded by the gene is the Serum amyoid A-5protein (koi Serum amyloid A5).

In this example, the open reading frame ORF of the koi Serum ammooid A-5 gene is further obtained by PCR amplification using koi spleen cDNA as a template, 372 nucleotides in total are encoded to obtain a protein consisting of 123 amino acids, and no signal peptide is contained. The amino acid sequence of the Serum amyloid A5 of the koi is shown as SEQ ID NO.1, and the nucleotide sequence of the open reading frame ORF of the Serum amyloid A-5 gene of the koi is shown as SEQ ID NO. 2.

In the embodiment, the relative expression amounts of the fancy carp Serum amyloid A-5 gene in different tissues of the healthy fancy carp are further researched by adopting fluorescent real-time quantitative PCR, and the tissue distribution of the fancy carp Serum amyloid A5 mRNA is researched, wherein the specific flow is as follows:

1. 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 the tissues was extracted by Trizol method and reverse transcribed into cDNA using Takara reverse transcription kit.

2. According to cDNA sequence of the koi Serum ammooid A-5 gene, designing a primer for fluorescent quantitative PCR, wherein the primer sequence is as follows:

q SAA5F：5’-ATTCTTGCTGTGCTGGTGCT-3’；

q SAA5R：5’-CCAATGGCTTGTCCTGGGTA-3’；

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

qS11F：5’-CCGTGGGTGACATCGTTACA-3’；

qS11R：5’-TCAGGACATTGAACCTCACTGTCT-3’。

3. and detecting the relative expression quantity of the fancy carp Serum ammooid A-5 genes in the 12 tissues by using an ABI7500 real-time fluorescence quantitative PCR instrument. The reaction procedure is: 95 ℃ for 15s;95℃for 5s,59.6℃for 30s,72℃for 30s,40 cycles.

As shown in FIG. 1, the koi Serum ammonioid A-5 gene was expressed in all of the above 12 tissues. Wherein the expression level of the koi Serum amyoid A-5 gene in the skin is highest, the expression level of the koi Serum amyoid A-5 gene is next to the expression level of the koi Serum amyoid A-5 gene in the skin, the koi Serum amyoid A-5 gene is next to the koi Serum amyoid A-5 gene, and the expression level of the koi Serum amyoid A-5 gene is remarkably different from the expression level of the koi Serum amyoid A-5 gene in the other 8 tissues (P < 0.05). The protein plays an important role in mucosal immune organ-skin besides secretory expression in liver, and has higher expression level in main immune tissue-spleen of fish, which indicates that the gene can be used as a first defense line for pathogen invasion and participate in the immune regulation function of koi.

Example 2 expression Change of the Serum amyoid A-5 Gene of koi after infection with Aeromonas veronii

Aeromonas veronii (Aeromonas veronii, A.v) was purchased from China general microbiological culture collection center (CGMCC), strain number: 1.927). After being taken out from the ultra-low temperature refrigerator, the LB plate is scribed and thenCulturing in 28 deg.C incubator, resuscitating strain, picking single colony, culturing in LB liquid medium, centrifuging to collect thallus, re-suspending with PBS, and adjusting to 5×10 ⁸ CFU/mL。

60 healthy fancy carp (about 20 g) are randomly selected, and the average is divided into 2 groups of virus attack groups and control groups, and 3 parallel cylinders are arranged in each 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 L of PBS buffer solution per tail koi abdominal cavity. Before infection (0 h) and 6h, 12h, 24h, 48h, 96h and 7d, 6 tail koi is randomly selected from a virus attack group and a control group respectively, spleen, head and kidney and liver tissues are collected, and the koi is placed in liquid nitrogen for quick freezing and then stored at-80 ℃ for RNA extraction. To reduce individual errors, mix samples were performed every 2 fish. And detecting the relative expression quantity of the cyprinus carpio Serum ammooid A-5 gene by a real-time fluorescence quantitative PCR method.

As shown in FIGS. 2 to 4, the expression of the koi Serum ammonioid A-5 gene in the head and kidney tissues was rapidly up-regulated 6 hours after infection with Aeromonas verrucosa, and peaked 12 hours after infection, and then decreased to the level of the control group. In spleen and liver, the Serum amyoid A-5 gene of koi was significantly up-regulated at 12h post infection and up to 96h, significantly higher than the control level (P < 0.05). The result shows that the fancy carp Serum amyoid A-gene is taken as a A.v infection early response gene and possibly participates in the antibacterial immune process of the fancy carp.

Example 3 verification of the ability of serum amyloid A5 of koi to be infected with an anti-pathogenic bacterium

1. Construction of koi Serum amyoid A-5 eukaryotic expression plasmid

(1) According to the sequencing result, designing amplification primers carrying KpnI and XhoI restriction enzyme sites respectively according to mRNA sequences of the koi Serum ammooid A-5 gene:

F：5’-GGGGTACCGGGATGGAGCTTATTCTTGCT-3’，

R：5’-CCCTCGAGTCAGTGGTGGTGGTGGTGGTGGTACTT-3’。

(2) PCR amplification is carried out by taking the spleen cDNA of the koi as a template and F and R as primers, so as to obtain a cDNA fragment containing the ORF of the SAA-5 gene.

The PCR amplification reaction (25. Mu.L) contained 2.5. Mu.L of 10 XEx buffer, 2. Mu.L of dNTPs, 0.3. Mu. Mol each of F and R primers, 1U of Ex taq enzyme (Takara), 500ng of cDNA template;

the PCR reaction procedure was: 95 ℃ for 5min;92℃15s,53℃25s,72℃30s (10 cycles); 95℃15s,60℃25s,72℃30s (25 cycles); 7min at 72 ℃.

(3) The amplified product was analyzed by 1% agarose gel electrophoresis, and then recovered and purified by using a DNA gel recovery kit (Takara).

(4) Eukaryotic expression vector pCDNA3.1 (Invitrogen) was digested with restriction enzymes KpnI and XhoI, and the resultant was ligated overnight with the purified PCR product of step (3) at 4℃with T4DNA ligase, the ligation product was transformed into E.coli DH 5. Alpha. And cultured on LB agar plates containing ampicillin (50. Mu.g/mL) for 18 hours, positive clones were selected for sequencing, and plasmids were extracted from positive bacteria, which had correctly recombined the gene fragment of the fancy starch A-5 of fancy carp, and designated pCDNA3.1-SAA5.

2. Effect of pcDNA3.1-SAA5 for eliminating pathogenic bacteria of koi

(1) Plasmid injection: the pCDNA3.1-SAA5 was diluted to 200. Mu.g/mL in PBS, i.e., pCDNA3.1-SAA5 injection. The blank plasmid pCDNA3.1 was diluted to 200. Mu.g/mL in PBS, the control plasmid injection. 10 koi (about 20 g) were randomly divided into 2 groups of 5 tails each, two groups being control and test groups, respectively. Each fish of the test group was respectively injected with 100 μl of plasmid injection (injection of rich muscle site in front of dorsal fin base of koi) and each fish of the control group was respectively injected with 100 μl of control plasmid injection.

(2) Preparation of pathogenic bacteria suspension: LB medium cultures aeromonas veronii (CGMCC, 1.927) to OD ₆₀₀ 0.6-0.8, centrifuging (8000 g,2 min), pouring the supernatant, suspending the cells in PBS buffer to a final concentration of 1X 10 ⁷ CFU/mL。

(3) Attack of toxic infection: injecting 100 mu L of the bacterial suspension in the step (2) into each fish of the control group and the test group 72h after the injection in the step (1). At 24h 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, 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 incubation of the plates at 28℃for 24 hours, and statistical analysis was performed. As shown in Table 1, the number of colonies (30/mg) of the spleens of the test group koi was significantly (P < 0.05) lower than the number of colonies (79/mg) of the spleens of the control group koi.

TABLE 1 fancy carp (20 g) spleen colony count (individual/mg)

3. Protection effect of the Cyprinus Carpio pCDNA3.1-SAA5 on the infection of the Cyprinus Carpio against pathogenic bacteria

(1) Plasmid injection: and diluting the pCDNA3.1-SAA5 to 200 mug/mL in PBS to obtain the pCDNA3.1-SAA5 injection. The blank plasmid pCDNA3.1 is diluted to 200 mug/mL in PBS buffer solution to obtain 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 of the plasmid injection, and each fish of the control group was injected with 100 μl of the control plasmid injection.

(2) Preparation of pathogenic bacteria suspension: LB medium cultures aeromonas veronii (CGMCC, 1.927) to OD ₆₀₀ After centrifugation (8000 g,2 min) at 0.6-0.8, the supernatant was poured and the cells were suspended in PBS buffer to a final concentration of 5X 10 ⁷ CFU/mL。

(3) At 72h after the injection in step (1), 100. Mu.L of the bacterial suspension in (2) above was injected into each fish of the control group and the test group. Observing and counting the death condition of each group of koi within 10d, finding out dead fish in time, and drawing a survival curve by using Graphpad.

As shown in FIG. 5, the survival rate of the test group on the 10 th day of the koi is 87.5 percent, which is obviously higher than that of the control group on the 10 th day (50 percent), and the result shows that the koi Serum amyoid A-5 gene can enhance the infection of the koi against pathogenic bacteria.

Experimental example 4 Effect of Cyprinus Carpio pCDNA3.1-SAA5 on spleen load after Aeromonas hydrophila infection

(1) Plasmid injection: the plasmid pCDNA3.1-SAA5 of example 3 was diluted to 200. Mu.g/mL in PBS buffer to give pCDNA3.1-SAA5 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: LB medium culture of Aeromonas hydrophila (Aeromonas hydrophila, A.h) NX830 (deposited in aquatic animal pathogen library of China department of agriculture, accession number: BYK 20130805) to OD ₆₀₀ 0.6-0.8, centrifuging (8000 g,2 min), pouring the supernatant, suspending the cells in PBS buffer to a final concentration of 1X 10 ⁸ CFU/mL。

(3) Attack of toxic infection: and (2) injecting 100 mu L of the bacterial suspension prepared in the step (2) into each fish of the control group and the test group 72h after the injection flow of the step (1). At 96h post infection, koi was anesthetized with MS222, 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 shown in Table 2, the number of colonies (56/mg) of the spleen of the test group koi was significantly (P < 0.05) lower than the number of colonies (81/mg) of the spleen of the control group koi.

TABLE 2 spleen colony count (individual/mg) of koi (30 g)

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

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

<120> application of serum amyloid A5 of koi or encoding gene thereof in regulation and control of koi's disease-resistant pathogenic bacteria infection

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Claims

1. Application of koi serum amyloid A5 or encoding gene thereof in preparing medicines for regulating and controlling pathogenic bacteria infection of koi;

the amino acid sequence of the koi serum amyloid A5 is shown as SEQ ID NO. 1;

the pathogenic bacteria are one or more of aeromonas veronii or aeromonas hydrophila.

2. Application of serum amyloid A5 of koi or its coding gene in preparing medicine for culturing variety of koi with pathogenic bacteria resisting;

the amino acid sequence of the koi serum amyloid A5 is shown as SEQ ID NO. 1;

3. The use according to claim 1 or 2, wherein the use is to increase the anti-pathogenic bacterial infection capacity of koi by increasing the expression level of serum amyloid A5 of koi.

4. The use according to claim 1 or 2, wherein said koi serum amyloid A5 is encoded by a nucleotide sequence shown as the nucleotide sequence shown as SEQ ID No. 2.