CN104725504B - A kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody and its application - Google Patents
A kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody and its application Download PDFInfo
- Publication number
- CN104725504B CN104725504B CN201510150327.1A CN201510150327A CN104725504B CN 104725504 B CN104725504 B CN 104725504B CN 201510150327 A CN201510150327 A CN 201510150327A CN 104725504 B CN104725504 B CN 104725504B
- Authority
- CN
- China
- Prior art keywords
- mrnv
- macrobrachium rosenbergii
- open country
- pgex
- yolk antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to biotechnologies, and in particular to a kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody and its application.The Yolk antibody is prepared by following methods：By in Macrobrachium rosenbergii open country field viral capsid proteins genetic recombination to GST integrative gene expression vectors pGEX-5x-1, it is built into pGEX-5x-1-MrNV-CP recombinant plasmids；By pGEX-5x-1-MrNV-CP recombinant plasmid transformeds Escherichia coli and induced fusion gene expression；It is extracted using inclusion body purification technique and purifies to obtain GST-MrNV-CP recombinant proteins；With GST-MrNV-CP recombinant protein immune hens, collects egg and extract Yolk antibody from egg.Yolk antibody prepared by the present invention can effectively inhibit Macrobrachium rosenbergii open country field viral, and potency is high, have good immune protection effectiveness, can be added in Macrobrachium rosenbergii feed as Macrobrachium rosenbergii open country field viral vaccine in the shrimp seedling phase of shrimps.
Description
Invention field
The invention belongs to biotechnologies, and in particular to a kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody and
It is applied.
Technical background
Macrobrachium rosenbergii originates in the India Pacific region, lives in various types of fresh water or salt-fresh water waters.1976
It introduces the kind for the first time from China, mainly promotes cultivation in the more a provinces (city, area) of southern l0 at present.Growth speed is fast, recipe
Extensively, full of nutrition, it is a kind of Important Economic shrimps；To 2000, oneself reached nearly 20,000,000,000 tail to China's Macrobrachium rosenbergii seed yield, produces
More than 20 hundred million yuan of value.Since cultivation density is continuously improved, breeding water body pollution getting worse and breed variety germplasm are degenerated, Roche natural pond
Shrimp disease increased popularity in nursery and breeding process.In the later stage nineties, Macrobrachium rosenbergii muscle necrosis is in succession in south China
Each province was broken out, and in pandemic in 20001.The infection object of the virus is Macrobrachium rosenbergii seedling, and principal pathogenetic period is
After Desalination in 1-2 weeks, the death rate is up to 60% or more, up to 100% when serious.Serious economic loss is caused, so far
Still without the method that can effectively control this disease.Studies have shown that the disease cause of disease is nodavirus, it is named as Macrobrachium rosenbergii open country field
Viral (Macrobranchium rosenbergii nodavirus, MrNV).Virion is in twenty face body, no cyst membrane, diameter
For 23-25nm, viral nucleic acid is single stranded RNA, is made of 2 segments, respectively RNA1 (3.20kb) and RNA2 (1.2kb).Its
Middle RNA2 encoding capsid proteins are important albumen and important antigen of the virus infection with pathogenicity.Since shrimps can not be voluntarily
Antibody is generated, therefore common vaccine can not effectively prevent the disease.Yolk antibody has the characteristics that stability and high efficiency, is
Prevent the important means of such disease.
Relevant patent about Macrobrachium rosenbergii open country field virus at present, mainly there is following report：Macrobrachium rosenbergii muscle gonorrhoea
Virus enzyme-linked immunologic diagnosis kit and its detection method (application publication number：CN1603830；Application number：031434282 Shen
It please publication No.：CN1603830)；Macrobrachium rosenbergii nodavirus detection kit and detection method (application publication number：
CN103122393A；Application number：2012105024179)；Method (the application of Nuoda Virus mucleic-acid point cross-breeding of luoshi shrimp in natural pond detection
Publication No.：CN1438330；Application number：031187331)；The side of the compound RT polymerization chain reaction of Ro s pond shrimp viral
Method (application publication number：CN1451764；Application number：031190898).The above patent is all the detection about virus, without reference to
The preparation of Yolk antibody.
Invention content
The purpose of the present invention is to provide a kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody and its applications.
In order to solve the above technical problems, the present invention uses following technical scheme：
A kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody, is prepared by following methods：
(1) DNA of Macrobrachium rosenbergii open country field viral capsid proteins gene is cloned by RT-PCR method, paddy Guang is arrived in then recombination
In sweet peptide S- transferases (GST) integrative gene expression vector pGEX-5x-1, pGEX-5x-1-MrNV-CP recombinant plasmids are built into,
Contain glutathione S-transferase and Macrobrachium rosenbergii open country field viral capsid proteins in the pGEX-5x-1-MrNV-CP recombinant plasmids
Fusion；
(2) expression of the recombinant plasmid pGEX-5x-1-MrNV-CP in Escherichia coli：By pGEX-5x-1-MrNV-CP weights
Group plasmid converts Escherichia coli, and the Escherichia coli single bacterium that next day is selected on conversion tablet is fallen in the culture medium containing kanamycins
Expand culture, induces the fusion of glutathione S-transferase and Macrobrachium rosenbergii open country field viral capsid proteins big using IPTG
It is expressed in enterobacteria；Thalline is collected, is extracted using inclusion body purification technique and purifies to obtain GST-MrNV-CP recombinant proteins；
(3) it uses GST-MrNV-CP recombinant proteins that bird inlay is immunized, the produced egg of immune hen is collected, from collected
It is extracted in egg and purifies Yolk antibody.
By said program, the molecular weight of the GST-MrNV-CP recombinant proteins is 66kDa~68kDa.
By said program, the RT-PCR method is：It, will using specific primer using Macrobrachium rosenbergii open country field virus as template
The viral capsid proteins gene reverse transcription of Macrobrachium rosenbergii open country field obtains double-strand cDNA at the first chain cDNA, then PCR amplification.
By said program, the construction method of the pGEX-5x-1-MrNV-CP recombinant plasmids is：By cDNA and pGEX-5x-
1 carrier carries out double digestion respectively, then is attached and is obtained by the reaction containing glutathione S-transferase and Macrobrachium rosenbergii open country field virus
The pGEX-5x-1-MrNV-CP recombinant plasmids of the fusion of capsid protein.
By said program, the recombinant plasmid pGEX-5x-1-MrNV-CP is expressed as in Escherichia coli：pGEX-5x-
1-MrNV-CP recombinant plasmid transformed Escherichia coli, the single bacterium that next day is selected on conversion tablet fall within the LB liquid containing kanamycins
In body culture medium, carries out constant-temperature table culture in 37 DEG C, 200 revs/min and add when the OD values of Escherichia coli reach 0.4-0.6
The IPTG for entering final concentration of 1mmol/L is induced 4-8 hours at 25-37 DEG C.
It is described to be immunized as two wings and the injection of both sides chest muscle by said program.
By said program, the immune number is 3 times, and immune time interval is 2 weeks every time.
By said program, the immunization ways are：First immunisation injection by GST-MrNV-CP recombinant proteins in equal volume it is complete
The vaccine that full Freund's adjuvant is emulsified into, for the second time and third time inoculation is by GST-MrNV-CP recombinant proteins and not exclusively not
Family name's adjuvant emulsion at vaccine.
By said program, is extracted from egg described in step (3) and the method for purifying Yolk antibody is：By collected egg
Egg white and yolk separation, take yolk, remove vitellinae membrana, sterile purified water is then added, fully shake up, be placed under the conditions of 4 DEG C
After overnight, it is Yolk antibody to collect supernatant.
Above-mentioned Macrobrachium rosenbergii open country field virus Yolk antibody is preparing shrimp in the virus vaccine preparation of anti-Macrobrachium rosenbergii open country field
Using.
Beneficial effects of the present invention：The Yolk antibody expression quantity that the method for the present invention is prepared is continual and steady, through Roche natural pond
Shrimp seedling Immunoprotection test confirms that prepared Yolk antibody can effectively inhibit Macrobrachium rosenbergii open country field viral, and potency is high, has
Good immune protection effectiveness can be added to Roche in the shrimp seedling phase of shrimps as Macrobrachium rosenbergii open country field viral vaccine
In pond crayfish feed, possibility is provided for the commercialization popularization of Macrobrachium rosenbergii open country field viral vaccine, is had broad application prospects.
Specific implementation mode
For a better understanding of the present invention, with reference to the embodiment content that the present invention is furture elucidated, but the present invention
Content is not limited solely to the following examples.
Embodiment 1：The structure of Macrobrachium rosenbergii open country field viral capsid proteins gene pGEX-5x-1-MrNV-CP recombinant plasmids
Using the total serum IgE of the pond crayfish muscle extraction of Macrobrachium rosenbergii open country field virus infection as template, using downstream primer MrNV-
NotI-BW：5 '-ATAGCGGCCGCCTAATTATTGCCGACGATAG-3 ', by Macrobrachium rosenbergii open country field viral capsid proteins gene
It carries out RT-PCR reactions and generates the first chain cDNA, the reagent that RT-PCR reacts is：Reverse transcriptase, dNTP, buffer solution；Condition is：
75℃,5min；42 DEG C, 1 hour；80℃,10min.Then, using the cDNA of acquisition as template, sense primer MrNV- is used
SalI-FW:5 '-CATGTCGACATGGCTAGAGGT AAACAA AA-3 ' and downstream primer MrNV-NotI-BW：5'-
TAGCGGCCGCCTAATTATTGCCGACGATAG-3 ', PCR amplification obtain double-stranded DNA.PCR reaction reagent be：High-fidelity
Archaeal dna polymerase, dNTP, buffer solution；Pcr amplification reaction condition is：95 DEG C of 5min of pre-degeneration；Subsequent 95 DEG C, 40s；61℃,40s；
72℃,l min；Totally 35 cycles then extend 5min, 4 DEG C of preservations at 72 DEG C.Reaction product is powered in 1% agarose
Swimming separation, purifies target DNA fragment.The DNA fragmentation of purification and pGEX-5x-1 carriers are then used into the bis- enzymes of SalI and NotI respectively
It cuts, uses T4The DNA fragmentation of digestion is connected on the pGEX-5x-1 carriers of digestion by DNA ligase, is acquired containing GST and sieve
The pGEX-5x-1-MrNV-CP recombinant plasmids of family name pond crayfish open country field viral capsid proteins fusion.The coupled reaction system
For：DNA 2 μ l, pGEX-5x-16 μ l, 10 × connection buffer solution 2 μ l, T4DNA ligase 1 μ l, 16 DEG C 30 minutes.
Embodiment 2：Expression of the recombinant plasmid pGEX-5x-1-MrNV-CP in Escherichia coli
By pGEX-5x-1-MrNV-CP recombinant plasmid transformed Escherichia coli, the single bacterium that next day is selected on conversion tablet is fallen within
In LB liquid medium containing 50g/mL kanamycins, in 37 DEG C, 200 revs/min of progress constant-temperature table cultures, when large intestine bar
When the OD values of bacterium reach 0.4-0.6, the IPTG of final concentration of 1mmol/L is added, is induced 4-8 hours at 25-37 DEG C.6000rpm
10min is centrifuged, thalline is collected.High pressure is crushed thalline, and the suspension after bacterial cell disruption divides through 4 DEG C, 12,000rpm centrifugation 10min
It Shou Ji not supernatant precipitation.Albumen is extracted using inclusion body purification technique to the precipitation of collection, concrete operation step is as follows：
(1) precipitation is fully hanged, mixing using 20mL buffer A (50mM Tris-HCl, 5mM EDTA, pH 8.0),
4 DEG C of centrifugation 10,000rpm 20min, remove supernatant；It is repeated once；
(2) precipitation is fully hanged using 20mLbuffer B (50mM Tris-HCl, 5mM EDTA, 2M ureas, pH8.0), is mixed
Even, 4 DEG C of refrigerated centrifuges 10,000rpm 20min remove supernatant；It is repeated once；
(3) precipitation is not washed with the 1% Qula reduction of fractions to a common denominator, 4 DEG C of refrigerated centrifuges 10,000rpm 20min, removes supernatant；It repeats
Once；
(4) precipitation is fully hanged using 20mL buffer C (0.1M Tris-HCl, 10mM DTT, 8M ureas, pH8.0),
Mixing, is placed on 37 DEG C of constant-temperature tables and quickly shakes 1h with 200rpm, and 4 DEG C of centrifugation 10,000rpm 10min retain supernatant, go
Except precipitation；
(5) supernatant is fitted into bag filter, is placed in 50 times of volume dialyzates (0.1M Tris-HCl, 5mM EDTA, 5mM
Cysteins, pH8.0) in, 4 DEG C of dialysis 16h or more；It is placed in 4 DEG C of dialysis 16h or more, 4 DEG C of freezings in above-mentioned dialyzate (1L) again
Centrifugation 10,000rpm10min retains supernatant, removal precipitation.Then SDS-PAGE Detection and Extraction GST-CP recombinant proteins are used
The molecular weight of concentration, purity and recombinant protein, testing result display extraction recombinant protein concentration can reach 5 milligrams/
Milliliter or more, for purity up to 99% or more, the molecular weight of recombinant protein is 68kD.It is extracted using inclusion body combination SDS-PAGE methods
The method of recombinant protein can be quickly obtained the recombinant protein of large-scale purification, for subsequently preparing Yolk antibody provides facility.This
Outside, since GST molecular weight is bigger (26kD), there is better immunogenicity compared with other fusion small peptides (such as His-tag).
Embodiment 3：The preparation of Macrobrachium rosenbergii open country field virus Yolk antibody
1. the preparation of antigen：By recombinant protein with isometric adjuvant is fully emulsified (is placed in emulsification product after fully emulsified
In water, see whether it scatters, not scattering, this illustrates that emulsification is abundant), first immunisation injection by GST-MrNV-CP recombinant proteins with etc.
The vaccine that volume complete Freund's adjuvant is emulsified into, for the second time and third time inoculation is by GST-MrNV-CP recombinant proteins and not
The vaccine that complete Freund's adjuvant is emulsified into.
2. immune：10 plumage of healthy hens is chosen, 1ml vaccines is taken to be injected in two wing of hen and both sides chest muscle, every injection
0.25ml is immunized once every 2 weeks, and specific immune programme, which see the table below, to be started to collect egg after 1, head exempts from, and 4 DEG C save backup,
It is negative control to choose the egg before being immunized.
1 immune programme of table
3. the extraction purification of Yolk antibody water-soluble component (WSF)：By the egg of collection, sterilized with 5% bromogeramine
30min dries；Yolk is detached with Yolk separator, is rolled on filter paper, removes vitellinae membrana as possible, yolk is drawn with syringe
In graduated cylinder, the sterile purified water of 9 times of volumes precooling is added, fully shakes up, 4 DEG C overnight, 4 DEG C, 10000rpm/min centrifugations
30min, it is Yolk antibody water-soluble component (WSF) to collect supernatant.
Embodiment 4：The titration of Macrobrachium rosenbergii open country field virus Yolk antibody
By the carbonate buffer solution (Na of GST-MrNV-CP recombinant proteins pH=9.62CO30.15g, NaHCO30.293g,
Add the HCl tune pH to 9.6 for using 1mol/L after distilled water 90mL, be finally settled to 100mL) be diluted to 10ng/mL be coated with 100 μ L in
96 hole elisa Plates, 4 DEG C overnight；With 100 μ L, 5% skimmed milk powers 2h, 200 μ L washing buffer PBST (PBS+ are closed in 37 DEG C
It 0.05%Tween-20) washs 4 times, is incubated 3min every time, pats dry.The phosphate of WSF pH=7.6 prepared by embodiment 3
Buffer solution presses 1:6400,1:12800,1：25600,1:100 μ L, 37 DEG C of incubation 2h are added per hole for the dilution of 51200 constant gradients, with phase
The negative WSF of extension rate is answered to compare；PBST is washed 4 times, and 100 μ of rabbit anti-chicken IgG of 1: 5000 diluted HRP labels is added
L, 37 DEG C of incubation 1h；TMB (3', 3', 5', 5'- tetramethyl benzidine) substrate colour reagent box colour developing 20min is finally used, is added
The H of 2mol/L2SO4Color development stopping reads OD in microplate reader (Infinite M200PRO)450Value.P/N >=2.1, as potency
Value, titration is the results show that Macrobrachium rosenbergii open country field virus Yolk antibody valence value is 102400.
Embodiment 5：The Western blot specific detections of Macrobrachium rosenbergii open country field virus Yolk antibody
GST-MrNV-CP recombinant proteins are detached by SDS-PAGE, adhesive tape is cut to suitable size after electrophoresis, by glue
It is handled with filter paper transferring film buffer solution, cutting out and being immersed in transferring film buffer solution with the pvdf membrane that methyl alcohol process is crossed in advance, with half
Dry method is by protein delivery to pvdf membrane, 80mA, transferring film 9min.Pvdf membrane is taken out after the completion of transferring film to be dipped in containing 5% skimmed milk power
TBST in, liquid must cover film whole, close 1h on room temperature shaker, wash film 3 times with TBST, each 5min.Film is soaked
Enter the WSF (1 of the Macrobrachium rosenbergii open country field viral capsid proteins of the preparation of embodiment 3:400 dilutions) in, it is incubated at room temperature 1h, TBST washes film
3 each 5min are added 1:The rabbit anti-chicken IgG of 3000HRP labels, 37 DEG C of incubation 1h, is shown after washing with DAB colour reagent boxes
Color.The result shows that there is the Yolk antibody of specificity.
Embodiment 6：Luo pond crayfish fries immunoprotection experiment
(1) prepared by bait：By beating eggs, 50-100 milliliters of water are added according to an egg, yolk and egg white is abundant
Stirring, steams 10-15 minutes in boiling water, is prepared into the bait of shrimp seedling；Experimental group adds in above-mentioned bait according to every 1 kilogram of bait
Enter 200 microlitres of serum amount additions containing Yolk antibody, control group is not added with Yolk antibody；
(2) it feeds：Bait feeding Luo pond crayfish fries are continuously fed one week 6 times a day；
(3) poison is attacked：Shrimp seedling is divided into 2 groups, one group is the shrimp seedling for having fed the not bait of Yolk antibody, and one group is
Lecithality antibody one week shrimp seedling of bait has been fed, has been added in water containing Macrobrachium rosenbergii open country field virus, impregnate and attacks poison.
As a result：By infection in 2 weeks, there is muscular death symptom and reaches in the shrimp seedling for feeding the not bait of Yolk antibody
65%；It has fed lecithality antibody one week shrimp seedling of bait and muscular death symptom has not occurred, immune protective rate reaches
100%, show that the Yolk antibody prepared has protection shrimp body, protects it from poisoning intrusion.
Claims (5)
1. a kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody, which is characterized in that be prepared by following methods：
（1）The cDNA of Macrobrachium rosenbergii open country field viral capsid proteins gene is cloned by RT-PCR method, gluathione is arrived in then recombination
In peptide S- transferase integrative gene expression vectors pGEX-5x-1, pGEX-5x-1-MrNV-CP recombinant plasmids are built into, it is described
Containing glutathione S-transferase and Macrobrachium rosenbergii open country field viral capsid proteins in pGEX-5x-1-MrNV-CP recombinant plasmids
Fusion；The molecular weight of the GST-MrNV-CP recombinant proteins is 66kDa ~ 68kDa；The pGEX-5x-1-MrNV-CP weights
Group plasmid construction method be：DNA and pGEX-5x-1 carriers are subjected to double digestion respectively, then be attached be obtained by the reaction containing
The pGEX-5x-1-MrNV-CP of the fusion of glutathione S-transferase and Macrobrachium rosenbergii open country field viral capsid proteins recombinates matter
Grain；
（2）Expression of the recombinant plasmid pGEX-5x-1-MrNV-CP in Escherichia coli：PGEX-5x-1-MrNV-CP is recombinated into matter
Grain conversion Escherichia coli, next day, which selects the Escherichia coli single bacterium on conversion tablet and falls in the culture medium containing kanamycins, to be expanded
Culture induces the fusion of glutathione S-transferase and Macrobrachium rosenbergii open country field viral capsid proteins in large intestine bar using IPTG
It is expressed in bacterium；Thalline is collected, is extracted using inclusion body purification technique and purifies to obtain GST-MrNV-CP recombinant proteins；
（3）Bird inlay is immunized with GST-MrNV-CP recombinant proteins, the produced egg of immune hen is collected, from collected egg
Middle extraction simultaneously purifies Yolk antibody；It is described to be immunized as two wings and the injection of both sides chest muscle；The immune number is 3 times, is exempted from every time
The time interval of epidemic disease is 2 weeks；The immunization ways are：First immunisation injection by GST-MrNV-CP recombinant proteins in equal volume it is complete
The vaccine that full Freund's adjuvant is emulsified into, for the second time and third time inoculation is by GST-MrNV-CP recombinant proteins and not exclusively not
Family name's adjuvant emulsion at vaccine.
2. Macrobrachium rosenbergii open country according to claim 1 field viral capsid proteins Yolk antibody, which is characterized in that the RT-
PCR method is：Using Macrobrachium rosenbergii open country field virus as template, by the viral capsid proteins gene reverse transcription of Macrobrachium rosenbergii open country field at first
Chain cDNA, then obtain double-stranded DNA with primer amplified.
3. Macrobrachium rosenbergii open country according to claim 1 field viral capsid proteins Yolk antibody, which is characterized in that the recombination
Plasmid pGEX-5x-1-MrNV-CP is expressed as in Escherichia coli：PGEX-5x-1-MrNV-CP recombinant plasmid transformed large intestine bars
Bacterium, the single bacterium that next day is selected on conversion tablet is fallen in the LB liquid medium containing kanamycins, in 37 DEG C, 200 revs/min
Constant-temperature table culture is carried out, when the OD values of Escherichia coli reach 0.4 ~ 0.6, the IPTG of final concentration of 1 mmol/L is added,
25 ~ 37 DEG C induce 4 ~ 8 hours.
4. Macrobrachium rosenbergii open country according to claim 1 field viral capsid proteins Yolk antibody, which is characterized in that step（3）
The method extracted from egg and purify Yolk antibody is：The egg white of collected egg and yolk are detached, yolk is taken, goes
Fall vitellinae membrana, sterile purified water be then added, fully shake up, be placed in 4 DEG C under the conditions of overnight after, it is that yolk is anti-to collect supernatant
Body.
5. any Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody of claim 1 ~ 4 is preparing the anti-Macrobrachium rosenbergii of shrimp
Application in wild field virus vaccine preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510150327.1A CN104725504B (en) | 2015-03-31 | 2015-03-31 | A kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510150327.1A CN104725504B (en) | 2015-03-31 | 2015-03-31 | A kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104725504A CN104725504A (en) | 2015-06-24 |
| CN104725504B true CN104725504B (en) | 2018-10-30 |
Family
ID=53449978
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510150327.1A Active CN104725504B (en) | 2015-03-31 | 2015-03-31 | A kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104725504B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003070258A1 (en) * | 2002-02-21 | 2003-08-28 | Lee & Joe Biotech Co. | Anti-white spot syndrome virus igy |
| CN1583792A (en) * | 2004-06-05 | 2005-02-23 | 大连理工大学 | Vitellus immune globulin for preventing prawn virus, tis preparing method and use thereof |
| CN1603830A (en) * | 2003-09-30 | 2005-04-06 | 浙江省淡水水产研究所 | Macrobrachium rosenbergii muscle turbid urine virus enzyme-linked immunologic diagnosis kit and detection method thereof |
| CN103122393A (en) * | 2012-11-30 | 2013-05-29 | 杭州三合创新科技有限公司 | Macrobrachium rosenbergii nodavirus detection kit and detection method |
-
2015
- 2015-03-31 CN CN201510150327.1A patent/CN104725504B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003070258A1 (en) * | 2002-02-21 | 2003-08-28 | Lee & Joe Biotech Co. | Anti-white spot syndrome virus igy |
| CN1603830A (en) * | 2003-09-30 | 2005-04-06 | 浙江省淡水水产研究所 | Macrobrachium rosenbergii muscle turbid urine virus enzyme-linked immunologic diagnosis kit and detection method thereof |
| CN1583792A (en) * | 2004-06-05 | 2005-02-23 | 大连理工大学 | Vitellus immune globulin for preventing prawn virus, tis preparing method and use thereof |
| CN103122393A (en) * | 2012-11-30 | 2013-05-29 | 杭州三合创新科技有限公司 | Macrobrachium rosenbergii nodavirus detection kit and detection method |
Non-Patent Citations (2)
| Title |
|---|
| 罗氏沼虾Nodavirus的分离、致病性与检测技术研究;钱冬;《中国博士学位论文全文数据库 农业科技辑》;20070115(第1期);第二部分第二章 * |
| 罗氏沼虾诺达病毒单克隆抗体的制备及应用;刘问，等;《水产学报》;20050830;第29卷(第4期);摘要，第3节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104725504A (en) | 2015-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103585625B (en) | A kind of porcine epizootic diarrhea recombinant baculovirus genetic engineering subunit vaccine and preparation method thereof and application | |
| CN111632137B (en) | Triple vaccine for feline calicivirus disease, feline infectious rhinotracheitis and feline panleukopenia as well as preparation method and application thereof | |
| CN103436553B (en) | A kind of method preparing restructuring coxsackie virus A 16-type virus-like particle | |
| CN101905021B (en) | Trigeminal live vaccine of canine distemper viruses, canine parvoviruses and Type I canine adenoviruses and preparation method thereof | |
| CN105801707A (en) | Oral vaccine for treating grass carp hemorrage as well as preparation and application thereof | |
| CN107653260A (en) | A kind of preparation method and application of Recombinant Lactococcus lactis | |
| CN103525855B (en) | A kind of method preparing restructuring enterovirus EV 71 virus-like particle | |
| CN104725503B (en) | A kind of Fugu rubripes nervous necrosis virus capsid protein Yolk antibody and its application | |
| CN106540249A (en) | A kind of bird flu (H5N1) or the antigen concentrating and purifying process of Porcine reproductive and respiratory syndrome (PRRS) viral vaccine | |
| CN106139140B (en) | A kind of Muscovy duck parvovirus subunit vaccine | |
| CN105198989B (en) | Anti-yellowing sea Shewanella Yolk antibody and preparation method thereof | |
| CN106011084A (en) | Porcine epidemic diarrhea virus local variant and application thereof | |
| CN101016541A (en) | Method of producing brucella vaccine antigen protein | |
| CN114561366B (en) | Goat kuraria virus isolate and application thereof | |
| CN101380469A (en) | Production technique of turbot reddish body iridovirus inactivated vaccine | |
| CN103013897A (en) | Lactobacillus casei ghost, preparation method and application thereof | |
| CN105349562A (en) | Recombinant vector and recombinant strain for expressing PPV (porcine parvovirus) VP2 protein and applications of recombinant vector and recombinant strain | |
| CN102727879A (en) | Spinner bottle production method of inactivated singapore grouper iridovirus (SGIV) vaccine | |
| CN110124025A (en) | A kind of bird flu and 4 type bigeminy genetic engineering subunit vaccine of aviadenovirus and preparation method thereof | |
| CN108079287A (en) | A kind of subunit vaccine of grouper irido virus and its preparation method and application | |
| CN104725504B (en) | A kind of Macrobrachium rosenbergii open country field viral capsid proteins Yolk antibody and its application | |
| CN1411378A (en) | Method for production of egg containing anti-pathogenic bacteria specific antibodies (IgY) and yogurt and ice cream containing IgY | |
| CN103993032A (en) | Method for preparing recombinant poliovirus-like particles | |
| CN101979502A (en) | Recombinant brucella expressing VP1 gene of O-type foot-and-mouth disease virus and method for producing vaccines thereof | |
| CN107129530A (en) | A kind of extracting method of bursopoietin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |