CN109381697A

CN109381697A - A kind of reagent and application for preventing or treating infection of marine fishes CyHV-2

Info

Publication number: CN109381697A
Application number: CN201811308463.9A
Authority: CN
Inventors: 温智清; 刘莹; 贾鹏; 刘荭; 黄志坚; 简廷涵; 王寒
Original assignee: GONGLIN INDUSTRY (SHENZHEN) Co Ltd; Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry Exit Inspection and Quarantine Bureau
Current assignee: GONGLIN INDUSTRY (SHENZHEN) Co Ltd; Kyorin Industry Shenzhen Co Ltd; Animal and Plant Inspection and Quarantine Technology Center of Shenzhen Entry Exit Inspection and Quarantine Bureau
Priority date: 2018-11-05
Filing date: 2018-11-05
Publication date: 2019-02-26
Anticipated expiration: 2038-11-05
Also published as: CN112321685B; CN112279896A; CN112321685A; CN112279896B; CN112316113B; CN112279897A; CN109381697B; CN112279898A; CN112279897B; CN112279898B; CN112316113A

Abstract

The reagent and application that this application discloses a kind of for preventing or treating infection of marine fishes CyHV-2.The reagent that the application is used to prevent or treat infection of marine fishes CyHV-2 includes at least one of first polypeptide to the 8th polypeptide；First polypeptide to the 8th polypeptide is sequentially sequence shown in Seq ID No.1 to 8.The application is used to prevent or treat the reagent of infection of marine fishes CyHV-2, its first polypeptide to the 8th polypeptide is capable of the immune in conjunction with CyHV-2 of specificity, and the immune resistance that can be caught by the enhancing fish body of modes specificity such as feeding or impregnating to CyHV-2, to improve the survival rate for the shoal of fish for infecting CyHV-2, a kind of new scheme and approach are provided for the prevention and treatment of CyHV-2 infection.

Description

A kind of reagent and application for preventing or treating infection of marine fishes CyHV-2

Technical field

This application involves carp herpesvirusⅡtype infection prevention and treatment field, more particularly to one kind for prevent or Reagent and its application for treating infection of marine fishes CyHV-2.

Background technique

CyHV-2 is the abbreviation of carp herpesvirusⅡtype, also referred to as the sick virus (goldfish of goldfish Hematopoietic Necrosis Haematopoietic necrosis virus, abridge GFHNV), it is goldfish Hematopoietic Necrosis's disease (goldfish Haematopoietic necrosis, abridge GFHN), the cause of diseases of the diseases such as crucian carp fishy fulminant hemorrhage.

CyHV-2 infectiousness with higher, but the pattern of infection of CyHV-2 is smaller only infects goldfish, crucian and its common Mutation, becoming the virus as the hybrid of Hedrick (2006) research discovery goldfish and carp can also infect CyHV-2 Carrier.Fish-egg, fry, fingerling and parent population can infect, but juvenile fish is more susceptible than adult fish, and cause the usual of fulminant death It is less than the juvenile fish in 1 age, adult fish, parent population also have dead Case report.Jung and Miyazaki (1995) pass through intraperitoneal injection The virus infects the fancy carp at 4 monthly ages, and the fancy carp of infection does not have death, without pathological change, shows that the virus is not yet The cause of disease of fancy carp.Jeffrey etc. (2007) is also reported with graining of the morbidity goldfish under same cultivating system, tench not by shadow It rings.Show with fish similar in goldfish germline such as carp, graining, tench etc., even if being supported altogether with sick fish for a long time in preference temperature Also it does not fall ill.The disease occurs mainly in season in spring and autumn, but mainly by Water Temperature, 15-25 DEG C of susceptible disease.Water temperature is higher than 25 DEG C When, disease incidence reduces, and Goodwin etc. (2009) report occurs the phenomena of mortality and almost stop at once when water temperature is improved to 27 DEG C； When environment temperature sharply drops to the 15-25 DEG C of temperature range, the goldfish population for carrying the virus can generate typical disease Disease and mortality occurs, and when temperature slowly declines, disease slows down, this may have with immune response It closes.Show that temperature is the key factor for influencing infection goldfish tissue inner virus duplication.Goodwin etc. (2009) is infecting Fish-egg that the parent population of CyHV-2 hatches and the virus is had found in fry, to confirm that the virus is passed there is vertical It broadcasts.

The nucleocapsid of CyHV-2 virus is in hexagon or spherical shape, and diameter 100-110nm has the virion of cyst membrane in ellipse Circle, diameter are 175-200nm (Groffet al., 1998).CyHV-2 is isolated from the virus of cyprinid fish with other two kinds, 3 type of 1 type of Cyprinidae herpesviral and Cyprinidae herpesviral (Cyprinid herpesvirus3, CyHV-3), relationship is very close, Yu 1 type of Channel-catfish herpesviral (Ictalurid herpesvirus 1, IcHV-1) relationship relatively far away from (Waltzek et al., 2005).Virus is all more sensitive to idoxene (IUdR), acidity and ether, in the IUdR and pH that concentration is 10-4mol/L When value is 3, virus not reproducible, but when pH value is 11, the titre of experimental group virus-culturing fluid is close with control group；It is deposited in ether In the case that, virus loses to the appeal of FHM cell (Jung andMiyazaki, 1995).The capsid of CyHV-2 at present The partial or complete nucleotide of three protein gene, DNA polymerase gene, Helicase gene, end enzyme gene etc. between body Sequence it has been reported that but the research of other genes about CyHV-2 have not been reported, complete genome sequence and gene map Spectrum need further study and it is perfect.

The typical clinical symptom for infecting the goldfish of CyHV-2 includes depressed spirit, lethargic sleep, loss of appetite or anorexia, breathing frequency Rate increases, and illness fish rests on pond or water tank bottom, and the visible gill is pale after dissection, spleen and kidney swelling and be in pale asphyxia, once in a while Can see many places white lesion, liver is in pale asphyxia, enteron aisle sky without food (Jung and Miyazaki, 1995；Groffet al., 1998；Chang et al.,1999).In addition to this, bleeding on the case gill of Philbey (2007) report, Goodwin etc. (2006) describe have ecchymosis bleeding on the fish glue of sick fish, Jeffrey etc. (2007), which is observed, bubble shape purulence on illness goldfish fin Blister, some fishes also show abdomen and expand, eyes exophthalmos.

The typical histopathologic change of CyHV-2 has kidney hematopoietic tissue, spleen, pancreas, enteron aisle and gill tissue to be sent out by more lesions Open up dispersivity necrosis (Jung and Miyazaki, 1995；Groffet al.,1998；Chang et al., 1999), the gill There is Focal necrosis in small pieces fusion, epithelial hyperplasia, oropharynx and epidermal cell degeneration necrosis, heart, and thymus gland dispersivity is bad Extremely, there is apparent karyopycnosis and karyorhexis necrosis in hematopoietic cell in head-kidney and body kidney, and the pulpa lienis and parteriole in spleen are big The necrosis of area, sometimes also with bleeding.Other histoorgans, including musculature, brain do not find that pathologic changes.Sense The cell nuclear swelling of CyHV-2 is contaminated, Electronic Speculum inspection finds nuclei dyeing chromaticness side collection and intranuclear inclusion, has into nucleus Virion in ripe and formation, and mature virion intersperses among in cytoplasm.

There are research and the report to the molecular detecting method of CyHV-2 at present, still, for how to prevent or treat Related disease caused by CyHV-2 infects, still without effective scheme.

Summary of the invention

The purpose of the application is to provide a kind of new reagent and its application for being used to prevent or treat infection of marine fishes CyHV-2.

The one side of the application discloses a kind of for preventing or treating the reagent of infection of marine fishes CyHV-2, which includes At least one of first polypeptide to the 8th polypeptide；First polypeptide is sequence shown in Seq ID No.1, and the second polypeptide is Seq ID Sequence shown in No.2, third polypeptide be Seq ID No.3 shown in sequence, the 4th polypeptide be Seq ID No.4 shown in sequence, the 5th Polypeptide is sequence shown in Seq ID No.5, and the 6th polypeptide is sequence shown in Seq ID No.6, and the 7th polypeptide is Seq ID No.7 Shown sequence, the 8th polypeptide are sequence shown in 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.

It should be noted that the application's it is critical that from phage display library screening obtain some positive colonies, After carrying out IPTG inducing expression to it, be finally obtained it is multiple can be specifically bound with CyHV-2, and have specifically to CyHV-2 The polypeptide of property Immune-enhancing effect, i.e. the first polypeptide to the 8th polypeptide.In a kind of implementation of the application, by injection CyHV-2 into Row challenge viral dosage, the results show that the fish for feeding the first polypeptide to the 8th polypeptide has certain survival rate, without feeding polypeptide Control group it is totally dead, especially feed the test group of the second polypeptide, the 5th polypeptide and the 7th polypeptide, survival rate is both greater than 70%.Therefore, the application proposes that these polypeptides can be used for preventing or treating infection of marine fishes CyHV-2；Especially the second polypeptide, 5th polypeptide and the 7th polypeptide effect are the most significant.

The another side of the application discloses the application for preventing or treating the reagent of infection of marine fishes CyHV-2 in CyHV-2 Application in detection or identification.

The another side of the application discloses the application and is preparing for preventing or treating the reagent of infection of marine fishes CyHV-2 Application in the kit or device of CyHV-2 detection or identification.

It should be noted that the polypeptide of the application can not only be by the enhancing fish body of modes specificity such as feeding, impregnating To the immunity of CyHV-2, make it that there is higher survival rate in infection CyHV-2；Moreover, the polypeptide of the application being capable of specificity Combination CyHV-2.In a kind of testing program of the application, the first polypeptide to the 8th polypeptide can specificity combination CyHV-2, without cross reaction occurs with other viruses, such as PFRV, STIV, VNNV, VHSV and IHNV.Therefore, the application Polypeptide can be equally used for CyHV-2 specific detection or identification, and also or the preparation of the polypeptide based on the application CyHV-2 is specific The kit or device of detection or identification, such as can use the specificity of the application polypeptide, prepare immuno-chromatographic test paper strip etc. Detection kit or device, are not specifically limited herein.

Disclosing on one side again for the application is a kind of for preventing or treating the feed of the disease of infection of marine fishes CyHV-2, the feeding It is used to prevent or treat the reagent of infection of marine fishes CyHV-2 in material containing the application.

Preferably, the application is for preventing or treating dosage of the reagent of infection of marine fishes CyHV-2 in feed to be every gram Contain at least 0.5 μ g reagent in feed.

Preferably, the disease of the so-called infection of marine fishes CyHV-2 of the application includes that goldfish Hematopoietic Necrosis disease and crucian are sudden and violent Hair property hemorrhage.

It should be noted that the reagent that the application is used to prevent or treat infection of marine fishes CyHV-2 can be carried out by feeding Prevention improves fish body to the specific immunity of CyHV-2, to improve the survival rate of the shoal of fish of infection CyHV-2；It can manage Solution, the mode fed are most directly exactly that the reagent of the application is entrained in feed, and therefore, the application is creative to be proposed For preventing or treating the reagent of infection of marine fishes CyHV-2 in feed.It is understood that, on the one hand, the feed can be various be applicable in In the feed of crucian or goldfish, the key of the application is not intended to feed, and is the polypeptide reagent of the application, therefore, feed Specific formula or type can with reference to existing crucian, goldfish or other fish feed；On the other hand, commonplace at present Disease with serious infection CyHV-2 is exactly the goldfish Hematopoietic Necrosis disease of goldfish and the crucian carp fishy fulminant hemorrhage of crucian, It is appreciated that the key of the polypeptide reagent of the application is to be capable of the combination CyHV-2 of specificity, thus caused by infecting it Disease has prevention or therapeutic effect, therefore, however not excluded that there are also the diseases of other infection CyHV-2, equally can be using the application's Polypeptide reagent is prevented or is treated, and is not specifically limited herein.

In addition, in every gram of feed contain at least 0.5 μ g reagent, this be the application a kind of testing program in use it is effective Dosage；As long as being appreciated that the polypeptide reagent containing the application in feed, more or less all there is specificity to exempt from CyHV-2 Epidemic disease effect；Therefore, the dosage of the application polypeptide reagent can require to be adjusted according to use demand or product design in feed, It is not specifically limited herein.

Disclosing on one side again for the application is a kind of for preventing or treating the soak of the disease of infection of marine fishes CyHV-2, should Soak includes at least one of first polypeptide to the 8th polypeptide.Likewise, the soak of the application is also especially suitable for gold Fish Hematopoietic Necrosis disease and crucian carp fishy fulminant hemorrhage.

It should be noted that the reagent that the application is used to prevent or treat infection of marine fishes CyHV-2 can be by by fish soaking Mode in the soak of polypeptide reagent is prevented, and improves fish body to the specific immunity of CyHV-2, to improve sense Contaminate the survival rate of the shoal of fish of CyHV-2；Therefore, the application is creative proposes for preventing or treating infection of marine fishes CyHV-2 Reagent in soak.It is appreciated that the soak of the application is suitable for the fish of various infection CyHV-2 and infection CyHV-2 makes At disease, include but are not limited to goldfish and its goldfish Hematopoietic Necrosis disease, crucian and its crucian carp fishy fulminant hemorrhage.

The beneficial effects of the present application are as follows:

The application is used to prevent or treat the reagent of infection of marine fishes CyHV-2, and the first polypeptide to the 8th polypeptide can be special Property it is immune in conjunction with CyHV-2, and disease can be infected to CyHV-2 by the enhancing fish body of modes specificity such as feeding or impregnating The immune resistance of disease, to improve the survival rate of the shoal of fish of infection CyHV-2, the prevention and treatment for CyHV-2 infection are provided A kind of new scheme and approach.

Detailed description of the invention

Fig. 1 is the PCR testing result of sick crucian carp tissue supernatant in the embodiment of the present application；

Fig. 2 is to be inoculated in the embodiment of the present application and the Electronic Speculum of the KF cell of non-virus inoculation observation result figure；

Fig. 3 is the PCR testing result of the culture of the KF cell of virus inoculation in the embodiment of the present application；

Fig. 4 is the PCR testing result that susceptible crucian carp is returned in the embodiment of the present application；

Fig. 5 be in the embodiment of the present application 8 polypeptides to the specific detection result of CyHV-2；

Fig. 6 is serum polypeptide Concentration Testing result in the fish body of 8 polypeptide feeding tests in the embodiment of the present application；

Fig. 7 is serum polypeptide Concentration Testing result in the fish body of 8 polypeptide soak tests in the embodiment of the present application.

Specific embodiment

The application is described in further detail below by specific embodiment.Following embodiment only to the application carry out into One step explanation, should not be construed as the limitation to the application.

Embodiment

One, crucian carp fishy fulminant hemorrhage attacks malicious model foundation

1. virus purification

(1) virus sampling and experimental animal

Huichun viremia virus (abbreviation SVCV) and Koi herpesvirus (abbreviation KHV) are by Shenzhen inspection and quarantining for import/export Office animals and plants inspection and quarantine technique center provides and saves.Illness crucian carp picks up from Yancheng hybridized prussian carp farm, the long 24 ± 1cm of body Left and right, 250 ± 10g of average weight.Fancy carp fin ray cell (Koi fin cell line, abridge KF) is entered and left the border by Shenzhen and is examined Quarantine Bureau's animals and plants inspection and quarantine technique center provides.It is visible that the air port dying fish of morbidity under slow pelagic fish pond is salvaged into disembarkation A large amount of blood overflow naturally from gill cover lower edge, body surface hyperemia abdomen swelling, and eye socket base portion is congested, and gill filament severe haemorrhage is simultaneously whitened. It tests the healthy hybridized prussian carp selected to be provided by the Wuxi fresh water research center base Nan Quan, the long 15 ± 1cm of body, weight 100 ± 10g.The healthy fish of all experiments is taken from the area of no GFHNV morbidity history, cultivates in this laboratory sink, and water temperature control exists 23-25℃。

It is sterile that illness crucian carp brain, spleen, kidney etc. is taken to organize, sea sand is added, pours into mortar and is fully ground, be prepared into homogenate, press The ratio of 1:10 is suspended in cell culture fluid, and after 5 DEG C are incubated overnight, 14000r/min is centrifuged 15min, takes supernatant, i.e., viral Supernatant, -80 DEG C of preservations, is used for virus purification.

(2) isolated viral is identified

It is detected respectively with the primer of GFHNV, KHV and SVCV.This test specifically uses two pairs of GFHNV primers, i.e., GFHNV-JPF/R and GFHNV-cefas/R and KHV, SVCV primer amount to four pairs of specific primers, to the disease of sick fish tissues Malicious supernatant carries out PCR amplification detection, is respectively provided with GFHNV virus-positive for GFHNV-JPF/R and GFHNV-cefas/R Control is provided with KHV positive control for KHV primer, is provided with SVCV positive control for SVCV primer, and draw for four pairs Object is provided with the negative control that four templates are DEPC water.2% agarose gel electrophoresis detection is carried out to pcr amplification product.Tool Body is as follows:

RNA and DNA extracting: this test use Dneasy Blood Tissue kit (Qiagen, Germany) and RNAmin (Qiagen, Germany) extracts nucleic acid, and the specific procedure is described in the kit instructions.

The reaction system and condition of SVCV RT-PCR is as follows:

Reaction system is 50 μ L, comprising: 10 × PCRbuffer 5L, 25mmol/LMgCl₂5 μ L, 2.5mmol/L dNTPs 4 μ L, 20mol/L primers Fs 1 and each 0.5 μ L, 5M/ μ L reverse transcriptase of 2.5 μ L, 5M/ μ L archaeal dna polymerase, 1 μ L, 40M/L RNA of R1 1 μ L of enzyme inhibitor, 5 μ L of total serum IgE add DEPC water to 50 μ L of total system.Two parallel, SVCV PCR are arranged in each reaction system With primers F 1 and R1 with OIE handbook.

Reaction condition: 50 DEG C of reverse transcription 30min；95 DEG C of initial denaturation 2min；It is recycled subsequently into 30: 95 DEG C of denaturation 30s, 50 DEG C of annealing 30s, 72 DEG C of extension 60s；After circulation terminates, 7min is re-extended for 72 DEG C；4 DEG C of preservations.

The reaction system and condition of KHV PCR is as follows:

The amplimer of KHV PCR is forward primer 09-KHV-TK-F and reverse primer 09-KHV-TK-R, 09-KHV- TK-F is sequence shown in Seq ID No.9, and 09-KHV-TK-R is sequence shown in Seq ID No.10.Pcr amplified fragment is 409bp, annealing temperature are 55 DEG C.

Seq ID No.9:5 '-GGGTTACCTGTACGAG-3 '

Seq ID No.10:5 '-CACCCAGTAGATTATGC-3 '

Reaction system is 100 μ L, comprising: 10 × Mg of 10 × buffer, 10 μ L, 0.2mM²⁺10 μ L, the primer of 0.8mM are each 2.5 μ L, 2 μ L of dNTPs of 10mM, 1 μ L of Taq enzyme, then add water to total volume to 100 μ L.

Reaction condition are as follows: 94 DEG C of 4min are recycled: 94 DEG C of 1min, 55 DEG C of 1min of annealing temperature, 71 DEG C subsequently into 32 1min；72 DEG C of 10min after circulation terminates, last 4 DEG C of heat preservations.

The reaction system and condition of GFHNV PCR is as follows:

GFHNV PCR is respectively adopted two pairs of amplimers and carries out, and first pair of amplimer is sequence shown in Seq ID No.11 The downstream primer JR of sequence shown in the upstream primer JF and Seq ID No.12 of column, pcr amplified fragment are that the DNA of 366bp untwists Enzyme gene segment；Second pair of amplimer is upstream primer GFHNV-CefasF and the Seq ID of sequence shown in Seq ID No.13 The downstream primer GFHNV-CefasR of sequence shown in No.14, pcr amplified fragment are the DNA helicase genetic fragment of 362bp.

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 '

Two pairs of amplimers expand in different PCR pipes respectively, and the reaction system and reaction condition of the two is identical.

By reaction tube as in PCR instrument.94 DEG C of 4min are recycled: 94 DEG C of 1min, 55 DEG C of annealing temperatures subsequently into 32 1min,71℃1min；72 DEG C of 10min after circulation terminates, last 4 DEG C of heat preservations.

The pcr amplification product of GFHNV, KHV and SVCV are detected using 2% agarose gel electrophoresis, as a result as shown in Figure 1. In Fig. 1, swimming lane 1 is DNAmarker DL2000, swimming lane 2-4 be sequentially primer GFHNV-JPF/R to sick fish tissues supernatant, The electrophoresis result of the pcr amplification product of GFHNV virus positive control and negative control, swimming lane 5-7 are sequentially GFHNV-cefasF/ The electrophoresis result of the pcr amplification product of R disease fish tissues supernatant, GFHNV virus positive control and negative control, swimming lane 8-10 is sequentially For the electrophoresis result of the pcr amplification product of KHV primer pair disease fish tissues supernatant, KHV virus positive control and negative control, swimming lane 11-13 is sequentially the electricity of the pcr amplification product of SVCV primer pair disease fish tissues supernatant, SVCV virus positive control and negative control Swimming result.

Fig. 1's the results show that four pairs of primer pair its positive controls have amplification, and all do not expand to negative control, say The PCR amplification system of bright four pairs of primers is normal, and for sick fish tissues supernatant, only the two of GFHNV detection primers, i.e., GFHNV-JPF/R and GFHNV-cefas/R is by amplified fragments, and SVCV and KHV are not expanded, and illustrates in disease fish tissues supernatant Contain GFHNV, i.e. CyHV-2.

The amplified production to Shenzhen Hua Da gene pairs GFHNV-JPF/R is sent to be sequenced pcr amplification product.GFHNV- The sequencing result of JPF/R amplified fragments is sequence shown in Seq ID No.15.

Seq ID No.15:

5’-ggacttgcgaagagtttgatttctacacgcctcgcatcatgcatcaggacaacgcggtcagacaa ctcaacgagtcttgtatgaaaaagactgtgggcgccgaacggatcttcaagcccaagatcaatcacaataacgtgc agaacccggacgagcgtagaaagtttgcagccgtggtccgtcaacggttcaagcacattgacttctttcaaggcgt ccgaatcaaggtcggatctctggtgtgcgtactaaaatatcaaactcaagtgtttgaaggctgtctgggaatagtg gaatcagtacaacccgtcatggtacgcctttttttgtttgtttgtttgtttgatgagacgatggtgactatgg-3’

Sequencing result shows that GFHNV-JPF/R amplification obtains 366 amplified fragments, is consistent with expected results.It will sequencing As a result it is compared with the GFHNV sequence logged in Genbank, as a result, it has been found that the gene sequence of sequencing result and GFHNV type strain Column homology is 99%.It is thus determined that sick fish tissues supernatant contains GFHNV really, i.e., the virus that sick fish is infected is GFHNV.

2. strain is established

The vial supernatant of 3 dilutions such as 1:10,1:100 and 1:1000 is taken, is inoculated into growth respectively with proper volume In fancy carp fin ray cell (Koi fin cell line, KF) cell monolayer about for 24 hours, this test specifically will be in the virus of 50 μ L Clear liquid is inoculated into cell monolayer, after 25 DEG C of absorption 1h, cell culture fluid is added, 20 DEG C are incubated overnight.40- is used daily in 7 days 100 times of inverted microscope inspections after suspicious induced cytopathic effect (abbreviation CPE) to appear, take the KF cell of culture to freeze repeatedly Melt 3 times, after 10 times of work is serially diluted, is inoculated into the KF cell monolayer of growth about for 24 hours and is passed on, while setting normal KF cell Control, and CPE is observed daily.Virus inoculation supernatant is collected by centrifugation and the KF cell of CPE just occurs, makes ultra-thin section, Electric microscopic observation；Meanwhile normal KF cell of the same period is taken, ultra-thin section is also made, Electronic Speculum observation is carried out；As a result such as Fig. 2 It is shown.

In Fig. 2, left figure is the electron-microscope scanning result of the KF cell of CPE occur, wherein circle show KF cell infection disease The CPE generated after poison；Right figure is the electron-microscope scanning result of normal cell.

Tissue supernatant is inoculated with single layer KF cell by experimental result discovery, after blind passage three generations, starts to occur typical and rule Cytopathy, CPE show as cell volume increase be rounded with a small amount of cytoplasmic vacuoles, as shown in Fig. 2, lesion is obvious after 8 days, Cell detachment, and the normal KF cell well-grown of control group.

The 3rd generation DNA for extracting KF cell culture detects the DNA of extraction with identification primer GFHNV-JPF/R, And 2% agarose gel electrophoresis is carried out to pcr amplification product.This test DNA extracting uses Dneasy Blood with kit Tissue kit (Qiagen, Germany) carries out freeze thawing to KF cell in advance before carrying out DNA extraction, takes supernatant after centrifugation Extract nucleic acid.GFHNV-JPF/R amplification refers to " identification of (2) isolated viral " step, pcr amplification product electrophoresis result such as Fig. 3 institute Show.

In Fig. 3, swimming lane 1 is DNAmarker DL2000, and swimming lane 2 is the pcr amplification product of GFHNV virus positive control Electrophoresis result, swimming lane 3 are the electrophoresis result of the pcr amplification product of normal KF cell negative control, and swimming lane 4 is the KF of virus infection The electrophoresis result of the pcr amplification product of the 3rd generation DNA of cell culture.Fig. 3's the results show that extract the 3rd of KF cell culture For DNA, gone out and the consistent 362bp target fragment of expection with identification primer GFHNV-JPF/R Successful amplification.

3. attacking malicious condition test

Sick fish is acquired, the histoorgans such as its gill are taken, grinding, centrifugation take supernatant, with 0.22 μm of membrane filtration degerming, with The dosage of every tail 1mL is inoculated in experiment fish body by intraperitoneal injection, and control group injects 1mL DMEM culture medium.This test setting Two infection experiment groups and a control group, every group of 10 tail health crucian.

Observation daily, the results show that the infection crucian carp of test group I, II is dead in appearances on the the 3rd and the 4th respectively, infection it is dead Dying peak period is all death after the 7th, 8, the 10th, and body surface hyperemia abdomen swelling characteristics are identical with natural occurrence crucian carp, dissection After find, the gill filament whitens, and the obvious enlargement of liver and spleen kidney meets the symptom of crucian carp fishy fulminant hemorrhage.And inject DMEM culture medium Control group is without significant change.

The tissue such as sterile illness crucian carp brain, spleen, kidney for taking test group death, is added sea sand, pours into mortar and be fully ground, and makes Standby to be suspended in cell culture fluid at homogenate in the ratio of 1:10, after 5 DEG C are incubated overnight, 14000r/min is centrifuged 15min, takes Supernatant carries out PCR amplification detection to it using primer GFHNV-JPF/R.At the same time, as control, the crucian carp of a tail control group is taken The tissue such as brain, spleen, kidney of fish, is prepared using same method and obtains supernatant, as negative control.The portion of pcr amplification product Divide gel electrophoresis result as shown in Figure 4.

In Fig. 4, swimming lane 1 is DNAmarkerDL2000, and swimming lane 2 is the electrophoresis result of the pcr amplification product of negative control, Swimming lane 3 is the electrophoresis result of the pcr amplification product of GFHNV virus positive control, and swimming lane 4 is back the pcr amplification product of susceptible crucian carp Electrophoresis result.PCR amplification is the results show that infection fish test group I, II amplifies the segment of 362bp, with expected size phase Symbol；GFHNV in this crucian carp that illustrates to fall ill can be with artificial challenge's health hybridized prussian carp, and control group is without target fragment, with expected phase Symbol.

Two, crucian carp fulminant bleeding virus specific immunity enhancing polypeptide screening

1. phage display library

Display technique of bacteriophage (phage display techniques, PDT) is the base by exogenous proteins or polypeptide Because expression product is merged with bacteriophage coat protein, and in phage display, while its genetic code information being integrated into In the genome of bacteriophage.

Phage antibody technology (phage display antibody library techniques), which refers to, utilizes PCR Technology expands body full set variable region gene, by display technique of bacteriophage Fab or single-chain antibody (scFv) and coding bacteriophage The gene of coat protein is connected, and by infecting host, the process of fusion protein is expressed as in phage surface, by target The specific binding screening process of molecule such as albumen, glycoprotein, virus and small-molecule substance etc., thus the mesh that enrichment is directed to Mark the phage displaying antibody of molecule.Antibody library directly utilizes antigen to obtain specific antibody from antibody library, indicates Antibody preparation enters a new era.

Foreign protein in phasmid display systems is shown on the surface of filobactivirus, but constructs its display libraries when institute Carrier is phasmid, rather than complete filamentous phage gene group.Phasmid (phagemid) be bacteriophage (phage) and The mixture of plasmid (plasmid), the advantages of possessing bacteriophage and plasmid.Phasmid is (double with M13 (single-stranded) and plasmid Chain) replication origin, can the same quick copy of image quality grain, can also be assembled with the help of helper phage such as M13KO7 Reassemble into recombinant phage particle.Phasmid contains acillin (Amp) resistant gene, and M13K07 contains kanamycins (Kan) resistant gene enables to pick out in the never infected cell of infected cell.

The phage antibody library of this test, e. coli tg1, helper phage M13KO7 are by Chinese Academy of Sciences aquatile Research institute wears peaceful researcher and provides.From document: Dai HP, Gao H, Zhao XY, Dai LF, Zhang XK, Xiao N, Zhao RH,Hemmingsen SM.Construction and characterization of a novel recombinant single-chain variable fragment antibody against White Spot Syndrome Virus from shrimp.Journal of Immunological Methods,2003,279(1-2): 267-275。

2. the specific polypeptide of anti-crucian carp fulminant bleeding virus screens

(1) phage display library screens

Three-wheel screening is carried out with CyHV-2 totivirus of the phage display library to purifying: being purified with the diluted 50 μ g of PBS The immune pipe of CyHV-2 coating, 4 DEG C are overnight；Next day, the coating buffer fallen in immune pipe of inclining are washed with PBS, remove unbonded egg It is white, then immune pipe is closed with 10%PBSM solution, in 37 DEG C of incubation 1H；It is washed with PBS, the phage display library that will be prepared It is added in immune pipe with 10%PBSM by 1:1,37 DEG C of incubation 2h；Respectively with the washing of PBST solution, it is added the 100mM's of 1mL Triethylamine acts on the 1M Tris-HCl (pH=7.5) of addition 0.5mL after 10min, the e. coli tg1 of 5mL is then added, O.D.600=0.3-0.5,37 DEG C of incubation 1h；The bacterium solution of infection is coated with SOBAG plate, 30 DEG C of inversion overnight incubations；Next day, The bacterium colony on plate is scraped with culture medium, and saves and carries out next round screening at bacteriophage.

The specific method is as follows for the phage library amplification and rescue of this test:

1) phage library frozen, about 1.5mL are taken from -70 DEG C, 37 DEG C of water-baths are thawed；

2) defrosting bacterium solution is added to 2 × YT-AG culture medium dilution of 13mL, mixes；

3) it takes 1.5mL to be coated on SOBAG massive plate, is coated with 10 altogether, 30 DEG C of overnight incubations after being coated with uniformly, about 16h；

4) bacterium on plate is scraped with 2 × YT-AG culture medium, then packing and cryopreservation tube, -70 DEG C save backup.

Anti- GFHNV single chain antibody phage library is stored in host strain in the form of phasmid, will before elutriation It saves the library at bacteriophage form.The specific method is as follows:

5) the second level phage library frozen, about 1.5mL are taken from -70 DEG C, 37 DEG C of water-baths are thawed, are then added to In 2 × YT-AG culture medium of 600mL, make OD₆₀₀For 0.3-0.4；

6) 37 DEG C, 200r/min shaking table culture to OD₆₀₀=0.5-0.8；

7) in Escherichia coli: helper phage, 37 DEG C of shaking tables are added in helperphage (M13K07)=1:5 ratio After culture 1 hour；

8) with 4000r/min, 4 DEG C of centrifugation 15min, supernatant is removed, precipitating is resuspended in the 2YT-AK of 200mL, 37 DEG C of shaking table trainings Support 2.5h；

9) culture precipitates the bacteriophage in supernatant with 10000g, 4 DEG C of centrifugation 20min with the PEG/NaCl of 1/5 volume, Ice bath 1 hour；

10) with 10000g, 4 DEG C of centrifugation 20min, the bacteriophage of precipitating is resuspended in the 2YT of 5mL, spare.

This test has carried out three-wheel screening to phage library, and the selectivity for having counted the single-chain antibody of every wheel screening is rich Collection, the results are shown in Table 1.

Table 1 respectively takes turns the selective enrichment of single-chain antibody in panning process

Bacteriophage number	The first round	Second wheel	Third round
				Input amount (CFU)	7.6×10¹⁰	7.7×10¹⁰	7.5×10¹⁰
Elution amount (CFU)	1.3×10⁴	4.2×10⁵	2.8×10⁵
				Output capacity (%)	1.7×10^-7	5.5×10^-6	3.7×10^-6

In table 1,4 wheel elutriations are carried out to phage antibody library using CyHV-2 as antigen, are counted with the ratio of input and output Calculate output capacity, i.e. output capacity (%)=(elution amount ÷ input amount) × 100%；

The calculation of input amount is the TG1 for taking 10 phage libraries of the μ L without elutriation to be in logarithmic growth phase in 90 μ L In bacterium solution, 10 times are prepared with 2 × YT and is serially diluted bacterial suspension, is coated with BOBAG plate, counts single colonie number.The calculating of quantum of output Mode is after taking 10 μ L elutriations and to have infected the bacteriophage of TG1 and be added to 2 × YT of 90 μ L, carries out 10 times and is serially diluted, is coated with BOBAG plate counts single colonie number.Then elutriation efficiency is calculated.

Table 1 the results show that third round screening output capacity be apparently higher than the first round, this illustrates the clone of higher affinity Effectively it is enriched with, and third round and the second wheel incalculability grade difference, illustrate that three-wheel screening is enough to ensure that and screens high-affinity Antibody.

(2) positive clone identification

Random picking single bacterium is fallen in 96 hole Bacteria Culture plates from the plate that third round is screened, the specific picking of this test 2 plates, the reserved positive and negative control hole of each culture plate, are added benzyl containing ammonia and culture medium containing helper phage are incubated overnight； Culture medium, 30 DEG C of culture 3h is added from the Bacteria Culture plate for drawing 30 μ L bacterium solution Yu Yixin in every hole in next day, and centrifugation is gone After clear, bacterium precipitates the culture medium for being resuspended in benzyl containing ammonia and kanamycins, and 30 DEG C are incubated overnight；Next day will be contained after centrifugation The supernatant of antibody is stored in 4 DEG C of spare, i.e. antibody supernatants.

The CyHV-2 of purifying is added in elisa plate with 0.5 hole μ g/, coating 2h is carried out in room temperature, after coating buffer of inclining It is washed with PBS, 4%PBSM carries out staying overnight closing；Next day is washed with PBS after the deblocking liquid that inclines, and 100 μ L are added with 4% in every hole The antibody supernatant that PBSM solution has been diluted with 1:1 ratio, in 30 DEG C of incubation 1h；After the washing of PBST solution, every hole is added 100 μ L press 30 DEG C of incubation 1h of secondary antibody of 1:5000 dilution with 4%PBSM solution, and the secondary antibody of this test is HRP/Anti- M13Monoclonal antibody；After the washing of PBST solution, the tmb substrate solution that 100 μ L are added in every hole develops the color, most It is terminated and is reacted with 2M sulfuric acid afterwards；Using the value of microplate reader measurement excitation wavelength 450nm, when the value of experiment sample is divided by blank control Value be greater than 2.1 when, be considered as positive colony.

This test picking simultaneously has detected 192 monoclonals, the results show that having 33 in 192 monoclonals after elisa assay ELISA value is the positive, positive clone rate 17.2%.

33 positive colonies filtered out are further identified, 7 dilutions are arranged in antigen, respectively with 0, 50,100,200,300,400, the hole 500ng/ coating elisa plate is identified.Test result shows in 33 positive colonies, have 13 clones have concentration dependant to antigen coat measurer.

After this 13 plants of positive colony amplification cultivations, sequencing is carried out, and sequence analysis is carried out to sequencing result.Specifically It is as follows:

1) in the Bacteria Culture plate for taking 30 μ L positive colony bacterium solution Yu Yixin, and 2YT-AG culture medium is supplied to 200 μ L/ Hole, 30 DEG C of culture 3h；Wherein, helperphage is added in 2YT-AG culture medium, the M13k07 of 100 μ L is added in 100mL culture medium.

2) 15min is centrifuged with 3500rpm, removes supernatant, bacterium precipitating is resuspended in the 2YT-AK of 200 μ L, 30 DEG C of 150rpm trainings It supports overnight；Wherein 2YT-AK includes 2YT, 100 μ g/mLAmp and 50 μ g/mL Kna.

3) next day, by Bacteria Culture plate with 4 DEG C of centrifugation 15min of 3500rpm, 4 DEG C of supernatant are saved backup.

PCR amplification is carried out to positive colony using primer sets.The primer sets upstream primer of this example is Seq ID No.16 institute Show that sequence, downstream primer are sequence shown in Seq ID No.17.

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

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

PCR reaction system is 100 μ L, including 10 × buffer, 10 μ L, 10 × Mg²⁺The primer of (0.2mM) 10 μ L, 0.8mM Each 2.5 μ L, 2 μ L of dNTPs of 10mM, 1 μ L of Taq enzyme, are then added the supernatant containing DNA, and moisturizing is to 100 μ L of total volume.

By reaction tube as PCR reaction is carried out in PCR instrument, reaction condition is set are as follows: 94 DEG C of 5min are followed subsequently into 35 Ring: 94 DEG C of 1min, 56 DEG C of annealing 1min, 72 DEG C of 1min, after circulation terminates, and 72 DEG C of 10min, last 4 DEG C of heat preservations.PCR amplification produces Object send Shenzhen Hua Da gene to be sequenced.

Sequencing result shows to have 3 plants as non-monoclonal, remaining 10 plants are monoclonal, there is 3 plants of CDR3 in 10 strain clones Area is identical, remaining 7 plants are all different.

Three, crucian carp fulminant bleeding virus specific immunity enhancing polypeptide nature analysis

1. polypeptide and CyHV-2 affinity analysis

From 10 plants of monoclonals that screening obtains, optional one plant of the identical monoclonal in 3 plants of areas CDR3 is tested, in addition its Remaining 7 plants, amounts to after carrying out IPTG inducing expressions using 8 plants of positive colonies, pass through prokaryotic expression system, i.e. Bacillus coli expression system System, obtaining crucian carp fulminant bleeding virus specific immunity enhances polypeptide.Eight crucian carp fulminant bleeding virus specific immunity enhancings In polypeptide, the first polypeptide is sequence shown in Seq ID No.1, and the second polypeptide is sequence shown in Seq ID No.2, and third polypeptide is Sequence shown in Seq ID No.3, the 4th polypeptide are sequence shown in Seq ID No.4, and the 5th polypeptide is sequence shown in Seq ID No.5 Column, the 6th polypeptide are sequence shown in Seq ID No.6, and the 7th polypeptide is sequence shown in Seq ID No.7, and the 8th polypeptide is Seq Sequence shown in ID No.8；

SeqIDNo.1:VVYYYAMDSSGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSM YASL

SeqIDNo.2:GGYDWMAVVWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSM YASL

SeqIDNo.3:SSLLYAMGYWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSM YASL

SeqIDNo.4:QYSWYTTVWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMY ASL

SeqIDNo.5:GMFTYAADSWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSM YASL

SeqIDNo.6:QWGWSLDGGGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMY ASL

SeqIDNo.7:MAGGLTAYWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMY ASL

SeqIDNo.8:QSQQGGAYWGQGTSVTVSTGGGGSGGGGSGGGGSDVVMAQSPSSMY ASL.

Wherein, IPTG inducing expression method particularly includes: bacterium solution will be incubated overnight in the ratio of 1:20 be inoculated into LB liquid In culture medium, 37 DEG C of shaken cultivations.As bacterium solution OD_600nmThe IPTG of final concentration of 1.0mmol/L is added when value is up to 0.6, induces 6h, Bacterium solution is collected every 1h, is saved backup, is analyzed for SDS-PAGE, observes the variation of expression quantity.Meanwhile by the bacterium solution after induction Ultrasonic disruption is carried out, 4 DEG C, 5000r/min is centrifuged after 5min supernatant precipitating progress SDS-PAGE electrophoresis, analysis expression egg White solubility.

Then specificity analysis is combined to this 8 polypeptides, and cell fragment is set as negative control and PBS blank pair According to.Wherein, cell fragment refers to Bacillus coli cells fragment.

This example is combined signature analysis using dot hybridization experiment, specifically includes:

The analysis of the binding characteristic of soluble single-chain antibody and CyHV-2

1) take the PBS, cell fragment and CyHV-2 point sample of 3 μ L on NC film；

2) to after natural drying, close 1h in room temperature with 3% BSA；

3) PBS is washed 3 times, film is put into the diluted single-chain antibody scFv solution of 1:5, room temperature 1h；Wherein dilution uses 3% BSA is carried out；

4) PBST, PBS are respectively washed 3 times, and then film is put into HRP/Anti E-Tag solution, react at room temperature 1h；HRP/ Anti E-Tag solution is diluted with 3%BSA by 1:5000；

5) PBST, PBS are respectively washed 3 times, then with the colour developing of DAB substrate solution.

The binding characteristic of soluble single-chain antibody and denaturation CyHV-2 are analyzed

1) 12% SDS-PAGE is carried out to CyHV-2 and cell fragment；

2) by the albumen electrotransfer to pvdf membrane of separator well, 100mA, 1.5h；

3) 1h is closed in room temperature with 3%BSA；

4) PBS is washed 3 times, and then pvdf membrane is put into the diluted single-chain antibody solution of 1:5, reacts at room temperature 1h；

5) PBST, PBS are respectively washed 3 times, and then pvdf membrane is put into HRP/Anti E-Tag solution, react at room temperature 1h； HRP/Anti E-Tag solution is diluted with 3%BSA by 1:5000；

6) PBST, PBS are respectively washed 3 times, then with the colour developing of DAB substrate solution.

Dot hybridization the results show that 8 polypeptides can react with CyHV-2, and negative control and blank control are equal Immaculate.

SDS-PAGE electrophoresis is carried out to cell fragment and CyHV-2 and then goes to pvdf membrane, respectively using 8 polypeptides as primary antibody, HRP/Anti E-TagAntibody is that secondary antibody carries out westernblot detection.

The glycoprotein of inducing expression is analyzed into its immunogenicity through Westernblot, the specific method is as follows:

(1) the 15 μ L loading of sample handled well is taken, SDS-PAG electrophoresis is carried out；

(2) after the completion of rear electrophoresis, adhesive tape is placed in transfer buffer and balances 10min；Two are cut out according to adhesive tape size Filter paper and a pvdf membrane, 100% methyl alcohol process of obtained pvdf membrane, and respectively by filter paper, treated pvdf membrane, foam It is put into transfer buffer and impregnates 20min；

(3) successively suitable according to cathode-foam-filter paper (two layers)-adhesive tape-pvdf membrane-filter paper (two layers)-foam-anode Sequence is put into clamping plate slot, and electrophoretic blotting liquid, 35V, 3h is added；

(4) after the completion of transferring film, the front and back sides of label film, PBST is rinsed three times, each 10min, and 2% confining liquid, envelope is added Close 10h；

(5) washing is same as above, and rabbit-anti IHNV totivirus serum is added as primary antibody, 1:20000 dilution, 37 DEG C of effect 1h；

(6) washing is same as above, and using the sheep anti-mouse igg of HRP label as secondary antibody, 1:2000 dilutes, and 37 DEG C of effect 1h are finally used DAB colour reagent box develops the color, photographic analysis.

Westernblot testing result shows that only the 8th polypeptide can identify the CyHV-2 of denaturation, indicates that the 8th polypeptide is known Other is linear epitope, and other 7 polypeptide identifications is comformational epitope.

2. polypeptid specificity test and affinity constant

This test uses the non-competing elisa assay method of solid phase, to the first polypeptide to the 8th polypeptide, the combination of this 8 polypeptides Specificity is detected.The material to be tested of specific detection includes CyHV-2, PFRV (pike juvenile rhabdovirus), STIV (first Fish rainbow virus), VNNV (fish virus nerve necrotic virus), VHSV (viral hemorrhagic septicemia, VHS virus) and IHNV (biography Metachromia Hematopoietic Necrosis's disease virus), these materials to be tested are by Shenzhen Entry-Exit Inspection and Quarantine Bureau animals and plants inspection and quarantine skill Art center provides and saves.

The non-competing elisa assay method of solid phase specifically includes, using 96 orifice plates measure the affinity costant (Kaff) of each polypeptide= (n-1)/2 (n [Ab2]-[Ab1]), when [Ab1] and [Ab2] respectively represents the 50% of different antigen concentration maximum absorbances in formula Corresponding antibody concentration, n are the multiple of two antigen diluents.3 envelope antigen concentration gradients and 10 μ are arranged in this test G/mL, 5 μ g/mL and 2.5 μ g/mL are serially diluted each polypeptide as primary antibody with 3%BSA after closing, carry out ELISA test, test As a result as shown in Figure 5.Wherein, method of the ELISA test with reference to (1987) such as Beatty, Beatty JD, Beatty BG, Vlahos WG.Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay.Journal of Immunological Methods,1987,100(1-2):173-179。

In Fig. 5, abscissa each group is sequentially the first polypeptide to the 8th polypeptide, i.e. it is the first polypeptide that No. 1 polypeptide, which corresponds to primary antibody, Test group as a result, No. 2 polypeptides correspond to primary antibody be the second polypeptide test group as a result, and so on；Ordinate is extinction Degree；It is from left to right sequentially CyHV-2, PFRV, STIV, VNNV, VHSV, IHNV, negative control in each test group of abscissa The testing result of PBS and negative control cell fragment.

Fig. 5's the results show that eight kinds of polypeptides can specificity combination CyHV-2, without intersecting with other viruses Reaction.The affinity constant of first polypeptide to the 8th polypeptide is sequentially respectively 7.16 ± 1.25 × 10⁷M^-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^-1With 1.15 ± 0.69 × 10⁵M^-1。

3. polypeptide and CyHV-2 indirect immunofluorescence are tested

KF cell is passed in six orifice plates, prepares inoculation CyHV-2 when cell growing way is preferable；It connects after poison about for 24 hours, is sucked out Cell culture fluid washs plate hole with PBS, with the fixer of pre-cooling in 4 DEG C of fixed 10min；After washing away fixer with PBS, it is added The diluted each polypeptide of 1:5, in 37 DEG C of incubation 1h；After washing away polypeptide dilution with PBS, the diluted rabbit anti-mouse igg-of 1:200 is added FITC labeling polypeptide, in 37 DEG C of incubation 1h；After PBS board-washing, with 50% glycerol-PBS mounting microscopy, in fluorescence inverted microscope Lower observation result.Meanwhile setting does not connect the normal KF cell of poison as negative control, negative control is not in addition to having inoculation CyHV-2 In addition, other processing modes and condition are all identical as the inoculation KF cell of CyHV-2.The observation result of fluorescence microscope (400 ×) It has been shown that, the first polypeptide to the 8th polypeptide, this 8 kinds of polypeptides can generate specificity fluorescent dye to the KF cell of infection CyHV-2 virus Color, and be then presented negative reaction being uninfected by area, and 8 kinds of single-chain antibodies and the KF cell that is not inoculated with CyHV-2 are reactionless.

Four, crucian carp fulminant bleeding virus specific immunity enhances polypeptide live test

1. toxotest

It is to fish body to confirm that this 8 kinds of CyHV-2 specific immunities of the first polypeptide to the 8th polypeptide of this test enhance polypeptide No have tight security and without adverse side effect, this test stop to bait for 24 hours after, by two groups of crucians respectively with effective concentration 10 times and the polypeptides of 20 multiple doses be injected intraperitoneally, at the same time, the control group of an intraperitoneal injection equivalent PBS is set, Every group of 25 crucians, 8 kinds of polypeptides are tested respectively；And observe 28 days day by day, confirmation has no adverse reaction and survival rate, to make For the standard for assessing 8 kinds of polypeptide safeties.

The results show that after being injected intraperitoneally with the polypeptide of 10 times and 20 multiple doses, depositing for 28 days all test groups is observed The case where motility rate is all 100%, does not have any adverse reaction, control group is identical, illustrates the first polypeptide of this test to Eight polypeptides are to the highly-safe of fish body.

3. polypeptide protection is tested

(1) feeding test

The feed that feeds during the polypeptide preparation test of 0.5 μ g is added in every gram of feed in proportion, in control group It is added without any polypeptide, this test devises 9 kinds altogether and feeds feed, i.e., adds the feed of the first polypeptide, the second polypeptide respectively Feed ... the feed of the 8th polypeptide, and the conventional feed of any polypeptide is not added.Therefore, the test of this test is divided into 9 groups, Every group includes 55 crucians, and body long 20 ± 1cm, 150 ± 10g of weight feed control group feed respectively and accordingly add polypeptide Feed, three meals in a day, every meal 50g feed are continuously fed 14 days, each random in every group at the 0th day, the 7th day and the 14th day respectively Peptide concentration in 5 crucian measurement serum is chosen, remaining 40 carry out challenge test after 14 days.

Serum polypeptide concentration determination:

The E-tag of purifying is added in elisa plate with 0.5 hole μ g/, coating 2h is carried out in room temperature, after coating buffer of inclining It is washed with PBS, 4%PBSM solution carries out staying overnight closing；Next day is washed with PBS after the deblocking liquid that inclines, every hole be added 150 μ L with 4%PBSM carries out twice of diluted crucian serum (1:100-1:12800), in 30 DEG C of incubation 1h；After the washing of PBST solution, often Hole be added 150 μ L with 4%PBSM by 1:5000 dilution rabbit-anti crucian IgM antibody as secondary antibody, in 30 DEG C of incubation 1h；With PBST After solution washing, every hole is added 150 μ L and is resisted by the HRP/ goat anti-rabbit igg antibody of 1:5000 dilution as three with 4%PBSM, in 30 DEG C be incubated for 1h；After the washing of PBST solution, the tmb substrate solution that 100 μ L are added in every hole develops the color, finally with the termination of 2M sulfuric acid Reaction.Then using the value of microplate reader measurement excitation wavelength 450nm, when the experiment sample value of dilution ratio is divided by blank value When greater than 3 times, the dilution ratio is regarded as serum concentration.Serum polypeptide concentration test result is as shown in Figure 6.

In Fig. 6, abscissa is the serum polypeptide concentration of measurement in the 0th day, the 7th day and the 14th day, and ordinate is serum polypeptide Concentration value；In 0th day, the 7th day and the 14th day three groups of data, each group is sequentially to feed the control that any polypeptide is not added from left to right The crucian serum polypeptide concentration of group feeds the crucian serum polypeptide concentration of the first polypeptide feed, feeds the crucian carp of the second polypeptide feed Fish serum peptide concentration, the crucian serum polypeptide concentration for feeding third polypeptide feed, the crucian serum for feeding the 4th polypeptide feed Peptide concentration, the crucian serum polypeptide concentration for feeding the 5th polypeptide feed, the crucian serum polypeptide for feeding the 6th polypeptide feed are dense It spends, feed the crucian serum polypeptide concentration of the 7th polypeptide feed, feed the crucian serum polypeptide concentration of the 8th polypeptide feed.

Fig. 6's the results show that it is continuous feed 7 days after, the peptide concentration in serum can be up to 1600-3200 times, and 14 It more can reach 6400-12800 times after it, it was demonstrated that by feeding the content that can effectively improve each polypeptide in fish.

Challenge test:

The virus liquid of purifying is taken, with 0.22 μm of membrane filtration degerming, intraperitoneal injection inoculation is passed through with the dosage of every tail 1mL In in experiment fish body, control group injects the DMEM culture medium of 1mL, control group and each 20 fishes of test group, and 14 days fishes are observed continuously Survival rate.

Test result shows, control group after attacking poison 12 days it is total dead, and have using the group of polypeptide in off-test When still at least 20% survival rate feed the survival rate of the 5th polypeptide wherein the survival rate for feeding the second polypeptide is up to 75% Up to 70%, the survival rate for feeding the 7th polypeptide is also up to 75%.Also, feed the group of the second polypeptide, the survival at the 9th day Rate is up to 90%, and occurring within several days fish successively later, only death, survival rate maintain 75%；The 7th other situation of polypeptide group and second Polypeptide group is similar；There is not fish only death after the 11st day in the group for feeding the 5th polypeptide, and survival rate is stablized.

(2) soak test

Soak used in the polypeptide preparation test in the process of 100mg, control group is added in every liter of water body in proportion For common fresh water.Test is divided into 9 groups, and every group includes 55 crucians, body long 20 ± 1cm, 150 ± 10g of weight, respectively at control group And carry out 1h immersion in the soak of corresponding addition polypeptide, once a day, be carried out continuously 14 days, respectively at the 0th day, the 7th day and Peptide concentration in 5 bars of measurement serum is randomly selected in every group within 14th day, remaining 40 carry out challenge test after 14 days.Its In, the acquisition methods of soak are that the fermentation liquid after expressing polypeptid induction carries out cell by homogenizer with 1000bar pressure It is broken, the fermentation liquid of broken wall is obtained into supernatant after 4 DEG C are centrifuged 30 minutes with 12000g, that is, is obtained containing soluble more The soak of peptide, also, peptide concentration is 3 μM -30 μM in soak.

Serum polypeptide concentration determination: test method is with " (1) feeding test ", and test results are shown in figure 7.In Fig. 7, horizontal seat It is designated as the serum polypeptide concentration of measurement in the 0th day, the 7th day and the 14th day, ordinate is serum polypeptide concentration value；0th day, the 7th day And in the 14th day three groups of data, each group be sequentially from left to right feed the control group that any polypeptide is not added crucian serum polypeptide it is dense It spends, feed the crucian serum polypeptide concentration of the first polypeptide feed, the crucian serum polypeptide concentration for feeding the second polypeptide feed, feed The crucian serum polypeptide concentration of third polypeptide feed, is fed more than the 5th the crucian serum polypeptide concentration for feeding the 4th polypeptide feed The crucian serum polypeptide concentration of peptide feed, feeds the 7th polypeptide feed at the crucian serum polypeptide concentration for feeding the 6th polypeptide feed Crucian serum polypeptide concentration, feed the crucian serum polypeptide concentration of the 8th polypeptide feed.

Fig. 7's the results show that it is continuous feed 7 days after, the peptide concentration in serum can be up to 800-3200 times, and 14 days It more can reach 6400-12800 times afterwards, it was demonstrated that it is consistent with mode is fed by impregnating, it can effectively improve each polypeptide in fish Content.

Challenge test: test method is the same as " (1) feeding test ".Test result shows, control group after attacking poison 11 days it is total Death, and have using the group of polypeptide in off-test still at least 15% survival rate, wherein feeding depositing for the second polypeptide Motility rate is up to 75%, feeds the survival rate of the 5th polypeptide up to 65%, feeds the survival rate of the 7th polypeptide also up to 70%, survival rate is imitated Fruit is the most significant.

The above test explanation, feeds or impregnates any polypeptide of the first polypeptide of this test into the 8th polypeptide, can Enhance fish body to the immunity of CyHV-2, to improve its survival rate；Especially the second polypeptide, the 5th polypeptide and the 7th polypeptide, Survival rate effect is the most significant.Although the application is only tested with carp, however, it will be understood that the application's is special Property immunopotentiating polypeptides for CyHV-2, therefore all CyHV-2 infection or the disease as caused by its infection can adopt Carp and its crucian are included but are not limited to the immunopotentiating polypeptides of the application to enhance fish body to the immunity of CyHV-2 Fulminant hemorrhage, goldfish and its goldfish Hematopoietic Necrosis's disease etc..

The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen Specific implementation please is only limited to these instructions.For those of ordinary skill in the art to which this application belongs, it is not taking off Under the premise of from the application design, a number of simple deductions or replacements can also be made.

Sequence table

<110>Gonglin Industry (Shenzhen) Co., Ltd.

Animal &. Plant Inspection and Quarantine Techn Center, Shenzhen Bureau of Impor

<120>a kind of reagent and application for preventing or treating infection of marine fishes CyHV-2

<130> 18I26892

<160> 17

<170> SIPOSequenceListing 1.0

<210> 1

<211> 50

<212> PRT

<213>crucian carp fulminant bleeding 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 fragments ()

<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. a kind of for preventing or treating the reagent of infection of marine fishes CyHV-2, it is characterised in that: the reagent includes the first polypeptide At least one of to the 8th polypeptide；

First polypeptide is sequence shown in Seq ID No.1, and the second polypeptide is sequence shown in Seq ID No.2, and third polypeptide is Seq Sequence shown in ID No.3, the 4th polypeptide are sequence shown in Seq ID No.4, and the 5th polypeptide is sequence shown in Seq ID No.5, 6th polypeptide is sequence shown in Seq ID No.6, and the 7th polypeptide is sequence shown in Seq ID No.7, and the 8th polypeptide is Seq ID Sequence shown in 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.

2. reagent according to claim 1, it is characterised in that: the reagent includes the second polypeptide, the 5th polypeptide and the 7th At least one of polypeptide.

3. application of the reagent according to claim 1 or 2 in CyHV-2 is detected or identified.

4. application of the reagent according to claim 1 or 2 in the kit or device for preparing CyHV-2 detection or identification.

5. a kind of for preventing or treating the feed of the disease of infection of marine fishes CyHV-2, it is characterised in that: contain in the feed Reagent of any of claims 1 or 2.

6. feed according to claim 5, it is characterised in that: dosage of the reagent in feed is, in every gram of feed Contain reagent described at least 0.5 μ g.

7. feed according to claim 5 or 6, it is characterised in that: the disease of the infection of marine fishes CyHV-2 includes goldfish Hematopoietic Necrosis's disease and crucian carp fishy fulminant hemorrhage.

8. a kind of for preventing or treating the soak of the disease of infection of marine fishes CyHV-2, it is characterised in that: the soak packet Containing at least one of first polypeptide to the 8th polypeptide.

9. soak according to claim 8, it is characterised in that: the disease of the infection of marine fishes CyHV-2 includes that goldfish is made Blood organ necrosis disease and crucian carp fishy fulminant hemorrhage.