CN104725503B - A kind of Fugu rubripes nervous necrosis virus capsid protein Yolk antibody and its application - Google Patents
A kind of Fugu rubripes nervous necrosis virus capsid protein Yolk antibody and its application Download PDFInfo
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Abstract
The invention belongs to biotechnologies, and in particular to a kind of Fugu rubripes nervous necrosis virus capsid protein Yolk antibody and its application.The Yolk antibody is prepared by following methods：By in the recombination to GST integrative gene expression vector pGEX 5x 1 of Fugu rubripes nervous necrosis virus capsid protein gene, it is built into 1 CP recombinant plasmids of pGEX 5x；By 1 CP recombinant plasmid transformeds Escherichia coli of pGEX 5x and induced fusion gene expression；It is extracted using inclusion body purification technique and purifies to obtain GST CP recombinant proteins；With GST CP recombinant protein immune hens, collects egg and extract Yolk antibody from egg.Yolk antibody prepared by the present invention can effectively inhibit Fugu rubripes nervous necrosis virus, and potency is high, have good immune protection effectiveness, can be added in fish feed as anti-nervous necrosis virus vaccine in the postlarva phase of fish.
Description
Invention field
The invention belongs to biotechnologies, and in particular to a kind of Fugu rubripes nervous necrosis virus capsid protein yolk
Antibody and its application.
Technical background
Nervous necrosis virus (Nervous necrosis virus, NNV) is a kind of fish of worldwide prevalence
Cause of disease, prevalence can cause 30 in countries and regions such as China's Mainland, Taiwan, Japan, Australia, Britain, Canada, Norway
The postlarva of a variety of Important Economic fish, prelarva even grouper adult fish massive mortality, seriously endanger the healthy aquaculture of fish.
The research of this virosis is the research hotspot of international aquatic products disease.Belong to nodaviridae in nervous necrosis virus classification
(Nodaviridae), B-mode nodavirus category (Betanodavirus).About 30-40 nanometers of virion diameter, 20 faces
Body, no cyst membrane.Nervous necrosis virus is single normal chain, 2 segment RNA virus.Viral genome is made of RNA1 and RNA2.RNA1
Encode viral rna polymerase；RNA2 encoding capsid proteins, and viral exclusive architecture albumen, and virus infection are closely related,
It is the important albumen for studying viral vaccine.Nervous necrosis virus main infection fry, illness fish are visible in brain and retina
Apparent vacuole can cause the fry death rate up to 90% or more.To this virosis, there are no specific medicaments to treat at present, epidemic disease
Seedling is the important means for preventing such disease.But up to the present, there are no the vaccines of commercialization.Patent
CN200410027186 discloses a kind of nervous necrosis virus recombinant protein vaccine, but the vaccine has the following disadvantages：(1) refreshing
It is in the postlarva phase through necrosis virus infection fish, postlarva generates the ability of antibody not yet at this time, therefore such vaccine can not be
The postlarva phase uses, therefore application value is little；(2) this vaccine needs to prevent epidemic by injection, for the mankind and herding,
Since fry is smaller, in water, injecting immune method takes, inconvenient, thus greatly limits the commercialization of the vaccine for life
It promotes.
Invention content
The purpose of the present invention is to provide a kind of Fugu rubripes nervous necrosis virus capsid protein Yolk antibody and its answer
With.
In order to solve the above technical problems, the present invention uses following technical scheme：
A kind of Fugu rubripes nervous necrosis virus capsid protein Yolk antibody, is prepared by following methods：
(1) DNA that Fugu rubripes nervous necrosis virus capsid protein gene is cloned by RT-PCR method, then recombinates
Into glutathione S-transferase integrative gene expression vector pGEX-5x-1, pGEX-5x-1-CP recombinant plasmids are built into, it is described
Fusion containing glutathione S-transferase and nervous necrosis virus capsid protein in pGEX-5x-1-CP recombinant plasmids；
(2) expression of the recombinant plasmid pGEX-5x-1-CP in Escherichia coli：By pGEX-5x-1-CP recombinant plasmid transformeds
Escherichia coli, next day, which selects the Escherichia coli single bacterium on conversion tablet and falls within, expands training in the culture medium containing ampicillin
It supports, using the fusion of IPTG induction glutathione S-transferases (GST) and nervous necrosis virus capsid protein in large intestine bar
It is expressed in bacterium；Thalline is collected, is extracted using inclusion body purification technique and purifies to obtain GST-CP recombinant proteins；
(3) it uses GST-CP recombinant proteins that bird inlay is immunized, the produced egg of immune hen is collected, from collected egg
It extracts and purifies Yolk antibody.
By said program, the molecular size range of the GST-CP recombinant proteins is 66kDa~68kDa.
By said program, the RT-PCR method is：Using Fugu rubripes nervous necrosis virus as template, using specificity
Fugu rubripes nervous necrosis virus capsid protein gene reverse transcription is obtained double-strand by primer at the first chain cDNA, then PCR amplification
cDNA。
By said program, the construction method of the pGEX-5x-1-CP recombinant plasmids is：By cDNA and pGEX-5x-1 carriers
Double digestion is carried out respectively, then is attached and is obtained by the reaction containing glutathione S-transferase and nervous necrosis virus capsid protein
The pGEX-5x-1-CP recombinant plasmids of fusion.
By said program, the recombinant plasmid pGEX-5x-1-CP is expressed as in Escherichia coli：pGEX-5x-1-CP
Recombinant plasmid transformed Escherichia coli, the single bacterium that next day is selected on conversion tablet fall within the LB liquid medium containing ampicillin
In, it is added dense eventually in 37 DEG C, 200 revs/min of progress constant-temperature table cultures when the OD values of Escherichia coli reach 0.4~0.6
Degree is the IPTG 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-CP recombinant proteins with it is isometric completely not
Family name's adjuvant emulsion at vaccine, second and third time inoculation emulsified by GST-CP recombinant proteins and incomplete Freund's adjuvant
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 Fugu rubripes nervous necrosis virus capsid protein Yolk antibody is preparing the anti-nervous necrosis virus epidemic disease of fish
Application in seedling product.
Beneficial effects of the present invention：The Yolk antibody expression that the method for the present invention is prepared is continual and steady, neutralized experiment
It confirms, the Yolk antibody prepared by the present invention can effectively inhibit Fugu rubripes nervous necrosis virus, and potency is high, have good
Immune protection effectiveness, can be added in fish feed as anti-nervous necrosis virus vaccine in the postlarva phase of fish,
Possibility is provided for the commercialization popularization of fish nervous necrosis virus 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 Fugu rubripes nervous necrosis virus capsid protein gene pGEX-5x-1-CP recombinant plasmids
Using the total serum IgE of the fish cell extraction of Fugu rubripes nervous necrosis virus infection as template, using downstream primer
CP-NotI-BW:GATGCGGCCGCTTAGTTTCCCGAGTCAACCC, by Fugu rubripes nervous necrosis virus capsid protein base
The first chain cDNA is generated because carrying out RT-PCR reactions, the reagent that RT-PCR reacts is：Reverse transcriptase, dNTP, buffer solution；Condition
For：75℃,5min；42 DEG C, 1 hour；80℃,10min.Then, using the cDNA of acquisition as template, sense primer CP- is used
SalI-FW:5 '-GGTCGACTCATGGTACGCAAGGGTGATAAG-3 ' and downstream primer CP-NotI-BW:
GATGCGGCCGCTTAGTTTCCCGAGTCAACCC, PCR amplification obtain double-stranded DNA.PCR reaction reagent be：High-fidelity 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.By reaction product on 1% agarose electrophoresis
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 god
PGEX-5x-1-CP recombinant plasmids through necrosis virus capsid protein fusion.The coupled reaction system is：2 μ l of DNA,
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-CP in Escherichia coli
By pGEX-5X-1-CP recombinant plasmid transformed Escherichia coli, the single bacterium that next day is selected on conversion tablet is fallen within containing ammonia
In the LB liquid medium of parasiticin, in 37 DEG C, 200 revs/min of progress constant-temperature table cultures, when the OD values of Escherichia coli reach
When to 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 centrifuges 10min,
Collect thalline.High pressure is crushed thalline, and the suspension after bacterial cell disruption collects supernatant respectively through 4 DEG C, 12,000rpm centrifugation 10min
And precipitation.Albumen largely is extracted using inclusion body purification technique to the precipitation of collection, 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, be placed in 50 times of volume dialyzates (0.1M Tris-HCl, 5mM EDTA,
5mMCysteins, pH8.0) in, 4 DEG C of dialysis 16h or more；It is placed in above-mentioned dialyzate (1L) 4 DEG C of dialysis 16h or more again, 4 DEG C
Refrigerated centrifuge 10,000rpm10min retains supernatant, removal precipitation.Then SDS-PAGE Detection and Extraction GST-CP is used to recombinate egg
The concentration of the molecular weight of white concentration, purity and recombinant protein, the recombinant protein of testing result display extraction can reach 5 millis
More than grams per milliliter, for purity up to 99% or more, the molecular weight of recombinant protein is 68kD.It is extracted using inclusion body combination SDS-PAGE
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 Fugu rubripes nervous necrosis 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 is by GST-CP recombinant proteins and in equal volume
The vaccine that complete Freund's adjuvant is emulsified into, for the second time and third time inoculation is by GST-CP recombinant proteins and incomplete Freund
The vaccine that agent 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, yolk is rolled on filter paper, removes vitellinae membrana as possible, is inhaled with syringe
It takes egg yolk liquid 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 centrifuges 30min, and it is Yolk antibody water-soluble component (WSF) to collect supernatant.
Embodiment 4：The titration of Fugu rubripes nervous necrosis virus Yolk antibody
By the carbonate buffer solution (Na of GST-CP recombinant proteins pH=9.62CO30.15g, NaHCO30.293g adds double
The HCl tune pH to 9.6 that 1mol/L is used after steaming water 90mL, is finally settled to 100mL) 100 μ L of 10ng/mL coatings are diluted in 96 holes
ELISA Plate, 4 DEG C overnight；With 100 μ L, 5% skimmed milk powers 2h, 200 μ L washing buffer PBST (PBS+0.05% are closed in 37 DEG C
Tween-20 it) washs 4 times, is incubated 3min every time, pats dry.WSF prepared by embodiment 3 is pressed with the phosphate buffer of pH=7.6
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, accordingly to dilute again
Several negative WSF are compareed；PBST is washed 4 times, the 100 μ L of rabbit anti-chicken IgG of 1: 5000 diluted HRP labels is added, 37 DEG C incubate
Educate 1h；TM B (3', 3', 5', 5'- tetramethyl benzidine) substrate colour reagent box colour developing 20min is finally used, is added 2mol/L's
H2SO4Color development stopping reads OD450 values in microplate reader (Infinite M200PRO).P/N >=2.1, as valence value, potency are surveyed
Determine the results show that Fugu rubripes nervous necrosis virus Yolk antibody valence value is 51200.
Embodiment 5：The Western blot specific detections of Fugu rubripes nervous necrosis virus Yolk antibody
GST-CP recombinant proteins are detached by SDS-PAGE, adhesive tape is cut to suitable size after electrophoresis, by glue and filter
Paper is handled with 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, uses semidry method
By on 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
In TBST, liquid must cover film whole, close 1h on room temperature shaker, wash film 3 times with TBST, each 5min.Film is immersed
The WSF (1 of Fugu rubripes nervous necrosis virus capsid protein prepared by embodiment 3:400 dilutions) in, it is incubated at room temperature 1h, TBST
3 each 5min of film are washed, are added 1:The rabbit anti-chicken IgG of 3000HRP labels, 37 DEG C of incubation 1h, uses DAB colour reagent boxes after washing
Colour developing.The result shows that the Yolk antibody of specificity can be generated.
Embodiment 6：The neutralization test of Fugu rubripes nervous necrosis virus Yolk antibody
This experiment is detected with experiment for WB in 6 orifice plates, 12 orifice plates are examined with IF using the method for fixed virus dilution antibody
It surveys, 3 repetitions of each group setting.
(1) antibody dilutes：When neutralization activity detects, WSF is done 1:20、1:40、1:80、1:160、1:320、1:640、1:
1280、1:2560、1:5120 dilutions.
(2) Fugu rubripes nervous necrosis virus and antibody incubation：From -80 DEG C of taking-up Fugu rubripes nervous necrosis virus
Virus takes 200 μ l virus liquids and antibody, and 1.5h is incubated in 28 DEG C.
(3) SSN-1 cells are infected：Above-mentioned antibody-viral incubation mixed liquor is taken out from 28 DEG C, shakes mixing.With shifting
Liquid rifle sops up the culture medium in orifice plate, and 200 μ l antibody virus mixed liquors are added in 6 orifice plates, add 5%FBS-MEM culture mediums mend to
1ml, 12 orifice plates are added 100 μ l antibody virus mixed liquors, add 5%FBS-MEM culture mediums to mend to 500ul, while totivirus is arranged
It organizes and virus-free group.
(4) it is incubated：Fugu rubripes nervous necrosis virus infection cell is in 28 DEG C of CO2It is incubated, observes in cell incubator
The lesion situation in each hole.
The result shows that：Antibody is being diluted to 1:When 2560, still there is the ability for neutralizing virus infection.This shows system of the present invention
Standby Fugu rubripes nervous necrosis virus Yolk antibody has higher potency.
Claims (5)
1. a kind of Fugu rubripes nervous necrosis virus capsid protein Yolk antibody, which is characterized in that be prepared by following methods
It arrives：
（1）The DNA of Fugu rubripes nervous necrosis virus capsid protein gene is cloned by RT-PCR method, paddy is arrived in then recombination
In the sweet peptide S- transferases integrative gene expression vector pGEX-5x-1 of Guang, pGEX-5x-1-CP recombinant plasmids are built into, it is described
Fusion containing glutathione S-transferase and nervous necrosis virus capsid protein in pGEX-5x-1-CP recombinant plasmids；Institute
The construction method for stating pGEX-5x-1-CP recombinant plasmids is：DNA and pGEX-5x-1 carriers are subjected to double digestion respectively, then are carried out
The pGEX-5x-1- of the fusion containing glutathione S-transferase and nervous necrosis virus capsid protein is obtained by the reaction in connection
CP recombinant plasmids；
（2）Expression of the recombinant plasmid pGEX-5x-1-CP in Escherichia coli：By pGEX-5x-1-CP recombinant plasmid transformed large intestines
Bacillus, next day, which selects the Escherichia coli single bacterium on conversion tablet and falls within, expands culture in the culture medium containing ampicillin, profit
Induce the fusion of glutathione S-transferase and nervous necrosis virus capsid protein in expression in escherichia coli with IPTG；It receives
Collect thalline, is extracted using inclusion body purification technique and purify to obtain GST-CP recombinant proteins；Point of the GST-CP recombinant proteins
Son amount size is 66kDa ~ 68kDa；
（3）Bird inlay is immunized with GST-CP recombinant proteins, collects the produced egg of immune hen, is extracted from collected egg
And purify 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, when immune every time
Between between be divided into 2 weeks；The immunization ways are：First immunisation injection is by GST-CP recombinant proteins and isometric complete Freund's adjuvant breast
The vaccine of chemical conversion, second and the epidemic disease that is emulsified by GST-CP recombinant proteins and incomplete Freund's adjuvant of third time inoculation
Seedling.
2. Fugu rubripes nervous necrosis virus capsid protein Yolk antibody according to claim 1, which is characterized in that institute
Stating RT-PCR method is：Using Fugu rubripes nervous necrosis virus as template, by Fugu rubripes nervous necrosis virus capsid egg
White gene reverse transcription obtains double-stranded DNA at the first chain cDNA, then with primer amplified.
3. Fugu rubripes nervous necrosis virus capsid protein Yolk antibody according to claim 1, which is characterized in that institute
Recombinant plasmid pGEX-5x-1-CP is stated to be expressed as in Escherichia coli：PGEX-5x-1-CP recombinant plasmid transformed Escherichia coli,
The single bacterium that next day is selected on conversion tablet is fallen in the LB liquid medium containing ampicillin, 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. Fugu rubripes nervous necrosis virus capsid protein Yolk antibody according to claim 1, which is characterized in that step
Suddenly（3）The method extracted from egg and purify Yolk antibody is：The egg white of collected egg and yolk are detached, egg is taken
Huang removes vitellinae membrana, and sterile purified water is then added, fully shake up, be placed in 4 DEG C under the conditions of overnight after, collect supernatant be ovum
Yellow antibody.
5. any Fugu rubripes nervous necrosis virus capsid protein Yolk antibody of claim 1 ~ 4 is preparing fish with anti-god
Through the application in necrosis virus vaccine product.
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| CN106591328B (en) * | 2016-12-15 | 2019-07-05 | 斯澳生物科技（苏州）有限公司 | A kind of preparation method and applications of grouper nervous necrosis virus capsid protein |
| CN108251356B (en) * | 2017-12-29 | 2021-06-22 | 中山大学 | Fish egg cleaning solution for resisting fish nervous necrosis virus |
| CN110540996B (en) * | 2019-08-29 | 2022-01-28 | 华中农业大学 | Procambarus clarkii i-type lysozyme gLysi2 gene, gLysi2 protein coded by same and application thereof |
| CN113683665A (en) * | 2021-04-29 | 2021-11-23 | 山东农业大学 | Preparation method of circovirus type 3 egg yolk antibody |
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