CN106350475A - Novel bacterial artificial chromosome of turkey herpes virus, construction method and application - Google Patents
Novel bacterial artificial chromosome of turkey herpes virus, construction method and application Download PDFInfo
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Abstract
The invention provides a novel bacterial artificial chromosome of a turkey herpes virus, a construction method and application and relates to the field of animal medicine. The bacterial artificial chromosome is obtained by knocking out gC gene in HVT genome and replacing with a mini-F plasmid sequence. The construction method includes: amplifying upstream and downstream homologous arms of the gC gene in the HVT genome, and inserting into a pUC19 vector to obtain a recombinant vector A; inserting a mini-F plasmid segment into the recombinant vector A to obtain a recombinant transfer vector B, performing cotransfection (CEF) with DNA of HTV, and screening to obtain mini-F recombinant HVT; extracting DNA of the mini-F recombinant HVT to convert DH10B cells, adopting chloromycetin for screening to obtain positive recombinant bacteria, culturing, and then extracting plasmid DNA. The bacterial artificial chromosome has high stability, and the construction method is simple, efficiency and applicable to construction of recombinant vector live vaccines.
Description
Technical field
The present invention relates to field of animal medicine and in particular to a kind of bacterial artificial chromosome of new herpes turkey viruss,
Construction method and application.
Background technology
Herpes turkey viruss (herpesvirus of turkey, hvt) are belonging to chicken Marek's disease serum virus iii
A kind of α herpesviruss of type.Early stage research finds, on hereditism and serology, hvt and marek's disease viruses (marek ' s
Disease virus, mdv) there is dependency, and hvt does not have pathogenic to chicken, therefore, hvt fc-126 strain is widely used in
The Marek (marek ' s disease, md) of prevention chicken.
Hvt is extremely safe to chicken, does not produce any side reaction；It can carry out inoculation in embryo or the early stage inoculation of 1 age in days is exempted from
Epidemic disease, is not only avoided that the interference of maternal antibody but also starts immunity soon；Duration of immunity is long, the virus that virus can last up to 72 weeks
Mass formed by blood stasis.Hvt virion size is about 160nm, contains double-strand dna that size is about 158kbp in icosahedral nucleocapsid
Genome (genbank:af291866).Hvt genome is big, the characteristic of non-pathogenic and life-long infection already causes people couple
It is as the interest of virus live vector purposes.At present, abroad successful Application hvt expresses multiple heterologous antigens, such as chicken new city
Epidemic disease poison (newcastle disease, ndv) f antigen (- nd-sb (msd animal health) andHvt-ndv (ceva)) and chicken infectivity bursa of Fabricius virus (infectious bursal disease
Virus, ibdv) vp2 antigen (Hvt+ibd (merial) andHvt-ibd (ceva)) etc., exhibition
Show hvt as the applications well prospect of live vaccine vectors.
The common method building herpesviruss recombinant vector live vaccine has homologous recombination or sticking grain regeneration, and screening technique is usual
It is by lac-z or gfp labelling etc..However, during the viral dna of application is recombinated, Virus culture is loaded down with trivial details, and different
The viral genome of generation usually can morph, and leads to antigenicity unstable.
The bacterial artificial chromosome (bacterial artificial chromosome, bac) of herpesviruss is in recent years
The new technique growing up, can greatly improve the efficiency building recombinant herpesvirus.The structure of the hvt bac of report is
The us2 area that mini-f sequence is inserted hvt genome has infective clone although obtaining, but mini-f restructuring disease
Poison is very unstable, and some restructuring poison lose multiplication capacity after passing on, and save, from infection clones, the virus obtaining
Growth characteristics are also inconsistent, and some viral multiplication capacities weaken significantly, and these results show can by mini-f insertion us2 site
Can there is interference effect to the normal replication of hvt.
Content of the invention
The present invention provides a kind of bacterial artificial chromosome of herpes turkey viruss, and this bacterial artificial chromosome has higher
Stability, can apply to the structure of recombinant vector live vaccine.
It is a further object of the present invention to provide the bacterial artificial chromosome of herpes turkey viruss, this construction method is simple, high
Effect.
Another object of the present invention is to provide described bacterial artificial chromosome building herpes turkey viruss carrier live
Application in strain.
The purpose of the present invention adopts the following technical scheme that realization.
A kind of bacterial artificial chromosome of new herpes turkey viruss, is by the gc gene knockout in hvt genome, and replaces
It is changed to mini-f plasmid sequence.
The present invention also provides the method building described bacterial artificial chromosome, comprises the steps:
(1) expand gc gene upstream and downstream homology arm in hvt genome respectively, homology arm insertion puc19 in upstream and downstream is carried
Internal acquisition recombinant vector a；
(2) fragment that mini-f plasmid obtains after pac i enzyme action is inserted in recombinant vector a, obtain restructuring transfer and carry
Body b；
(3) by the dna cotransfection cef of recombinant transfer vector b and hvt, carry out homologous recombination, picking sends green fluorescence
Cytopathy plaque, then through stress medium screening, obtain mini-f restructuring hvt；Extract the dna conversion of mini-f restructuring hvt
Dh10b cell, using chloromycetin screening, obtains positive restructuring bacterium, after culture, picking plasmid dna, and obtain described bacteria artificial dye
Colour solid.
In the present invention, described stress medium be add in dmem final concentration of 10% serum, the green grass or young crops of 100u/ml
After mycin, the streptomycin of 100 μ g/ml, the hypoxanthine of the mycophenolic acid of 350 μ g/ml, the xanthine of 80 μ g/ml and 100 μ g/ml
Obtain.
In the present invention, with hvt genome as template, with seq id no:1 and seq id no:2 for primer amplification gc base
Because of upstream homology arm；With seq id no:3 and seq id no:4 for primer amplification gc downstream of gene homology arm.
In the present invention, the sequence of described upstream homology arm is that the sequence of downstream homology arm is such as shown in seq id no:5
Shown in seq id no:6.
The present invention also provides application in building herpes turkey viruss carrier live strain for the described bacterial artificial chromosome.
In the present invention, the method building herpes turkey viruss carrier live strain using described bacterial artificial chromosome is such as
Under:
(1) pass through the sequence of the method acquisition exogenous gene of pcr method or synthetic, then connect at its upstream and open
Mover, terminator sequence in downstream connection, it is built into exogenous gene expression box；
(2) bacterial artificial chromosome described in claim 1 is proceeded to engineering bacteria gs1783, then by described exogenous gene table
Reach box to insert in described bacterial artificial chromosome, be built into restructuring hvt bac；
(3) the restructuring hvt bac transfection chick embryo fibroblast constructed by (2) is carried out virus rescue, simultaneously by gc base
Because recovering, obtain recombinant herpesvirus of turkeys carrier live strain by choosing speckle purification.
Preferably in technical scheme, described exogenous gene expression box insert described bacterial artificial chromosome noncoding region or
Dispensable gene site.
The bacterial artificial chromosome of herpes turkey viruss of the present invention has higher stability, and it recovers poison and parent's strain
There is consistent growth characteristics and immunogenicity, therefore, it can be applied to the structure of recombinant vector live vaccine.The present invention builds fire
The method of chicken herpetoviruses bacterial artificial chromosome is simple, efficiently.The bacterial artificial chromosome of herpes turkey viruss of the present invention is
By the gc gene knockout in hvt genome, and replace with mini-f plasmid sequence, be structure and the improvement of hvt live vector vaccine
Reliable technology platform is provided.This hvt live vector vaccine diva vaccine, can distinguish the vaccine of infection animal and immune animal,
Because the prevention and control difficulty of some poultry infectious disease is big, therefore carrying out purification is maximally effective measure, and such as bird flu etc. has many
Developed country has completed to purify, and no longer carries out immunity inoculation, in China, with the development of animal husbandry and veterinary hygiene cause,
The prevention and control of many animal epidemics inevitable from immunoprophylaxises progressively excessively to based on purifying, during this transfer to can distinguish
Immune animal is very urgent with the demand of the diva vaccine of infection animal.By applying the antibacterial of the herpes turkey viruss of the present invention
Artificial chromosome bac-hvt (c1) strain can efficiently build herpes turkey viruss carrier live.
Brief description
The structural representation of Fig. 1 transfer vector puc19 (hvt)-h1-h2-mini-f.
The digestion verification collection of illustrative plates of Fig. 2 transfer vector puc19 (hvt)-h1-h2-mini-f.Wherein sering 1 is restricted interior
Enzyme cutting sca i enzyme action result, stripe size is respectively 10165,3775bp；Sering 2 is that restricted enzyme ecor i enzyme action is tied
Really, stripe size be respectively 7507,5095,1338bp；Sering 3 is puc19 (the hvt)-h1-h2-minif plasmid of non-enzyme action
Dna compares；m.15000bp dna marker.
Fig. 3 mini-f restructuring hvt virus plaques.A. wavelength is the green fluorescence that sends observed under 488nm ultraviolet light
Virus plaques；B. the plaque observed under white light.
The rflp collection of illustrative plates of Fig. 4 bac-hvt (c1) plasmid dna.Swimming lane m is dna marker, and other swimming lanes are plasmid dnas
Digestion products.
Fig. 5: hvtbac-gc-r(c1)Gc gene differentiate electrophoretogram, wherein m is dna marke, other swimming lanes be respectively each poison
Strain pcr product.
Fig. 6: hvt bac recovers poison and gc gene-deleted strain and growth kineticses curve on cef for the parent plant.A. three plants of recoveries
Poison with hvt fc-126 andIt is digested to the virus titer of individual cells through pancreatin；B. three plants are recovered poison and hvtfc-
126 HesThe titre of the cell-associated virus through cracking；C. recover malicious hvtbac-gc-r(c1)F1 generation, f10 generation and
The titre through the cell-associated virus of cracking for the f20 generation.
Specific embodiment
To further illustrate the present invention by the following examples, but scope and spirit of the present invention are not limited to listed enforcement
Example.
The structure of embodiment 1 transfer vector puc19 (hvt)-h1-h2-minif and identification
1st, experiment material
Spf Embryo Gallus domesticus are purchased from Beijing Cimmeria Wei Tong laboratory animal Technology Co., Ltd..Hvt fc-126 Strain is purchased
From China Institute of Veterinary Drug Control (cvcc av19).Puc19 plasmid is purchased from takara company.Mini-f plasmid is by Freie Uni Berlin
Institute of viruses nikolaus professor osterriender give and (is disclosed in: tischer b, smith g, osterrieder
n.en passant mutagenesis:a two step markerless red recombination
system.methods mol biol.2010；634:421-430.).Enzyme ecor i, hind iii, sma i, sca i, bamh
I and pac i and ex taq dna polymerase, and ligase and pcr reagent purchase be purchased from takara company；Dmem culture medium,
Pancreatin and new-born calf serum are purchased from sigma company；Other reagent are purchased from Nanjing raw work biology company limited.Plasmid extraction tries
Agent: solutions i: be added with 25mmol/l tris-hcl (ph8.0) 50mmol/l glucose, 10mmol/l edta and 2 milligrams/
The lysozyme of milliliter.In the naoh aqueous solution of solutions i i:200mmol/l containing 1% sds.Solutions i ii:3mol/l naac
(ph4.8) solution.Te buffer: be added with the edta of 1mmol/l in 10mmol/l tris-hcl (ph 7.5).
2nd, experimental technique and result
The design of 2.1pcr primer and pcr react
According to hvt fc-126 strain full-length genome sequence (genbank:af291866) having delivered, apply vector
The pcr primer (table 1) of nti software design gc gene upstream and downstream homology arm amplification.Add ecor at the two ends of two pairs of primers respectively
I, pac i, sma i, bamh, pac i and hind iii site and protection base, particular sequence is shown in Table 1.
Table 1 pcr primer
The system of pcr reaction is dna template 1ul of hvt fc-126 strain, taq polymerase 0.5ul, forward primer 2ul
(10pm), downstream primer 2ul (10pm), dntp mixture 4ul, 10 times of diluent 5ul, 25mm mg2+4ul, add water benefit extremely
50ul.
Pcr response procedures are: 94 DEG C of denaturations 3min；94 DEG C of degeneration 30sec, 51 DEG C of annealing 40sec, 72 DEG C of reactions
1min20sec, 10 circulations；94 DEG C of degeneration 30sec, 60 DEG C of annealing 40sec, 72 DEG C of reaction 1min20sec, 27 circulations；72
DEG C extend 10 minutes.
The preparation of 2.2 chick embryo fibroblast
Select 9-10 age in days well-developed spf Embryo Gallus domesticus, aseptic taking-up Embryo Gallus domesticus, decaptitate, extremity and internal organs, use Chinese krebs solution
Washing idiosome, is cut into the piece of tissue of grain of rice size, then washs 2-3 time, then digests 20-30 minute with 0.25% trypsin solution,
Suction out pancreatin, washed 2-3 time with Chinese krebs solution, add the appropriate Chinese krebs solution piping and druming containing serum and antibiotic, through 4-6 layer yarn
Cloth filters, and makes the cell suspension of every 1ml 100-150 containing living cells ten thousand, adds 1ml, put co in the hole of each 6 orifice plate2Culture
In case, to grow up to monolayer within 24 hours standby for 37 DEG C of cultures.
2.3 the extraction of viral dna
The infection multiplicity that hvt fc-126 strain presses 0.01 infects chick embryo fibroblast, when cytopathy (cpe) reaches 60%-
When 70%, multigelation 3 times together with cell by supernatant, 8000rpm is centrifuged 15min, takes supernatant standby as tissue extract.
Set the chick embryo fibroblast being uninfected by herpes turkey viruss as compareing, at the cell identical method by infection virus simultaneously
Reason.
The extraction of viral dna: 1. take tissue extract 445 μ l, add 10% sds solution 50 μ l, 20mg/ml protease
K solution 2.5 μ l, mixes；Put 56 DEG C of water-bath 1h, 99 DEG C of water-bath 10min, be cooled to room temperature；2. add tris saturated phenol 200 μ l,
Mix, 4 DEG C, be centrifuged 5min under the conditions of 12000rpm, take supernatant.Repeat step 1. 2. 2 times；3. add tris saturated phenol 100 μ l
With chloroform 100 μ l, mix, 4 DEG C, be centrifuged 5min under the conditions of 12000rpm, take supernatant；4. add the vinegar of the 3mol/l of 1/10 volume
Acid sodium solution, and 1-2 times of volume dehydrated alcohol, mixing of turning upside down, under the conditions of putting -20 DEG C, 40min is to a couple of days；⑤4℃、
It is centrifuged 20min under the conditions of 12000rpm, abandons supernatant, precipitation is washed with 70% ethanol solution, 4 DEG C, centrifugation under the conditions of 12000rpm
1min, abandons supernatant, washing of precipitate 2 times；6. put in super-clean bench and air-dry, add the dissolving of 20-100 μ l sterile pure water, as hvt fc-
126 strain virus dna are standby.
The structure of 2.4puc19 (hvt)-h1
With the dna of hvt fc-126 strain as template, with hvt-b h1 f and hvt-b h1 r as primer, amplification gc gene
Upstream homology arm ul44 h1 (seq id no:5), carries out glue reclaim purification, and after demarcating concentration, application ecor i and sma i is carried out
Double digestion, recovery product is connected with the puc19 plasmid dna application ligase processing through same double digestion, conversion dh5 α impression
State cell, extracts dna to the clone obtaining and carries out sequencing, obtain recombiant plasmid puc19 (hvt)-h1.
The structure of 2.5p uc19 (hvt)-h1-h2
With the dna of hvt fc-126 strain as template, with hvt-b h2 f and hvt-b h2 r as primer, amplification gc gene
Downstream homology arm ul44 h2 (seq id no:6), carries out glue reclaim purification, applies bamh i and hind iii after demarcating concentration
Carry out double digestion, recovery product is connected with puc19 (the hvt)-h1 plasmid dna application ligase processing through same double digestion,
Conversion dh5 α competent cell, extracts dna to the clone obtaining and carries out sequencing identification, obtain recombiant plasmid puc19
(hvt)-h1-h2.
The structure of 2.6puc19 (hvt)-h1-h2-mini-f and identification
Puc19 (hvt)-h1-h2 plasmid dna application pac i is carried out enzyme action, recovery product is processed with through same enzyme action
Mini-f plasmid dna application ligase connect, by after mini-f plasmid enzyme restriction fragment insertion ul44 h1 and ul44 h2 it
Between, obtain transfer vector puc19 (hvt)-h1-h2-mini-f (structure such as Fig. 1), convert dh5 α competent cell.Transfer is carried
Body puc19 (hvt)-h1-h2-mini-f applies ecor i and sca i enzyme action respectively, applies vector nti software prediction enzyme action
Fragment afterwards should be respectively 3 of 7507bp, 5095bp and 1338bp and 2 band of 10165bp and 3775bp size.Enzyme action
Result is visible, and ecor i and sca i digestion verification meet theoretical prediction result (Fig. 2).
The structure of the bacterial artificial chromosome of embodiment 2hvt fc-126 strain
1st, experiment material
Spf Embryo Gallus domesticus are derived from purchased from Beijing Cimmeria Wei Tong laboratory animal Technology Co., Ltd..Hvt fc-126 Strain
From China Institute of Veterinary Drug Control (cvcc av19).Plasmid mini kit and midi kit is purchased from qiagen company.Dmem trains
Foster base, pancreatin and new-born calf serum are purchased from sigma company；Other reagent are purchased from Nanjing raw work biology company limited.Dh10b feels
It is purchased from invitrogen company by state cell.
2nd, the method for bacterial artificial chromosome and the identification of hvt fc-126 strain are built
The acquisition of 2.1mini-f restructuring hvt
Take the dna 10～20ul (about 1～3ug) and transfer vector puc19 (hvt)-h1-h2- of hvt fc-126 strain
3～5ul (about 3～5ug) mixing of mini-f, using calcium phosphate precipitation cotransfection primary chick embryo fibroblast (chicken
Embryofibroblast, cef).Transfectional cell Secondary Culture, observes under wavelength is for 488nm ultraviolet light, and picking sends green
The cytopathy plaque of fluorescence passes to be cultivated on the monolayer cef of fresh preparation, then continuous several times picking green statin (Fig. 3), inoculation
After purification and expanding propagation in the 6 orifice plate cef, the cell toxicant collected in 6 orifice plates takes ultrasonic treatment to carry out cell breakage, crushes thin
10 times of doubling dilutions of cellular virus liquid are inoculated on the cef flat board of the fresh preparation in 96 holes, and the restructuring poison of single green statin in screening single hole,
Carry out expanding propagation through accessing cef again, carry out pressurization culture simultaneously, that is, before accessing, 1h changes the cell culture fluid of cef into pressure training
Foster base, every 24h changes a stress medium, final 5 plants of mini-f restructuring hvt of acquisition.Stress medium is in dmem culture medium
The middle serum adding final concentration of 10% (mass percentage concentration), the penicillin of 100u/ml, the streptomycin of 100 μ g/ml, 350 μ
Obtain after the hypoxanthine of the mycophenolic acid of g/ml, the xanthine of 80 μ g/ml and 100 μ g/ml.
The acquisition of the bacterial artificial chromosome of 2.2 herpes turkey viruss
Take 3 plants of mini-f restructuring hvt, extract its dna, electricity is transformed in e.coli dh10b competent cell respectively, point
Do not take 5～10 clones, concrete grammar is as follows: take the dna about 5 μ g of mini-f restructuring hvt to be added to 50 μ l electricity transformed competence colibacillus thin
In born of the same parents e.coli dh10b.Gently mix, ice bath 5min about, being drawn into specification is in the pre-cooled electricity conversion cup of 0.1cm, should
With Harvard Apparatus company of U.S. ecm630 electroporation, electric conversion condition is 1500v, 200 ω, 25 μ f, after the completion of electric shock, is added to
In the soc culture medium of 32 DEG C of preheatings, 32 DEG C, 220r/min vibration activation 1h, 500g is centrifuged 5min, takes bacterial precipitation 150 μ l
It is spread evenly across after lb culture medium is resuspended on the lb agar plate containing 30 μ g/ml chloromycetin, 32 DEG C of culture more than 48h, if picking
Dry positive bac clone's single bacterium colony.After each strain mini-f restructuring hvt conversion e.coli dh10b, take a clone respectively, be named as
Bac-hvt (c1), bac-hvt (c2) and bac-hvt (c3).By bac-hvt (c1), bac-hvt (c2) and bac-hvt (c3) point
It is not inoculated in the lb culture fluid containing 30 μ g/ml chloromycetin, 32 DEG C of shaken cultivation overnight, use alkaline lysis method of extracting plasmid dna,
Obtain the bacterial artificial chromosome of each herpes turkey viruss.
The identification of the bacterial artificial chromosome of 2.3 herpes turkey viruss
Take the plasmid dna of bac-hvt (c1), bac-hvt (c2) and bac-hvt (c3), be respectively adopted ecor i and bamh
I carries out rflp identification, and the collection of illustrative plates of restriction enzyme mapping and the hvt fc-126 strain of application invitrogen software prediction is contrasted
Analysis, carries out Preliminary Identification to the bacterial artificial chromosome clone obtaining.Wherein, the rflp identification of bac-hvt (c1) plasmid dna
Result such as Fig. 4 it can be seen that playing restriction enzyme mapping is typical bac spectral pattern, coincide by band distribution and software analysis result.To bac-
Hvt (c2) is consistent with bac-hvt (c1) with the qualification result of bac-hvt (c3).
Embodiment 3 virus rescue
1st, material
Hvt fc-126 strain is derived from China Institute of Veterinary Drug Control (cvcc av19), the spf Embryo Gallus domesticus of preparation chick embryo fibroblast
Purchased from Beijing Cimmeria Wei Tong laboratory animal Technology Co., Ltd..Carry the bacterial artificial chromosome of herpes turkey viruss
The recombinant bacterium bac-hvt (c1) of body, bac-hvt (c2) and bac-hvt (c3) are to prepare in embodiment 2.plasmid mini
Kit and midi kit is purchased from qiagen company.Dmem culture medium, pancreatin and new-born calf serum are purchased from sigma company.Other reagent
It is purchased from Nanjing raw work biology company limited.
2nd, experimental technique and result
Extract the plasmid dna of bac-hvt (c1) using qiagen plasmid midi kit to specifications, take about
500ng dna adopts calcium phosphate precipitation to transfect the monolayer cef of fresh preparation.Next day changes culture fluid containing 10% hyclone into
Cell culture fluid, culture 48～72h after with pancreatin, cell dissociation is become individual cells, pass on the cef cell of fresh preparation
On flat board, after continuing culture 48～72h, picking sends the virus plaques of green fluorescence under wavelength is for 488nm ultraviolet excitation,
Obtain
The acquisition of gc recovered virus: application hvt-b h1 f and hvt-b h2 r is primer, with the dna of hvt fc-126 strain
The homologous recombination fragment being made up of upstream homology arm, gc gene and downstream homology arm for template, pcr amplification.Take homologous recombination piece
Section 2～3 μ g plasmid dna about 500ng cotransfection cef with bac-hvt (c1), bac-hvt (c2) and bac-hvt (c3) respectively,
After transfection, 96h takes infection cell to be inoculated into culture on the monolayer cef of fresh preparation, after 24～48h, uses methylcellulose medium
Cover, observe the pathological changes plaque (white macula) not sending green fluorescence under ultraviolet excitation, picking white macula is inoculated into new cef,
So repeat multiple circulations until purification is successful.Bac-hvt (c1), bac-hvt (c2) and antibacterial people entrained by bac-hvt (c3)
The recovery poison of work chromosome respectively obtains one plant, is respectively designated as hvtbac-gc-r(c1)、hvtbac-gc-r(c2)And hvtbac-gc-r(c3).
With hvtbac-gc-r(c1)、hvtbac-gc-r(c2)And hvtbac-gc-r(c3)Dna be template, with pair of primers hvt-gc f (5 '-
Tcgccatgtgcgccactagcat-3 ') and hvt-gc r (5 '-aacacacacaagacgcgagggctc-3 ') carry out pcr expansion
Increase, and amplified fragments are carried out with sequencing, whether checking gc gene is successfully recovered.Wherein hvtbac-gc-r(c1)With hvt fc-
Through sequencing, 126 plants of fragments all amplifying about 1600～1700bp size, prove that both sequences are completely the same, show gc base
Because becoming work recovery, andDo not amplify band (as Fig. 5).Application same procedure is to hvtbac-gc-r(c2)And hvtbac -gc-r(c3)Identified, result and hvtbac-gc-r(c1)Unanimously.
Embodiment 4 recovers the comparison of poison and parent's poison growth characteristics
1st, test material
、hvtbac-gc-r(c1)、hvtbac-gc-r(c2)And hvtbac-gc-r(c3)Strain virus are prepared for embodiment 3.Will
hvtbac-gc-r(c1)Cef is passed on, 1st generation is hvtbac-gc-r(c1)f1, the 10th on behalf of hvtbac-gc-r(c1)f10, the 20th on behalf of
hvtbac-gc-r(c1)f20, preserve seed culture of viruses respectively standby.Hvt fc-126 strain virus are derived from China Institute of Veterinary Drug Control (cvccav19), prepare Embryo Gallus domesticus
Fibroblastic spf Embryo Gallus domesticus are purchased from Beijing Cimmeria Wei Tong laboratory animal Technology Co., Ltd..Dmem culture medium, pancreatin and new
Raw Ox blood serum is purchased from sigma company.Other reagent are purchased from Nanjing raw work biology company limited.
2nd, experimental technique
By hvt fc-126,、hvtbac-gc-r(c1)、hvtbac-gc-r(c2)、hvtbac-gc-r(c3)、hvtbac -gc-r(c1)f1、hvtbac-gc-r(c1)f10And hvtbac-gc-r(c1)f20Press 0.01 infection multiplicity (multiplicity of respectively
Infection, moi) it is inoculated into and cultivate on the primary cef of monolayer of growth 24h.First measure inoculation before and inoculation after 6h, 12h,
24h, 36h, 48h and 72h infection cell is digested to the virus titer of individual cells through pancreatin.When collecting virus, remove thin first
Born of the same parents' supernatant, cell washs 3 times through pbs, uses 2mldmem re-suspended cell again with after 0.1% pancreatin digestion.Disease to individual cells
Malicious titre, taking 100 10 times of multiple proportions of μ l cell suspension to be serially diluted to dilution factor is 10-7, it is inoculated into the monolayer cef of fresh preparation
On, count plaque forming unit (plaque forming unit, pfu) after culture 72h.In addition, measuring the cell knot through cracking
Close the titre of sexually transmitted disease (STD) poison, receive the malicious time ibid, method be re-suspended cell is centrifuged after, incline supernatant, and precipitation is protected with spga
After liquid (Chinese veterinary pharmacopoeia: marek isease turkey herpes virus live vaccine manufactures and inspection procedure) is resuspended again, through 50 hertz
Hereby after 5min ultrasonic degradation, inoculation cef measures titre, and test is repeated 3 times altogether, draws viral growth according to pfu count results bent
Line.
3rd, result
Measure and compare hvt fc-126,、hvtbac-gc-r(c1)、hvtbac-gc-r(c2)And hvtbac -gc-r(c3)Strain and hvtbac-gc-r(c1)f1, hvtbac-gc-r(c1)f10And hvtbac-gc-r(c1)f20The multistep growth kineticses of infection cef,
After result display hvt fc-126 and each strain bacterial artificial chromosome recovery poison inoculation cef, either it is digested to single through pancreatin
The virus titer (Fig. 6 a) of cell, or titre (Fig. 6 b) all no significant differences of the cell-associated virus through cracking.And gc
Gene-deleted strainVirus titer is decreased obviously, and it have dropped about 3 times through the virus titer that pancreatin is digested to individual cells,
And the titre of the cell-associated virus through cracking then have dropped about 100 times.Although show the gc gene of hvt be not one required
Gene, but the propagation of hvt is played an important role.hvtbac-gc-r(c1)Pass the hvt of a generationbac-gc-r(c1)f1, pass 10 generations hvtbac -gc-r(c1)f10With the hvt passing for 20 generationsbac-gc-r(c1)f20After infection cef, the titre of the cell-associated virus through cracking is all no notable
Difference (Fig. 6 c), shows that bacterial artificial chromosome recovers the good stability of poison.
Embodiment 5 recovers the comparison with parent's poison to Marek Vaccine effectiveness for the poison
1st, test material
、hvtbac-gc-r(c2)And hvtbac-gc-r(c3)Strain virus are prepared for embodiment 3, hvtbac-gc-r(c1)f1、
hvtbac-gc-r(c1)f10And hvtbac-gc-r(c1)f20Prepare for embodiment 4.Hvt fc-126 strain virus are derived from China Institute of Veterinary Drug Control (cvcc
Av19), -1 plant of marek's disease viruses capital virulent strain is derived from China Institute of Veterinary Drug Control (cvcc av86), prepares chick embryo fibroblast and incubates
The spf Embryo Gallus domesticus changing chickling are purchased from Beijing Cimmeria Wei Tong laboratory animal Technology Co., Ltd..Dmem culture medium, pancreatin and new born bovine
Serum is purchased from sigma company.Other reagent are purchased from Nanjing raw work biology company limited.
2nd, experimental technique
Will、hvtbac-gc-r(c2)、hvtbac-gc-r(c3)、hvtbac-gc-r(c1)f1、hvtbac-gc-r(c1)f10With
hvtbac-gc-r(c1)f20Respectively 1 age in days spf chickling 20 is inoculated with 4000pfu, in latter 21 days of inoculation, compare chicken 20 with counteracting toxic substances
(not immune), -1 plant of capital of lumbar injection marek's disease viruses virulent strain (cvcc av86) 0.2ml, blank control group chicken is unavoidably
Epidemic disease not counteracting toxic substances, observe 2 months, to chicken and the survival chicken cut open inspection observation pathological changes of dying of illness, sciatic nerve, brachial plexus etc. and swell
Greatly, the lymphadenomatous pathological change of the internal organs such as liver, kidney, heart, gonad, lungs appearance is then judged to the Marek positive, system
Meter Marek disease rates, compare the immune protection effectiveness to md for 6 kinds of viruses.
3rd, experimental result
6 kinds of viruses are inoculated 1 Japanese instar chickling with 4000pfu respectively, any untoward reaction does not occur, apply horse in 21 ages in days
- 1 plant of Garrick disease virus capital virulent strain counteracting toxic substances, observe after counteracting toxic substances to 82 ages in days, to chicken and the survival chicken cut open inspection observation horse of dying of illness
The typical cytopathic of vertical creutzfeldt jakob disease.Wherein blank control group chicken is all healthy, no Marek pathological changes.Counteracting toxic substances matched group 85%
(17/20) md is positive, hvt fc-126 and hvtbac-gc-r(c1)f1、hvtbac-gc-r(c2)、hvtbac-gc-r(c3)And hvtbac-gc-r(c1)f20
The md positive rate of immune group is respectively 20% (4/20), 15% (3/20), 20% (4/20) 10% (2/20) and 15% (3/20),
The positive rate of md decreases 65%, 70%, 65%, 75% and 70% respectively, has reached the qualified standard of immunoprotection, shows this
Bacterial artificial chromosome entrained by invention bac-hvt (c1) is hvt genome cloning, can apply to recombinant vector work epidemic disease
The structure (table 2) of Seedling.
Table 2 mdv strong virus attack protective rate
Recover in malicious growth characteristics and the present embodiment, to recover the Vaccine effectiveness to Marek for the poison from embodiment 4,
It can be seen that by the gc gene knockout of hvt, and it is highly stable to be substituted for the bacterial artificial chromosome of mini-f sequence, therefore adopts
The bacterial artificial chromosome that the present invention builds can construct stability extraordinary recombinant vector live vaccine.
Embodiment 6 adopts bacterial artificial chromosome of the present invention to build recombinant vector live vaccine
The structure of 1 antigen gene (ha) expression cassette
The acquisition of 1.1 antigen genes
Herpes turkey viruss (hvt) both can serve as the live vector of chicken infectious disease it is also possible to be used as the work of the infectious disease of duck
Carrier, the infectious disease of chicken includes infectious bursal disease, newcastle disease, h5 type bird flu, h9 type bird flu, infectiousness larynx trachea
Inflammation and avian encephalomyelitises etc., the infectious disease of duck includes h5 type bird flu, h9 type bird flu, duck viral hepatitiss and duck Tan Busu disease
Viral disease etc..The gene of the protective antigen of these infectious disease pathogens, example can be obtained by the method for pcr method or synthetic
As the vp2 gene of infectious bursal disease viruses, hemagglutinin (ha) gene of bird flu etc..
The structure of 1.2 antigen gene expression boxes
For strengthening the expression to exogenous gene for the herpes turkey viruss live vector, add in the upstream of the exogenous gene of insertion
The upper strong promoter that can strengthen transcription, such as extreme early promoter of sv40 promoter or mcmv etc., also initial in exogenous gene
Codon front end adds kozak sequence, enables the correct accurate translation of exogenous gene.And add termination letter in the downstream of exogenous gene
Number, such as sv40 polya sequence etc., so that transcription is correctly terminated.
The structure of 2 herpes turkey viruss carrier live, this vaccine is a kind of diva vaccine.
The acquisition of 2.1 restructuring hvt bac and identification
Extract plasmid dna first from bac-hvt (c1), bac-hvt (c2) or bac-hvt (c3), proceed to gs1783 work
Journey bacterium, referring next to literature method (tischer b, smith g, osterriedern.en passant mutagenesis:a
two step markerless red recombination system.methods mol biol.2010；634:421-
430. or wang j, osterrieder n.generation of an infectious clone of duck
enteritis virus and generation of a vectored dev expressing hemagglutinin of
h5n1 avian influenza virus.virus res.2011；159 (1): 23-31.), application en passant method will
Antigen gene expression box is inserted into the noncoding region of hvt genome or dispensable gene site in bacterial artificial chromosome, builds
Restructuring hvt bac containing antigen gene expression box, is identified by rflp and pcr and gene order surveying method.
The screening of 2.2 herpes turkey viruss carrier live restructuring poison and identification
Apply midi-prep (qiagen) test kit according to embodiment 3, by specification methods described is extracted and contained antigenic site
Restructuring hvt bac because of expression cassette.According to embodiment 2 method, take the restructuring hvt bac 10 containing antigen gene expression box～
20ul (about 1～3ug), recovers gc gene while according to embodiment 3 method transfection chick embryo fibroblast rescue recombinant viruses,
Picking does not send the recombinant viruses of green fluorescence under ultraviolet excitation.Extract the dna of recombinant viruses, surveyed by pcr and gene
Whether sequence identification exogenous gene expression box is correctly inserted into, and whether identification gc gene correctly recovers simultaneously.By by recombinant viruses even
Resume generation 25 more than generation, detection passes on whether rear exogenous gene occurs to lose and make a variation, the stability of checking recombinant viruses.
The development of 2.3 herpes turkey viruss carrier live
The qualified recombinant viruses of empirical tests, according to the related request of national regulations, can be used for developing turkey
Herpesvirus vector live vaccine, this vaccine can provide protection to Marek, also can be to the cause of disease institute of contained exogenous gene
The epidemic disease causing provides protection.
2.4 difference immune animals and natural infected animal
A protection of cause of disease is only expressed after the herpes turkey viruss carrier live immune animal that the application present invention develops
Property antigen, after immunity, the cause of disease of contained exogenous gene is only produced with the antibody of this antigen, and in zoogenetic infection cause of disease, then not only
Produce the antibody for this protective antigen, also produce the antibody for other antigens, so applying the immunity of this carrier live
Animal can pass through the methods such as elisa and detect that the serum antibody of animal distinguishes immune animal and natural infected animal, and this is for epidemic disease
It is crucial technology that disease purifies, significant.
sequence listing
<110>Jiangsu Province Agriculture Science Institute
<120>a kind of bacterial artificial chromosome of new herpes turkey viruss, construction method and application
<130> 20160826
<160> 6
<170> patentin version 3.3
<210> 1
<211> 29
<212> dna
<213> artificial
<220>
<223> hvt-b h1 f
<400> 1
gtagaattcc agtgtatgtt ccttagtgt 29
<210> 2
<211> 38
<212> dna
<213> artificial
<220>
<223> hvt-b h1 r
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atcccgggct ttaattaagt ttcgatagat gcgtaata 38
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<213> artificial
<220>
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cgggatccag ttaattaaat cagcaccatc gcattg 36
<210> 4
<211> 32
<212> dna
<213> artificial
<220>
<223> hvt-b h2 r
<400> 4
gacaagcttg aaatgtctat ggtgtgattg ga 32
<210> 5
<211> 1297
<212> dna
<213> hvt fc-126
<400> 5
gtgtatgttc cttagtgtgc atcgctgcac atttagctgt taccgtgtcg ggagtggcat 60
taattccgct tatcgatcaa aacagagctt acggaaactg tacggtatgt gtaattgccg 120
gattcatcgc tacgtttgct gcacgactta cgataagact ttcggaaacg cttatgctag 180
tgggcaagcc ggcgcagttt atatttgcta taatcgcttc cgttgcggaa acactgatca 240
ataacgaggc gcttgccatc agtaatacta cttacaaaac tgcattgcga ataatcgaag 300
taacatcttt ggcgtgtttt gttatgctcg gggcaataat tacatcccac aactatgtct 360
gcatttcaac ggcaggggac ttgacttgga aggcgggatt tttcatgctt accaccggaa 420
cattactcgg tataacaata ccaaacatac acccaatccc tctcgcgggg tttcttgcag 480
tctatacaat attggctata aatatcgcta gagatgcaag cgctacatta ttatccactt 540
gctattatcg caattgccgc gagaggacta tacttcgccc ttctcgtctc ggacatggtt 600
acacaatccc ttctcccggt gccgatatgc tttatgaaga agacgtatat agttttgacg 660
cagctaaagg ccattattcg tcaatatttc tatgttatgc catggggctt acaacaccgc 720
tgattattgc gctccataaa tatatggcgg gcattaaaaa ttcgtcagat tggactgcta 780
cattacaagg catgtacggg cttgtcttgg gatcgctatc gtcactatgt attccatcca 840
gcaacaacga tgccctaatt cgtcccattc aaattttgat attgataatc ggtgcactgg 900
ccattgcatt ggctggatgt ggtcaaatta tagggcctac attatttgcc gcgagttcgg 960
ctgcgatgtc atgttttaca tgtatcaata ttcgcgctac taataagggt gtcaacaaat 1020
tggcagcagc cagtgtcgtg aaatctgtac tgggcttcat tatttccggg atgcttactt 1080
gcgtgctatt accactatcg tgatagatcg tcggtctgcg catcgcccat gctggcggaa 1140
cgctctttcg aaccgtgaat aaaactttgt atctactaaa caataacttt gtgttttatt 1200
gagcggtcga aaacaatgag gagctgcaat ttaaagctaa ccgcatacgc cgggcgggta 1260
aagaccattt tataccatat tacgcatcta tcgaaac 1297
<210> 6
<211> 1043
<212> dna
<213> hvt fc-126
<400> 6
atcagcacca tcgcattgtt tcgtagatga ctcatgttca gtccgcgtga tgtcaaaaat 60
acgtattttt ggtatcacgc agcggccaaa atgcccatta tgttattttt actccaaacg 120
cggtatttaa aacatcggga cgtacatcat gtggcgcacg ttaatcgtat acggtgccgc 180
tacattaaaa atcgcaagtc tccgaatatc aagctcacgg ccaaaacgtc ggtaataatc 240
ttacgcatcg aatgtgatac ggataccgta caatcgctga gtagatttcc tatatagtta 300
ctcagtagtg atacacaatc acaaaatcgc tggggtatat catataagaa tgatgtcgcc 360
cacccctgaa gatgatcgcg atctcgttgt ggttcgtgga cgtctccgaa tgatggatag 420
cggcacggaa acagatagag agcaacgaca tccacgtacg acttggcgat cgatctgttg 480
tgggtgtacg ataggaatgg tatttaccat attcgttctc gtagcggcag tattgttggg 540
atcactattc actgtttcat acatggccat ggaatcggga acatgtcccg atgaatggat 600
tggtttgggt tatagttgca tgcgcgtggc cgggaaaaat gcaactgatc ttgaggcgtt 660
ggatacatgt gctcggcata acagcaaact tattgacttc gcaaacgcca aagttctggt 720
tgaagctatc gccccattcg gtgtgccaaa tgcagcatat ggggaagtct tccggttaag 780
ggacagcaaa accacgtgta tacgacctac catgggagga cccgtgtcgg cagactgtcc 840
tgtaacatgt accgttatat gtcagcgacc caggcctcta agtaccatgt cttccatcat 900
tagagatgcc cgcgtgtatc ttcatttaga acgacgcgat tattatgaag tctacgcctc 960
tgtcctctct aatgcgatga gtaaataaaa acgcacctct aacggttact gtgttttatt 1020
tatccaatca caccatagac att 1043
Claims (8)
1. a kind of bacterial artificial chromosome of new herpes turkey viruss is it is characterised in that be by the gc gene in hvt genome
Knock out and replace with mini-f plasmid sequence.
2. build the method for bacterial artificial chromosome according to claim 1 it is characterised in that comprising the steps:
(1) expand gc gene upstream and downstream homology arm in hvt genome respectively, upstream and downstream homology arm is inserted in puc19 carrier
Obtain recombinant vector a；
(2) by mini-f plasmid warppacIn the fragment insertion recombinant vector a obtaining after i enzyme action, obtain recombinant transfer vector
b；
(3) by the dna cotransfection cef of recombinant transfer vector b and hvt, carry out homologous recombination, picking sends the thin of green fluorescence
Born of the same parents' pathological changes plaque, then through stress medium screening, obtain mini-f restructuring hvt；Extract the dna conversion of mini-f restructuring hvt
Dh10b cell, using chloromycetin screening, obtains positive restructuring bacterium, after culture, extracts plasmid dna, obtains described bacteria artificial dye
Colour solid.
3. according to claim 2 build bacterial artificial chromosome method it is characterised in that described stress medium be
Add in dmem culture medium final concentration of 10% serum, the penicillin of 100u/ml, the streptomycin of 100 g/ml, 350 g/ml
The hypoxanthine of mycophenolic acid, the xanthine of 80 g/ml and 100 g/ml after obtain.
4. build the method for bacterial artificial chromosome according to Claims 2 or 3 it is characterised in that with hvt genome as mould
Plate, with seq id no:1 and seq id no:2 for primer amplification gc upstream region of gene homology arm；With seq id no:3 and seq id
No:4 is primer amplification gc downstream of gene homology arm.
5. build the method for bacterial artificial chromosome according to claim 4 it is characterised in that the sequence of described upstream homology arm
It is classified as shown in seq id no:5, the sequence of downstream homology arm is as shown in seq id no:6.
6. application in building herpes turkey viruss carrier live strain for the bacterial artificial chromosome described in claim 1.
7. apply according to claim 6 it is characterised in that turkey is built using bacterial artificial chromosome described in claim 1
The method of herpesvirus vector live vaccine strain is as follows:
(1) pass through the sequence of the method acquisition exogenous gene of pcr method or synthetic, then connect at its upstream and start
Son, terminator sequence in downstream connection, it is built into exogenous gene expression box；
(2) bacterial artificial chromosome described in claim 1 is proceeded to engineering bacteria gs1783, then by described exogenous gene expression
Box inserts in described bacterial artificial chromosome, is built into restructuring hvt bac；
(3) the restructuring hvt bac transfection chick embryo fibroblast constructed by (2) is carried out virus rescue, simultaneously will be extensive for gc gene
Multiple, obtain recombinant herpesvirus of turkeys carrier live strain by choosing speckle purification.
8. apply according to claim 7 it is characterised in that described exogenous gene expression box inserts described bacterial artificial chromosome
The noncoding region of body or dispensable gene site.
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| CN107446880A (en) * | 2017-09-01 | 2017-12-08 | 铜仁职业技术学院 | A kind of method for preparing chicken embryo fibroblasts |
| CN110205308A (en) * | 2019-05-24 | 2019-09-06 | 华南农业大学 | It is a kind of express HA gene recombinant herpesvirus of turkeys and its application |
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