CN102988973A

CN102988973A - Nucleic acid vaccine for kio herpesvirus

Info

Publication number: CN102988973A
Application number: CN2012104188082A
Authority: CN
Inventors: 周井祥; 王好; 李新伟; 张东鸣; 朱霞; 祖岫杰
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2012-10-29
Filing date: 2012-10-29
Publication date: 2013-03-27

Abstract

The invention discloses a nucleic acid vaccine for kio hepesvirus. The nucleic acid vaccine has high immunogenicity. The nucleic acid vaccine for the kio hepesvirus is a recombinant plasmid which is obtained by cloning an ORF81 gene into a eucaryon expression vector pIRES-neo. The nucleic acid vaccine for the kio hepesvirus has an effective preventative effect on a kio herpesvirus disease.

Description

A kind of Koi herpesvirus nucleic acid vaccine

Technical field

The present invention relates to a kind of Koi herpesvirus nucleic acid vaccine, belong to biological technical field.

Background technology

Sick (the Kio herpesvirus disease of Koi herpesvirus, KHVD) be a kind of disease with hyperinfection and lethal that is caused the morbidities such as Cyprinus carpio L., Cyprinus carpio and common mutation thereof such as frame mirror Cyprinus carpio by Koi herpesvirus (Kio herpesvirus, KHV).

First in the Magan of Israel Michael area and U.S.'s outburst, this disease reached Britain, Germany and Belgian to this disease in 2000 in 1998; In April, 2002, primary disease occurs in Indonesia, and Chinese Guangdong is economized the doubtful generation primary disease of Cyprinus carpio L. simultaneously; The same year, December China Taiwan Province confirmed that Cyprinus carpio L. infects primary disease; In October, 2003, Japan confirmed that this disease exists home.At present, this disease is fallen ill more in the carp culture of China, causes the mortality of Cyprinus carpio, has already brought strike as almost destroying for carp culture.Although there is multiple traditional vaccine to can be used for the immunoprophylaxis of disease; but in production practices and our experimental study all show; utilize inactivated vaccine to come immunoprophylaxis Koi herpesvirus disease can not play complete immanoprotection action; simultaneously there is not yet the report of isolating or work out KHV low virulent strain virus in China, therefore demand this viral new generation vaccine urgently and come out.

Nucleic acid vaccine (Nucleic acid vaccine) claim again gene vaccine, dna vaccination; it be utilize the DNA recombinant technique with the protective antigen protein gene cloning to carrier for expression of eukaryon; and directly import it in body; make antigen protein endogenous expression submission to immune system, bring out body and produce specific humoral immunization and cell immune response.The main envelope protein of ORF81 gene expression is the important component part of KHV cyst membrane, can consist of viral major antigen determinant; Its function with induce virus and cell fusion, virus to the absorption of cell and penetrate and go out to be placed with the pass at nuclear membrane.This patent is take the ORF81 gene as genes of interest, and pIRES-neo is carrier for expression of eukaryon, has made up the pIRES-ORF81 Eukaryotic expression recombinant plasmid, and studies its immune efficacy, develops efficient, novel KHV nucleic acid vaccine.

Summary of the invention

The purpose of this invention is to provide a kind of good immunogenic Koi herpesvirus nucleic acid vaccine that has.

Koi herpesvirus nucleic acid vaccine provided by the present invention is that the ORF81 gene clone is obtained the pIRES-ORF81 recombiant plasmid in eukaryotic expression vector pIRES-neo.

Wherein, the nucleotide sequence of described ORF81 gene is shown in sequence table SEQ ID No.1; Its corresponding aminoacid sequence is shown in sequence table SEQ ID No.2.

Utilization of the present invention will be encoded the ORF81 gene directed cloning of Koi herpesvirus in eukaryotic expression vector pIRES-neo, be transformed into the bacillus coli DH 5 alpha competent cell, extract plasmid DNA and confirm to contain correct ORF81 gene through restriction endonuclease identification.With SDS alkaline lysis large quantity extracting plasmid, by Luo Shi FuGENE HD transfection reagent recombiant plasmid is imported in the frame mirror Cyprinus carpio caudal fin cell (MFC), collect the MFC cell of transfection after 48 hours, with the protein expression situation of Western-blot method check purpose gene, prove that constructed eukaryotic expression recombination plasmid success has obtained expression in the MFC cell.Recombinant expression plasmid carries out the healthy Cyprinus carpio of intramuscular injection, and immunity in per 14 days once is total to immunity 3 times, and weekly heart blood sampling utilizes the KHV specific antibody in the ELISA method detection Serum of Common Carp.Can detect the specific antibody of KHV in the Cyprinus carpio blood with the immunity of ORF81 recombiant plasmid, antibody horizontal increases with the increase of immune time behind the booster immunization.Adopt fixed virus dilute serum method, measure after the immunity of ORF81 recombiant plasmid in each test group Serum of Common Carp anti-KHV neutralization and tire, ORF81 recombiant plasmid immune group Serum of Common Carp has all produced obvious neutralizing antibody.Two weeks used 100TCID after the immunity for the third time ₅₀KHV to each group Cyprinus carpio thoracic cavity counteracting toxic substances, the Cyprinus carpio protective rate after the immunity significantly improves.Every group of Cyprinus carpio carried out pathological section, show immune Cyprinus carpio greatly improved infect after the pathological condition of the cheek, kidney,spleen,liver.

Wherein, the used Koi herpesvirus Jilin Province, China of the present invention strain (KHV-CJ) and frame mirror Cyprinus carpio tail fin primary cell (MFC) are preserved by Jilin Agriculture University's Animal Science And Technology laboratory provides.

Beneficial effect of the present invention: Koi herpesvirus nucleic acid vaccine of the present invention is produced easily, but in escherichia coli high-efficient cloning; Stability is strong, can strictly copy the clone in the escherichia coli midium or long term; In the fish body, use safety, so owing to be that eukaryotic vector expression protective antigen can not cause the virus diffusion; Immunizing dose is little, and after the nucleic acid vaccine of inventing carried out immunity to Cyprinus carpio, carrier high-efficiency was expressed, and has guaranteed the high-efficiency continuous expression of antigen and is secreted into outside the born of the same parents, so immunizing dose is minimum.

Nucleic acid vaccine of the present invention is tested Cyprinus carpio at present, has obtained and has very obviously protected efficient.If with this vaccine immunity fish, will greatly reduce fish and suffer from Cyprinus carpio L. herpes Expectancy, guarantee output and the quality of fish.The present invention can make fish set up for the sick effective disease-resistant mechanism of Koi herpesvirus, makes the raiser reduce economic loss.Reduce simultaneously the drug use amount, thereby guarantee the quality of aquatic products, safeguard ecologic stability.Use the present invention to have important economic benefit, social benefit and ecological benefits.

Description of drawings

Fig. 1: the ORF81 gene PCR amplification electrophoretogram of Koi herpesvirus Jilin Province, China strain (KHV-CJ), wherein M is the dna molecular quality standard, 2 is ORF81 gene PCR product;

Fig. 2: the PCR qualification result of recombiant plasmid pIRES-ORF81, wherein M is the dna molecular quality standard, the 4 PCR results for restructuring Plasmid pIRES-ORF81,5 positive contrasts, 6 negative contrasts;

Fig. 3: the double digestion analytical electrophoresis figure of recombiant plasmid pIRES-ORF81, wherein M is the dna molecular standard quality, 1 is the enzyme action result of pIRES-ORF81;

Fig. 4: contain the Western-blot analysis result of the MFC cellular expression albumen of pIRES-ORF81 expression vector, wherein M is albumen Marker, and 1 is the pIRES-ORF81 transfection group, 2 negative contrasts;

Fig. 5: with the microscopic section (* 100) of every group of Cyprinus carpio cheek tissue behind the counteracting toxic substances of herpesvirus KHV vein thoracic cavity, wherein A is injection PBS group, and B is blank group for injection pIRES group, C, and D is the immune group of injection pIRES-ORF81;

Fig. 6: with the microscopic section (* 100) of every group of Cyprinus carpio renal tissue behind the counteracting toxic substances of herpesvirus KHV vein thoracic cavity, wherein A is injection PBS group, and B is blank group for injection pIRES group, C, and D is the immune group of injection pIRES-ORF81;

Fig. 7: with the microscopic section (* 100) of every group of Cyprinus carpio spleen tissue behind the counteracting toxic substances of herpesvirus KHV vein thoracic cavity, wherein A is injection PBS group, and B is blank group for injection pIRES group, C, and D is the immune group of injection pIRES-ORF81;

Fig. 8: with the microscopic section (* 100) of every group of Carp Liver tissue behind the counteracting toxic substances of herpesvirus KHV vein thoracic cavity, wherein A is injection PBS group, and B is blank group for injection pIRES group, C, and D is the immune group of injection pIRES-ORF81;

The physical map of Fig. 9: eukaryotic expression vector pIRES-neo

The specific embodiment

Embodiment 1:ORF81 gene cloning and evaluation

1. design ORF81 gene amplification primer

According to the upper listed KHV-I strain ORF81 gene order of GenBank, utilize software Primer Premiers 5.0 design primers, forward primer is: F15 '- CCCGGGATGGCAGTCACCAAAGCT-3 ', downstream primer is: F25 '- TCTAGATCACCACATCTTGCCGG-3 ' introduces respectively two restriction enzyme sites of Sma I and Xba I (line position), and primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

2.ORF81 the pcr amplification of gene and evaluation

Take the KHV nucleic acid DNA as template, the ORF81 gene is carried out pcr amplification.Reaction system is: F1, F2(20pmol/ μ L) 0.5 μ L, 10 * ExBuffer, 2.5 μ L, dNTP 2 μ L, DNA 1 μ L, ExTaq enzyme 0.25 μ L, add aquesterilisa and complement to 25 μ L.The PCR reaction condition is: 95 ℃ of denaturations, 5min; 94 ℃ of degeneration, 40s; Anneal 59.3 ℃ 30s; Extend 72 ℃, 1min.The amplification cycles number average is 35 times; At last, reaction system is in 72 ℃ of overall elongation 10min, and reaction finishes rear 4 ℃ of preservations.

Get 5 μ L PCR products, in 1 * TAE buffer, do the molecular weight contrast with DL 2000Marker, 1% agarose gel electrophoresis, the size of detection amplified fragments, record gel imaging result.As shown in Figure 1, go out the dna fragmentation of expection through pcr amplification, be 771bp, illustrate successfully to amplify the ORF81 gene.The nucleotide sequence of described ORF81 gene is shown in sequence table SEQ ID No.1; Its corresponding aminoacid sequence is shown in sequence table SEQ ID No.2.

The Construction and identification of embodiment 2:pIRES-ORF81 eukaryotic expression recombinant plasmid

1.pIRES-neo the preparation of plasmid

Get 5 μ l and contain pIRES-neo plasmid (available from Jin Weike (China) biotechnology center) DH5 α bacterium liquid, join in the LB fluid medium that 5mL contains ampicillin, 37 ℃ of 180r/min concussion overnight incubation.Prepare in a small amount plasmid according to molecular cloning operating guidance (third edition) SDS alkaline lysis.

2.pIRES-neo the enzyme action of plasmid

The pIRES-neo plasmid is carried out Sma I and Xba I double digestion, and 1.0% sepharose electrophoresis reclaims the test kit description with reference to day root agarose gel DNA of company and reclaims the enzyme action sheet.

3.ORF81 the clone of gene in the pIRES-neo plasmid

Utilize day root agarose gel DNA of company recovery test kit that the PCR product of ORF81 is reclaimed purification, get the genetic fragment 5 μ l behind the purification, the large fragment 1 μ l that pIRES-neo plasmid double digestion reclaims, 10 times of T4 buffer1 μ l, T4DNA ligase 1 μ l, ddH ₂O2 μ l is mixed and puts 16 ℃ and spend the night.To connect product and go in the DH5 α competent cell, and obtain the positive colony bacterium colony, picking some positive bacterium colony is seeded in overnight incubation in the LB liquid training base at random, prepares in a small amount plasmid according to molecular cloning operating guidance (third edition) SDS alkaline lysis.

5. the PCR of recombiant plasmid pIRES-ORF81 and enzyme action are identified

With reference to the PCR condition of above-mentioned ORF81, the recombiant plasmid pIRES-ORF81 that gets an amount of a small amount of preparation is that template is carried out pcr amplification.The result has obtained length and has been the fragment of 771bp, with the genes of interest in the same size as shown in Figure 2.

The pIRES-ORF81 plasmid is carried out Sma I and Xba I double digestion, and the enzyme action system is as follows: each 0.5 μ l of Sma I and Xba I, 10 times of H buffer1 μ l, plasmid 2 μ l, ddH ₂O 6 μ l, cumulative volume are 10 μ l, 37 ℃ of water-bath 1h.Behind 1% agarose gel electrophoresis, gel imaging system gathers the enzyme action result.The result as shown in Figure 3, pIRES-ORF81 two bands occur after using Sma I and Xba I double digestion, one treaty 4419bp, with the large fragment in the same size behind pIRES Sma I, the Xba I double digestion, another treaty 771bp, with the ORF81 in the same size, show that ORF81 successfully is inserted in the PIRES carrier.

Embodiment 3: the expression of recombiant plasmid pIRES-ORF81 in the MFC cell

1. a large amount of extractions and the purification of recombiant plasmid

To be accredited as positive pIRES-ORF81 Plasmid Transformation to the bacillus coli DH 5 alpha competent cell, picking list bacterium colony; Be inoculated in the LB liquid medium that 5mL contains Amp+, 37 ℃ of concussions are cultured to exponential phase (OD600 ≈ 0.6); Get 1mL and be inoculated in the LB liquid medium amplification culture that 500mL contains Amp.With the centrifugal 10min of above-mentioned culture 5000r/min, abandon supernatant, after low temperature refrigerator is placed a few hours, STE (0.1mol/L NaCl with the pre-cooling of 200ml ice, 10m mol/L Tris-HCl pH8.0,1m mol/L EDTA) bacterial precipitation is resuspended, centrifugal collection antibacterial as stated above.

Prepare in a large number the plasmid method with reference to molecular cloning operating guidance (third edition) SDS alkaline lysis and extract plasmid, carry greatly with purification process as follows:

(1) with 18ml alkaline lysis liquid I, resuspended bacterial precipitation (place on the vortex oscillation device and vibrate);

(2) add the new 2ml lysozyme (10mg/ml) that disposes;

(3) add the new alkaline lysis liquid II that disposes of 40ml, cover and put upside down gently for several times after centrifuge tube covers, with thorough mixing, place 5-10min under the room temperature;

(4) add the solution III of 20ml pre-cooling, cover centrifuge tube gently behind the mixing, ice bath 10min;

(5) the centrifugal 30min of 11000r/min under 4 ℃ of conditions moves to supernatant in the graduated cylinder, discards the precipitation in the centrifuge tube;

(6) measure the volume of supernatant, be transferred in the new centrifuge tube after adding the isopropyl alcohol of 0.6 times of volume, fully behind the mixing, room temperature effect 10min;

(7) the centrifugal 15min of 8000r/min reclaims the nucleic acid precipitation under the room temperature;

(8) carefully abandon supernatant, centrifuge tube is opened wide lid, be inverted in and remove remaining supernatant on the clean filter paper, 70% ethanol is rinsed and is washed the pipe end and tube wall under the room temperature condition, outwells behind the ethanol centrifuge tube is inverted remaining ethanol is vapored away;

(9) with the moistening nucleic acid precipitation of 3ml TE dissolving, then transfer in the 15ml centrifuge tube, ice bath is cooled to 0 ℃;

(10) add the pre-ice-cold 5mol/L LiCL of 3ml, 4 ℃ of centrifugal 10min of 10000r/min behind the mixing;

(11) supernatant is transferred in the 30ml centrifuge tube, adds the equivalent volumes isopropyl alcohol, the centrifugal 10min of room temperature 10000r/min behind the mixing;

(12) carefully outwell supernatant, be inverted and make dried liquid stream, 70% washing with alcohol precipitation and tube wall, carefully outwelling behind the ethanol to be inverted to make in several minutes at clean filter paper does not have alcohol residue, but should make precipitation keep moistening;

(13) contain the TE dissolution precipitation of RNaseA with 500 μ l, be transferred to microcentrifugal tube, room temperature is placed 30min;

(14) with plasmid-RNase mixture phenol: after the chloroform extracting once, use the chloroform extracting once, then the ethanol precipitation with standard reclaims nucleic acid again;

(15) add the 1ml aquesterilisa plasmid is dissolved, add again 0.5ml PEG-MgCl ₂After solution, room temperature were placed and surpassed 10min, the centrifugal 20min of 12000r/min under the room temperature condition reclaimed precipitation;

(16) with the resuspended precipitation of 70% ethanol to remove the PEG of trace, the centrifugal 5min of 12000r/min reclaims precipitation;

(17) suck ethanol, repeat previous action, then centrifuge tube is placed and place 20min on the pipe support, ethanol is volatilized fully;

(18) with 500 μ lTE dissolution precipitation plasmids, dilute the rear OD of survey with 1:100 with TE ₂₆₀And the concentration of calculating plasmid.Press 1OD ₂₆₀=50 μ g plasmids/ml calculates.

(19)-20 ℃ of preservations after the packing.

2. the transfection of recombiant plasmid

Be cultured to the logarithmic growth after date after frame mirror Cyprinus carpio caudal fin cell (MFC) recovery with the laboratory preservation and be inoculated in 6 orifice plates, place 27 ° of C 5%CO ₂Cultivate in the cell culture incubator, treat to carry out when the cell coverage rate reaches 80% left and right sides transfection.

Use Luo Shi FuGENE HD transfection reagent to carry out transfection, undertaken by the test kit operation instruction, concrete steps are as follows:

First transfection reagent, plasmid and Optimal Medium temperature are bathed to 15-25 ℃; Plasmid is diluted to 2 μ g/100 μ L with not containing serum M199 culture fluid; Then add transfection reagent 5 μ L in the mixed liquor, transfection reagent is directly joined in the plasmid after the dilution, should avoid during operation contacting with the EP tube wall; Rotaring redyeing system is placed room temperature effect 20min; With the M199 culture fluid washed twice of Tissue Culture Plate with serum-free, add the culture fluid of 1.9mL serum-free; 100 μ L transfection drops are entered in the cell, and jog cell plates mixing places 27 ℃ of 5%CO ₂Cell culture incubator is outwelled culture fluid after cultivating 5h, and every hole adds the M199 culture fluid that 2mL contains 10% hyclone, 27 ℃ of 5%CO in the six porocyte culture plates ₂Cell culture incubator is cultivated.The cell that collection is hatched behind the 48h is for subsequent use, establishes simultaneously the contrast of PIRES-neo empty plasmid and normal cell contrast.

3.Western-blot evaluation protein expression

Collect the MFC cell of transfection after 48 hours, with the protein expression situation of Western-blot method check purpose gene.The result can be observed the protein band of about 28KD as shown in Figure 4, with the destination protein in the same size of expection acquisition; Negative control does not all have protein band to occur.Show that constructed Eukaryotic expression recombinant plasmid pIRES-ORF81 success has obtained expression in the MFC cell.

Experimental example 4: recombiant plasmid pIRES-ORF81 induction of immunity Cyprinus carpio and effect detection

Tested front 10 days, choose at random in same batch of healthy Cyprinus carpio of 248 tails (the long 20-30cm of body, body weight 200-300g) 8 tails use PCR method detect do not infect KHV after, remain 480 tails and be divided into 6 groups, every group of 40 tails are fed under the normal condition in the laboratory aquarium.

With Oleum Caryophylli Cyprinus carpio is anaesthetized during experiment, stop to move about until fish and rapidly nucleic acid vaccination is carried out at the muscle position afterwards, and put back in the clear water at once.To be divided into be 6 processed group in experiment: 1. not injection group; 2. injecting normal saline (autoclaving); 3. inject pIRES-neo empty carrier group (dosage is 10 μ g); 4. injection recombiant plasmid pIRES-ORF81(dosage is 1 μ g); 5. injection recombiant plasmid pIRES-ORF81(dosage is 10 μ g); 6. injection recombiant plasmid pIRES-ORF81(dosage is 50 μ g).

More than 6 groups of carp muscle injected dose be 200 μ L, immunity in per 14 days once, altogether immunity is 3 times.Blood sampling before the immunity, for the first time immunity two weeks to three immunity, weekly heart blood sampling.Blood is placed 1h, the centrifugal 15min of 4500r/min behind 4 ℃ of placement 2h, collection upper serum in 37 ℃.Adopt salting out method slightly to carry, recombinant protein A (HiTrap rProteinA Sepharose) affinity chromatography essence is carried the IgM in the Serum of Common Carp, makes the rabbit sera anti-IgM of high-titer behind the protein immunization experimental rabbit with purification.

1.ELISA antibody horizontal in the detection Serum of Common Carp

Coated: dilute according to 600ng/mL with the ORF81 albumen of coating buffer with purification, the every hole of elisa plate is coated with 100 μ L, acts on 1h in 37 ℃ of incubators, and 4 ℃ are spent the night;

Washing: every hole adds 300 μ L PBST washing elisa plate, washs altogether 3 times, and each 5min dries after patting on the filter paper;

Sealing: every hole adds 100 μ L, 5% skim milk, acts on 1.5h in 37 ℃ of incubators;

Washing: the same;

Add test serum: every hole adds 10 μ L test serums, 37 ° of C incubator effect 1.5～2h;

Washing: the same;

Add ELIAS secondary antibody: after ELIAS secondary antibody was pressed the working concentration dilution, every hole added 100 μ L, 37 ℃ of incubation 1.5～2h;

Washing: the same;

Colour developing: every hole adds substrate nitrite ion 100 μ L, and lucifuge colour developing 15min observes chromogenic reaction under the room temperature condition;

Stop: every hole adds stop buffer 50 μ L, measures OD450nm absorption photometric value with microplate reader immediately.

The result judges: when test serum OD450nm value/negative control OD450nm value 〉=2.0, be judged to be the positive.

After each experimental group immunity, weekly heart blood sampling utilizes the KHV specific antibody in the ELISA method detection Serum of Common Carp.The result is by shown in the table 1, Cyprinus carpio is carried out immunity after, all can detect the specific antibody of KHV in the Cyprinus carpio blood of injection recombiant plasmid, antibody horizontal increases with the increase of immune time behind the booster immunization; Matched group does not detect the specific antibody of KHV, compares antibody horizontal significant difference (p＜0.05) with immune recombiant plasmid group; The experiment of three groups of immunizing dose gradients, the immunizing dose of 1 μ g/ tail just can obtain preferably antibody horizontal, and 10 μ g/ tails and 50 μ g/ tail immunizing doses can slightly improve antibody horizontal, but three's difference not significantly (p＞0.05);

Table 1 Serum of Common Carp ELISA antibody horizontal detects (OD450)

2. detect antibody titer with experiment in

Adopt fixed virus dilute serum method, measure after the recombiant plasmid immunity in each test group Serum of Common Carp anti-KHV neutralization and tire.Before Cyprinus carpio is just exempted from and just exempt from booster immunization during weekly heart blood sampling, take a blood sample altogether 7 times, at the clear neutralization test of the enterprising promoting the circulation of blood of MFC cell, experimental result is as shown in table 2, intramuscular injection PBS, empty carrier and blank group Cyprinus carpio do not produce neutralizing antibody, and recombiant plasmid immune group Cyprinus carpio has all produced corresponding neutralizing antibody.

Table 2 neutralization test detects Serum of Common Carp KHV Antibody Results

Group	PBS	PIRES	Blank group	pIRES ORF81
					Before the immunity	﹤1:2	﹤1:2	﹤1:2	﹤1:2
One exempts from a rear week	﹤1:2	﹤1:2	﹤1:2	﹤1:4
					One exempts from rear two weeks	﹤1:2	﹤1:2	﹤1:2	﹤1:8
Two exempt from a rear week	﹤1:2	﹤1:2	﹤1:2	﹤1:8
					Two exempt from rear two weeks	﹤1:2	﹤1:2	﹤1:2	﹤1:32
Three exempt from a rear week	﹤1:2	﹤1:2	﹤1:2	﹤1:64
					Three exempt from rear two weeks	﹤1:2	﹤1:2	﹤1:2	﹤1:64

3. the counteracting toxic substances experiment detects antibody protection effect

(1) statistics protective rate

Two weeks used 100TCID after three immunity ₅₀KHV to each group Cyprinus carpio thoracic cavity counteracting toxic substances, with heating rod Shui nationality the temperature inside the box is transferred to about 25 ℃,

Two weeks used 100TCID after three immunity ₅₀KHV to each group Cyprinus carpio thoracic cavity counteracting toxic substances, Shui nationality the temperature inside the box is transferred to about 25 ℃, observe the also dead number of record Cyprinus carpio every day, calculate protective rate.As can be seen from Table 3, injection PBS, pIRES and blank experiment group Cyprinus carpio be dead 40 tails, 38 tails and 39 tails respectively, and protective rate is respectively 0%, 5% and 2.5%; And inject dead 8 of 40 Cyprinus carpios of pIRES-81 experimental group, protective rate is 80%.Illustrate that the nucleic acid vaccine pIRES-81 that makes up has preferably KHV protection effect.

Protective rate statistical result behind table 3 counteracting toxic substances

(2) preparation pathological section

Behind the virus inoculation, when treating that the first three groups matched group has fish to begin death, choose the gill, kidney, liver and spleen tissue after in every group, selecting at random three Cyprinus carpios to put to death and fix the making paraffin section with 0.4% neutral formalin, after H.E dyeing, in the pathological change of 100 times of tissues observed of microscopically.

Research matched group Cyprinus carpio and immune Cyprinus carpio cheek histopathology situation behind counteracting toxic substances, the result as shown in Figure 5, the Cyprinus carpio gill tissue structure of injection PBS group, the unloaded group of injection pIRES-neo and blank group almost disappears, and the gill non-evident sympton of the immune group Cyprinus carpio of injection pIRES-ORF81;

Research contrast Cyprinus carpio and immune Cyprinus carpio renal tissues pathology situation behind counteracting toxic substances, as shown in Figure 6, interstitial inflammation appears in the Cyprinus carpio kidney of injection PBS group, injection pIRES group and blank group, and the kidney non-evident sympton of the immune group Cyprinus carpio of injection pIRES-ORF81;

Research contrast Cyprinus carpio and immune Cyprinus carpio spleen tissue pathologic condition behind counteracting toxic substances, as shown in Figure 7, the Cyprinus carpio spleen cell generation blister degeneration of injection PBS group, injection pIRES group and blank group hyporrhea is arranged, and the spleen cell of the immune group Cyprinus carpio of injection pIRES-ORF81 increases;

Research contrast Cyprinus carpio and immune Cyprinus carpio liver organization pathologic condition behind counteracting toxic substances, as shown in Figure 8, the Carp Liver cell generation blister degeneration of injection PBS group, injection pIRES group and blank group, and the liver cell non-evident sympton of the immune group Cyprinus carpio of injection pIRES-ORF81.

Claims

1. a Koi herpesvirus nucleic acid vaccine is characterized in that, is the ORF81 gene clone is obtained the pIRES-ORF81 recombiant plasmid in eukaryotic expression vector pIRES-neo.

2. Koi herpesvirus nucleic acid vaccine according to claim 1 is characterized in that, the nucleotide sequence of described ORF81 gene is shown in sequence table SEQ ID No.1.

3. Koi herpesvirus nucleic acid vaccine according to claim 1 is characterized in that, the aminoacid sequence that described ORF81 gene pairs is answered is shown in sequence table SEQ ID No.2.