CN105861555A - Efficient bidirectional transcription/expression plasmid and application thereof in influenza virus reverse genetics - Google Patents
Efficient bidirectional transcription/expression plasmid and application thereof in influenza virus reverse genetics Download PDFInfo
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Abstract
The invention discloses an efficient bidirectional transcription/expression plasmid and application thereof in influenza virus reverse genetics. The bidirectional transcription/expression plasmid is obtained by transformation by taking a eukaryotic expression vector pEF4/myc-His B as a skeleton. In order to create a reverse genetic system of EIVs (Equine influenza viruses), the bidirectional transcription/expression plasmid obtained by structuring is used for respectively expressing EIVs PB2, PB1, PA, HA, NP, NA and NS and M genes, and a strain of the EIV and mutants thereof are successfully saved by means of cotransfection, sequencing, drawing of growth curves and enzyme digestion are obtained. Compared with existing pBD systems, the created reverse genetic system is superior to the pBD systems in toxigenic quantity. A novel technical means is provided for research on reverse genetics of the influenza viruses, and a solid foundation is laid for future in-depth study on transmission of the EIVs, pathogenesis and even screening of vaccine candidates.
Description
Technical field
The present invention relates to a kind of bidirectional transcription plasmid and the application in influenza virus reverse genetics, the present invention
Belong to biological technical field.
Background technology
Influenza virus belongs to orthomyxovirus section (Orthomyxoviridae), anti-according to NP albumen and M albumen
Originality can be divided into, A, B and c-type influenza virus.Wherein, c-type influenza virus only temperate infection people, almost without
Symptom.A type and Type B influenza virus all may result in seasonal influenza, and influenza A can cause entirely
Ball is very popular.Additionally, the genome of influenza virus is individual segmented by 7 (c-types) or 8 (A type and Type Bs)
Mononegavirale RNA forms, and is susceptible to reset, produces new strain, and this makes the prevention and control of influenza encounter to choose
War.The prevention and control of the understanding and influenza for promoting influenza virus of studying flu virus have positive effect.
Reverse genetics is one of instrument of being most widely used in influenza virus research.At present, there is many
Virus rescue system is applied in the correlational study of influenza virus, wherein the most simple and easy to do with 8 pUC pUCs,
Its core is bidirectional transcription/expression vector.This kind of carrier typically have the relative polI promoter of transcriptional orientation and
PolII promoter, it is possible to for template Retroviral strand RNA respectively and express virus protein with same cDNA,
And then assemble virus.2000, Hoffman etc. utilized pHW2000 carrier to establish the 8 of H1N1 first
PUC pUC.2005, Li Zejun etc. used 8 pUC pUC research H5N1 fowl based on pBD carrier first
Influenza virus.Although pHW2000 and pBD all uses strong CMV promoter, the f1 disease of rescue
The titre of poison is the most relatively low, needs, by mdck cell or Embryo Gallus domesticus amplification culture, to waste time and energy, it is difficult to adapt to
Many Research Requirements.But, it has been reported that EF1-α promoter is higher than CMV promoter activity.The most permissible
Use EF1-α promoter to build bidirectional transcription/expression vector, set up 8 plasmid reverse genetics systems, save out more
The virus of high titre.
Based on above-mentioned it is assumed that verify through scientific experiments, the special proposition present invention.
Summary of the invention
The technical problem to be solved is to provide a kind of efficient bidirectional transcription/expression plasmid and in influenza
Application in virus reverse hereditism.
In order to achieve the above object, present invention employs techniques below means:
A kind of bidirectional transcription/expression vector, it is characterised in that with carrier for expression of eukaryon pEF4/myc-His B as skeleton
Transform: between KpnI and the BclI restriction enzyme site of described pEF4/myc-His B carrier, insert Mus source
The polI promoter sequence in polI terminator, BsmBI restriction enzyme site and people source；At itself SphI and BspQI enzyme action
Insert the Linker sequence of a section short between site, replace on original vector from f1ori to SV40poly a-signal it
Between nonessential element, described Linker sequence is 5 '-CTGGGGATGCGGTGGGCTCTATG-
3 ', by named for improved carrier pEZ.
In the present invention, it is preferred to, the nucleotide sequence such as SEQ ID of described bidirectional transcription/expression vector
Shown in NO.1.
Further, the invention allows for the reverse genetic operating system of a kind of equine influenza virus, it contains 8
The recombiant plasmid built based on bidirectional transcription/expression plasmid of the present invention, expresses wild type or sudden change respectively
Equine influenza virus PB2, PB1, PA, HA, NP, NA, NS and M gene segment of type.
In the present invention, it is preferred to, described influenza virus PB2, PB1, PA, HA, NP, NA, NS
And the nucleotide sequence of M gene segment is respectively as shown in SEQ ID NO.2-9.
In one particular embodiment of the present invention, it is preferred that HA gene segment is with HindIII and NdeI enzyme
Cutting site, its nucleotide sequence is as shown in SEQ ID NO.10.
Further, the invention allows for the reverse genetic operating system described in any of the above item in equine influenza
Purposes in virus rescue.
A kind of method utilizing reverse genetic operating system of the present invention rescue equine influenza virus, including following
Step:
(1) structure of bidirectional transcription/expression vector
Carrier for expression of eukaryon pEF4/myc-His B is that skeleton is transformed: at described pEF4/myc-His B carrier
KpnI and BclI restriction enzyme site between insert the polI terminator in Mus source, BsmBI restriction enzyme site and people source
PolI promoter sequence；Between itself SphI and BspQI restriction enzyme site, insert the Linker sequence of a section short, replace
Changing the nonessential element between f1ori to SV40poly a-signal on original vector, described Linker sequence is
5 '-CTGGGGATGCGGTGGGCTCTATG-3 ', by named for improved carrier pEZ；
(2) structure and the introducing of molecular marker of the recombiant plasmid of virus cDNA are carried
Extract the viral RNA of A/equine/Jilin/1/1989 strain, with uni12 as reverse transcription primer, save with each
Duan Xiangying primer to for amplimer, uses RT-PCR method 8 gene segments of amplification: PB2, PB1,
PA, HA, NP, NA, M and NS；Each sections, after BsmBI or BsaI enzyme action, distinguishes directed cloning
Among the BsmBI of pEZ, build and obtain 8 recombiant plasmid；
Described primer sequence is as follows:
(3) rescue of recombinant virus
Use step (2) to build the recombiant plasmid cotransfection cell of 8 sections obtained, save recombinant virus.
In method of the present invention, it is preferred that also include using following primer to enter to wild type HA sections
Row T449C and C983The nonsense point mutation of T, builds the recombiant plasmid containing saltant type HA sections so that it is HA
Sections contains single HindIII and NdeI restriction enzyme site, use HA sections containing sudden change recombiant plasmid and
Contain the recombiant plasmid cotransfection cell of other 7 sections respectively, save recombinant virus；
5’-CTTCGCTGAAAGCTTCACTTGGACA-3’
5 '-TGTCCAAGTGAAGCTTTCAGCGAAG-3 ' and
5’-CAAGATCACATATGGAGCATGTCCC-3’
5’-GGGACATGCTCCATATGTGATCTTG-3’
It is furthermore preferred that the nucleotide sequence of the HA sections of sudden change is as shown in SEQ ID NO.10.
In method of the present invention, it is preferred that step (1) builds the bidirectional transcription/expression vector obtained
The nucleotide sequence of pEZ is as shown in SEQ ID NO.1.
In method of the present invention, it is preferred that influenza virus PB2, PB1, PA, HA, NP,
The nucleotide sequence of NA, NS and M gene segment is respectively as shown in SEQ ID NO.2-9.
Compared to prior art, the beneficial effects of the present invention is:
The EF1-α and the polI promoter that present invention employs people source have createed the bidirectional transcription/expression optimized and have carried
Body, EF1-α promoter and BGH poly a-signal are positioned at outside polI promoter and terminator and two promoteres
Transcriptional orientation relative, and between polI promoter and terminator containing two BsmBI restriction enzyme sites so that
Insert exogenous dna fragment, there is the function utilizing same template to transcribe out mRNA and strand RNA, permissible
It is applied in the research of minus-stranded rna virus, sets up reverse genetics system.The present invention is applied to EIV JL89
In the reverse genetics of strain and variant thereof, successfully save EIV JL89.And this system is existing with domestic
PBD system is compared, and this system can save first generation virus in a large number, and its titre is 19 times of pBD system, aobvious
Write higher than pBD system, produce poison amount higher.
Accompanying drawing explanation
Fig. 1 is the structure schematic diagram of bidirectional transcription/expression vector pEZ；
Fig. 2 is the structure schematic diagram of pEZ-vEGFP；
Fig. 3 is the testing result of green fluorescence；
Fig. 4 is strand RNA integrity detection result；
Fig. 5 is the electrophoresis detection result of 8 fragments of amplification；
Fig. 6 is the enzyme action qualification result of HA sections；
Fig. 7 is the growth kinetics curve in mdck cell；
Fig. 8 is to use pEZ system and the measurement result of pBD rescuing system virus titer.
Detailed description of the invention
Further describe the present invention, advantages of the present invention and feature below in conjunction with specific embodiment to will be with retouching
State and apparent.But these embodiments are only exemplary, the scope of the present invention is not constituted any limit
System.It will be understood by those skilled in the art that can be to this under without departing from the spirit and scope of the present invention
The details of bright technical scheme and form are modified or replace, but these amendments and replacement each fall within the guarantor of the present invention
In the range of protecting.
Main agents involved in following example of the present invention and experiment material
1, Strain and cell
A/equine/Jilin/1/1989 (H3N8) Strain (being called for short JL89) has been documented in document (Heilongjiang Province
The genome sequencing of one strain equine influenza virus and homology analysis, Zhao Zhao etc., " China's animal inspection
Epidemic disease ", 12 phases in 2015), existing by Harbin Veterinary Medicine Inst., China Academy of Agriculture's preservation, offer；HEK
293T cell and mdck cell are preserved by this laboratory.
2, main agents
PEF4/myc-His B plasmid, SuperScript III reverse transcription, RNase inhibitor,
Lipofectamine 2000 transfection reagent is purchased from invitrogen company；T4RNA ligase, Phusion DNA
Polymerase, restriction endonuclease are purchased from New England Biolabs company；Viral RNA extracts test kit
Purchased from Qiagen company；PMD18-T carrier is purchased from TaKaRa company；PBD carrier is by being aged blue researcher's favour
Give；SPF Embryo Gallus domesticus is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture.
The structure of embodiment 1 bidirectional transcription/expression vector and Function Identification
1, the structure of bidirectional transcription/expression vector
As it is shown in figure 1, transform with carrier for expression of eukaryon pEF4/myc-His B for skeleton: at its KpnI and
Required element (Insert 1, sequence is as shown in SEQ ID NO.11) Mus source is inserted between BclI restriction enzyme site
PolI terminator (TpolI, 33bp), BsmBI restriction enzyme site and people source polI promoter (PpolI,
236bp) sequence；Linker sequence (the Insert of one section short is inserted between itself SphI and BspQI restriction enzyme site
2, sequence is 5 '-CTGGGGATGCGGTGGGCTCTATG-3 '), replace on original vector from f1ori to
Nonessential element between SV40poly a-signal.The named pEZ of improved carrier, its nucleotide sequence is such as
Shown in SEQ ID NO.1.
2, the construction of recombinant plasmid of reporter gene
Reporter gene is inserted to verify its bidirectional transcription/expressive function in pEZ carrier.Use primer to (5 '-
TATTCGTCTCAGGGAGCAAAAGCAGGTAGATATTTAAAGATGGTGAGCAAGG
GCGAGGA-3’(BsmBI)/5’-
ATATCGTCTCGTATTAGTAGAAACAAGGTAGTTTTTTACTTGTACAGCTCGTC
CA-3 ' (BsmBI)), with EGFP gene as template, utilize PCR method obtain reporter gene (vEGFP,
765bp), its 5 ' end and 3 ' ends merge 5 ' noncoding regions of upper EIV JL89 strain M sections respectively
(5 ' UTR) and 3 ' noncoding regions (3 ' UTR), orients after restricted enzyme BsmBI enzyme action and is inserted in
Among the BsmBI of pEZ carrier, construction recombination plasmid (pEZ-vEGFP) (Fig. 2).
3, the detection of green fluorescence
According to Lipofectamin 2000 description, recombiant plasmid pEZ-vEGFP transfection abundance is reached more than 90%
293T cell, change liquid after 6h, 48h be placed under inverted fluorescence microscope observation green fluorescence.In experiment together
Time empty vector control is set.
4, the detection of strand RNA integrity
TRIzol is used to extract the total serum IgE in cell after plasmid transfection 48h.With uni12 (5 '-
AGCAAAAGCAGG-3 ') it is reverse transcription primer, with primer to M-F/M-R (table 1) as amplimer,
Expand vEGFP by RT-PCR method, be cloned in pMD18-T carrier sequence verification.
Meanwhile, utilize RNA interconnection technique to make RNA molecule be cyclized, with O-vEGFP F (5 '-
CTGCTGCCCGACAACCACTACCTGA-3 ') it is reverse transcription primer, with primer to O-vEGFP F
/ O-vEGFP R (5 '-GTGAACAGCTCCTCGCCCTTGCTC-3 ') it is amplimer, pass through
The noncoding region of RT-PCR method amplification vEGFP, is cloned in pMD18-T carrier sequence verification.
Result:
Bidirectional transcription/expression vector pEZ is adapted from carrier for expression of eukaryon pEF4/myc-His B, starts at its polI
Containing two BsmBI restriction enzyme sites so that directed cloning foreign DNA sheet between son and polI terminator sequence
Section, its EF1-α promoter and BGH poly a-signal are positioned at outside polI promoter and polI terminator and two
The transcriptional orientation of promoter is relatively (Fig. 1).Wherein EF1-α promoter and BGH poly a-signal can instruct
MRNA transcribes, and then translates albumen；And polI promoter and polI terminator can control strand RNA
Transcribe.
The function utilizing same template to transcribe out mRNA and strand RNA to verify pEZ carrier to have, structure
Building recombiant plasmid pEZ-vEGFP, its vEGFP is formed (Fig. 2) by 5 ' UTR, EGFP and 3 ' UTR.
PEZ-vEGFP and pEZ transfects 293T cell respectively, observes green fluorescence after 48h.PEZ-vEGFP transfects
Cell it is observed that green fluorescence, and the cell of pEZ transfection does not has green fluorescence (Fig. 3).This shows
PEZ-vEGFP transcribes out corresponding mRNA with vEGFP for template intracellular, and then translates EGFP egg
In vain.
On the one hand, extract cell total rna after plasmid transfection and carry out RT-PCR amplification (RT group) and direct
PCR expands (No RT group).Wherein, can amplify and sun through RT-PCR after only pEZ-vEGFP transfection
Property the comparison (+Ctrl) band that is consistent, and other process can not amplify respective strap (Fig. 4 A), and surveys
Sequence result is consistent with vEGFP sequence.On the other hand, the RNA molecule of extraction is laggard through RNA ligase effect
Row RT-PCR expands.Wherein can amplify and the bar expecting that (212bp) is consistent after pEZ-vEGFP transfection
Band, and respective strap (Fig. 4 B) can not be amplified after pEZ transfection.In addition sequencing result (figure consistent with expection
4C).This shows that pEZ-vEGFP complete has accurate 5 ' intracellular transcribe out with vEGFP for template
The strand RNA of UTR and 3 ' UTR.
Result above shows that pEZ carrier has bidirectional transcription/expressive function, it is possible to use same template gives expression to phase
The albumen answered and transcribe out complete strand RNA.
The foundation of embodiment 2EIV reverse genetics system
1, structure and the introducing of molecular marker of the recombiant plasmid of virus cDNA are carried
Extract test kit operation instructions by viral RNA and extract the viral RNA of EIV JL89 strain, with uni12
For reverse transcription primer, with the corresponding primer of each sections to (table 1) as amplimer, RT-PCR method is used to expand
Increase 8 gene segments (PB2, PB1, PA, HA, NP, NA, M and NS).Each sections is through corresponding
Restricted enzyme (BsmBI or BsaI) enzyme action after, respectively directed cloning is among the BsmBI of pEZ,
Structure obtains 8 recombiant plasmid, contained 8 gene segment PB2, PB1, PA, HA, NP,
The nucleotide sequence of NA, NS and M gene segment is respectively as shown in SEQ ID NO.2-9.
Table 1 expands the primer of 8 sections
Additionally, use primer to 5 '-CTTCGCTGAAAGCTTCACTTGGACA-3 '/5 '-
TGTCCAAGTGAAGCTTTCAGCGAAG-3 ' and 5 '-
CAAGATCACATATGGAGCATGTCCC-3’/5’-
GGGACATGCTCCATATGTGATCTTG-3 ', carries out nonsense point and dashes forward wild type (wt) HA sections
Become (T449C and C983T), the recombiant plasmid containing saltant type (mutant) HA sections is built so that it is HA
Sections contains single HindIII and NdeI restriction enzyme site, the nucleotide sequence such as SEQ of the HA sections after sudden change
Shown in ID NO.10.
Result:
Extract viral RNA, expand 8 sections (Fig. 5) through RT-PCR, it is cloned into respectively pEZ and carries
Among the BsmBI of body, build 8 recombiant plasmid.
2, the rescue of recombinant virus
Use the recombiant plasmid of the HA sections containing sudden change and contain the recombiant plasmid corotation of other 7 sections respectively
Dye cell, saves recombinant virus.According to Lipofectamin 2000 description, by 8 each 0.5 μ of recombiant plasmid
G (totally 4 μ g) and 10 μ L liposomees are dissolved in 250 μ L Opti-MEM respectively, the two mixing, dropwise add
Entering in six orifice plates in the 293T cell that abundance reaches more than 90%, changing liquid after transfection 6h is nonreactive serum-free
Opti-MEM, adds the TPCK-pancreatin of final concentration of 250ng/mL, gathers in the crops after transfection 48h after transfection 30h
Supernatant, as first generation recombinant virus (rJL89).Use 9～11 age in days SPF Embryo Gallus domesticus amplification culture
Gather in the crops allantoic fluid after rJL89,72h, use 1% chicken red blood cell to measure its hemagglutinative titer.
Result:
Site-directed mutagenesis technique is utilized to build the recombiant plasmid containing HindIII and NdeI restriction enzyme site on HA sections.
By this plasmid and other 7 plasmid co-transfection 293T cellular rescue recombinant viruses, HA effect after replicating in Embryo Gallus domesticus
Valency reaches 28.Recombinant virus (rJL89) is saved out in this explanation.
The qualification of embodiment 3 recombinant virus
1, the sequencing of recombinant virus and the detection of its molecular marker
Expand 8 sections of rJL89 according to primer described in embodiment 2 method 1 and method, be cloned into pMD
18T carrier, measures each sections sequence, and compares with each sections sequence of JL89.Meanwhile, make
With restricted enzyme HindIII and NdeI, HA sections is carried out enzyme action qualification.
Result:
8 sections of clone rJL89, and measure its sequence.Each sections of rJL89 shows with the comparison of JL89
Showing, HA sections contains nonsense mutation (T449C and C983T), the HA sections concordance with JL89 reaches
99.9%；And PB2, PB1, PA, NP, NA, M and NS sections concordance corresponding to JL89 reaches
100%；The hereditary information almost all of this explanation rJL89 is derived from JL89.
The HA sections of rJL89 contains HindIII and NdeI restriction enzyme site, distinguishes rescue as molecular marker
Recombinant virus and parent's strain.Utilize restricted enzyme HindIII and this saltant type of NdeI enzyme action
(mutant) HA sections and wild type (wt) HA sections.Result shows, saltant type (mutant) HA can
To be cut into two bands (about 446bp and 1 319bp) by HindIII, it is cut into two bands (about by NdeI
785bp and 980bp), it is three bands (about 446bp, 534bp and 785 by HindIII and NdeI double digestion
bp)；And wild type (wt) HA can not be cut, it is consistent with negative control (-Ctrl) banding pattern (Fig. 6).This
Illustrate that recombinant virus rJL89 the most stably carries this molecular marker.
2, viral growth kinetic determination
Infect mdck cell with the virus quantity of 0.01moi, after infecting 3,6,9,12,24,36,
48,60 and 72h results supernatant.Use TCID50Method measures the virus titer of each time point, by virus liquid
Carry out doubling dilution (10-1～10-11, totally 11 dilution factors), each dilution factor 100 μ L inoculates 8 hole MDCK
Cell, adds up after 72h and occurs cytopathic hole count under each dilution factor, calculates virus titer
(TCID50/mL).With infection time (Hours post infection) as abscissa, with virus titer
(lgTCID50/ mL) it is vertical coordinate, draw viral growth curves.And utilize statistical method to analyze each time
The difference of point rJL89 and JL89 virus titer.
Result:
RJL89 is after Embryo Gallus domesticus amplification culture, and its hemagglutinative titer reaches 28, identical with the hemagglutinative titer of JL89.This
Show that rJL89 saves the blood clotting characteristic of JL89.
RJL89 and JL89 infects different time points results supernatant after mdck cell, and measures its virus titer,
Draw growth curve (Fig. 7).From Fig. 7 result it can be seen that rJL89 infects after mdck cell,
In 0～12h, its virus titer slowly increases；In 12～48h, its virus titer rises rapidly and reaches peak value；It
After maintain near this titre.This is identical with the growth characteristics of JL89.Although the two is in the virus of each time point
Titre has difference, but difference is the most notable.This shows that this system successfully saves the virion providing infectious,
And rJL89 has the growth characteristics identical with JL89 on mdck cell.
The assessment of embodiment 4pEZ rescuing system virus capable
Method:
PEZ system and pBD system is utilized (to be built by this laboratory, also contain the full-length genome of JL89, reference
Document: Li Z, Chen H, Jiao P, Deng G, Tian G, Li Y, Hoffmann E, Webster RG, Matsuoka
Y,Yu K.Molecular basis of replication of duck H5N1influenza viruses in a mammalian
mouse model.J Virol.2005Sep；79(18):12058-64.PubMed PMID:16140781；PubMed
Central PMCID:PMC1212590.), use method Revive virus described in embodiment 2 method 2.Use
Method described in embodiment 3 method 2 measures the titre of first generation virus.Statistical method is utilized to analyze the two difference.
Result:
Use pEZ system and pBD system Revive virus simultaneously, and measure the titre of first generation virus.Result is divided
It is not 2.38 × 105With 1.25 × 104Individual TCID50/ mL, illustrates that the titre of pEZ rescuing system virus is pBD
19 times of system, the two significant difference (Fig. 8).
Claims (10)
1. bidirectional transcription/expression vector, it is characterised in that with carrier for expression of eukaryon pEF4/myc-His B as bone
Frame is transformed: insert Mus source between KpnI and the BclI restriction enzyme site of described pEF4/myc-His B carrier
The polI promoter sequence in polI terminator, BsmBI restriction enzyme site and people source；At itself SphI and BspQI enzyme action
Insert the Linker sequence of a section short between site, replace on original vector from f1 ori to SV40poly a-signal it
Between nonessential element, described Linker sequence is 5 '-CTGGGGATGCGGTGGGCTCTATG-
3 ', by named for improved carrier pEZ.
2. bidirectional transcription/expression vector as claimed in claim 1, it is characterised in that described bidirectional transcription/table
Reach the nucleotide sequence of carrier as shown in SEQ ID NO.1.
3. the reverse genetic operating system of an equine influenza virus, it is characterised in that containing 8 based on claim
Bidirectional transcription/expression plasmid described in 1 or 2 and the recombiant plasmid that builds, express wild type or the horse of saltant type respectively
Influenza virus PB2, PB1, PA, HA, NP, NA, NS and M gene segment.
4. reverse genetic operating system as claimed in claim 3, it is characterised in that described influenza virus
The nucleotide sequence of PB2, PB1, PA, HA, NP, NA, NS and M gene segment is respectively such as SEQ
Shown in ID NO.2-9.
5. reverse genetic operating system as claimed in claim 3, it is characterised in that HA gene segment with
HindIII and NdeI restriction enzyme site, its nucleotide sequence is as shown in SEQ ID NO.10.
6. the use in equine influenza virus is saved of the reverse genetic operating system according to any one of claim 3-5
On the way.
7. the reverse genetic operating system rescue equine influenza virus that a kind utilizes described in any one of claim 3-5
Method, it is characterised in that comprise the following steps:
(1) structure of bidirectional transcription/expression vector
Carrier for expression of eukaryon pEF4/myc-His B is that skeleton is transformed: at described pEF4/myc-His B carrier
KpnI and BclI restriction enzyme site between insert the polI terminator in Mus source, BsmBI restriction enzyme site and people source
PolI promoter sequence；Between itself SphI and BspQI restriction enzyme site, insert the Linker sequence of a section short, replace
Changing the nonessential element between f1ori to SV40poly a-signal on original vector, described Linker sequence is
5 '-CTGGGGATGCGGTGGGCTCTATG-3 ', by named for improved carrier pEZ；
(2) structure and the introducing of molecular marker of the recombiant plasmid of virus cDNA are carried
Extract the viral RNA of A/equine/Jilin/1/1989 strain, with uni12 as reverse transcription primer, save with each
Duan Xiangying primer to for amplimer, uses RT-PCR method 8 gene segments of amplification: PB2, PB1,
PA, HA, NP, NA, M and NS；Each sections, after BsmBI or BsaI enzyme action, distinguishes directed cloning
Among the BsmBI of pEZ, build and obtain 8 recombiant plasmid；
Described primer sequence is as follows:
(3) rescue of recombinant virus
Use step (2) to build the recombiant plasmid cotransfection cell of 8 sections obtained, save recombinant virus.
8. method as claimed in claim 7, it is characterised in that also include using following primer to wild type
HA sections carries out T449C and C983The nonsense point mutation of T, builds the recombiant plasmid containing saltant type HA sections,
Make its HA sections contain single HindIII and NdeI restriction enzyme site, use the weight containing the HA sections suddenlyd change
Organize plasmid and contain the recombiant plasmid cotransfection cell of other 7 sections respectively, saving recombinant virus；
5’-CTTCGCTGAAAGCTTCACTTGGACA-3’
5 '-TGTCCAAGTGAAGCTTTCAGCGAAG-3 ' and
5’-CAAGATCACATATGGAGCATGTCCC-3’
5’-GGGACATGCTCCATATGTGATCTTG-3’
Preferably, the nucleotide sequence of the HA sections of sudden change is as shown in SEQ ID NO.10.
9. method as claimed in claim 7, it is characterised in that step (1) builds the bidirectional transcription/table obtained
Reach the nucleotide sequence of carrier pEZ as shown in SEQ ID NO.1.
10. method as claimed in claim 7, it is characterised in that influenza virus PB2, PB1, PA, HA,
The nucleotide sequence of NP, NA, NS and M gene segment is respectively as shown in SEQ ID NO.2-9.
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