CN110295148A - A kind of method of 3 type duck hepatitis A virus reverse genetic strain of rapid build - Google Patents
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Abstract
The present invention relates to a kind of methods of 3 type duck hepatitis A virus reverse genetic strain of rapid build.3 type duck hepatitis A virus full-length genomes are divided into three segments similar in size and carry out PCR amplification, the end 5' addition cytomegalovirus early promoter of first segment, same sense mutation is introduced in the 2A gene of second segment as genetic marker site, hepatitis delta virus ribozyme sequence and vacuolating virus of monkey early stage mRNA polyadenylation signal sequence are added at the end 3' of viral genome the third fragment, complete the building of 3 type duck hepatitis A virus infectivity Subgenomic replicons, Transfected primary duck embryo fibroblasts after being mixed with transfection reagent, the spontaneous recombination of replicon, it is final to obtain the 3 type duck hepatitis A virus containing genetic marker.3 type duck hepatitis A virus mutant strains can be quickly obtained using this method, this is that the pathogenesis for studying 3 type duck hepatitis A virus, exploitation new generation vaccine etc. provide advantageous tool.
Description
Technical field
The invention belongs to Veterinarian virus technical field of molecular biology, and in particular to a kind of 3 type duck hepatitis A of rapid build
The method of malicious reverse genetic strain.
Background technique
Duck virus hepatitis (Duck viral hepatitis, DVH) is by duck hepatitis virus (Duck hepatitis
Virus, DHV) one kind is acute, highly contagious disease caused by infection duckling.Current duck culturing area main in the world has
The presence of this disease has the characteristics that interval outburst, endemicity, is to endanger one of principal disease of duck culturing industry.The disease is main
The duckling within four week old is encroached on, has morbidity anxious, is propagated rapidly, the features such as course of disease is short and the death rate is high；Clinical main table
It is now the dead preceding generation spasm of duckling, it is in " opisthotonos " that pathological change is mainly that the visible liver of dissect is swollen that head is swung back to back
Big inflammation and a large amount of haemorrhagic puncta.The disease is mainly by belonging to the duck hepatitis A virus of Picornaviridae fowl hepatovirus
(Duck hepatitis A virus, DHAV) causes.There are three types of serotype, i.e. 1 type, 2 types and 3 types for DHAV tool.In recent years,
The DHAV of China's prevalence is mainly 1 type duck hepatitis A virus (DHAV-1) and 3 type duck hepatitis A virus (DHAV-3).
Reverse genetics manipulation technology is a kind of Important Platform for carrying out viral molecular biology research, can be right in vitro
Rna virus cdna group carries out the manual operations such as gene knockout, direct mutagenesis, in illustrating viral pathogenesis mechanism and vaccine development
It can play a significant role and have the advantage shorter than the natural mutagenesis period.The key of traditional picornavirus infection cloning process is
Obtain full-length cDNA full length fragment clone, and viral genome be converted to cDNA after need to be cloned into suitable load
In body, in order to avoid instability problem of the virus sequence in bacterium, cDNA clones method is usually taken in researcher, by small
Segment connects into large fragment, and large fragment is finally obtained full length cDNA clone by the method that digestion connects.But this method enzyme
It is more that enzyme site chooses limitation, multiple large fragments are attached efficiency in vitro lower, other rdrp gene cDNA
Being cloned in bacterium has unstability, therefore not only operating procedure is numb for the whole process of acquisition full length viral genome cDNA
It is tired, and take a long time, success rate is not high, meanwhile, although the full-length cDNA of some viruses can not be cloned or can be cloned into load
It body but easily morphs in host strain and leads to the Revive virus that cannot succeed.Currently, a kind of be referred to as " infectious sub-gene
The technology of group replicon (Infectious Subgenomic Amplicons) " has been shown in mammal or mosquito
The artificial rescue to single strand plus RNA virus is realized in cell, and is applied to japanese encephalitis virus, west nile virus, stockaded village's card
In the reverse genetics research of the virus such as virus, flavivirus, dengue virus and people's coxsackie virus.The technology is a kind of
Novel " no bacterium " reverse-genetics approach does not need to obtain virus full length cDNA plasmid and obtains virus in vitro
Rna transcription sheet can directly have the DNA segment Revive virus of homology region by transfection.It is " infectious for specific
The technology path that Subgenomic replicon " uses is: being generated first by PCR method comprising entire virus genomic overlapping
Non-infectious subgenomic dna segment, these overlapping Subgenomic replicon quantity can be 3 to 10, it is adjacent it
Between segment between the overlapping region with 100bp or so, meanwhile, the end 5' of first segment and the 3' of the last one segment
End flank respectively cytomegalovirus early promoter (Cytomegalovirus immediate early promoter,
PCMV) sequence, hepatitis delta virus ribozyme (Hepatitis delta virus ribozyme, HDVR) sequence and monkey vacuole
Viral early stage mRNA polyadenylation signal sequence (SV40 early mRNA polyadenylation signal, SV40pA)
Sequence, these elements can help Subgenomic replicon be mixed be transfected into permissive cell after start to transcribe, and utilize place
The spontaneous recombination of the homologous recombination machinery of chief cell, being formed has infective intact virus transcript, then leads to virus
Duplication and proliferation, final obtain have infective Revive virus.
Although at present about the building of 3 type duck hepatitis A virus infection clones and save method report,
Be that researcher uses is still the strategy for constructing virus full length infectious CDNA clone, while having result of study to show containing 3
The plasmid of type duck hepatitis A virus genome cloning mutation rate with higher in succeeding generations, it may be possible to because of virus portion
Sub-sequence has genotoxic potential and unstability.Therefore, if " infectious Subgenomic replicon " technology can be used to establish
A kind of easy, efficient 3 type duck hepatitis A virus " no bacterium " reverse genetics system, this by for the pathogenesis of the research virus,
Exploitation new generation vaccine etc. provides advantageous tool.
Summary of the invention
The object of the present invention is to provide a kind of method of 3 type duck hepatitis A virus reverse genetic strain of rapid build, this method makes
With " infectious Subgenomic replicon " technology, avoid in the prior art 3 type hepatitis A virus virus full length cDNA clone of duck thin
The problem of preservation cannot be stablized in bacterium, the lower problem of traditional digestion joint efficiency, and caused transcript is transcribed in vitro
Heterogeneous problem；It can quick, 3 type duck hepatitis A virus mutant strains of easy building using this method.
In order to achieve the above technical purposes, sentence of the present invention is achieved through the following technical solutions:
A kind of method of 3 type duck hepatitis A virus reverse genetic strain of rapid build, specifically includes the following steps:
1) parental virus strain full-length genome is divided into three segments similar in size and carries out PCR amplification, in viral genome
Add cytomegalovirus early promoter (Cytomegalovirus immediate early in the end 5' of first segment
Promoter, pCMV), same sense mutation is introduced in the 2A gene of second segment as genetic marker site, in third piece
The end 3' addition hepatitis delta virus ribozyme (Hepatitis delta virus ribozyme, the HDVR) sequence of section and monkey are empty
Steep viral early stage mRNA polyadenylation signal (SV40 early mRNA polyadenylation signal, SV40pA) sequence
Column obtain three DNA fragmentations, constitute 3 type duck hepatitis A virus infectivity Subgenomic replicons；
2) duck embryo fibroblasts are transfected after mixing the infectious Subgenomic replicon of building with transfection reagent, it is multiple
System transcribes in host cell and using the spontaneous recombination of homologous recombination machinery of cell, and being formed has infective complete disease
Malicious transcript then leads to the duplication and proliferation of virus, obtains the 3 type duck hepatitis A virus containing genetic marker and reversely loses
Pass strain virus.
Further, first segment and the third fragment contain 74 and 83 with second segment respectively
The overlapping region of base-pair.
Further, it is introduced in the 2A gene of 3 second segment of type duck hepatitis A virus genome with fusion DNA vaccine method
One synonymous base mutation, so that the G of the 3403rd nucleotide of reverse genetic strain virus sports T, the site is as sense
The genetic molecule label of metachromia clone, can distinguish reverse genetic strain virus and parent plant by PCR method combination DNA sequencing
And street strain.
The 3 type duck hepatitis A virus (Duck Hepatitis A Virus type 3, DHAV-3) are preserved in China
Type Tissue Collection, deposit number are CCTCC NO:V201305, preservation address: Wuhan City, Hubei Province Wuchang District eight
All the way No. 299 Wuhan Universitys in the school, preservation date: on March 25th, 2013.
Proliferative capacity and virus titer of the Revive virus containing genetic marker in duck embryos that the method for the present invention obtains,
It is similar to parental virus to the pathogenicity of duckling, and genetic stability is good.
The method of the present invention can carry out mutation or fragment deletion/replacement/insertion to any base of 3 type duck hepatitis A virus, obtain
Obtain mutation recombinant virus newly.
The invention has the benefit that
1, prove that " infectious Subgenomic replicon " technology can be used in the reverse genetic rescue of avian viral for the first time
In.
2, the Revive virus obtained in the present invention has characteristic identical with parental virus, can stablize increasing in duck embryos
Passage is grown, and is had and proliferative capacity similar in parental virus and antigenicity.
3, invention provide method it is easy to operate, quick, any base of 3 type duck hepatitis A virus can be carried out mutation or
Fragment deletion/replacement/insertion and obtain new mutation recombinant virus, for study 3 type duck hepatitis A virus pathogenesis etc. provide
Effective tool, and have great importance for the exploitation for accelerating new generation vaccine.
Detailed description of the invention
Fig. 1 is the ideograph of 3 type duck hepatitis A virus reverse genetic strains " infectious Subgenomic replicon "；
Fig. 2 is pCMV-F1, F2 and F3-HDVR/SV40pA segment agarose gel electrophoresis figure；M is DL5,000DNA
Marker, No. 1 to No. 3 swimming lane are followed successively by pCMV-F1, F2 and F3-HDVR/SV40pA segment；
Fig. 3 is that 3 type duck hepatitis A virus reverse genetic strains " infectious Subgenomic replicon " transfect duck embryo fibroblasts
Result figure；
Fig. 4 is Revive virus and parental virus F2 genetic fragment BlnI digestion result electrophoretogram；M is DL5,000DNA
Marker, No. 1 and 2 swimming lanes are respectively Revive virus and parental virus；
Fig. 5 is the molecular genetic marker position nucleotide sequencing result of Revive virus；
Fig. 6 is the ideograph of 3 type duck hepatitis A virus mutant strains " infectious Subgenomic replicon "；
Fig. 7 is that 3 type duck hepatitis A virus mutant strains " infectious Subgenomic replicon " transfect duck embryo fibroblasts result
Figure.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail, is not limitation of the invention further.Under
It states experimental method used in embodiment unless otherwise specified, is conventional method.Material as used in the following examples,
Reagent etc., is commercially available unless otherwise specified.
Material and reagent as used in the following examples are specific as follows:
Strain:
3 type duck hepatitis A virus velogen strains, this laboratory is isolated, has been preserved in the Chinese Typical Representative of Wuhan, China university
Culture collection, deposit number are as follows: CCTCCNO:V201305, classification naming: 3 type of fowl hepatovirus Picornaviridae
Duck hepatitis A virus (Duck Hepatitis A Virus type3, DHAV-3).
Reagent and instrument:
TaKaRa MiniBEST Universal RNA Extraction Kit, PrimeSTAR Max DNA
Polymerase, DNA Marker etc. is purchased from precious bioengineering (Dalian) Co., Ltd；Plastic recovery kit, plasmid extraction examination
Agent box etc. is purchased from U.S. Omega company；Lipofectamine box Lipofectamine 3000 is public purchased from Invitrogen
Department；Other reagents are that domestic analysis is pure.
Nucleic acid-protein detector (Bio Rad, Smartspec 3000), grads PCR instrument (Biometra,
Tgradient), electrophoresis apparatus (Bio Rad, Powerpac 300) and gel imaging system (Bio Rad Versa Doc Model
2000)。
The building of 13 type duck hepatitis A virus of embodiment " infectious Subgenomic replicon " and virus rescue
1.1. the design and synthesis of primer
According to the whole genome sequence of 3 type duck hepatitis A virus in GenBank, 6 pairs of primers are devised for expanding viral base
Because of a group sequence, pCMV and SV40pA sequence, particular sequence information is shown in Table 1, and primer is closed by Shanghai Sheng Gong bioengineering Co., Ltd
At.
The sequence of primer needed for table 1 constructs 3 type duck hepatitis A virus reverse genetic strains " infectious Subgenomic replicon "
1.2. viral RNA extracts
It is operated referring to the operation instructions of TaKaRa MiniBEST Universal RNA Extraction kit,
Extracted from duck embryos allantoic fluid virus full-length genome RNA, and using nucleic acid-protein detector (BioRad,
Smartspec3000 after the nucleic acid concentration and the purity that) measure it, -70 DEG C are saved backup.
1.3. gene fragment amplification is cloned
(1) using PrimeScript II 1st Strand cDNA Synthesis kit that the total serum IgE of extracting is anti-
It is transcribed into cDNA template, then uses DNA High fidelity PCR enzyme PrimeSTAR Max DNA Polymerase, with parent plant disease
The reverse transcription product of malicious total serum IgE is template, expands to obtain DHAV-3-F1 segment using primers F 1-F and F1-R, uses primer
F2-1-F and F2-1-R expands to obtain DHAV-3-F2-1 segment, expands to obtain DHAV-3- using primers F 2-2-F and F2-2-R
F2-2 segment expands to obtain DHAV-3-F3-HDVR segment using primers F 3-HDVR-F and F3-HDVR-R；With eukaryotic expression matter
Grain pEGFP-C1 is template, expands to obtain pCMV segment using primer pCMV-F and pCMV-R, uses primer HDVR-SV40pA-
F and HDVR-SV40pA-R expands to obtain HDVR-SV40pA segment；
(2) by fusion DNA vaccine technology, as shown in Figure 1, be pCMV-F1 by pCMV and DHAV-3-F1 segment composition, it will
DHAV-3-F2-1 and DHAV-3-F2-2 segment composition is F2 segment, DHAV-3-F3-HDVR and HDVR-SV40pA segment is melted
Be combined into F3-HdvRz/SV40pA segment, agarose gel electrophoresis results as shown in Fig. 2, amplified fragments through 1% Ago-Gel
Gel reclaims kit (Omega) gel extraction target fragment is used after electrophoretic separation, and DNA fragmentation is sent to the raw work in Shanghai
Bioengineering Co., Ltd is sequenced, and sequencing result shows to successfully complete reverse genetic strain " infectious sub-genome duplication
The building of son ".
1.4. reverse genetic strain " infectious Subgenomic replicon " Revive virus is transfected
Primary duck embryo fibroblasts are prepared using 9 age in days duck embryos, when the cell in 3.5cm culture dish grows to 90%
When fusion, after pCMV-F1, F2 and F3-HdvRz-SV40pA genetic fragment of equivalent (1.5 μ g) are mixed, use
(Invitrogen) by specification of Lipofectamine 3000 transfects cell.Cell is placed in 37 DEG C of 5%CO2In incubator into
Row culture observation, replaced culture medium after 16 hours, and after transfection 72 hours, cell fragmentation phenomenon occurs in transfection group cell, and
Cellular control unit upgrowth situation is good.Transfection duck embryo fibroblasts 120 hours after, cell growth condition as shown in figure 3,
By cell multigelation 3 times after photographing to record, cell culture supernatant is then inoculated with 5 piece of 9 age in days duck by allantoic cavity approach
Embryo, is put into after paraffin sealing and continues to be incubated in incubator by 0.2mL/ pieces, and every 8 hours photograph embryo 1 time, observation duck embryos are being inoculated with
Death condition afterwards discards dead duck embryos in 24 hours.Duck embryos are dead between 48 hours to 120 hours after inoculation as the result is shown
It dies, duck embryos death idiosome bleeding is serious, collects allantoic fluid and is saved as first generation reverse genetic strain.
The identification and characteristic of 2 reverse genetic strain virus of embodiment
2.1. the passage of reverse genetic strain virus
To observe whether the Revive virus for saving out can be proliferated passage in duck embryos, by the Revive virus of 1st generation
1:100 dilution is done with sterile saline, is inoculated with 5 piece of 9 age in days duck embryos.The duck embryos death time concentrates after inoculation as the result is shown
Between 48 hours to 120 hours, idiosome lesion is obvious, collects dead duck embryos allantoic fluid, and continuous passage 5 times, the equal lesion of duck embryos
Obviously, similar to parental virus.
2.2. in reverse genetic strain virus genetic marker identification
It may be from parental virus or street strain's pollution in transfection or succeeding generations to exclude reverse genetic strain virus
Possibility, in replicon building process, we introduce one using fusion DNA vaccine method in the 2A gene of F2 genetic fragment
Base mutation (G sports T) generates the genetic marker BlnI restriction enzyme position that 1 virus itself does not have after mutation
Point, while the mutation does not change the composition of the corresponding amino acid of 2A albumen.Revive virus passes on 5 times in duck embryos.From allantoic fluid
Middle extraction total serum IgE, with F2-F and F2-R primer amplification DNA fragmentation containing mutation sites, and according to restriction enzyme BlnI
The digestion system of recommendation, agarose gel electrophoresis results are as shown in Figure 4 after F2 genetic fragment is carried out digestion, the results showed that,
The F2 segment of DNA transfection Revive virus can produce two segments of 0.9kb and 1.7kb with BlnI digestion, and parental virus
2.6kb amplified fragments can not use BlnI digestion.Amplified fragments are after the separation of 1% agarose gel electrophoresis using gel recycling examination
Agent box (Omega) gel extraction target fragment, and DNA fragmentation is sent to Shanghai Sheng Gong bioengineering Co., Ltd and is sequenced,
The product of sequencing result display amplification contains the silent mutation (G3403T) of introducing, as shown in figure 5, showing that we obtain just
True Revive virus, rather than wild type strains pollute.
2.3. virus multiplication and assay
With the 3 type duck hepatitis A virus reverse genetic strain of sterile saline solution doubling dilution and parent plant, 10 are selected-3、
10-4、10-5、10-6、10-7、10-8This 6 dilutions are inoculated with 5 piece of 9 age in days through allantoic cavity approach with the amount of every embryo 0.2mL respectively
Duck embryos, separately set sterile saline and compare 5 pieces, inoculation is placed in 37 DEG C of constant incubators and is incubated for, dead in 24 hours
Duck embryos are disregarded, and the death and survival condition that duck embryos are inoculated in 7 days are observed and recorded, and calculate ELD50, knot by Reed-Muech method
Fruit shows Revive virus and parent's strain virus has similar proliferative capacity, and viral level is respectively in every 0.2mL allantoic fluid
10-4.33ELD50 and 10-4.50ELD50, Revive virus proliferative capacity in duck embryos are similar to parental virus.
2.4. the antigenicity of serum neutralization test detection virus
Using fixed virus diluted blood heat-clearing method measurement 3 type duck hepatitis A virus serum of rabbit-anti to reverse genetic strain and parent plant
Neutralization titer, the rabbit-anti DHAV-3 standard serum (potency >=1:128) by preparation laboratory early period where inventor first uses
Sterile saline makees 10 times of doubling dilutions from 2-1To 2-9This 9 dilutions, at the same by the viral dilution titrated through malicious valence at
Both then each unit dose (0.2mL) contains 200ELD50, mixed in equal amounts and is added dual anti-37 DEG C of penicillin and streptomycin of 5%
It water-bath l hours, is then inoculated in 9 age in days health duck embryos allantoic cavities with the metering of every embryo 0.2mL, each dilution is inoculated with 5 pieces of ducks
Embryo.Separately setting negative serum control (healthy rabbit anteserum with virus mix) and blank control group, (sterile saline is mixed with virus
Close), the case where discarding duck embryos dead in 24 hours, observe and record the duck embryos death and survival in 7 days, then calculate disease
The neutralization titer of poison.
As a result the blank control group and negative serum control group duck embryos of Revive virus and parent's strain virus at 48 hours extremely
96 small times are all dead；When serum dilution is 2-1~2-6Between when, in Revive virus and parental virus and the duck embryos of group
It is all strong to live, when dilution is 2-7When duck embryos start to lose protection, start at this time, higher with dilution, duck embryos protective rate is got over
It is low, until dilution is 2-9When duck embryos thoroughly lose protection, show Revive virus and parent's strain virus antigen having the same
Property.
2.5. to the Virulence detection of susceptible duckling
Virulence detection is carried out using parent plant and rescue strain, acquires the liver organization of dead duck embryos in embodiment 2.3
Homogenized, by volume 1:100 ratio be added sterile phosphate buffer solution, grinding, multigelation 3 times, 12000g from
Heart 10min, supernatant are inoculated with 9 age in days duck embryos through allantoic cavity approach after 0.22 μm of filter filtration sterilization and measure its ELD50, so
Virus liquid is diluted to 10 afterwards3.0ELD50/0.4mL.In addition 1 age in days health duckling 30 is only randomly divided into 3 groups, test group
Duckling is inoculated with 0.4mL parent plant through intramuscular injection path or rescue strain, the duckling of control group are then inoculated with isometric sterile physiological
Salt water, difference group duckling isolated rearing in different animals room, free water feeding are observed daily after inoculation, record duckling
Morbidity, death condition, timely dissect death duckling, dissect survival duckling, records duckling liver, kidney and other organs after observation 7 days
Lesion situation.
7 days internal reference group ducklings do not occur clinical symptoms, and duckling feed and drinking-water situation are normal；Revive virus and parent
Strain virus has similar pathogenicity to 1 age in days duckling, and duckling disease incidence is 80% after inoculation, and the death rate is 60%, together
When, duckling dissect liver also equal visible part needle point size blutpunkte of surviving.
Application 3 type duck hepatitis A virus " infectious Subgenomic replicon " the method rescue ISA-A117C mutation of embodiment 3
Strain virus
3.1. the design and synthesis of primer
According to the whole genome sequence of 3 type duck hepatitis A virus in GenBank, it is viral complete for expanding to devise 7 pairs of primers
Genome sequence, pCMV and SV40pA sequence, particular sequence information are shown in Table 2, and primer is by Shanghai Sheng Gong bioengineering Co., Ltd
Synthesis.
The sequence of primer needed for table 2 constructs 3 type duck hepatitis A virus mutant strain ISA-A117C " infectious Subgenomic replicon "
Column
3.2. virus extracting
It is operated referring to the operation instructions of TaKaRa MiniBEST Universal RNA Extraction kit,
The viral full-length genome RNA of 3 type duck hepatitis A virus separation strains is extracted from duck embryos allantoic fluid, and uses nucleic acid-protein detector
After (BioRad, Smartspec3000) measures its nucleic acid concentration and purity, -70 DEG C are saved backup.
3.3. gene fragment amplification is cloned
(1) using PrimeScript II 1st Strand cDNA Synthesis kit that the total serum IgE of extracting is anti-
It is transcribed into cDNA template, DNA High fidelity PCR enzyme PrimeSTAR Max DNA Polymerase is then used, uses primers F 1-
F and A117C-R, A117C-F and F1-R obtain DHAV-3- as template amplification using the reverse transcription product of parent plant virus total RNA
F1-A117C-F and DHAV-3-F1-A117C-R segment, using primers F 2-1-F and F2-1-R, F2-2-F and F2-2-R with parent
The reverse transcription product of this strain virus total serum IgE expands respectively for template and obtains DHAV-3-F2-1 segment and DHAV-3-F2-2 segment,
DHAV- is obtained as template amplification using the reverse transcription product of parent plant virus total RNA using primers F 3-HDVR-F and F3-HDVR-R
3-F3-HDVR segment；Using eukaryon expression plasmid pEGFP-C1 as template, expand to obtain using primer pCMV-F and pCMV-R
PCMV segment expands to obtain HDVR-SV40pA segment using primer HDVR-SV40pA-F and HDVR-SV40pA-R.(2) pass through
Fusion DNA vaccine technology, as shown in fig. 6, being first by DHAV-3-F1-A117C-F and DHAV-3-F1-A117C-R segment composition
Then pCMV and DHAV-3-F1-A117C segment composition is pCMV-F1 by DHAV-3-F1-A117C；By DHAV-3-F2-1 and
DHAV-3-F2-2 segment composition is F2 segment；It is F3-HdvRz/ by DHAV-3-F3-HDVR and HDVR-SV40pA segment composition
SV40pA segment, three DNA fragmentations finally obtained constitute mutant strain ISA-A117C " infectious Subgenomic replicon ".
Gel reclaims kit (Omega) gel extraction purpose is respectively adopted after the separation of 1% agarose gel electrophoresis in amplified fragments
Segment.DNA fragmentation is sent to Shanghai Sheng Gong bioengineering Co., Ltd and is sequenced.
3.4. the transfection rescue of mutant strain ISA-A117C " infectious Subgenomic replicon "
Primary duck embryo fibroblasts are prepared using 9 age in days duck embryos, when the cell in 3.5cm culture dish is grown to
When 90% fusion, by pCMV-F1, F2 and F3-HdvRz-SV40pA genetic fragment and Lipofectamine of equivalent (1.5 μ g)
90% duck embryo fibroblasts are covered in transfection after 3000 (Invitrogen) mixing, and Lipofectamine is used only in control group
3000 (Invitrogen) are transfected.Cell is placed in 37 DEG C of 5%CO2Culture observation is carried out in incubator, after 16 hours more
Culture medium is changed, after transfection 72 hours, cell fragmentation phenomenon occurs in transfection group cell, and cellular control unit upgrowth situation is good.
After transfection duck embryo fibroblasts 120 hours, cell growth condition is as shown in fig. 7, by cell multigelation after photographing to record
3 times, cell culture fluid is inoculated with 5 piece of 9 age in days duck embryos by allantoic cavity approach, 0.2mL/ pieces, is put into incubator after paraffin sealing
Inside continue to be incubated for, every 8 hours photograph embryo 1 time, the death condition of observation duck embryos after inoculation discards dead duck embryos in 24 hours.
Duck embryos are dead between 24 hours to 48 hours after inoculation as the result is shown, and duck embryos death idiosome bleeding is serious, collect allantoic fluid and make
It is saved for first generation reverse genetic Strain.
3.5. in reverse genetic mutant strain genetic marker and mutational site identification
It may be from parental virus or street strain in transfection or succeeding generations to exclude reverse genetic mutant virus
The possibility of pollution, using reverse-genetics approach, 3403 bit bases of mutant strain genome sport T by being mutated G, and the mutation is not
Change the composition of the corresponding amino acid of 2A albumen, which can be surveyed as molecular genetic marker site by PCR method combination DNA
Sequence distinguishes mutant strain and parent plant and street strain.Revive virus passes through limiting dilution assay passage purifying 5 times in duck embryos.
Total serum IgE is extracted from allantoic fluid, expands the DNA piece containing genetic marker site using F2-F and F2-R primer PCR after reverse transcription
Section expands the DNA fragmentation containing targeted mutagenesis site using F1-F and F1-R primer PCR, and amplified fragments are solidifying through 1% agarose
Then gel electrophoresis separation uses gel reclaims kit (Omega) gel extraction, and DNA fragmentation is sent to the raw work biology in Shanghai
Engineering Co., Ltd is sequenced, sequencing result display amplification product contain introducing genetic marker site (G3403T) and
Targeted mutagenesis site (A117C) shows we obtain correct Revive virus, rather than parent plant or wild type strains are dirty
Dye.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understand without departing from the principles and spirit of the present invention can to these examples carry out it is a variety of variation, modification, replacement and
Modification, the scope of the present invention is defined by the appended.
Sequence table
<110>Sichuan Agricultural University
<120>a kind of method of 3 type duck hepatitis A virus reverse genetic strain of rapid build
<160> 17
<170> SIPOSequenceListing 1.0
<210> 1
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tagttattaa tagtaatcaa ttacggggtc a 31
<210> 2
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
acaccacagc cgctttcaaa cggttcacta aaccagctct 40
<210> 3
<211> 40
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
agagctggtt tagtgaaccg tttgaaagcg gctgtggtgt 40
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
caacctgcca aaagtcaaac ca 22
<210> 5
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
attctgttac acctttacgc cccaca 26
<210> 6
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
caacctaggt aagtgagcac gat 23
<210> 7
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gtgctcactt acctaggttg gtt 23
<210> 8
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tggcaacttc ctgtctaacc tg 22
<210> 9
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ccttgaacac tggaacccaa 20
<210> 10
<211> 69
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
aagtagccca ggtcggaccg cgaggaggtg gagatgccat gccgaccctt tttttttttt 60
ttagggtgg 69
<210> 11
<211> 59
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
cggtccgacc tgggctactt cggtaggcta agggagaaga acttgtttat tgcagctta 59
<210> 12
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
taagatacat tgatgagttt gga 23
<210> 13
<211> 583
<212> DNA
<213>cytomegalovirus early promoter is sub (pCMV)
<400> 13
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cgt 583
<210> 14
<211> 67
<212> DNA
<213>hepatitis delta virus ribozyme sequence (HDVR)
<400> 14
gggtcggcat ggcatctcca cctcctcgcg gtccgacctg ggctacttcg gtaggctaag 60
ggagaag 67
<210> 15
<211> 122
<212> DNA
<213>vacuolating virus of monkey early stage mRNA polyadenylation signal (SV40pA)
<400> 15
aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60
aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120
ta 122
<210> 16
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
gcctagtcct agcgctatag gactccc 27
<210> 17
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
gggagtccta tagcgctagg actaggc 27
Claims (5)
1. a kind of method of 3 type duck hepatitis A virus reverse genetic strain of rapid build, which comprises the following steps:
1) parental virus strain full-length genome is divided into three segments similar in size and carries out PCR amplification, in viral genome first
The end 5' addition cytomegalovirus early promoter of a segment, introduces same sense mutation as something lost in the 2A gene of second segment
Marker site is passed, adds hepatitis delta virus ribozyme sequence and vacuolating virus of monkey early stage mRNA polyadenous at the end 3' of the third fragment
Nucleotide signal sequence obtains three DNA fragmentations, constitutes 3 type duck hepatitis A virus infectivity Subgenomic replicons；
2) duck embryo fibroblasts are transfected after mixing the infectious Subgenomic replicon of building with transfection reagent, replicon exists
The spontaneous recombination of homologous recombination machinery for transcribing and utilizing cell in host cell, being formed has the transcription of infective intact virus
This, then leads to the duplication and proliferation of virus, obtains the 3 type duck hepatitis A virus reverse genetic strain diseases containing genetic marker
Poison.
2. a kind of method of 3 type duck hepatitis A virus reverse genetic strain of rapid build according to claim 1, feature exist
Contain the overlay region of 74 and 83 base-pairs with second segment respectively in, first segment and the third fragment
Domain.
3. a kind of method of 3 type duck hepatitis A virus reverse genetic strain of rapid build according to claim 1, feature exist
In it is prominent that a synonymous base is introduced in the 2A gene of 3 second segment of type duck hepatitis A virus genome with fusion DNA vaccine method
Become, so that the G of the 3403rd nucleotide of reverse genetic strain virus sports T.
4. a kind of method of 3 type duck hepatitis A virus reverse genetic strain of rapid build according to claim 1, feature exist
In the parental virus strain is 3 type duck hepatitis A virus velogen strains, is preserved in China typical culture collection center, and preservation is compiled
Number be CCTCCNO:V201305.
5. a kind of method of 3 type duck hepatitis A virus reverse genetic strain of rapid build according to claim 1, feature exist
In the method can carry out mutation or fragment deletion/replacement/insertion to any base of 3 type duck hepatitis A virus, obtain new
It is mutated recombinant virus.
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