CN104726409A - Preparation method and applications of immortalized duck embryo hepatic cell line - Google Patents
Preparation method and applications of immortalized duck embryo hepatic cell line Download PDFInfo
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Abstract
The invention provides a preparation method of an immortalized duck embryo hepatic cell line. The preparation method comprises following steps: (1) duck embryo liver is subjected to digestion with trypsase, and an obtained digestive juice is subjected to culturing so as to obtain primary cultured duck embryo hepatic cells; (2) lipidosome transfection is adopted, and eukaryotic expression plasmids used for coding hTERT and Marker genes are introduced into the primary cultured duck embryo hepatic cells; (3) obtained transfected duck embryo hepatic cells are subjected to culturing, and hTERT positive clones are selected with G418; and (4) selected hTERT positive clones are subjected to culturing, and the immortalized duck embryo hepatic cell line is obtained after more than 50 generations of continuous culturing. The immortalized duck embryo hepatic cell line is capable of maintaining relatively excellent activity after more than 50 generations of in vitro continuous cell culture; division and proliferation can be maintained; no aging or apoptosis is caused; and immortalization of the immortalized duck embryo hepatic cell line is realized. The immortalized duck embryo hepatic cell line is regular in cell morphology; culturing conditions are simple and are easy to control; using is convenient; and an excellent carrier is provided for research on duck disease, especially on duck hepatitis virus.
Description
Technical field
The present invention relates to a kind of preparation method and application of duck embryo hepatic cell line of immortalization.
Background technology
Duck liver is the dominant digestion organ of duck, and duck liver cell is the chief functional cells of duck liver, and many duck cause of diseases can cause duck liver cell to produce pathology, so set up duck embryo hepatic cell line can provide a kind of carrier for research duck disease, sick cell levels research duck.
Duck viral hepatitis causes the one of duckling to propagate rapidly and height lethal infectious diseases by duck hepatitis virus, duck virus hepatitis yolk antibody can effective this disease of prevention and therapy, domestic is at present with duck embryo breeding virulent duck enteritis virus, then antigen is made, immunity laying ducks, high with duck embryo breeding virulent duck enteritis virus cost, operating process is loaded down with trivial details.
The method that current mensuration duck virus hepatitis yolk antibody adopts is neutralization test, after yolk antibody doubling dilution, with standard antigen neutralization, then inoculates duck (chicken) embryo, calculates medium lethal dose (LD
50), the drawbacks such as it is high that the method exists cost, and error is larger, can not the level of Accurate Determining duck viral hepatitis antibody.So there is people to attempt the method that will primary duck hepatocyte utilized to set up neutralization test, as " research of duck embryo liver cell culture " (Gansu Agriculture University's journal, 02 phase in 1994), " foundation of duck liver cell primary culture method " (Medical University Of Tianjin's journal, 02 phase in 1996) disclose the hepatocellular isolation cultivation method of duck embryo.But the hepatocellular separation and Culture of primary duck embryo must use duck embryo, and production process is loaded down with trivial details, and cost is high, easily pollutes at every turn, cell state is not easy to control.In addition, and containing heteroproteose cell, between batch, difference is large, and cytopathy is not obvious, is difficult in this way promote.
Summary of the invention
For solving the deficiencies in the prior art, the present invention adopts gene recombination technology, by telomerase reverse transcriptase gene (hTERT) Transfected primary duck embryo liver cell, set up the method preparing the duck embryo hepatic cell line of immortalization first, prepare the duck embryo liver cell of immortalization, and virulent duck enteritis virus can be bred on cell, there is obvious pathology, the virus titer being used for breeding is tired higher than duck embryo propagative viruses, easy to use, can be used for producing duck viral hepatitis antigen.
Main purpose of the present invention is the preparation method of the duck embryo hepatic cell line providing a kind of immortalization, and described method comprises: (1), with trypsinization digestion duck embryo liver, is cultivated described Digestive system, obtained the duck embryo liver cell of original cuiture; (2) adopt lipofection, the eukaryon expression plasmid of coding hTERT and Marker gene is imported the duck embryo liver cell of described original cuiture; (3) cultivate the duck embryo liver cell of described transfection, screen described hTERT positive colony with G418; (4) cultivate the described hTERT positive colony of screening, cultured continuously, more than 50 generations, obtains the clone of immortalization.
Preferably, described Marker gene is neo gene.
Particularly, described method comprises: 1. with trypsinization digestion duck embryo liver, M199 culture medium culturing is after 48 hours, and cell covers with individual layer, sets up the method for duck embryo Primary Hepatocyte Culture;
2. adopt lipofection, the eukaryon expression plasmid pCI-neo-hTERT of coding hTERT and neo gene is imported the duck embryo liver cell of original cuiture;
3. cell transfecting pCI-neo-hTERT eukaryon expression plasmid 48 hours, cell attachment and in good condition, add 450 μ g/ml G418 to screen, within 4th day, cell starts death, within every 3 days, change a subculture afterwards and add 450 μ g/ml G418 and continue screening, 10 days a large amount of Vacuole formations afterwards, 18th day, untransfected group cell is all dead under G418 effect, transfection group still has comparatively many cells, and existing G418 resistant cell starts growth, after screening 4 weeks, G418 concentration is down to 100 μ g/ml and is maintained screening, visible larger positive colony under treating mirror, use filter paper enzyme by after positive clone digestion, and planted in 48 orifice plates, obtain anti-G418 positive cell.
4. cultivated in CO2 incubator by the cell of screening, went down to posterity once every 2 ~ 3 days, cultured continuously, more than 50 generations, obtains the clone of immortalization.
Another object of the present invention is to the duck embryo hepatic cell line of a kind of immortalization providing described method to prepare.
The duck embryo liver cell form rule of immortalization, culture condition simply, easily controls, easy to use, for research duck is sick, particularly studies duck hepatitis virus and provides good carrier.
Another object of the present invention is to provide a kind of method preparing duck virus hepatitis vaccine, and described method comprises: (1) cultivates the duck embryo hepatic cell line of the described immortalization of propagation; (2) in the duck embryo hepatic cell line of the described immortalization cultivated, inoculate virulent duck enteritis virus, cultivate the described virulent duck enteritis virus of propagation; (3) virulent duck enteritis virus of described cultivation is gathered in the crops, deactivation.
The duck embryo hepatic cell line inoculation virulent duck enteritis virus that the present invention sets up can produce obvious pathology, and can be used for measuring duck virus hepatitis yolk antibody, this method cost is low, easy to use, and accuracy is high, can promote on a large scale.
Preferably, described virulent duck enteritis virus is DHV-1-LY strain.
Another object of the present invention is the duck virus hepatitis vaccine providing described method to prepare.
An also object of the present invention is to provide a kind of method measuring duck hepatitis virus yolk antibody and tire, and described method comprises:
(1) described duck embryo hepatic cell line is inoculated in cell bottle, after covering with individual layer, goes down to posterity in 96 orifice plates, every hole 1.5 ~ 2.0 × 10
5individual cell, continues to cultivate;
(2) virulent duck enteritis virus is diluted to 100TCID
50/ 1.0ml, mixes with the tested yolk antibody of 2 times of serial dilutions of equivalent, reaction;
(3), after reaction terminates, each extent of dilution inoculation 1 row (8 holes) cover with hepatocellular 96 orifice plates of described duck embryo, are placed in 37 DEG C of CO
2continue in incubator to cultivate, establish feminine gender and positive control simultaneously;
(4) latter 3 ~ 4 days of inoculation, records the hole count that cytopathy (CPE) appears in each extent of dilution, calculates the Neutralizing titer of described yolk antibody.
Preferably, described in step (2), virulent duck enteritis virus is DHV-1-LY strain.
The method that current inspection duck hepatitis virus is tired is inoculation embryo alive or agar diffusion method, this method error is larger, many mechanisms all at the measuring method of tiring by duck embryo liver cell research duck hepatitis, utilize the duck embryo hepatic cell line set up to greatly facilitate the researchs such as duck hepatitis virus production, inspection.
The present invention also aims to provide the duck embryo hepatic cell line of described immortalization preparing duck virus hepatitis vaccine, measure duck hepatitis virus yolk antibody tire in application.
The duck embryo hepatic cell line of a kind of immortalization provided by the invention, using duck embryo liver primary cell as host cell, transfection pCI-neo-hTERT carrier for expression of eukaryon, the expression human telomerase reverse transcriptase obtained through G418 resistance screening and the clone of tool height titers virus.This clone goes down to posterity in vitro more than 50 generations and still has good vigor, and keep division growth, aging and apoptosis do not occur, and are a kind of clone of immortalization.
Accompanying drawing explanation
Fig. 1 is the duck embryo hepatic cell line cell growth curve of immortalization.
Fig. 2 detects Transfected Cells source property hTERT for using RT-PCR, the 1st, 2,3 swimming lanes be respectively to the 30th generation after transfection and the 65th generation cell, there is situation in what before transfection, 2nd generation cell carried out that RT-PCR measures hTERT in genome.
Fig. 3 be after transfection the 30th generation and the 65th generation cell, the detection of 2nd generation cell inside end telomerase activity before transfection.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Below by way of gene and the virus multiplication qualification of the structure of the duck embryo hepatic cell line to immortalization, cell morphological characteristic, mark, and measure at virus titer and should be used for elaborating, the explanation of the invention is not limited.
The structure of embodiment 1 duck embryo hepatic cell line
1.pCI-neo-hTERT eukaryotic expression system
PCI-neo-hTERT can connect structure by carrier or be purchased, and the pCI-neo-hTERT carrier that the present invention specifically uses is purchased from Addgene company.
2. the hepatocellular separation and ientification of duck embryo
The aseptic duck embryo liver taking 15 ages in days, is cut into about 1mm
3tissue block, clean 3 times by the PBS solution of 37 DEG C of preheatings, go out blood stains.Add appropriate EDTA-trypsin solution (containing pancreatin 0.05%, containing EDTA0.02%, 10ml/ embryo), 37 DEG C of digestion 5min, supernatant discarded.Again add EDTA-trypsin solution, add magnetic stir bar, be put on magnetic stirring apparatus, 120 turns/min, stir digestion 10min.Collect supernatant liquor, at 2 ~ 8 DEG C, the centrifugal 10min of 600g, resuspended with the M199 substratum containing appropriate 15% serum, after 8 layers of filtered through gauze, cell counting, is adjusted to 1.0 ~ 1.5 × 10
6individual/ml, adds in 25T cell bottle, is put in CO
2in incubator, cultivate 2 ~ 3 days, can individual layer be grown up to for 37 DEG C.
The transfection of 3.pCI-neo-hTERT eukaryotic expression system and screening
First 48 hours of transfection, utilizes ATV solution digestion primary duck embryo liver cell to be passaged to 2nd generation, and is inoculated in 12 orifice plates, 1.0 × 10
6/ hole, transfection reached 80 ~ 90% cell confluency degree the same day.First 2 hours of transfection, is replaced with the M199 substratum of serum-free by substratum, every hole 400 μ l.With the M199 solution of serum-free, pCI-neo-hTERT plasmid is diluted to 50 μ l, 0.02 μ g/ μ l, mixes, room temperature effect 5min gently.The liposome (Lipofectamine2000, invitrogen company) of 4 μ l is added in the serum-free M199 substratum of 46 μ l, mixes gently, room temperature effect 5min.The plasmid of mixing is mixed with liposome dilution, shakes up gently, room temperature effect 20min.
By the plasmid liposome solutions of mixing dropwise join in 12 orifice plates, limit edged mixes gently, and 10 μ l/ holes, stay 2 holes to contrast.Be placed in 37 DEG C, 5%CO
2cultivate 5 ~ 6 hours in incubator, then change the M199 solution containing 15% serum, continue to cultivate.
After transfection 48 hours, add (450 μ g/ml) G418 and carry out screening 4 weeks, the cell mortality of untransfected, the cell of survival starts growth, after 4 weeks, G418 concentration is reduced to 100 μ g/ml, when there being large stretch of positive cell clone, picked out by monoclonal cell and be passaged in 48 orifice plates, the substratum added containing 100 μ g/ml continues to cultivate, and goes down to posterity once every 2 ~ 3 days.Cultivation, more than after 50 generations, is the duck embryo hepatic cell line of immortalization.
4. the qualification of the duck embryo hepatic cell line of immortalization
The mensuration of 4.1 cell morphology characteristic and growth curve
Duck embryo hepatic cell line is compared with the primary cell be separated, and cell is Polygons, evenly the pebbles paving stone shape arrangement of rule, and nucleus is obvious, and iuntercellular exists contact inhibition.
By after transfection 65 generation duck embryo liver cell with 1.0 × 10
4individual cells/well is inoculated in 24 well culture plates, and 3d changes liquid 1 time, and every day counts with 0.25% trypsin solution peptic cell, blood cell counting plate.Each time point establishes 3 parallel holes, and each hole counts 3 times, gets its mean value and draws cell growth curve, as Fig. 1.Fig. 1 illustrates that this cell starts propagation on the 2nd day, and within 3rd ~ 5 days, be increased logarithmic phase, this clone has very strong fecundity as seen.
4.2RT-PCR detects Transfected Cells source property hTERT
(1) according to the hTERT gene order delivered in GenBank, design pair of primers: upstream: 5 '-TATGCCGTGGTCCAGAAG-3 ', downstream: 5 '-TATGCCGTGGTCCAGAAG-3 ', amplified fragments size is 416bp.
(2) extraction of cell total rna: the duck embryo liver cell of getting the 30th generation, 65 generations after 2nd generation and transfection before transfection, discard substratum, PBS washing 2 ~ 3 times, add 1mL Trizol and mix, room temperature leaves standstill 5min and carries out cracking, lysate is transferred to centrifuge tube, and in centrifuge tube, adding 0.2mL chloroform, concussion, room temperature leaves standstill 10min, 4 DEG C, the centrifugal 15min of 10000r/min; Get top section after centrifugal and move into another centrifuge tube, add 0.2mL Virahol, turn upside down, mix gently, left at room temperature 30min, 4 DEG C, the centrifugal 15min of 10000r/min; Abandon supernatant, add the vibration of 1mL75% ethanol, the centrifugal 5min of 7500r/min; Precipitation ambient temperatare is put about 10min seasoning, resuspended with 30 μ L DEPC water, obtain cell total rna.
The synthesis of (3) first chain cDNA: reaction system is, 5 × AMV buffer4.0 μ l, dNTP5.0 μ l, random primer 1.0 μ l, RNA template 1.0 μ l, RNase Inhibitor0.5 μ l, ThermoScript II 1.0 μ l, 42 DEG C of effect 90min.
(4) pcr amplification: amplification program is 94 DEG C of 5min, 94 DEG C of sex change 30s, 56 DEG C of annealing 45s, 72 DEG C extend 45s, totally 30 circulations, and 72 DEG C extend 10min again, 4 DEG C of preservations.
(5) gel electrophoresis: the PCR primer 10 μ l electrophoresis in the sepharose of 0.1% obtained, result is as Fig. 2.1st, 2 swimming lanes be after transfection the 30th generation and the 65th generation cell, the 3rd swimming lane is 2nd generation cell before transfection, and the hTERT gene of visible transfection exists in the cell in the 30th generation and the 65th generation, and does not have hTERT gene in the cell of untransfected.
4.3 telomerase activations detect
Carry out according to the operation instructions bought in the TeloTAGGG Telomerase PCR ELISA kit of Roche company.First the duck embryo liver cell each about 2.0 × 10 in the 30th generation, 65 generations after 2nd generation and transfection before transfection is collected
5individual cell, with Lysis buffer at cracking 30min on ice.The centrifugal 20min of 12000rpm/min, gets 1 μ l cracking supernatant liquor as Telomerase template, increases according to the pcr amplification reaction condition of test kit, negative control RNAase process.After amplification terminates, develop the color by ELISA requirement, carry out absorbance measurement at 450nm and 690nm place, result is as Fig. 3.Visible, very high activity is had in Telomerase the 30th generation after transfection and the 65th generation cell, and activity is very low in 2nd generation cell before transfection, high expression in the cell in hTERT gene is described the 30th generation and the 65th generation after transfection, and before transfection, 2nd generation cell hTERT gene is expressed hardly.
Embodiment 2 measures duck hepatitis virus yolk antibody with duck embryo liver cell and tires
1. the preparation of duck virus hepatitis refined yolk antibody
Conventionally, prepare duck virus hepatitis refined yolk antibody, basic step is as follows:
(1) adopt duck viral hepatitis DHAV-1 type strain to inoculate 10 age in days susceptible chicken embryos, then gather in the crops allantoic fluid, formalin-inactivated also prepares vaccine;
(2) use the vaccine immunity laying hen of described preparation, after immunity, sampling measures anti-DHAV-1 type antigen-antibody Neutralizing titer >=1 in chicken high-immunity egg Huang: 8192, collects the high-immunity egg of described chicken afterwards;
(3) by described high-immunity egg eggshell sterilization, collect yolk, described yolk adds equal-volume distilled water, stirring and evenly mixing, low temperature pasteurization; Acidifying distillation water law is purified, sad method is purified; Micro-filtration, ultrafiltration.Prepare three batches of duck virus hepatitis refined yolk antibodies (1301,1302,1303)
2. conventional vaccination duck embryo mensuration duck hepatitis virus yolk antibody is tired
DHV-1-LY strain is diluted to every 0.1ml containing 200ELD
50, mix with the antibody to be checked of equivalent 2 times of serial dilutions, 37 DEG C effect 1 hour, period shaken several times.Each extent of dilution inoculates 5 piece of 10 age in days susceptible duck embryo, every embryo 0.2ml, the virus control group of separately establishing virus liquid to mix with equivalent sterilizing PBS and each 5 pieces of PBS blank group, puts 37 DEG C and cultivates 168 hours, record the duck embryo death toll of 48 ~ 168 hours, result of determination.Blank group all should be good for and be lived, and virus control group should be all dead.
The highest antibody extension rate that can make 50% chicken (duck) embryo that death does not occur is the Neutralizing titer of this antibody.
3. inoculate duck embryo liver cell mensuration duck hepatitis virus yolk antibody to tire
(1) the duck liver cell system of foundation is inoculated in 25T cell bottle, after covering with individual layer, goes down to posterity in 96 orifice plates, every hole 1.5 ~ 2.0 × 10
5individual cell, continues to cultivate.
(2) DHV-1-LY strain is diluted to 100TCID
50/ 1.0ml, mixes with the tested yolk antibody of 2 times of serial dilutions of equivalent, 37 DEG C of reaction 60min.
(3), after reaction terminates, each extent of dilution inoculation 1 row (8 holes) cover with 96 orifice plates of duck liver cell, are placed in 37 DEG C of CO
2continue in incubator to cultivate, if negative and positive control.
(4) latter 3 ~ 4 days of inoculation, records the hole count that cytopathy (CPE) appears in each extent of dilution, calculates the Neutralizing titer of yolk antibody by Reed Muench method.
4. inoculate the comparison that duck embryo is tired with inoculation duck embryo liver cell mensuration duck hepatitis virus yolk antibody
By three batches of duck virus hepatitis refined yolk antibodies (1301,1302,1303) of preparation, with reference to the method for " Chinese veterinary pharmacopoeia (three) " annex neutralization test, duck virus hepatitis refined yolk antibody after neutralization is inoculated duck embryo and duck embryo liver cell respectively, all establish 3 groups of parallel controls, result is as table 1.Because the duck embryo hepatic cell line form set up and vigor are stablized, and very responsive to duck viral hepatitis, pathology typical case, so tire with the yolk antibody that duck embryo liver cell measures, to tire height than the yolk antibody measured with duck embryo, and stablize, error is little.
Table 1
The preparation of embodiment 3 duck virus hepatitis vaccine
After the duck embryo liver cell of transfection is covered with individual layer, discard substratum, inoculation sterilizing PBS solution makes the DHV-1-LY strain virus liquid of 1: 100 dilution, after 37 DEG C of absorption 1h, discards virus liquid, adds the M199 substratum of 10%, put into CO
2in incubator, 37 DEG C are continued to cultivate, establish control group simultaneously.To connect after poison observation of cell pathology every day, compared with control group, connecing latter 24 hours of poison, there is CPE in cell, and 72h pathology is obvious, and cavity appears in sick cell, draws in the net phenomenon, part lysis.Receive poison, survey malicious valency.
Duck hepatitis virus DHV-1-LY strain seed culture of viruses sterilizing PBS solution is done 1: 100 dilution, through allantoic cavity inoculation 10 ~ 12 age in days susceptible duck embryos, every embryo 0.2ml.Put 37 DEG C to continue to hatch, discard the dead germ in 48 hours, gather in the crops 48 ~ 96 hours dead duck embryos, put 2 ~ 8 DEG C of coolings 12 ~ 24 hours.After duck blastochyle aseptic for inspection mixing, quantitative separating, freezen protective, surveys malicious valency.
Virus liquid virus titer prepared by result conventional vaccination duck embryo is 10
5.21tCID
50/ ml, and to inoculate the hepatocellular virus titer of duck embryo be 10
6.43tCID
50/ ml, visible duck viral hepatitis I C-type virus C DHV-1-LY can breed in a large number on duck embryo liver cell, and is better than the method for conventional vaccination duck embryo.
The virus liquid above-mentioned two kinds of methods prepared, respectively with after 0.2% formaldehyde solution deactivation, prepares duck hepatitis I C-type virus C inactivated vaccine with mineral oil adjuvant emulsification.Use duckling to measure, vaccine minimum immune dosage is 0.15ml, determines that using dosage is 0.3ml/ plumage.Single dose, single dose repeat and overdose proof test result is: vaccine prepared by cell does not cause the systemic adverse reactions of duckling and obvious local reaction, and generation more than 90% can be induced to protect; Vaccine 1/10-2/10 local reaction in various degree prepared by duck embryo, injection site easily forms lump, and generation more than 90% can be induced to protect.After two kinds of vaccine difference primary immune response 3 age in days ducklings, different time induces the HI antibody titers (GMT) of generation respectively: cell vaccine 7 days is 1:256, within 14 days, is 1:1024, within 21 days, is 1:1151; Duck embryo seedling 7 days is 1:227, within 14 days, is 1:896, within 21 days, is 1:1024; Antibody horizontal is all higher, exempts within latter 14 days, to use virulent strain LY strain to attack poison, all can provide >=the protection of 90%.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. a preparation method for the duck embryo hepatic cell line of immortalization, described method comprises:
(1) with trypsinization digestion duck embryo liver, cultivate described Digestive system, obtain the duck embryo liver cell of original cuiture;
(2) adopt lipofection, the eukaryon expression plasmid of coding hTERT and Marker gene is imported the duck embryo liver cell of described original cuiture;
(3) cultivate the duck embryo liver cell of described transfection, screen described hTERT positive colony with G418;
(4) cultivate the described hTERT positive colony of screening, cultured continuously, more than 50 generations, obtains the clone of immortalization.
2. method according to claim 1, wherein, described Marker gene is neo gene.
3. a kind of duck embryo hepatic cell line of immortalization prepared of method according to claim 1.
4. prepare a method for duck virus hepatitis vaccine, described method comprises:
(1) the duck embryo hepatic cell line of the described immortalization of propagation is cultivated;
(2) in the duck embryo hepatic cell line of the described immortalization cultivated, inoculate virulent duck enteritis virus, cultivate the described virulent duck enteritis virus of propagation;
(3) virulent duck enteritis virus of described cultivation is gathered in the crops, deactivation.
5. preparation method according to claim 4, wherein, described virulent duck enteritis virus is DHV-1-LY strain.
6. the duck virus hepatitis vaccine that according to any one of claim 4 ~ 5 prepared by method.
7. measure the method that duck hepatitis virus yolk antibody is tired, described method comprises:
(1) duck liver cell system according to claim 1 is inoculated in cell bottle, after covering with individual layer, goes down to posterity in 96 orifice plates, every hole 1.5 ~ 2.0 × 10
5individual cell, continues to cultivate;
(2) virulent duck enteritis virus is diluted to 100TCID
50/ 1.0ml, mixes with the tested yolk antibody of 2 times of serial dilutions of equivalent, reaction;
(3), after reaction terminates, each extent of dilution inoculation 1 row (8 holes) cover with hepatocellular 96 orifice plates of described duck embryo, are placed in 37 DEG C of CO
2continue in incubator to cultivate, establish feminine gender and positive control simultaneously;
(4) latter 3 ~ 4 days of inoculation, records the hole count that cytopathy (CPE) appears in each extent of dilution, calculates the Neutralizing titer of described yolk antibody.
8. method according to claim 7, wherein, described in step (2), virulent duck enteritis virus is DHV-1-LY strain.
9. according to claim 3 the duck embryo hepatic cell line of immortalization preparing duck virus hepatitis vaccine, measure duck hepatitis virus yolk antibody tire in application.
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