CN112279897B

CN112279897B - Reagent for preventing or treating fish infection CyHV-2 and application

Info

Publication number: CN112279897B
Application number: CN202011212638.3A
Authority: CN
Inventors: 贾鹏; 温智清; 刘莹; 刘荭; 黄志坚; 简廷涵; 王寒
Original assignee: Kyorin Industry Shenzhen Co Ltd; Shenzhen Technology University; Shenzhen Academy of Inspection and Quarantine; Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center
Current assignee: Kyorin Industry Shenzhen Co Ltd; Shenzhen Technology University; Shenzhen Academy of Inspection and Quarantine; Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center
Priority date: 2018-11-05
Filing date: 2018-11-05
Publication date: 2021-12-28
Anticipated expiration: 2038-11-05
Also published as: CN112321685B; CN112279896A; CN112321685A; CN112279896B; CN112316113B; CN112279897A; CN109381697B; CN112279898A; CN112279898B; CN109381697A; CN112316113A

Abstract

The application discloses a reagent for preventing or treating fish infection CyHV-2 and application thereof. The agent for preventing or treating fish infection with CyHV-2 comprises a third polypeptide; the third polypeptide is the sequence shown in Seq ID No. 3. According to the reagent for preventing or treating fish infected by CyHV-2, the third polypeptide can be specifically and immunologically combined with CyHV-2, and the immunity resistance of fish bodies to CyHV-2 infection diseases can be specifically enhanced by feeding or soaking, so that the survival rate of fish groups infected by CyHV-2 is improved, and a new scheme and way is provided for preventing and treating CyHV-2 infection.

Description

Reagent for preventing or treating fish infection CyHV-2 and application

Technical Field

The application relates to the field of prevention and treatment of carp herpesvirus II infection, in particular to a reagent for preventing or treating fish infection CyHV-2 and application thereof.

Background

CyHV-2 is an abbreviation for carp herpes virus type II, also called goldfish hematopoietic necrosis virus (GFHNV), and is a pathogen of diseases such as goldfish hematopoietic necrosis (GFHN) and crucian fulminant hemorrhagic disease.

CyHV-2 has high infectivity, but the infection spectrum of CyHV-2 is small, and only goldfish, crucian and common variants thereof are infected, and as proved by Hedrick et al (2006), hybrids of goldfish and carp can also infect CyHV-2 to become carriers of the virus. Roe, fry, fingerling and parent fish can all be infected, but juvenile fish are more susceptible than adult fish, and the juvenile fish usually less than 1 year old causing explosive death, and adult fish and parent fish also have cases of death reported. Jung and Miyazaki (1995) infected a koi 4 months old by intraperitoneal injection of the virus, and the infected koi did not die nor have pathological changes, indicating that the virus is not pathogenic to koi. Jeffrey et al (2007) also reported that Atlantic fries and Pagrus major were not affected in the same culture system as the diseased goldfish. It is shown that fishes similar to goldfish species such as carps, eleutherine, bungy and the like do not develop diseases even if being co-cultured with diseased fishes for a long time at a proper temperature. The disease mainly occurs in spring and autumn, but is mainly affected by water temperature, and is easy to occur at 15-25 ℃. The incidence of disease decreases at water temperatures above 25 ℃, Goodwin et al (2009) report that when water temperature is increased to 27 ℃, the occurrence of death stops almost immediately; when the ambient temperature drops sharply to the temperature range of 15-25 ℃, the goldfish population carrying the virus can develop typical diseases and undergo massive deaths, while when the temperature drops slowly, the occurrence of diseases is slowed, which may be associated with the development of immune responses. Indicating that temperature is a key factor affecting viral replication in infected goldfish tissues. Goodwin et al (2009) found the virus in both eggs and fry from parent fish hatched with CyHV-2, confirming that there is vertical transmission of the virus.

The nucleocapsid of the CyHV-2 virus is hexagonal or spherical, with a diameter of 100-110nm, and the enveloped virion is ellipsoidal, with a diameter of 175-200nm (Groff et al, 1998). CyHV-2 is closely related to two other viruses isolated from Cyprinid fish, Cyprinid herpesvirus type 1 and Cyprinid herpesvirus type 3 (CyHV-3), and is relatively distant from Ictaluri herpesvirus type 1 (Ictaluri herpesvirus 1, IcHV-1) (Waltzek et al, 2005). The virus is sensitive to iodine deoxyuridine (IUdR), acidity and ether, the virus cannot replicate at the IUdR concentration of 10-4mol/L and the pH value of 3, but the titer of the virus culture solution in an experimental group is similar to that of a control group at the pH value of 11; in the presence of ether, the virus lost infectivity on FHM cells (Jung and Miyazaki, 1995). At present, partial or complete nucleotide sequences of the capsid-capsid triplex protein gene, DNA polymerase gene, helicase gene, terminal enzyme gene and the like of CyHV-2 are reported, but researches on other genes of CyHV-2 are not reported, and the complete genome sequence and gene map of the CyHV-2 are still to be further researched and perfected.

Typical clinical symptoms of goldfish infected with CyHV-2 include mental depression, lethargy, poor or anorexia, increased respiratory rate, diseased fish staying at the bottom of the pond or tank, pale gills, swollen and pale spleen and kidneys, occasional multiple white lesions, pale liver, and empty food in the intestine (Jung and Miyazaki, 1995; Groff et al, 1998; Chang et al, 1999). In addition, Philbey (2007) reports bleeding on gill, Goodwin et al (2006) describes ecchymosis bleeding on swim bladder of diseased fish, Jeffrey et al (2007) observes that vesicular pustule is on fin of diseased goldfish, and some fish also show abdominal distension and two-eye eyeball protrusion.

The histopathological changes typical of CyHV-2 are the progression of renal hematopoietic tissue, spleen, pancreas, intestinal tract and gill tissue from multifocal to diffuse necrosis (Jung and Miyazaki, 1995; Groff et al, 1998; Chang et al, 1999), gill platelet fusion, epithelial cell proliferation, degenerative necrosis of oropharynx and epidermal cells, focal necrosis of the heart, diffuse necrosis of the thymus, marked nuclear and lytic necrosis of hematopoietic cells in the head and body kidneys, extensive necrosis of the spleen marrow and arterioles in the spleen, sometimes accompanied by bleeding. No pathological changes were found in other tissue organs, including muscle tissue, brain. CyHV-2-infected nuclei were swollen, nuclear chromatin aggregates and nuclear inclusion bodies were found by electron microscopy, mature and forming virus particles were present in the nuclei, and the mature virus particles were scattered in the cytoplasm.

At present, a molecular detection method of CyHV-2 is researched and reported, but no effective scheme is available for preventing or treating related diseases caused by CyHV-2 infection.

Disclosure of Invention

The application aims to provide a novel agent for preventing or treating fish infection CyHV-2 and application thereof.

One aspect of the application discloses an agent for preventing or treating fish infection CyHV-2, which comprises a third polypeptide, wherein the third polypeptide has a sequence shown in Seq ID No. 3;

Seq ID No.3：SSLLYAMGYWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL。

it is important to note that, some positive clones are screened from the phage display library, and after IPTG induced expression, a plurality of polypeptides which can be specifically combined with CyHV-2 and have specific immunity enhancement on CyHV-2, namely the first polypeptide to the eighth polypeptide, are finally obtained. In one implementation of the present application, the challenge experiment was performed by injecting CyHV-2, and the results showed that the fish fed the first to eighth polypeptides had a certain survival rate, while the control group that was not fed the polypeptide was all dead. Therefore, the application proposes that the polypeptides can be used for preventing or treating fish infection with CyHV-2.

In another aspect of the application, the application discloses the application of the reagent for preventing or treating fish infection CyHV-2 in the detection or identification of CyHV-2.

The application also discloses application of the reagent for preventing or treating fish infection CyHV-2 in preparation of a kit or device for detecting or identifying CyHV-2.

It is noted that the polypeptide of the application can specifically enhance the immunity of fish bodies to CyHV-2 by feeding, soaking and the like, so that the fish bodies have higher survival rate when infected with CyHV-2; furthermore, the polypeptides of the present application are capable of specifically binding to CyHV-2. In one assay protocol of the present application, the first to eighth polypeptides are all capable of specifically binding to CyHV-2 without cross-reacting with other viruses, such as PFRV, STIV, VNNV, VHSV and IHNV. Therefore, the polypeptide of the present application can also be used for specific detection or identification of CyHV-2, or a kit or a device for specific detection or identification of CyHV-2 can be prepared based on the polypeptide of the present application, for example, a detection kit or a device such as an immunochromatographic test strip can be prepared by utilizing the specificity of the polypeptide of the present application, and is not limited specifically herein.

In another aspect, the present application discloses a feed for preventing or treating a disease that fish are infected with CyHV-2, comprising an agent for preventing or treating CyHV-2 infection in fish according to the present application.

Preferably, the agent for preventing or treating CyHV-2 infection in fish according to the invention is present in the feed in an amount of at least 0.5. mu.g of agent per gram of feed.

Preferably, the fish infected CyHV-2 diseases include goldfish hematopoietic necrosis and crucian fulminant hemorrhagic disease.

It is noted that the agent for preventing or treating the fish infected with CyHV-2 can prevent the fish from being infected with CyHV-2 by feeding, improve the specific immunity of fish bodies to CyHV-2, and further improve the survival rate of fish infected with CyHV-2; it will be appreciated that the most straightforward way of feeding is to incorporate the agents of the present application in feed, and thus the present application inventively proposes feed for the prevention or treatment of fish infection with CyHV-2. It is to be understood that, in one aspect, the feed may be any feed suitable for crucian carp or goldfish, and the key to the present application is not the feed, but the polypeptide agent of the present application, so that the specific formulation or type of feed may refer to the existing feed for crucian carp, goldfish or other fish; on the other hand, the current common and serious diseases infected with CyHV-2 are goldfish hematopoietic necrosis of goldfish and crucian fulminant hemorrhage of crucian, and it can be understood that the key of the polypeptide reagent of the present application is that the polypeptide reagent can specifically bind to CyHV-2, thereby having a prevention or treatment effect on the diseases caused by the infection, therefore, other diseases infected with CyHV-2 are not excluded, and the polypeptide reagent of the present application can be used for prevention or treatment as well, and is not specifically limited herein.

In addition, at least 0.5 μ g of agent per gram of feed is an effective dose for use in one protocol of the present application; it will be appreciated that the polypeptide agents of the present application, as long as they are contained in the feed, are more or less specific for the immunological action of CyHV-2; therefore, the dosage of the polypeptide agent of the present application in the feed can be adjusted according to the use requirement or product design requirement, and is not specifically limited herein.

In another aspect, the present application discloses a soaking solution for preventing or treating a disease of fish infected with CyHV-2, which contains the reagent of the present application. Similarly, the soaking solution is also particularly suitable for the hemogenic organ necrosis of goldfish and the fulminant hemorrhagic disease of crucian.

The agent for preventing or treating the fish infected with the CyHV-2 can prevent the fish from being infected with the CyHV-2 by soaking the fish in a soaking solution of a polypeptide agent, so that the specific immunity of the fish to the CyHV-2 is improved, and the survival rate of fish infected with the CyHV-2 is improved; therefore, the application creatively provides the soaking solution for preventing or treating the fish infected with CyHV-2. It is understood that the soaking solution of the present application is suitable for various kinds of fishes infected with CyHV-2 and diseases caused by the infected CyHV-2, including but not limited to, hemogenic necrosis of goldfish and goldfish, fulminant hemorrhagic disease of crucian and crucian.

The beneficial effect of this application lies in:

according to the reagent for preventing or treating fish infected by CyHV-2, the third polypeptide can be specifically and immunologically combined with CyHV-2, and the immunity resistance of fish bodies to CyHV-2 infection diseases can be specifically enhanced by feeding or soaking, so that the survival rate of fish groups infected by CyHV-2 is improved, and a new scheme and way is provided for preventing and treating CyHV-2 infection.

Drawings

FIG. 1 is the PCR detection result of the supernatant of diseased crucian carp tissue in the example of the application;

FIG. 2 is a graph showing the results of electron microscope observation of KF cells inoculated with and not inoculated with viruses in the examples of the present application;

FIG. 3 shows the results of PCR detection of cultures of virus-inoculated KF cells in the examples of the present application;

FIG. 4 is the PCR detection result of the allodynia crucian carp in the embodiment of the present application;

FIG. 5 shows the result of the specificity test of 8 polypeptides for CyHV-2 in the example of the present application;

FIG. 6 shows the results of the measurement of serum polypeptide concentration in fish in 8 polypeptide feeding experiments in the examples of the present application;

FIG. 7 shows the results of the measurement of serum polypeptide concentration in fish bodies in 8 polypeptide soaking tests in the examples of the present application.

Detailed Description

The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.

Examples

Establishment of model for attacking toxin in fulminant hemorrhagic disease of crucian carp

1. Virus isolation

(1) Virus sampling and experimental animals

Spring viremia of carp virus (abbreviated SVCV) and Koi herpesvirus (abbreviated KHV) are provided and stored by Shenzhen entry and exit inspection and quarantine agency animal and plant inspection and quarantine technical center. The diseased crucian carps are collected from a certain carassius auratus gibelio farm in the Yancheng, the body length is about 24 +/-1 cm, and the average body weight is 250 +/-10 g. Koi fin streak cells (Koi fin cell line, abbreviated KF) are provided by Shenzhen entry and exit inspection and quarantine agency animal and plant inspection and quarantine technology center. The endangered fish which is attacked by diseases at the lower air opening of the slow floating fish pond are salvaged to the upper bank, a large amount of blood naturally overflows from the lower edge of the gill cover, the body surface is congested, the abdomen is swollen, the orbit base is congested, and the gill silk is seriously hemorrhagic and whitened. The healthy allogynogenetic crucian carp selected in the experiment is provided by a south spring base of a tin-free freshwater research center, the body length is 15 +/-1 cm, and the body weight is 100 +/-10 g. All experimental healthy fish were taken from areas without GFHNV history and cultured in the laboratory water tank with water temperature controlled at 23-25 ℃.

Aseptically taking diseased crucian carp brain, spleen, kidney and other tissues, adding sea sand, pouring into a mortar for full grinding, preparing into homogenate, suspending in a cell culture solution according to a ratio of 1:10, incubating overnight at 5 ℃, centrifuging for 15min at 14000r/min, taking supernatant, namely virus supernatant, storing at-80 ℃ for virus separation.

(2) Identification of isolated viruses

Detection was performed with primers for GFHNV, KHV and SVCV, respectively. The test specifically adopts two pairs of GFHNV primers, namely GFHNV-JPF/R and GFHNV-cefas/R, KHV and SVCV primers, and four pairs of specific primers are totally adopted, PCR amplification detection is carried out on virus supernatant of diseased fish tissues, GFHNV virus positive controls are respectively set for GFHNV-JPF/R and GFHNV-cefas/R, KHV positive controls are set for KHV primers, SVCV positive controls are set for SVCV primers, and four templates are set for four pairs of primers as DEPC water negative controls. And (3) carrying out 2% agarose gel electrophoresis detection on the PCR amplification product. The method comprises the following specific steps:

RNA and DNA extraction: nucleic acids were extracted using the Dneasy Blood Tissue kit (Qiagen, Germany) and RNAmin (Qiagen, Germany) for this assay, as detailed in the kit instructions.

The reaction system and conditions for SVCV RT-PCR are as follows:

the reaction system is 50 μ L, and comprises: 10 XPCR buffer 5L, 25mmol/L MgCl₂mu.L of 5. mu.L, 4. mu.L of 2.5mmol/L dNTPs, 2.5. mu.L of each of 20mol/L primers F1 and R1, 0.5. mu.L of 5M/. mu.L DNA polymerase, 1. mu.L of 5M/. mu.L reverse transcriptase, 1. mu.L of 40M/. mu.L RNase inhibitor, 5. mu.L total RNA, and 50. mu.L of DEPC water. Two primers F1 and R1 for SVCV PCR were set in parallel for each reaction system, as in the OIE manual.

Reaction conditions are as follows: reverse transcription is carried out for 30min at 50 ℃; pre-denaturation at 95 ℃ for 2 min; then 30 cycles were entered: denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 60 s; after the circulation is finished, the extension is carried out for 7min at 72 ℃; storing at 4 ℃.

The reaction system and conditions of KHV PCR are as follows:

the KHV PCR amplification primer is a forward primer 09-KHV-TK-F and a reverse primer 09-KHV-TK-R, the 09-KHV-TK-F is a sequence shown in Seq ID No.9, and the 09-KHV-TK-R is a sequence shown in Seq ID No. 10. The PCR amplified fragment is 409bp, and the annealing temperature is 55 ℃.

Seq ID No.9：5’-GGGTTACCTGTACGAG-3’

Seq ID No.10：5’-CACCCAGTAGATTATGC-3’

The reaction system is 100 mu L and comprises: 10 XBuffer 10. mu.L, 0.2mM 10 XMG²⁺mu.L of each of 10. mu.L, 2.5. mu.L of 0.8mM primer, 1. mu.L of 10mM dNTPs 2. mu. L, Taq enzyme, and then adding water to a total volume of 100. mu.L.

The reaction conditions are as follows: 94 ℃ for 4min, then 32 cycles were entered: 94 ℃ for 1min, annealing temperature of 55 ℃ for 1min, and annealing temperature of 71 ℃ for 1 min; after the circulation is finished, the temperature is 72 ℃ for 10min, and finally the temperature is kept at 4 ℃.

The reaction system and conditions of GFHNV PCR were as follows:

GFHNV PCR respectively adopts two pairs of amplification primers, the first pair of amplification primers are an upstream primer JF with a sequence shown in Seq ID No.11 and a downstream primer JR with a sequence shown in Seq ID No.12, and the PCR amplification fragment is a DNA helicase gene fragment of 366 bp; the second pair of amplification primers are an upstream primer GFHNV-CefasF with a sequence shown in Seq ID No.13 and a downstream primer GFHNV-CefasR with a sequence shown in Seq ID No.14, and the PCR amplification fragment is a DNA helicase gene fragment of 362 bp.

Seq ID No.11：5’-GGACTTGCGAAGAGTTTGATTTCTAC-3’

Seq ID No.12：5’-CCATAGTCACCATCGTCTCATC-3’

Seq ID No.13：5’-CCCAGCAACATGTGCGACGG-3’

Seq ID No.14：5’-CCGTARTGAGAGTTGGCGCA-3’

The two pairs of amplification primers are respectively amplified in different PCR tubes, and the reaction systems and the reaction conditions of the two pairs of amplification primers are the same.

The reaction tube was placed in a PCR apparatus. 94 ℃ for 4min, then 32 cycles were entered: 94 ℃ for 1min, 55 ℃ for 1min, 71 ℃ for 1 min; after the circulation is finished, the temperature is 72 ℃ for 10min, and finally the temperature is kept at 4 ℃.

The PCR amplification products of GFHNV, KHV and SVCV were detected by 2% agarose gel electrophoresis, and the results are shown in FIG. 1. In FIG. 1, lane 1 is DNA marker DL2000, lanes 2-4 are electrophoresis results of PCR amplification products of GFHNV-JPF/R primers for supernatant of diseased fish tissue, GFHNV virus positive control and negative control, lanes 5-7 are electrophoresis results of PCR amplification products of GFHNV-cefasF/R diseased fish tissue, GFHNV virus positive control and negative control, lanes 8-10 are electrophoresis results of PCR amplification products of KHV primers for supernatant of diseased fish tissue, KHV virus positive control and negative control, and lanes 11-13 are electrophoresis results of PCR amplification products of SVCV primers for supernatant of diseased fish tissue, SVCV virus positive control and negative control.

The results in FIG. 1 show that the four primers are amplified for the positive control and not amplified for the negative control, indicating that the PCR amplification system with the four primers is normal, while for the supernatant of diseased fish tissue, only two detection primers of GFHNV, namely GFHNV-JPF/R and GFHNV-cefas/R, are amplified from the amplified fragments, and SVCV and KHV are not amplified, indicating that the supernatant of diseased fish tissue contains GFHNV, namely CyHV-2.

And (4) sending the PCR amplification product to Shenzhen Huada gene to sequence the amplification product of GFHNV-JPF/R. The sequencing result of the amplified fragment of GFHNV-JPF/R is the sequence shown in Seq ID No. 15.

Seq ID No.15：

5’-ggacttgcgaagagtttgatttctacacgcctcgcatcatgcatcaggacaacgcggtcagacaactcaacgagtcttgtatgaaaaagactgtgggcgccgaacggatcttcaagcccaagatcaatcacaataacgtgcagaacccggacgagcgtagaaagtttgcagccgtggtccgtcaacggttcaagcacattgacttctttcaaggcgtccgaatcaaggtcggatctctggtgtgcgtactaaaatatcaaactcaagtgtttgaaggctgtctgggaatagtggaatcagtacaacccgtcatggtacgcctttttttgtttgtttgtttgtttgatgagacgatggtgactatgg-3’

Sequencing results showed that amplification of GFHNV-JPF/R yielded an amplified fragment of 366, consistent with expected results. The sequencing result was compared with the GFHNV sequence registered in Genbank, and as a result, it was found that the homology of the sequencing result with the gene sequence of the GFHNV standard strain was 99%. Therefore, the supernatant of the diseased fish tissue is determined to contain GFHNV, i.e., the virus infected by the diseased fish is GFHNV.

2. Strain establishment

The virus supernatant was inoculated into a monolayer of Koi fin line (KF) cells grown for about 24 hours at appropriate volumes at 3 dilutions of 1:10, 1:100, and 1:1000, respectively, and 50 μ L of the virus supernatant was specifically inoculated into the monolayer, adsorbed at 25 ℃ for 1 hour, and then added to the cell culture medium and cultured overnight at 20 ℃. After the suspected cytopathic effect (abbreviated as CPE) appears, the cultured KF cells are taken and repeatedly frozen and thawed 3 times within 7 days by using a 40-100-fold inverted microscope for examination every day, and after 10-fold serial dilution, the KF cells are inoculated into a KF cell monolayer which grows for about 24 hours for passage, and meanwhile, a normal KF cell control is set, and the CPE is observed every day. Centrifugally collecting supernatant inoculated with virus and KF cells with CPE, manufacturing ultrathin sections, and observing under an electron microscope; meanwhile, taking normal KF cells at the same period, manufacturing ultrathin sections, and observing by an electron microscope; the results are shown in FIG. 2.

In fig. 2, the left image is the result of electron microscope scanning of KF cells showing CPE, wherein the circles show CPE produced after KF cells were infected with virus; the right image shows the scanning results of normal cells by electron microscopy.

Experimental results show that after three blind passages, typical and regular cytopathic effects begin to appear after tissue supernatants are inoculated with a monolayer of KF cells, CPE shows that cell volume increases and becomes round and a small amount of cytoplasm vacuoles, as shown in fig. 2, after 8 days, the effects of cytopathic effects are obvious, cells drop off, and normal KF cells in a control group grow well.

Extracting the 3 rd generation DNA of KF cell culture, detecting the extracted DNA by using an identifying primer GFHNV-JPF/R, and performing 2% agarose gel electrophoresis on the PCR amplification product. In this test DNA extraction kit, a Dneasy Blood Tissue kit (Qiagen, Germany) was used, and KF cells were freeze-thawed before DNA extraction, and after centrifugation, the supernatant was taken to extract nucleic acids. GFHNV-JPF/R amplification reference "(2) virus isolation identification", and the electrophoresis result of PCR amplification products is shown in FIG. 3.

In FIG. 3, lane 1 is DNAmarker DL2000, lane 2 is the electrophoresis of the PCR amplification product of the GFHNV virus positive control, lane 3 is the electrophoresis of the PCR amplification product of the normal KF cell negative control, and lane 4 is the electrophoresis of the PCR amplification product of the 3 rd generation DNA of the virus-infected KF cell culture. The results in FIG. 3 show that the 3 rd generation DNA of KF cell culture was extracted and a 362bp target fragment consistent with that expected was successfully amplified using the identifying primer GFHNV-JPF/R.

3. Testing of challenge conditions

Collecting diseased fish, collecting tissue organs such as gill, grinding, centrifuging, collecting supernatant, filtering with 0.22 μm filter membrane for sterilization, inoculating into experimental fish body by intraperitoneal injection at dosage of 1mL per tail, and injecting 1mL DMEM medium into control group. Two infection test groups and a control group are set in the test, and each group contains 10 healthy crucian carps.

The results of daily observation show that the infected crucian carps of the test groups I and II die in

days

3 and 4 respectively, the death peak periods of the infection are

days

7 and 8, all the crucian carps die after day 10, the characteristics of the congestion and abdominal swelling of the body surface are the same as those of the naturally-occurring crucian carps, and after the crucian carps are dissected, the gill hairs are white, the liver, spleen and kidney are obviously swollen, and the crucian carp fulminant hemorrhagic disease signs are met. While the control group injected with DMEM medium had no significant change.

Aseptically taking dead diseased crucian carp tissues such as brain, spleen and kidney of a test group, adding sea sand, pouring into a mortar for full grinding, preparing into homogenate, suspending in a cell culture solution according to the proportion of 1:10, incubating overnight at 5 ℃, centrifuging for 15min at 14000R/min, taking supernatant, and carrying out PCR amplification detection on the supernatant by adopting a primer GFHNV-JPF/R. Meanwhile, as a control, tissues of the brain, spleen, kidney and the like of the crucian in one control group are taken, and the obtained supernatant is prepared by the same method and used as a negative control. The results of the partial gel electrophoresis of the PCR amplification products are shown in FIG. 4.

In fig. 4, lane 1 is DNA marker DL2000, lane 2 is the electrophoresis result of the PCR amplification product of the negative control, lane 3 is the electrophoresis result of the PCR amplification product of the positive control of the GFHNV virus, and lane 4 is the electrophoresis result of the PCR amplification product of the alloresponsive crucian carp. The PCR amplification result shows that 362bp fragments are amplified from the infected fish test groups I and II and are consistent with the expected size; this indicates that GFHNV in diseased crucian carp can artificially infect healthy carassius auratus gibelio, while the control group has no target fragment, consistent with expectations.

II, crucian carp fulminant hemorrhage virus specific immunity enhancing polypeptide screening

1. Phage display libraries

The Phage Display Technology (PDT) is a technique in which a gene expression product of a foreign protein or polypeptide is fused with a phage coat protein and displayed on the surface of a phage while integrating its genetic code information into the genome of the phage.

Phage antibody technology (phage display antibody library technologies) refers to a process of amplifying a whole set of variable region genes of a body by using a PCR technology, connecting Fab or single-chain antibody (scFv) with a gene coding phage coat protein by using a phage display technology, infecting a host to form a fusion protein on the surface of the phage body, and enriching and obtaining a phage display antibody of a target molecule through a specific binding screening process of the target molecule such as protein, glycoprotein, virus, small molecular substance and the like. The antibody library technology directly utilizes the antigen to obtain the specific antibody from the antibody library, and marks that the preparation of the antibody enters a brand new era.

The foreign proteins in the phagemid display system are displayed on the surface of the filamentous phage, but the vector used to construct the display library is the phagemid, not the entire filamentous phage genome. Phagemids (phagemid) are a mixture of phages (phage) and plasmids (plasmid), which possess the advantages of both phages and plasmids. The phagemid carries the replication origin of both M13 (single stranded) and plasmid (double stranded) and can replicate as rapidly as a plasmid or can be weighted with the aid of a helper phage such as M13KO7 to form recombinant phage particles. The phagemid contained the ampicillin (Amp) resistance gene, while M13K07 contained the kanamycin (Kan) resistance gene, allowing infected cells to be selected from uninfected cells.

The phage antibody library, Escherichia coli TG1 and helper phage M13KO7 of the test were provided by the institute of aquatic organisms, national academy of sciences, and peace researchers. From the literature: dai HP, Gao H, ZHao XY, Dai LF, ZHang XK, Xiao N, ZHao RH, Hemmingsen SM.Construction and characterization of a novel reactive group against inorganic particles, 2003,279(1-2): 267-275.

2. Screening of specific polypeptide for resisting crucian fulminant hemorrhagic virus

(1) Phage display library screening

Three rounds of screening of purified CyHV-2 whole virus with phage display library were performed: 50 μ g of purified CyHV-2 diluted in PBS was used to coat the immune tubes overnight at 4 ℃; the next day, the coating solution in the immune tube was decanted, washed with PBS to remove unbound protein, the immune tube was further blocked with 10% PBSM solution, and incubated for 1H at 37 ℃; washing with PBS, adding the prepared phage display library and 10% PBSM into an immune tube according to a ratio of 1:1, and incubating for 2h at 37 ℃; washing with PBST solution, adding 1mL of 100mM triethylamine, reacting for 10min, adding 0.5mL of 1M Tris-HCl (pH 7.5), adding 5mL of Escherichia coli TG1 (O.D. 600: 0.3-0.5), and incubating at 37 deg.C for 1 h; coating the infected bacterial liquid on an SOBAG plate, and performing inverted culture at 30 ℃ overnight; the next day, colonies on the plates were scraped with medium and rescued into phage for the next round of screening.

The specific methods of phage library amplification and rescue in this assay were as follows:

1) taking a frozen phage library from-70 ℃, thawing the phage library in a water bath at 37 ℃ by about 1.5 mL;

2) adding the unfrozen bacterial liquid into 13mL of 2 XYT-AG culture medium for dilution, and uniformly mixing;

3) coating 1.5mL of the solution on a large SOBAG plate, coating 10 solutions, uniformly coating, and culturing at 30 ℃ overnight for about 16 hours;

4) the bacteria on the plate are scraped by 2 XYT-AG culture medium, and then subpackaged and frozen in tubes to be preserved at-70 ℃ for later use.

The anti-GFHNV single-chain antibody phage library is stored in the form of phagemid in the host bacteria, and is rescued into the phage-form library before panning. The specific method comprises the following steps:

5) a frozen secondary phage library was removed from-70 ℃ and thawed in about 1.5mL of 37 ℃ water bath, and then added to 600mL of 2 XYT-AG medium to make OD₆₀₀0.3-0.4;

6) shaking culture at 37 deg.C and 200r/min to OD₆₀₀＝0.5-0.8；

7) Adding the helper phase according to the proportion of 1:5 of the escherichia coli (M13K07), and carrying out shake culture at 37 ℃ for 1 hour;

8) centrifuging at 4000r/min at 4 deg.C for 15min, removing supernatant, and resuspending in 200mL of 2YT-AK, and shake culturing at 37 deg.C for 2.5 h;

9) centrifuging the culture at 10000g and 4 ℃ for 20min, precipitating phage in supernatant by 1/5 volume of PEG/NaCl, and carrying out ice bath for 1 hour;

10) centrifugation was carried out at 10000g, 4 ℃ for 20min, and the precipitated phage were resuspended in 5mL of 2YT for further use.

The test performed three rounds of screening of phage libraries and counted the selective enrichment of single chain antibodies for each round of screening, with the results shown in table 1.

TABLE 1 Selective enrichment of Single chain antibodies during each round of panning

Number of phages	First wheel	Second wheel	Third wheel
				Input quantity (CFU)	7.6×10¹⁰	7.7×10¹⁰	7.5×10¹⁰
Elution volume (CFU)	1.3×10⁴	4.2×10⁵	2.8×10⁵
				Yield (%)	1.7×10^-7	5.5×10^-6	3.7×10^-6

In table 1, the phage antibody library was subjected to 4 rounds of panning using CyHV-2 as an antigen, and the ratio of input to output was used to calculate the yield (%) — (elution amount ÷ input amount) × 100%;

the input was calculated by taking 10. mu.L of the phage library without panning in 90. mu.L of TG1 bacterial broth in logarithmic phase, preparing 10-fold serial diluted bacterial suspensions with 2 XYT, coating BOBAG plates, and counting the number of single colonies. The yield was calculated by taking 10. mu.L of panning and TG1 infected phage and adding to 90. mu.L of 2 XYT, serial 10-fold dilutions were made, BOBAG plates were coated and single colony counts were counted. The panning efficiency is then calculated.

The results in table 1 show that the yield of the third round of screening is significantly higher than the first round, indicating that the higher affinity clones are effectively enriched, while the third round is orders of magnitude different from the second round, indicating that the three rounds of screening are sufficient to ensure that high affinity antibodies are screened.

(2) Positive clone identification

Randomly picking single bacterial colony from the plate screened in the third round into a 96-hole bacterial culture plate, specifically picking 2 plates in the experiment, reserving positive and negative control holes in each culture plate, and adding culture medium containing ampicillin and helper phage to perform overnight culture; the next day, sucking 30 μ L of bacterial liquid from each well into a new bacterial culture plate, adding culture medium, culturing at 30 deg.C for 3h, centrifuging to remove supernatant, suspending the bacterial precipitate in a culture medium containing ampicillin and kanamycin, and culturing at 30 deg.C overnight; the next day, after centrifugation, the antibody-containing supernatant was stored at 4 ℃ for use, i.e., antibody supernatant.

Purified CyHV-2 was added to ELISA plates at 0.5. mu.g/well, coated for 2h at room temperature, decanted, washed with PBS, and blocked overnight with 4% PBSM; the next day, the blocking solution was decanted and washed with PBS, 100. mu.L of antibody supernatant diluted with 4% PBSM at a ratio of 1:1 was added to each well, and incubated at 30 ℃ for 1 h; after washing with PBST solution, adding 100 μ L of secondary antibody diluted with 4% PBSM solution at a ratio of 1:5000 to each well, and incubating for 1h at 30 ℃, wherein the secondary antibody in the test is HRP/Anti-M13 Monoclonal antibody; after washing with PBST solution, 100. mu.L of TMB substrate solution was added to each well for color development, and the reaction was terminated with 2M sulfuric acid; and (3) measuring the value of the excitation wavelength of 450nm by using a microplate reader, and when the value of the experimental sample divided by the value of the blank control is more than 2.1, determining that the experimental sample is a positive clone.

192 monoclonals are picked and detected in the test, and the result after ELISA analysis shows that 33 ELISA values in the 192 monoclonals are positive, and the positive cloning rate is 17.2%.

The 33 positive clones selected were further identified by setting 7 dilutions of the antigen and coating the ELISA plate with 0, 50, 100, 200, 300, 400, 500 ng/well. The results of the assay showed that 13 out of 33 positive clones had concentration dependence on antigen coating.

After the 13 positive clones were amplified and cultured, sequencing was performed, and the sequencing results were subjected to sequence analysis. The method comprises the following specific steps:

1) taking 30 mu L of positive clone bacterial liquid to a new bacterial culture plate, supplementing 2YT-AG culture medium to 200 mu L/hole, and culturing for 3h at 30 ℃; wherein, the 2YT-AG culture medium is added with helper phase, and 100 mu L of M13k07 is added into 100mL culture medium.

2) Centrifuging at 3500rpm for 15min, removing supernatant, resuspending the bacterial pellet in 200 μ L2 YT-AK, and culturing at 30 deg.C and 150rpm overnight; wherein the 2YT-AK comprises 2YT, 100 mu g/mL Amp and 50 mu g/mL Kna.

3) The next day, the bacterial culture plates were centrifuged at 3500rpm at 4 ℃ for 15min, and the supernatant was stored at 4 ℃ for further use.

And carrying out PCR amplification on the positive clones by adopting a primer group. In this example, the forward primer of the primer set is represented by Seq ID No.16, and the reverse primer is represented by Seq ID No. 17.

Seq ID No.16：5’-CCATGATTACGCCAAGCTTTGGAGCC-3’

Seq ID No.17：5’-CGATCTAAAGTTTTGTCGTCTTTCC-3’

The PCR reaction system is 100. mu.L, including 10 XBuffer 10. mu.L, 10 XMG²⁺mu.L (0.2mM), 2.5. mu.L each of 0.8mM primers, 1. mu.L of 10mM dNTPs 2. mu. L, Taq enzyme, then DNA-containing supernatant was added and water was replenished to a total volume of 100. mu.L.

Placing the reaction tube into a PCR instrument for PCR reaction, and setting the reaction conditions as follows: 94 ℃ for 5min, then 35 cycles were entered: annealing at 94 deg.C for 1min, annealing at 56 deg.C for 1min, and annealing at 72 deg.C for 1min, and after circulation, maintaining at 72 deg.C for 10min, and keeping at 4 deg.C. And (5) sending the PCR amplification product to the Shenzhen Huada gene for sequencing.

Sequencing results show that 3 strains are non-monoclonal, the other 10 strains are monoclonal, 3 strains in 10 clones have the same CDR3 region, and the other 7 strains are different.

Characteristic analysis of specific immune enhancement polypeptide of crucian carp fulminant hemorrhage virus

1. Analysis of binding of Polypeptides to CyHV-2

And (3) selecting one of 10 monoclonals obtained by screening, optionally selecting one of the monoclonals with the same CDR3 region as 3 strains for testing, adding the other 7 strains, performing IPTG (isopropyl-beta-thiogalactoside) induced expression by adopting 8 strains of positive clones in total, and then obtaining the crucian fulminant hemorrhagic virus specific immune enhancement polypeptide through a prokaryotic expression system, namely an escherichia coli expression system. Among the eight crucian fulminant hemorrhagic virus specific immunity enhancing polypeptides, a first polypeptide is a sequence shown by Seq ID No.1, a second polypeptide is a sequence shown by Seq ID No.2, a third polypeptide is a sequence shown by Seq ID No.3, a fourth polypeptide is a sequence shown by Seq ID No.4, a fifth polypeptide is a sequence shown by Seq ID No.5, a sixth polypeptide is a sequence shown by Seq ID No.6, a seventh polypeptide is a sequence shown by Seq ID No.7, and an eighth polypeptide is a sequence shown by Seq ID No. 8;

Seq ID No.1：VVYYYAMDSSGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL

Seq ID No.2：GGYDWMAVVWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL

Seq ID No.3：SSLLYAMGYWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL

Seq ID No.4：QYSWYTTVWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL

Seq ID No.5：GMFTYAADSWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL

Seq ID No.6：QWGWSLDGGGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL

Seq ID No.7：MAGGLTAYWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL

Seq ID No.8：QSQQGGAYWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL。

the specific method for IPTG inducible expression is as follows: inoculating the overnight culture liquid into LB liquid culture medium according to the proportion of 1:20, and performing shaking culture at 37 ℃. Bacteria solution OD_600nmWhen the value reaches 0.6, IPTG with the final concentration of 1.0mmol/L is addedInducing for 6h, collecting bacterial liquid every 1h, storing for later use, and observing the change of expression quantity by SDS-PAGE analysis. Meanwhile, the induced bacterial liquid is subjected to ultrasonic crushing, centrifuged at 4 ℃ and 5000r/min for 5min, and then the supernatant and the precipitate are subjected to SDS-PAGE electrophoresis to analyze the solubility of the expressed protein.

The 8 polypeptides were then analyzed for binding properties and cell debris was set as a negative control and a PBS blank. Wherein the cell debris is Escherichia coli cell debris.

In this example, binding profile analysis was performed using a dot blot assay, which specifically included:

analysis of binding characteristics of soluble Single-chain antibody to CyHV-2

1) Spotting 3 μ L of PBS, cell debris and CyHV-2 on NC membrane;

2) after natural airing, blocking the mixture for 1 hour at room temperature by using 3% BSA;

3) washing with PBS for 3 times, and placing the membrane into a single-chain antibody scFv solution diluted by 1:5 at room temperature for 1 h; wherein the dilution is performed with 3% BSA;

4) PBST and PBS are washed for 3 times respectively, and then the membrane is put into HRP/Anti E-Tag solution to react for 1h at room temperature; diluting HRP/Anti E-Tag solution with 3% BSA at a ratio of 1: 5000;

5) PBST and PBS were washed 3 times each, and then developed with DAB substrate solution.

Analysis of binding characteristics of soluble Single-chain antibody to denatured CyHV-2

1) 12% SDS-PAGE of CyHV-2 and cell debris;

2) electrically transferring the separated protein to a PVDF membrane at 100mA for 1.5 h;

3) blocking with 3% BSA at room temperature for 1 h;

4) washing with PBS for 3 times, then putting the PVDF membrane into a single-chain antibody solution diluted by 1:5, and reacting for 1h at room temperature;

5) PBST and PBS are respectively washed for 3 times, and then the PVDF membrane is put into HRP/Anti E-Tag solution to react for 1h at room temperature; diluting HRP/Anti E-Tag solution with 3% BSA at a ratio of 1: 5000;

6) PBST and PBS were washed 3 times each, and then developed with DAB substrate solution.

The dot hybridization results showed that 8 polypeptides reacted with CyHV-2, and that both the negative control and the blank control had no spots.

SDS-PAGE electrophoresis is carried out on cell debris and CyHV-2, then the cell debris and CyHV-2 are transferred to a PVDF membrane, and western blot detection is carried out by respectively taking 8 polypeptides as primary antibodies and HRP/Anti E-Tag Antibody as a secondary Antibody.

The immunogenicity of the glycoprotein induced to be expressed is analyzed by Western blot, and the specific method is as follows:

(1) taking 15 mu L of processed sample, and carrying out SDS-PAG electrophoresis;

(2) after the electrophoresis is finished, placing the adhesive tape in a transfer buffer solution for balancing for 10 min; cutting two pieces of filter paper and one piece of PVDF membrane according to the size of the adhesive tape, treating the obtained PVDF membrane with 100% methanol, and respectively soaking the filter paper, the treated PVDF membrane and the foam in a transfer buffer solution for 20 min;

(3) sequentially putting the cathode, the foam, the filter paper (two layers), the adhesive tape, the PVDF membrane, the filter paper (two layers), the foam and the anode into a splint groove, adding transfer electrophoresis liquid, and keeping the temperature for 35V and 3 hours;

(4) marking the front and back sides of the membrane after the membrane is transferred, carrying out PBST rinsing for three times, each time for 10min, adding 2% of sealing liquid, and sealing for 10 h;

(5) washing the above, adding rabbit anti-IHNV whole virus serum as a primary antibody, diluting the primary antibody at a ratio of 1:20000, and acting the primary antibody at a temperature of 37 ℃ for 1 h;

(6) washing the sample, taking goat anti-mouse IgG marked by HRP as a secondary antibody, diluting the secondary antibody at a ratio of 1:2000, acting the secondary antibody at a temperature of 37 ℃ for 1 hour, finally developing the secondary antibody by using a DAB developing kit, and photographing and analyzing the secondary antibody.

The results of the western blot analysis showed that only the eighth polypeptide recognized denatured CyHV-2, indicating that the eighth polypeptide recognized a linear epitope and the other 7 recognized conformational epitopes.

2. Polypeptide specificity assay and affinity constants

The test uses solid-phase non-competitive ELISA assay to detect the binding specificity of the 8 polypeptides from the first to the eighth polypeptide. The test materials for specific detection comprise CyHV-2, PFRV (pike fry rhabdovirus), STIV (turtle rainbow virus), VNNV (fish viral nervous necrosis disease virus), VHSV (viral hemorrhagic septicemia virus) and IHNV (infectious hematopoietic necrosis disease virus), and are provided and stored by Shenzhen Exit and entry quarantine animal and plant quarantine technical center.

The solid-phase non-competitive ELISA assay specifically involves measuring the affinity constant (Kaff) ═ n-1)/2(n [ Ab2] - [ Ab1]) of each polypeptide using a 96-well plate, where [ Ab1] and [ Ab2] represent the corresponding antibody concentrations at 50% of the maximum absorbance for different antigen concentrations, and n is the fold of dilution of the two antigens. In the test, 3 concentration gradients of the coating antigen, 10 mug/mL, 5 mug/mL and 2.5 mug/mL are set, each polypeptide is serially diluted by 3% BSA after blocking to be used as a primary antibody, and an ELISA test is carried out, wherein the test result is shown in FIG. 5. Among them, the ELISA assay is described in Beatty et al (1987), Beatty JD, Beatty BG, Vlahos WG. measurement of monoclonal antibody affinity by non-reactive enzyme immunoassay. journal of Immunological Methods,1987,100(1-2): 173. sup. -. 179.

In FIG. 5, the abscissa shows the first to eighth polypeptides in the order of the groups, i.e., polypeptide No.1 corresponds to the results of the test group in which the first antibody is the first polypeptide, polypeptide No.2 corresponds to the results of the test group in which the first antibody is the second polypeptide, and so on; the ordinate is absorbance; the results of detection of CyHV-2, PFRV, STIV, VNNV, VHSV, IHNV, negative control PBS and negative control cell debris were shown in each test group on the abscissa in order from left to right.

The results in FIG. 5 show that all eight polypeptides are capable of specifically binding CyHV-2 without cross-reacting with other viruses. The affinity constants of the first polypeptide to the eighth polypeptide are respectively 7.16 +/-1.25 multiplied by 10 in sequence⁷M^-1、6.7±2.39×10⁷M^-1、6.93±3.55×10⁷M^-1、5.36±1.67×10⁷M^-1、1.63±0.43×10⁶M^-1、1.83±0.59×10⁶M^-1、4.6±1.75×10⁵M^-1And 1.15. + -. 0.69X 10⁵M^-1。

3. Indirect immunofluorescence assay for polypeptides and CyHV-2

KF cells are passaged in a six-hole plate, and CyHV-2 is prepared to be inoculated when the cells grow well; sucking out cell culture fluid about 24h after inoculation, washing a plate hole by PBS, and fixing for 10min at 4 ℃ by using precooled fixing fluid; washing away the fixing solution with PBS, adding each polypeptide diluted by 1:5, and incubating for 1h at 37 ℃; washing the polypeptide diluent with PBS, adding rabbit anti-mouse IgG-FITC labeled polypeptide diluted at a ratio of 1:200, and incubating at 37 ℃ for 1 h; after washing the plate with PBS, the plate was mounted with 50% glycerol-PBS for microscopic examination and observed under a fluorescent inverted microscope. Meanwhile, non-sterilized normal KF cells were set as a negative control, which was identical to CyHV-2-inoculated KF cells except that no CyHV-2 was inoculated. The observation by fluorescence microscope (400X) shows that the 8 polypeptides from the first to the eighth polypeptides can generate specific fluorescence staining on KF cells infected with CyHV-2 virus, but show negative reaction in uninfected areas, and 8 single-chain antibodies have no reaction with KF cells not inoculated with CyHV-2.

IV, crucian carp fulminant hemorrhage virus specific immunity enhancing polypeptide in vivo test

1. Toxicity testing

In order to confirm whether the 8 CyHV-2 specific immunity enhancing polypeptides from the first polypeptide to the eighth polypeptide in the test have high safety and no adverse side effect on fish bodies, the test stops feeding for 24 hours, then the two groups of crucian carps are subjected to intraperitoneal injection by the polypeptides with the dosage of 10 times and 20 times of effective concentration respectively, meanwhile, a control group with the same amount of PBS (phosphate buffer solution) for intraperitoneal injection is arranged, and the test is carried out on 25 crucian carps and 8 polypeptides in each group respectively; and observed for 28 days one by one to confirm whether adverse reactions exist and the survival rate, so as to be used as a standard for evaluating the safety of 8 polypeptides.

The results show that the survival rates of all test groups are 100% after the intraperitoneal injection is carried out on the polypeptides with 10-fold and 20-fold doses for 28 days, no adverse reaction occurs, and the conditions of the control groups are the same, which indicates that the first to eighth polypeptides in the test have high safety on fish bodies.

3. Polypeptide protection test

(1) Feeding test

0.5 mu g of polypeptide is added into each gram of feed according to a proportion to prepare the feed fed in the test process, no polypeptide is added into a control group, and 9 kinds of feed fed in the test are designed in total, namely the feed added with the first polypeptide, the feed added with the second polypeptide, the feed added with the eighth polypeptide and the conventional feed added with no polypeptide. Therefore, the experiment of the experiment is divided into 9 groups, each group comprises 55 crucian carps, the body length is 20 +/-1 cm, the body weight is 150 +/-10 g, the control group feed and the corresponding feed added with the polypeptide are respectively fed, three meals a day and 50g of feed are taken, the control group feed and the corresponding feed added with the polypeptide are continuously fed for 14 days, 5 crucian carps are randomly selected from each group respectively on the 0 th day, the 7 th day and the 14 th day to measure the concentration of the polypeptide in the serum, and the rest 40 crucian carps are subjected to the toxicity attack experiment after 14 days.

Serum polypeptide concentration test:

adding the purified E-tag into an ELISA plate at 0.5 mu g/hole, coating at room temperature for 2h, pouring out the coating solution, washing with PBS, and blocking with 4% PBSM solution overnight; the next day, after pouring off the blocking solution, washing with PBS, adding 150 μ L of crucian serum (1:100-1:12800) diluted twice with 4% PBSM per well, and incubating at 30 deg.C for 1 h; after being washed by PBST solution, 150 mu L of rabbit anti-crucian IgM antibody diluted by 4% PBSM according to a ratio of 1:5000 is added into each hole as a secondary antibody, and the mixture is incubated for 1h at 30 ℃; after washing with PBST solution, 150 μ L of HRP/goat anti-rabbit IgG antibody diluted with 4% PBSM at a ratio of 1:5000 was added to each well as a tertiary antibody, and incubated at 30 ℃ for 1 h; after washing with PBST solution, 100. mu.L of TMB substrate solution was added to each well for color development, and the reaction was finally stopped with 2M sulfuric acid. And then, measuring the value of the excitation wavelength of 450nm by using a microplate reader, and when the experimental sample value of the dilution ratio is divided by the blank control value to be more than 3 times, considering the dilution ratio as the serum concentration value. The results of the serum polypeptide concentration test are shown in FIG. 6.

In FIG. 6, the abscissa represents the serum polypeptide concentration at day 0, day 7 and day 14, and the ordinate represents the serum polypeptide concentration value; in the three groups of data of the 0 th day, the 7 th day and the 14 th day, the serum polypeptide concentration of the crucian fed with a control group without any polypeptide, the serum polypeptide concentration of the crucian fed with the first polypeptide feed, the serum polypeptide concentration of the crucian fed with the second polypeptide feed, the serum polypeptide concentration of the crucian fed with the third polypeptide feed, the serum polypeptide concentration of the crucian fed with the fourth polypeptide feed, the serum polypeptide concentration of the crucian fed with the fifth polypeptide feed, the serum polypeptide concentration of the crucian fed with the sixth polypeptide feed, the serum polypeptide concentration of the crucian fed with the seventh polypeptide feed and the serum polypeptide concentration of the crucian fed with the eighth polypeptide feed are sequentially arranged from left to right in each group.

The results in FIG. 6 show that the concentration of the polypeptide in the serum can reach up to 1600-3200 times after 7 days of continuous feeding, and can reach 6400-12800 times after 14 days, which proves that the content of each polypeptide in the fish can be effectively increased by feeding.

And (3) toxin counteracting test:

and (3) taking the purified virus liquid, filtering and sterilizing the virus liquid by using a 0.22 mu m filter membrane, inoculating the virus liquid into experimental fish bodies by intraperitoneal injection at the dosage of 1mL per tail, injecting 1mL of DMEM medium into a control group, and continuously observing the survival rate of the fish for 14 days by using 20 fish in each of the control group and the experimental group.

The test results show that the control group died all 12 days after challenge, while the group with the polypeptides still had at least 20% survival at the end of the test, with up to 75% survival when fed with the second polypeptide, 70% survival when fed with the fifth polypeptide and 75% survival when fed with the seventh polypeptide. Moreover, the survival rate of the group fed with the second polypeptide on the 9 th day is up to 90%, the fish die in the next few days, and the survival rate is maintained at 75%; the seventh polypeptide group is similar to the second polypeptide group; the group fed the fifth polypeptide had no fish death after day 11 and the survival rate was stable.

(2) Immersion test

100mg of the soaking solution used in the preparation test process of the polypeptide is added into each liter of water according to the proportion, and the control group is common fresh water. The test is divided into 9 groups, each group comprises 55 crucian carps, the body length is 20 +/-1 cm, the body weight is 150 +/-10 g, the crucian carps are respectively soaked in the control group and the soaking solution added with the polypeptide for 1h, the soaking solution is used for soaking once a day for 14 days continuously, 5 crucian carps are respectively selected randomly for each group on the 0 th day, the 7 th day and the 14 th day for determining the concentration of the polypeptide in serum, and the rest 40 crucian carps are subjected to toxicity attack test after 14 days. Wherein the soaking solution is obtained by subjecting fermentation broth induced and expressed by polypeptides to cell disruption with a homogenizer at 1000bar pressure, centrifuging the fermentation broth with disrupted cell wall at 12000g at 4 deg.C for 30min, and collecting supernatant to obtain soaking solution containing soluble polypeptides, wherein the concentration of polypeptides in the soaking solution is 3-30 μ M.

Serum polypeptide concentration test: the test method was the same as that of the feeding test (1), and the test results are shown in FIG. 7. In FIG. 7, the abscissa represents the serum polypeptide concentration at day 0, day 7 and day 14, and the ordinate represents the serum polypeptide concentration value; in the three groups of data of the 0 th day, the 7 th day and the 14 th day, the serum polypeptide concentration of the crucian fed with a control group without any polypeptide, the serum polypeptide concentration of the crucian fed with the first polypeptide feed, the serum polypeptide concentration of the crucian fed with the second polypeptide feed, the serum polypeptide concentration of the crucian fed with the third polypeptide feed, the serum polypeptide concentration of the crucian fed with the fourth polypeptide feed, the serum polypeptide concentration of the crucian fed with the fifth polypeptide feed, the serum polypeptide concentration of the crucian fed with the sixth polypeptide feed, the serum polypeptide concentration of the crucian fed with the seventh polypeptide feed and the serum polypeptide concentration of the crucian fed with the eighth polypeptide feed are sequentially arranged from left to right in each group.

The results in FIG. 7 show that the concentration of the polypeptide in the serum can reach up to 800-3200 times after continuous feeding for 7 days, and can reach 6400-12800 times after 14 days, which proves that the content of each polypeptide in the fish can be effectively increased by the consistent manner of soaking and feeding.

And (3) toxin counteracting test: the test method was the same as in "(1) feeding test". The test result shows that the control group dies in 11 days after the toxicity attack, the survival rate of the group using the polypeptide is at least 15% at the end of the test, wherein the survival rate of the group fed with the second polypeptide is as high as 75%, the survival rate of the group fed with the fifth polypeptide is 65%, the survival rate of the group fed with the seventh polypeptide is 70%, and the survival rate effect is most obvious.

The above experiments show that any polypeptide in the first to eighth polypeptides fed or soaked in the test can enhance the immunity of the fish body to CyHV-2, thereby improving the survival rate of the fish body; particularly the second polypeptide, the fifth polypeptide and the seventh polypeptide, the survival rate effect is most remarkable. Although the application is only tested on carps, it can be understood that the specific immune-enhancing polypeptide is directed to CyHV-2, so that the immune-enhancing polypeptide can be used for enhancing the immunity of fish bodies to CyHV-2 in diseases caused by CyHV-2 infection or CyHV-2 infection, including but not limited to fulminant hemorrhagic disease of carps and crucian carps thereof, hematopoietic necrosis of goldfish and goldfish thereof, and the like.

The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

SEQUENCE LISTING

<110> scaling-sharing actual industry (Shenzhen) Limited

Shenzhen Customs Animal and Plant Inspection and Quarantine Technology Center

<120> reagent for preventing or treating fish infection CyHV-2 and application

<130> 20I30779-D26892

<160> 17

<170> PatentIn version 3.3

<210> 1

<211> 50

<212> PRT

<213> crucian carp fulminant hemorrhage virus specific immunity enhancing polypeptide

<400> 1

Val Val Tyr Tyr Tyr Ala Met Asp Ser Ser Gly Gln Gly Thr Ser Val

1 5 10 15

Thr Val Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Asp Val Val Met Ala Gln Ser Pro Ser Ser Met Tyr Ala

35 40 45

Ser Leu

50

<210> 2

<211> 50

<212> PRT

<400> 2

Gly Gly Tyr Asp Trp Met Ala Val Val Trp Gly Gln Gly Thr Ser Val

1 5 10 15

Thr Val Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Asp Val Val Met Ala Gln Ser Pro Ser Ser Met Tyr Ala

35 40 45

Ser Leu

50

<210> 3

<211> 50

<212> PRT

<400> 3

Ser Ser Leu Leu Tyr Ala Met Gly Tyr Trp Gly Gln Gly Thr Ser Val

1 5 10 15

Thr Val Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Asp Val Val Met Ala Gln Ser Pro Ser Ser Met Tyr Ala

35 40 45

Ser Leu

50

<210> 4

<211> 49

<212> PRT

<400> 4

Gln Tyr Ser Trp Tyr Thr Thr Val Trp Gly Gln Gly Thr Ser Val Thr

1 5 10 15

Val Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

20 25 30

Gly Ser Asp Val Val Met Ala Gln Ser Pro Ser Ser Met Tyr Ala Ser

35 40 45

Leu

<210> 5

<211> 50

<212> PRT

<400> 5

Gly Met Phe Thr Tyr Ala Ala Asp Ser Trp Gly Gln Gly Thr Ser Val

1 5 10 15

Thr Val Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Asp Val Val Met Ala Gln Ser Pro Ser Ser Met Tyr Ala

35 40 45

Ser Leu

50

<210> 6

<211> 49

<212> PRT

<400> 6

Gln Trp Gly Trp Ser Leu Asp Gly Gly Gly Gln Gly Thr Ser Val Thr

1 5 10 15

Val Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

20 25 30

Gly Ser Asp Val Val Met Ala Gln Ser Pro Ser Ser Met Tyr Ala Ser

35 40 45

Leu

<210> 7

<211> 49

<212> PRT

<400> 7

Met Ala Gly Gly Leu Thr Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr

1 5 10 15

Val Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

20 25 30

Gly Ser Asp Val Val Met Ala Gln Ser Pro Ser Ser Met Tyr Ala Ser

35 40 45

Leu

<210> 8

<211> 49

<212> PRT

<400> 8

Gln Ser Gln Gln Gly Gly Ala Tyr Trp Gly Gln Gly Thr Ser Val Thr

1 5 10 15

Val Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

20 25 30

Gly Ser Asp Val Val Met Ala Gln Ser Pro Ser Ser Met Tyr Ala Ser

35 40 45

Leu

<210> 9

<211> 16

<212> DNA

<213> Artificial sequence

<400> 9

gggttacctg tacgag 16

<210> 10

<211> 17

<212> DNA

<213> Artificial sequence

<400> 10

cacccagtag attatgc 17

<210> 11

<211> 26

<212> DNA

<213> Artificial sequence

<400> 11

ggacttgcga agagtttgat ttctac 26

<210> 12

<211> 22

<212> DNA

<213> Artificial sequence

<400> 12

ccatagtcac catcgtctca tc 22

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence

<400> 13

cccagcaaca tgtgcgacgg 20

<210> 14

<211> 20

<212> DNA

<213> Artificial sequence

<400> 14

ccgtartgag agttggcgca 20

<210> 15

<211> 366

<212> DNA

<213> GFHNV-JPF/R amplified fragment

<400> 15

ggacttgcga agagtttgat ttctacacgc ctcgcatcat gcatcaggac aacgcggtca 60

gacaactcaa cgagtcttgt atgaaaaaga ctgtgggcgc cgaacggatc ttcaagccca 120

agatcaatca caataacgtg cagaacccgg acgagcgtag aaagtttgca gccgtggtcc 180

gtcaacggtt caagcacatt gacttctttc aaggcgtccg aatcaaggtc ggatctctgg 240

tgtgcgtact aaaatatcaa actcaagtgt ttgaaggctg tctgggaata gtggaatcag 300

tacaacccgt catggtacgc ctttttttgt ttgtttgttt gtttgatgag acgatggtga 360

ctatgg 366

<210> 16

<211> 26

<212> DNA

<213> Artificial sequence

<400> 16

ccatgattac gccaagcttt ggagcc 26

<210> 17

<211> 25

<212> DNA

<213> Artificial sequence

<400> 17

cgatctaaag ttttgtcgtc tttcc 25

Claims

1. An agent for preventing or treating fish infection CyHV-2, wherein the agent is a polypeptide with a sequence shown in Seq ID No. 3;

Seq ID No.3：SSLLYAMGYWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMYASL。

2. use of a reagent according to claim 1 in the manufacture of a kit or device for the detection or identification of CyHV-2.

3. A feed for preventing or treating a disease in fish infected with CyHV-2, characterized in that: the feed contains the agent according to claim 1.

4. The feed of claim 3, wherein: the amount of said agent in the feed is such that at least 0.5 μ g of said agent is contained per gram of feed.

5. The feed according to claim 3 or 4, characterized in that: the fish infected with CyHV-2 diseases comprise goldfish hematopoietic organ necrosis and crucian fulminant hemorrhagic disease.

6. A soaking solution for preventing or treating fish diseases infected with CyHV-2, which is characterized in that: the soaking solution contains the reagent according to claim 1.

7. The soak solution according to claim 6, wherein: the fish infected with CyHV-2 diseases comprise goldfish hematopoietic organ necrosis and crucian fulminant hemorrhagic disease.