CN105483139B - A kind of DNA molecular and application thereof comprising chikungunya virus full-length genome cDNA - Google Patents
A kind of DNA molecular and application thereof comprising chikungunya virus full-length genome cDNA Download PDFInfo
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- CN105483139B CN105483139B CN201410471749.4A CN201410471749A CN105483139B CN 105483139 B CN105483139 B CN 105483139B CN 201410471749 A CN201410471749 A CN 201410471749A CN 105483139 B CN105483139 B CN 105483139B
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Abstract
The invention discloses a kind of nucleotide sequence DNA moleculars as shown in SEQ ID NO:1 and application thereof, infection clones and its construction method including chikungunya virus DRDE-06 plants of label of method, nucleotide sequence chikungunya virus as shown in SEQ ID NO:1 of the recombinant plasmid containing the DNA molecular and its corresponding cDNA of construction method, geneome RNA.DNA molecular or recombinant plasmid of the invention can be used in constructing the infection clones of chikungunya virus similar in performance and wild-type virus.
Description
Technical field
The present invention relates to a kind of DNA moleculars and application thereof, and in particular, to one kind includes chikungunya virus full-length genome
The DNA molecular of cDNA, method, the recombinant plasmid containing the DNA molecular and its building for marking chikungunya virus DRDE-06 plants
The infection clones and its construction method of method, chikungunya virus.
Background technique
Chikungunya virus (Chikungunya virus, CHIKV) belongs to Togaviridae (Togaviridae) onychonosus
Poison belongs to (Alphavirus), and the member of the category further includes ross river virus (Ross River viruses), Semliki Forest
Virus (Semliki forest virus) and sindbis alphavirus (Sindbis virus) etc..The primary vehicle of CHIKV
It is Aedes aegypti (Aedes aegypti) and aedes albopictus (Ae.albopictus), can lead to Chikungunya fever, morbidity is special
Sign shows as generating heat, courbature and arthralgia.Although the death rate of Chikungunya fever is lower, its spread scope in the past 50 years
It expands rapidly, propagates to Europe, asia and ocenia area rapidly via originally popular East Africa area.Especially Southeast Asia
The India in area, Indonesia, Thailand and Vietnam etc. are national, there is the eruption and prevalence of Chikungunya fever repeatly in the past 10 years.China
The report of introduced cases only distributed between 2006-2008, but Dongguan, Guangdong area in 2010 broken out it is primary small
The Chikungunya fever epidemic situation of scale.Therefore, Chikungunya fever have become it is a kind of seriously threaten human health send out infectious disease again, but
It is at present still without effective therapeutic agent and prevention vaccine.
CHIKV is a kind of enveloped virus, and genome is the single-stranded positive RNA that length is about 12 kb, containing there are two reading codes
Frame is separately encoded 3 structural proteins (capsid protein C, envelope protein E1 and E2) and 4 non-structural proteins (nsP1-4).RNA disease
The full-length infectious CDNA clones of poison can get after being transcribed in vitro has infective viral RNA, then obtains after transfecting cell
Revive virus.This greatly facilitates us and is oriented transformation to virus genome sequence, so that obtaining has the prominent of new traits
Become virus.By comparing the variation of wild virus and mutated viruses biological property, the virulence site of viral key can be determined,
The epitope for filtering out viral key, provides target spot for vaccine design.Therefore, viral infectious full-length cDNA clone is deep
Enter to study the basis of virus replication and pathogenic mechanism.
The basic principle of synthetic biology is to obtain the target gene information being fitted to by genome database, then use
Iii vitro chemical synthetic method obtains corresponding gene order, and then is conducted into specific cells or tissue, and final rescue obtains
There must be the life entity of biological activity.Synthetic biology is that the construction method of the infection clones of virus opens new side
To such as obtaining the genome sequence for causing 1918 big strains of influenza viruses by full genome synthetic method, and successfully save
The influenza virus of biological activity is provided, the research of the biological property of influenza virus has effectively been pushed.
The research of current life science is had become by the gene order that chemical synthesis process obtains a life entity
Hot spot also brings many problems while greatly facilitating research life quintessence.An artificial synthesized nature is simultaneously not present
Completely new sequence, biological property is unknown, and Evaluation of Biocompatibility is urgently to be resolved.Once biological leakage accident occurs,
The sequence for how distinguishing artificial synthesized sequence and natural evolution is just particularly important, therefore obtains effective differentiating method meaning
It is great.
Summary of the invention
The object of the present invention is to provide a kind of Chickungunya virus genome cDNA of mutation, thus obtain can with it is wild
The infection clones mutually distinguished of chikungunya virus.
In order to distinguish artificial synthesized and natural evolution biological gene sequence, the present inventor has carried out a large amount of realities
It tests, as a result, it has been found that, two coding mutations (7522-7523 sport TT) is introduced in specific site, will not influence disease
The features such as the infectivity of poison.Further being found by sequence alignment, above-mentioned mutation does not occur in the sequence of natural evolution, because
This is to discriminate between artificial synthesized and natural evolution label, convenient for the evaluation to the sequence biological safety.
Therefore, to achieve the goals above, in a first aspect, including chikungunya virus full genome the present invention provides one kind
The DNA molecular of group cDNA, the nucleotide sequence of the DNA molecular is as shown in SEQ ID NO:1.DNA molecular provided by the invention can
Genome infectious full length cDNA clone as chikungunya virus, and the infectious and comparable base of wild virus can be obtained
Agree refined virus in hole.
Second aspect, the present invention provides a kind of recombinant plasmid, which contains nucleosides shown in SEQ ID NO:1
Acid sequence.
The recombinant plasmid may include the elements such as promoter, according to the present invention preferred embodiment, the recombination matter
Grain is to be connected with promoter sequence, SEQ ID in turn between the Sbf I restriction enzyme site and Sap I restriction enzyme site of plasmid pUC18
The weight of nucleotide sequence, polyadenylation sequence shown in NO:1 and the distinguished sequence for PCR identification selectively contained
Group plasmid.
It is highly preferred that the promoter is Sp6 promoter (5 '-GATTCCAATCTCGAGATTTAGGTGACAC of sequence
TATAG-3 ', SEQ ID NO:2).
Distinguished sequence for PCR identification is the Duan Xulie for facilitating PCR to detect, and belongs in recombinant plasmid and selectively contains
Sequence, it is further preferred that for PCR identification distinguished sequence such as SEQ ID NO:3 (5 '-
CCTCGACTGTGCCTTCTAAAT-3 ') shown in.
The polyadenylation sequence can be conventional polyadenylation sequence, such as SEQ ID NO:4 (5 '-AAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATAAT-3 ') shown in sequence.
The third aspect, the present invention provides a kind of methods of construction recombination plasmid, as shown in Figure 1, this method includes following
Step:
(1) DNA fragmentation F1 and DNA fragmentation F2 is obtained by chemical synthesis, wherein the sequence of DNA fragmentation F1 is by starting
The composition of sequence shown in subsequence and SEQ ID 1-7733 nucleotide of NO:1, the sequence of DNA fragmentation F2 is by SEQ ID NO:
Sequence shown in 1 7310-11774 nucleotide, polyadenylation sequence and selectively contain for PCR identification it is special
Sequence composition；
(2) digestion is carried out to DNA fragmentation F1 using Sbf I and Sap I, so that the DNA fragmentation F1 after digestion is inserted into matter
Between the Sbf I restriction enzyme site and Sap I restriction enzyme site of grain pUC18, pUC18-5 is obtained, using Sbf I and Sap I to DNA piece
Section F2 carries out digestion, thus by the Sbf I restriction enzyme site of the DNA fragmentation F2 insertion plasmid pUC18 after digestion and Sap I digestion position
Between point, pUC18-3 is obtained；
(3) digestion is carried out to pUC18-5 and pUC18-3 using BssH II and Sap I, it is resulting further connects digestion
Large fragment obtains recombinant plasmid pCHIKV-FL.In recombinant plasmid pCHIKV-FL of the invention, in the Sbf I enzyme of plasmid pUC18
It is connected with nucleotide sequence shown in promoter sequence, SEQ ID NO:1, more between enzyme site and Sap I restriction enzyme site in turn
Polyadenylic acid sequence and the distinguished sequence for PCR identification selectively contained.
Wherein, the specific choice of promoter sequence, polyadenylation sequence and the distinguished sequence for PCR identification is as before
It is described.The method of chemical synthesising DNA segment includes liquid phase synthesizing method and solid-phase synthesis, usually can entrust special company (such as
Shanghai Ying Jun Bioisystech Co., Ltd) it is synthesized, details are not described herein.By enzyme cutting method plasmid specific restriction enzyme site
Between be inserted into target fragment concrete operation step be well known to those skilled in the art, also repeat no more herein.
Fourth aspect, the present invention provides a kind of infection clones of chikungunya virus, the genes of the infection clones
The nucleotide sequence of the corresponding cDNA of group RNA is as shown in SEQ ID NO:1.
5th aspect, the present invention provides a kind of method of infection clones for constructing chikungunya virus, this method packets
It includes and recombinant plasmid described in second aspect is transcribed in vitro, the transcription RNA transfection sensitive cells that will be obtained.Preferably,
The sensitive cells is BHK-21 cell.
6th aspect, the present invention provides a kind of method of chikungunya virus DRDE-06 plants of label, this method includes will
The 7522-7523 nucleotide of the corresponding cDNA of chikungunya virus DRDE-06 pnca gene group RNA sport GG by TT.Base
Refined DRDE-06 plants of virus is agreed in " Santhosh, S.R.et al, Comparative full genome analysis in hole
revealed E1:A226V shift in 2007 Indian Chikungunya virus isolates,Virus
Res.135 (1): 36-41 (2008) " it is disclosed in.
The present invention has the advantage that
(1) the present invention is based on chemical synthesising technologies to construct chikungunya virus full-length infectious CDNA clones, and successfully
The chikungunya virus of infectious is provided in rescue.The method that the present invention describes has building process short, loyal viral genome
The features such as former sequence, can be used as the universal means of building single strand plus RNA virus infection clones, be virus replication mechanism and cause
The research of the interpretation of the cause, onset and process of an illness provides strong tool.The present invention chooses the full base of one plant of chikungunya virus from GenBank database
Because of sequence, corresponding DNA fragmentation is obtained by iii vitro chemical synthetic method, and is connected into common cloned plasmids pUC18, most
The clone containing Chickungunya virus genome full-length cDNA is obtained eventually.Compared with the method for conventional construction cDNA clone, this
The full genome synthetic technology used is invented based entirely on the virus sequence in gene database, without with viral genome template into
Row PCR or RT-PCR amplification, avoid the mutation that the process may introduce, therefore can obtain the virus of completely faithful to former sequence
Genetic fragment.Meanwhile the present invention can disposable composition length be about 7000nt length dna segment, avoid conventional infection
Short-movie section is connected as to the cumbersome process of middle long segment in clone's building process, significantly improves the effect of building full length cDNA clone
Rate.
(2) Chickungunya virus genome full length cDNA sequence of the present invention to synthesize in vitro, obtaining has infectivity
Chikungunya virus.The recovered virus contains chikungunya virus specific RNA sequences, sensitive cells can be made to result from wild
The similar lesion of type virus, while the plaque similar with wild-type virus can also be formed.This shows full genome used in the present invention
The full length cDNA clone that synthesizing mean obtains can successfully save out virus, imply this method in chikungunya virus infectivity
Clone's building aspect is feasible and efficient, is that the building of the infection clones of other chikungunya disease strains opens new side
To.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is that chikungunya virus full-length infectious CDNA clones (recombinant plasmid) constructs schematic diagram；
Fig. 2 is the RT-PCR testing result of chikungunya recovered virus (infection clones of chikungunya virus) genome
Figure, wherein swimming lane 1: using recovered virus cDNA as DNA fragmentation obtained by template amplification, size is about 1200bp；Swimming lane 2: it is not added
The blank control of cDNA template；Swimming lane 3: using wild-type virus cDNA as DNA fragmentation obtained by template amplification, size is about
1200bp；Swimming lane 4: with full-length infectious CDNA clones (recombinant plasmid) for the resulting DNA fragmentation of template amplification；Swimming lane M:DNA
Marker DL 2000；
Fig. 3 is the induced cytopathic effect result figure of chikungunya recovered virus；
Fig. 4 is the plaque aspect graph of chikungunya recovered virus.
Specific embodiment
Detailed description of the preferred embodiments with reference to embodiments.It should be understood that this place
The specific embodiment of description is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
In following embodiment, GD05/2010 plants of chikungunya wild-type virus is in " Li, X.F.et al, Complete
genome sequence of a chikungunya virus isolated in guangdong,China.J.Virol.86
(16): 8904-8905 (2012) it is disclosed in "；Sp6 promoter sequence is as shown in SEQ ID NO:2；For the special of PCR identification
Sequence is as shown in SEQ ID NO:3；Polyadenylation sequence is as shown in SEQ ID NO:4；The restriction enzyme used is purchased
From NEB company；Competent E.coli DH5 α is purchased from TIANGEN Biotech (Beijing) Co., Ltd., catalog number CB101-
01；BHK-21 cell is purchased from ATCC, catalog number CCL-10；The culture solution (OPTI-MEM and DMEM) used is purchased from
Life Technologies；Chemical synthesising DNA segment and DNA sequencing commission Shanghai Ying Jun Bioisystech Co., Ltd complete.
Embodiment 1
The method that the present embodiment is used to illustrate present invention building Chickungunya virus genome full length cDNA clone.
(1) DNA fragmentation F1 and DNA fragmentation F2 is obtained by chemical synthesis, wherein the sequence of DNA fragmentation F1 is by starting
The composition of sequence shown in subsequence and SEQ ID 1-7733 nucleotide of NO:1, the sequence of DNA fragmentation F2 is by SEQ ID NO:
Sequence shown in 1 7310-11774 nucleotide, polyadenylation sequence and the distinguished sequence composition for PCR identification；
(2) digestion is carried out to DNA fragmentation F1 using Sbf I and Sap I, so that the DNA fragmentation F1 after digestion is inserted into matter
Between the Sbf I restriction enzyme site and Sap I restriction enzyme site of grain pUC18, pUC18-5 is obtained, using Sbf I and Sap I to DNA piece
Section F2 carries out digestion, thus by the Sbf I restriction enzyme site of the DNA fragmentation F2 insertion plasmid pUC18 after digestion and Sap I digestion position
Between point, pUC18-3 is obtained, pUC18-5 and pUC18-3 are converted to competent E.coli DH5 α, plasmid reflects through DNA sequencing
It is set to correct sequence；
(3) digestion is carried out to pUC18-5 and pUC18-3 using BssH II and Sap I, it is resulting further connects digestion
Large fragment obtains recombinant plasmid pCHIKV-FL.In obtained recombinant plasmid pCHIKV-FL, in the Sbf I digestion of plasmid pUC18
It is connected with nucleotide sequence, poly shown in promoter sequence, SEQ ID NO:1 between site and Sap I restriction enzyme site in turn
Adenosine acid sequence and the distinguished sequence identified for PCR.Recombinant plasmid pCHIKV-FL is converted to competent E.coli
DH5 α is accredited as the sequence (Chickungunya virus genome of mutation as shown in SEQ ID NO:1 containing sequence through DNA sequencing
Full-length cDNA) recombinant plasmid.
Embodiment 2
The present embodiment is used to illustrate the method using full length cDNA clone rescue chikungunya recovered virus.
(1) in-vitro transcription of Chickungunya virus genome full length cDNA clone
Plasmid pCHIKV-FL is extracted with extraction reagent kit in plasmid (Qiagen Products), uses phenol again after Sac I digestion
Chloroform freezes for use for -20 DEG C after linearization plasmid is quantified and dispensed.It is with the full length cDNA clone plasmid after linearizing
Template, with SP6 RiboMAX Express Large ScaleRNA Production Systems (Promega Products)
It is carried out according to kit specification in-vitro transcription.It is said according further to RNeasy Mini Kit (Qiagen Products) operation
After bright purifying In vitro transcripts RNA and quantitative, -80 DEG C of separating device freeze for use.
(2) rescue of chikungunya recovered virus
The transcription RNA for being obtained step (1) with liposome Lipofectamin 2000 (being purchased from Invitrogen company)
It transfects single layer BHK-21 cell (convergence degree 80%), the method is as follows: first mix the OPTI-MEM of 50 μ l and 4 μ l liposomes
It is even, it is mixed again with the OPTI-MEM of the RNA of 10 μ l (5 μ g) and 50 μ l after being placed at room temperature for 5min, is placed at room temperature for 20min.It will be upper
1 hole that mixed liquor is added in 6 orifice plates is stated, while adding the OPTI-MEM of 450 μ l.37 DEG C, 5%CO2It is incubated for 6h.It removes
Clearly, the DMEM culture medium for adding the FBS containing 2 volume %, sets 37 DEG C, 5%CO2In incubator.The 4th day after transfection, cell occurs
Culture solution supernatant is collected by centrifugation in lesion.Supernatant is reinoculated on BHK-21 cell, there is lesion in the 3rd day cell after inoculation, from
The heart collects supernatant.It freezes in -80 DEG C, as recovered virus seed liquor, is named as R-CHIKV.
Test case 1
This test case is used to detect the genome sequence of chikungunya recovered virus.
(1) extraction of chikungunya recovered virus RNA
Illustrate to extract recovered virus seed liquor R-CHIKV genome according to RNeasy kit (Qiagen Products)
RNA.Extraction step is as follows: by β-mercapto of 210 μ l viral suspensions and RLT (the RNeasy Lysis Buffer) and 7 μ l of 700 μ l
The mixed liquor of base ethyl alcohol (β-mercaptoethanol), which shakes, to be mixed, and is acutely mixed, is cracked after adding 490 μ l dehydrated alcohols
Adsorption column is added in lysate by liquid, and 12000rpm is centrifuged 15s；It is primary that the RW1 (RNeasy Wash Buffer) of 700 μ l washes column,
12000rpm is centrifuged 15s；500 μ l RPE (RPE is the cleaning solution in RNeasy kit) are washed column 2 times, 10000rpm centrifugation
30s；Void column 10000rpm is centrifuged 1min.Add 50 water of the μ l without RNase, stand 2min, 12000rpm is centrifuged 1min.Add 2 μ l's
RNase inhibitor.Mix -80 DEG C of postposition for use.
(2) the RT-PCR amplification of chikungunya recovered virus RNA and sequencing
With reverse transcription primer R-11777 (sequence 5 '-TTTGTGTGGTTTCGAAATCGT-3 ', SEQ ID NO:5) system
The cDNA of standby chikungunya recovered virus R-CHIKV genome.The component of reverse transcription reaction is as follows: R-CHIKV geneome RNA
15μl；R-11777(10μM)1.25μl；dNTP(2.5mM each)10μl；Water (being free of RNase) 6.25 μ l.By above-mentioned reaction
Component is uniformly mixed, and sets 5min at 65 DEG C, ice bath 2min.Add following component: 5 × first-strand buffer, 10 μ l；
DDT(0.1mM)2.5μl；2.5 μ l of RNase inhibitor；Super Script III 2.5μl.60min at 55 DEG C；At 70 DEG C
15min.Add 1 μ l (2U) RNase H, 20min at 37 DEG C.- 20 DEG C save for use.
It uses the cDNA of acquisition as template, carries out PCR amplification with LA Taq archaeal dna polymerase (TaKaRa Products).Institute
It is respectively CHIKV-F6 (5 '-GACAATGGCAGAAGGAATTTC-3 ', SEQ ID NO:6) and CHIKV-R6 with upstream and downstream primer
(5 '-TGTGTGTCGCCCCGTCGTCTA-3 ', SEQ ID NO:7).The non-structural protein of above-mentioned primer amplification chikungunya virus
White code area.PCR reaction system composition is as follows: 10 × LA Buffer, 5 μ l；2 μ l of upstream primer (10 μM)；Downstream primer (10 μ
M)2μl；dNTP(2.5mM each)8μl；cDNA 3μl；0.5 μ l of LA Taq archaeal dna polymerase；29.5 μ l of water.Reaction condition is such as
Under: 94 DEG C of denaturation 4min, 94 DEG C of 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 3min 50s, 30 circulations, then be placed at 72 DEG C
10min.1% Ago-Gel detects PCR product length, recycles resulting PCR fragment with PCR purification kit and is sequenced.Fine jade
The testing result of sepharose is shown, with recovered virus cDNA, wild-type virus cDNA and full-length infectious cloned plasmids are
Template can expand the special DNA fragmentation for obtaining that size is about 1200bp, with expected consistent (as shown in Figure 2).Sequencing
As a result it also shows, above-mentioned segment is the special code area of the non-structural protein of chikungunya virus.These results indicate that being obtained
Recovered virus contain the special gene order of chikungunya virus.
Test case 2
This test case is used to illustrate the induced cytopathic effect of chikungunya recovered virus.
By chikungunya recovered virus seed liquor R-CHIKV and GD05/2010 plants of chikungunya wild-type virus
(CHIKV-GD05/2010) it is inoculated in the single layer BHK-21 cell (convergence degree 90%) for being laid on 6 orifice plates respectively.After adsorbing 1h,
Virus liquid is removed, virus liquid is discarded, the DMEM culture medium of FBS of the addition containing 2 volume % is placed in 37 DEG C, 5%CO2Incubator in
Culture.The variation of observation cellular morphology daily.The results show that BHK-21 cell is after infection recovered virus or wild-type virus
There is within 3rd day the contracting of cell circle, crack and fall off (as shown in Figure 3).It is thin that this shows that recovered virus can be such that sensitive cells generates
Born of the same parents' lesion, lesion process are similar to wild-type virus.
Test case 3
This test case is used to illustrate the plaque form of chikungunya recovered virus.
With 2% FBS DMEM by viral seed liquor with 10 doubling dilutions be 10-4, it is inoculated in 500 holes μ l/ and is laid on 6 holes
The single layer BHK-21 cell (convergence degree 90%) of plate.After adsorbing 2h, virus liquid is discarded, addition (contains 2 bodies containing DMEM culture medium
Product % FBS) 1% (1g/100mL) agar lid, be placed in 37 DEG C, 5%CO2Incubator in cultivate.With 4% formaldehyde after 3 days
Room temperature fixes 1h, abandons agar lid, and crystal violet room temperature dyes 10min, observes plaque form.It is wild to chikungunya in the same way
The plaque that GD05/2010 plants of type virus is analyzed.The result of plaque assays is as shown in figure 4, chikungunya recovered virus and open country
It is uniform that raw type virus can form size, sharp-edged plaque.The above results show chikungunya recovered virus and wild type
Virus has similar Proliferation Characteristics.
From the result of the above test case can be seen that DNA molecular or recombinant plasmid of the invention can be used in rescue obtain
With chikungunya virus similar in wild-type virus.
It is described the prefered embodiments of the present invention in detail above in conjunction with attached drawing, still, the present invention is not limited to above-mentioned realities
The detail in mode is applied, within the scope of the technical concept of the present invention, a variety of letters can be carried out to technical solution of the present invention
Monotropic type, these simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. a kind of DNA molecular comprising chikungunya virus full-length genome cDNA, which is characterized in that the nucleotide of the DNA molecular
Sequence is as shown in SEQ ID NO:1.
2. a kind of recombinant plasmid, which is characterized in that the recombinant plasmid contains nucleotide sequence shown in SEQ ID NO:1.
3. recombinant plasmid according to claim 2, wherein the recombinant plasmid is in the Sbf I digestion position of plasmid pUC18
Nucleotide sequence, poly gland shown in promoter sequence, SEQ ID NO:1 are connected in turn between point and Sap I restriction enzyme site
The recombinant plasmid of nucleotide sequence and the distinguished sequence for PCR identification selectively contained；
Wherein, the distinguished sequence for PCR identification is as shown in SEQ ID NO:3.
4. recombinant plasmid according to claim 3, wherein the promoter is Sp6 promoter.
5. a kind of method of construction recombination plasmid, which is characterized in that method includes the following steps:
(1) DNA fragmentation F1 and DNA fragmentation F2 is obtained by chemical synthesis, wherein the sequence of DNA fragmentation F1 is by promoter sequence
Sequence shown in column and 1-7733 nucleotide of SEQ ID NO:1 forms, and the sequence of DNA fragmentation F2 is by SEQ ID NO:1 the
Sequence shown in 7310-11774 nucleotide, polyadenylation sequence and the special sequence for PCR identification selectively contained
Column composition；
(2) digestion is carried out to DNA fragmentation F1 using Sbf I and Sap I, so that the DNA fragmentation F1 after digestion is inserted into plasmid
Between the Sbf I restriction enzyme site and Sap I restriction enzyme site of pUC18, pUC18-5 is obtained, using Sbf I and Sap I to DNA fragmentation
F2 carries out digestion, thus by the Sbf I restriction enzyme site and Sap I restriction enzyme site of the DNA fragmentation F2 insertion plasmid pUC18 after digestion
Between, obtain pUC18-3；
(3) digestion is carried out to pUC18-5 and pUC18-3 using BssH II and Sap I, further connects the resulting sheet of digestion
Section, obtains recombinant plasmid pCHIKV-FL.
6. a kind of infection clones of chikungunya virus, which is characterized in that the geneome RNA of the infection clones is corresponding
The nucleotide sequence of cDNA is as shown in SEQ ID NO:1.
7. a kind of method for the infection clones for constructing chikungunya virus, which is characterized in that this method includes by claim
Recombinant plasmid described in any one of 2-4 is transcribed in vitro, the transcription RNA transfection sensitive cells that will be obtained.
8. according to the method described in claim 7, wherein, the sensitive cells is BHK-21 cell.
9. a kind of method of chikungunya virus DRDE-06 plants of label, which is characterized in that this method includes by chikungunya virus
The 7522-7523 nucleotide of the corresponding cDNA of DRDE-06 pnca gene group RNA sport GG by TT.
10. a kind of method for the infection clones for constructing chikungunya virus, which is characterized in that this method comprises: from gene number
According to the complete genome sequence for choosing chikungunya virus in library, is obtained by iii vitro chemical synthetic method and have markd DNA fragmentation, and
It is connected into cloned plasmids, obtains the clone containing Chickungunya virus genome full-length cDNA, be transcribed in vitro and will be obtained
Transcription RNA transfection sensitive cells；
Wherein, the markd DNA fragmentation of tool is by the corresponding cDNA's of chikungunya virus DRDE-06 pnca gene group RNA
7522-7523 nucleotide are sported the DNA fragmentation of GG acquisition by TT.
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