CN109536464A

CN109536464A - It is a kind of lack capsid protein gene chikungunya virus infection clones and construction method and preparation attenuated vaccine in application

Info

Publication number: CN109536464A
Application number: CN201811504486.7A
Authority: CN
Inventors: 张波; 邓成林; 张亚南; 李嘉琪; 李娜; 李晓; 张秋艳; 叶寒青; 袁志明
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2018-12-10
Filing date: 2018-12-10
Publication date: 2019-03-29
Anticipated expiration: 2038-12-10
Also published as: CN109536464B

Abstract

The invention belongs to field of biotechnology, specifically disclose it is a kind of lack capsid protein gene chikungunya virus infection clones and construction method and preparation attenuated vaccine in application, it, which clones the CHIKV- Δ C virus saved out, can be used as attenuated vaccine, the virus saved out is similar to wild type CHIKV Virus plaque form, growth tendency, and genetic stability is high.It confirms sufficiently to be attenuated in A129 and C57BL/6 mouse model, safety is very high, and can generate the immunoprotection for CHIKV virus.The virus can be used as a kind of safely and effectively attenuated vaccine prevention chikungunya virus infection, while the virus is also used as a kind of expression vector, for the expression of foreign gene, has a good application prospect.

Description

It is a kind of lack capsid protein gene chikungunya virus infection clones and building side Method and the application in preparation attenuated vaccine

Technical field

The invention belongs to field of biotechnology.Specifically, the present invention relates to a kind of datum holes for lacking capsid protein gene Agree refined virus infection clones and construction method and the application in preparation attenuated vaccine, can be used as expression using the infectivity and carry Body, cloning the CHIKV- Δ C virus saved out can be used as attenuated vaccine.

Background technique

Chikungunya virus (Chikungunya virus, CHIKV) belongs to Togaviridae (Togaviridae) onychonosus Poison belongs to (Alphavirus).Virion is spherical in shape, and diameter about 60~70nm has coating.The genome of CHIKV is single-stranded positive RNA, length are about 11800bp.Whole gene group include 5 ' end noncoding regions, 2 independent open reading frames (ORF), with And 3 ' end noncoding region.Cap sequence is contained at 5 ' ends, and Poly (A) tail is contained at 3 ' ends, and 2 ORF are separately encoded the non-structural of virus Albumen and structural proteins are successively 4 kinds of non-structural proteins (nsP1, nsP2, nsP3 and nsP4) and 5 kinds from 5 ' ends to 3 ' ends Structural proteins (capsid protein C, envelope protein E3, E2,6K and E1 albumen), there is one section between nonstructural gene and structural gene The bonding pad of 66bp is non-translational region.Meanwhile its minus strand can copy one section and account for about whole gene group leader in virus replication Identical subgenome structure, referred to as the 26S RNA with 3 ' ends of degree 1/3 are turned in the later period of virus replication as structural proteins The template translated.

CHIKV is the pathogen for causing Chikungunya fever (Chikungunya fever, CHIK), is mainly stung by yellow-fever mosquito Sting propagation.Chikungunya fever clinical manifestation is fever, fash and violent arthralgia, is a kind of zoonosis, Major Epidemic In Africa and south east asia.People infects CHIKV, and incubation period is generally 3~7d.Although the disease lethality is lower, in mosquito matchmaker The higher area of density be easy to cause large-scale outbreak and prevalence.CHIKV is derived from Africa, and breaking out known to first time is nineteen fifty-two Betide African Tanzania, the state Ni Wala, south.Nineteen fifty-three is separated to chikungunya disease from the serum of women of this area's illness Poison, while the virus is also separated to from yellow-fever mosquito.After the 1960s, the east CHIK moves to south east asia.Nineteen sixty-five exists India is very popular Madras, about 300,000 people infection.Since 2005, CHIKV is in each island in the Indian Ocean and Indian continent Successively extensive popular, only the Chikungunya fever suspected case of the report of India in 2006 is more than 1,390,000, the disease incidence of some areas More than 45%.As population mobility enhances, the factors such as ecological-environment differention, popular frequency and Epidemic Scope have day in the whole world The widened trend of benefit.According to WHO Report, there are 37 countries and regions in the whole world in endemic conditions or have potentially Side's property prevalence risk.China's the first Introduced cases Chikungunya fever case in 2008 detects in Guangdong.In September, 2010, China Dongguan City breaks out Chikungunya fever, and rapidly results in hundreds of people morbidity, this is that the Chikungunya fever that China first appears is popular, as one Kind emerging infectious disease attracts wide attention.Yellow-fever mosquito is widely distributed on China south China, southwest and other places, becomes the Disease code Potential inducement.This prompt with personnel in global range and economic interaction increasingly frequently, China, which exists, to be inputted again, breaks out A possibility that it is larger.

Chikungunya fever is still without effective antiviral treatment at present, due to infection, the pathogenesis etc. to CHIKV Understand it is less, vaccine research and development still rest on laboratory stage.However CHIKV mainly passes through bite by mosquitos and propagates in crowd, passes It is fast to broadcast speed, harm is big, easily causes the common people panic, it has also become to endanger the sanitarian significant problem of the mankind.Therefore it carries out in a deep going way It is of great significance to the vaccine research of CHIKV.

CHIKV capsid protein Capsid includes two functional domains, respectively N-terminal RNA binding domain and C-terminal serine protease Domain.Wherein C-terminal serine protease mediates the cutting of polyprotein Capsid-E2-E1-6k, releases mature capsid protein. Mature capsid protein specifically binds the nucleocapsid to form virus by N-terminal RNA binding domain and CHIKV genome 49S RNA, Subsequent nucleocapsid is transported to cell membrane, promotes virion to sprout with envelope protein interaction, is formed with infective Virion.Therefore, Capsid is composition virus nucleocapsid, virion is promoted to be completed the indispensable egg of life cycle It is white.In the research in the past for flavivirus, Capsid gene delection can be kept to the duplication energy of geneome RNA Power, and virion cannot be packed out.

Traditional attenuated live vaccine be by toxicity variation or the method for artificial selection culture by strain become attenuated strain or Avirulent strain, immune effect is better than inactivated vaccine, but not easy to maintain, and the risk replied with virulence.Therefore it develops a kind of new Safer chikungunya virus attenuated live vaccine have important public health meaning and application prospect.

Summary of the invention

The object of the present invention is to provide a kind of chikungunya virus infection clones for lacking capsid protein gene CHIKV- Δ C, sequence are shown in SEQ ID NO.1.

It is another object of the present invention to provide the applications of infection clones CHIKV- Δ C, which can Protein expression vector as a purpose, the recombinant virus saved out are alternatively arranged as attenuated vaccine.

In order to achieve the above object, the present invention takes following technical measures:

A kind of chikungunya virus infection clones CHIKV- Δ C lacking capsid protein gene, is with full-length infectious Clone CHIKV-WT is skeleton, and by Capsid gene, all missing is obtained, and sequence is shown in SEQ ID NO.1.

The application of infection clones CHIKV- Δ C, including the infection clones as the application in expression vector, or rescue Recombinant virus is prepared into the attenuated vaccine of chikungunya virus out.

Compared with prior art, the present invention having the following advantages that and effect:

1. reverse Genetics Technique used by the chikungunya virus of present invention rescue missing capsid protein gene, has It is advanced and mature, facilitate it is simple and direct, positioning it is controllable the advantages that.

2. the side that the chikungunya virus CHIKV- Δ C for the missing capsid protein gene that the present invention saves can use cell culture Method carrys out massive amplification, and acquisition modes are fairly simple.

Plaque and growth curve do not change after 3. CHIKV- Δ C virus provided by the invention is passed on by 5 generations, Structural protein gene sequencing still lacks Capsid gene.Show that CHIKV- Δ C virus has good genetic stability, replys A possibility that for wild-type virus, is very low, improves safety of the virus as vaccine.

4. CHIKV- Δ C attenuated virus provided by the invention confirms to be sufficiently to subtract in A129 and C57BL/6 mouse model Poison, safety is very high, and can generate the immunoprotection for CHIKV virus.

5. CHIKV- Δ C infection clones provided by the invention can be used as a kind of expression vector, construct on this basis EGFP-CHIKV- Δ C reporter virus infection clones can be convenient efficiently for eGFP reporter gene to be substituted for other The expression of foreign gene such as interleukin I L-15 etc..

In conclusion the chikungunya virus CHIKV- Δ C of missing capsid protein gene of the invention, can be used as one kind Safely and effectively attenuated vaccine prevention chikungunya virus infection, has a good application prospect and highly important theory significance And realistic meaning.

Detailed description of the invention

Fig. 1 is to lack the building schematic diagram of the chikungunya virus infection clones of capsid protein gene and by infectious gram The indirect immunofluorescence schematic diagram of the grand recombinant virus saved out；

A: missing capsid protein RNA binding domain (CHIKV- Δ C-115aa) and missing capsid protein full gene (CHIKV- Δ C) infection clones building schematic diagram；

B:CHIKV-WT, CHIKV- Δ C-115aa and CHIKV- Δ C are transfected after BHK-21 cell respectively between different time points Connect immunofluorescence；

72h is immunized indirectly after C:CHIKV-WT, CHIKV- Δ C-115aa and CHIKV- Δ C infect BHK-21 cell respectively Fluorogram.

Fig. 2 is that CHIKV-WT and CHIKV- Δ C Virus plaque form, growth curve and infectious viral particle number compare Schematic diagram；

A:CHIKV-WT and CHIKV- Δ C plaque form compares；

B:CHIKV-WT and CHIKV- Δ C growth curve compares；

C:CHIKV-WT and CHIKV- Δ C infectious viral particle number compares.

Fig. 3 is CHIKV- Δ C viral genetic Detection of Stability schematic diagram；

A:RT-PCR compares the CHIKV- Δ C cell in P0, P5 generation and the purpose band size of supernatant sample；

The plaque form of the CHIKV- Δ C virus in B:P0, P5 generation compares；

The growth curve of the CHIKV- Δ C virus in C:P0, P5 generation compares；

The E2/Capsid protein expression situation of the CHIKV- Δ C virus in D:P0, P5 generation；

Peak figure is sequenced for the structural proteins of tri- plants of CHIKV- Δ C viruses of A/B/C in E:P5.

Fig. 4 is application schematic diagram of the CHIKV- Δ C attenuated vaccine in the evaluation of A129 mouse model virulence；

A: the mouse survival situation of immune CHIKV- Δ C attenuated vaccine；

B: the mouse insole swelling situation of immune CHIKV- Δ C attenuated vaccine；

C: the mouse weight situation of change of immune CHIKV- Δ C attenuated vaccine；

D: the small sodoku blood situation of immune CHIKV- Δ C attenuated vaccine；

E: the mouse insole swelling photo of immune CHIKV- Δ C attenuated vaccine.

Fig. 5 is application schematic diagram of the CHIKV- Δ C attenuated vaccine in the protection of A129 mouse model immunization；

A:CHIKV- Δ C attenuated vaccine is immunized in A129 mouse model, attacks malicious flow chart；

B: the mouse generation neutralizing antibody titers that experiment detects immune CHIKV- Δ C attenuated vaccine are neutralized；

C: survival condition of the mouse of immune CHIKV- Δ C attenuated vaccine after attacking poison；

D: footpad swelling situation of the mouse of immune CHIKV- Δ C attenuated vaccine after attacking poison；

E: changes of weight situation of the mouse of immune CHIKV- Δ C attenuated vaccine after attacking poison；

F: malicious blood situation of the mouse of immune CHIKV- Δ C attenuated vaccine after attacking poison.

Fig. 6 is application schematic diagram of the CHIKV- Δ C attenuated vaccine in the protection of C57BL/6 mouse model immunization；

A: the mouse generation neutralizing antibody titers that experiment detects immune CHIKV- Δ C attenuated vaccine are neutralized；

B: footpad swelling situation of the mouse of immune CHIKV- Δ C attenuated vaccine after attacking poison；

C: malicious blood situation of the mouse of immune CHIKV- Δ C attenuated vaccine after attacking poison.

Fig. 7 is missing capsid protein gene, and the eGFP-CHIKV- Δ C reporter virus for being inserted into foreign gene eGFP is infectious Clone's building and application schematic diagram；

A: missing capsid protein gene is inserted into the eGFP-CHIKV- Δ C reporter virus infection clones of foreign gene eGFP Building schematic diagram；

B:eGFP-CHIKV- Δ C reporter virus eGFP expression after the passage of BHK-21 cell.

Specific embodiment

PCR involved in this part, digestion connect, conversion, the extraction of RNA, the experimental methods such as RT-PCR, such as without spy Different explanation, is all made of the conventional method of this field.What is be exemplified below is only several specific embodiments of the invention.Obviously, originally Invention is not limited to following embodiment, and acceptable there are many deformations.Therefore those skilled in the art is in the disclosure of invention On the basis of do modify or improve, should belong to the scope of protection of present invention.

Embodiment 1:

A kind of virus of building and the rescue of the chikungunya virus infection clones lacking capsid protein gene:

The present embodiment explores the property of the infection clones of its building by exploring the different zones of missing capsid protein:

1. lacking capsid protein RNA binding domain (CHIKV- Δ C-115aa) and missing capsid protein full gene (CHIKV- Δ C) infection clones building: according to CHIKV-WT sequence (GenBank accession no.KC488650), synthesize six F1 and R1 in primer, sequence such as table 1；F2 and R2；Shown in F3 and R3.

CHIKV- Δ C-115aa segment is using CHIKV-WT as template, and respectively with F1 and R2, F2 and R1 are primer, uses PrimeSTAR HS enzyme (being purchased from Takara company) PCR amplification recycles PCR product, with above-mentioned resulting 2 sections of PCR recovery products For template, fusion DNA vaccine amplification, primer is F1 and R1, recycles PCR product.

CHIKV- Δ C segment is using CHIKV-WT as template, and respectively with F1 and R3, F3 and R1 are primer, uses PrimeSTAR HS enzyme PCR amplification recycles PCR product, using above-mentioned resulting 2 sections of PCR recovery products as template, fusion DNA vaccine amplification, and primer F1 And R1, recycle PCR product.

The PCR reaction system of two above amplification is equal are as follows: 94 DEG C of 2min, 94 DEG C of 20s, 55 DEG C of 10s, 68 DEG C of 3min, and 68 DEG C 10min, 30 circulations.

Two fusion segments after the recovery carry out double digestion with RsrII and NdeI respectively, with the overall length with identical enzymatic treatment Infection clones CHIKV-WT is ligated and transformed into E. coli competent HB101；Plasmid passes through DNA sequencing identification and is positive Really, infection clones CHIKV- Δ C-115aa and infection clones CHIKV- Δ C are respectively designated as.CHIKV- Δ C-115aa is lacked Capsid protein RNA binding domain is lost, CHIKV- Δ C lacks capsid protein full gene, and clone constructs schematic diagram A as shown in figure 1.

2. the linearisation and phenol chloroform of plasmid: with BamHI difference 10 μ g plasmid CHIKV- Δ C-115aa of digestion and CHIKV- Δ C, 37 DEG C digestion two hours, after the identification digestion of 0.8% agarose gel electrophoresis is complete, be added into digestion products 100 μ l saturated phenols (are purchased from Chinese medicines group chemical reagents corporation), and oscillation mixes, and 17000g is centrifuged 5min, draw supernatant in new In centrifuge tube, and the oscillation of 100 μ l sterile waters is added into former centrifuge tube and mixes, 17000g is centrifuged 5min；Draw supernatant and upper one Step gained supernatant merges (about 200 μ l of total volume), and 200 μ l chloroforms (being purchased from Chinese medicines group chemical reagents corporation) are added and mix, 17000g is centrifuged 5min；It draws supernatant (about 150~200 μ l) in the centrifuge tube of no RNAase, 1/10th volumes is added PH5.2,3mol/l sodium acetate (being purchased from Chinese medicines group chemical reagents corporation) and 2.5 times of volume dehydrated alcohols (are purchased from Chinese medicines group Chemical reagents corporation) it mixes, -20 DEG C of placements 30min, 17000g are centrifuged 5min；It inhales and abandons supernatant, 70% ethyl alcohol of 1ml is added and washes It washs, 17000g is centrifuged 5min, inhales and abandons supernatant；It is placed at room temperature for 15min, 11 μ l is eventually adding and is dissolved without the water of RNAase, used Thermo Scientific NanoDrop 2000 measures the concentration of DNA, uses 0.8% agarose gel electrophoresis detection DNA's Quality is saved backup in -20 DEG C.

3. RNA is transcribed in vitro: taking the linearisation product of the 1 above-mentioned phenol chloroform of μ g as template, tried using being transcribed in vitro Agent box T7mMESSAGE mMACHINE kit (is purchased from U.S. Ambion company), is recombinated according to kit specification The RNA of CHIKV- Δ C-115aa and CHIKV- Δ C.The dense of RNA is measured with Thermo Scientific NanoDrop 2000 Degree is saved backup using the quality of 0.8% freshly prepared agarose gel electrophoresis detection RNA in -80 DEG C.

4. the side that the RNA that obtained recombinant C HIKV- Δ C-115aa and CHIKV- Δ C is transcribed in vitro uses liposome transfection Method is transfected in BHK-21 cell, with the RNA of transfected wild-type virus CHIKV-WT as positive control: in the day before transfection, connecing Kind 2 × 10⁵For a BHK-21 cell in 35mm Tissue Culture Dish, every ware places three 10mm × 10mm coverslips, and cell is made to exist The transfection same day reaches about 80%；When transfection, culture medium in culture dish is first abandoned, is washed once with 1ml Opti-MEM, then plus 1ml Opti-MEM (makes cell be in wet face state)；1ml Opti-MEM is added in 1.5ml EP pipe, adds 4ul DMRIE-C (DMRIE-C is first mixed before) mixing of first turning upside down, then it is separately added into the RNA for the recombinant C HIKV that 1ug is transcribed in vitro (RNA is not added in control group), mixing of turning upside down；Opti-MEM in culture dish is abandoned rapidly, by (movement is wanted in homomixture addition ware Gently, it is not blown and beaten against cell)；After cultivating 4h in 37 DEG C of carbon dioxide incubators, culture is abandoned, then respectively 2mL is added to contain 2%FBS DMEM culture medium.The cell state of microscopically observation experimental group and control group, and 24 after transfection, 48,72h take one A slide is fixed cell with 5% acetone fixer (being purchased from Chinese medicines group chemical reagents corporation), and room temperature fixes 15 minutes, It after PBS washes three times, is saved in 4 DEG C, and collect viral supernatants when cytopathy is obvious, obtains CHIKV- Δ C-115aa virus With CHIKV- Δ C virus, saved in -80 DEG C.

5. the viral protein expression feelings of indirect immunofluorescence (IFA) detection recombinant C HIKV- Δ C-115aa and CHIKV- Δ C Condition: being incubated for primary antibody for the slide of 4 DEG C of preservations in above-mentioned steps 4 under room temperature environment, and antibody is 1:250 times of diluted rabbit source CHIKV E2 protein polyclone antibody.It is incubated at room temperature 1~2h of primary antibody and is protected from light incubation secondary antibody in room temperature after PBS is rinsed 10 times, resist Body is 1:125 times of diluted coupling fluorescein isothiocynate (FITC) goat anti-rabbit antibody.After secondary antibody is incubated for 1h, PBS is rinsed 10 times, Glass slide is taken out, is marked, each mark, 95% glycerol of a dot is put, coverslip has to cell is face-down, be placed in glycerol In drop, (B in Fig. 1) is observed using 200 × amplification factor under fluorescence microscope.

The positive rate of the CHIKV- Δ C virus recombinated as the result is shown at every point of time is lower than CHIKV-WT, but With the extension of incubation time after transfection, positive cell number gradually increases CHIKV- Δ C, consistent with CHIKV-WT trend；And For the CHIKV- Δ C-115a of recombination with the extension of incubation time after transfection, positive cell number has no increase, and positive cell Dispersion exists, and shows that CHIKV- Δ C is successfully saved with CHIKV-WT as and provides the virion of infectious, and CHIKV- Δ C-115a normal replication but can cannot be generated with infective virion.

The infection conditions for recombinant C HIKV- Δ C-115aa and CHIKV- Δ the C virus that 6.IFA detection rescue obtains: will be upper CHIKV-WT, CHIKV- Δ C-115aa and CHIKV- Δ the C virus collected in step 4 are stated, after infecting BHK-21 cell respectively 72h carries out IFA detection (C in Fig. 1), and method is the same.The result shows that the CHIKV- Δ C-115aa of recombination does not have infectivity, and The CHIKV- Δ C of recombination can produce with infective virion, similar to the CHIKV-WT of recombination.

The chikungunya virus infection clones that table 1. lacks capsid protein gene construct the primer

Embodiment 2:

CHIKV- Δ C virus provided by the invention and CHIKV-WT plaque form, growth curve and infectious virus Grain number compares:

1. embodiment 1 is saved obtained CHIKV- Δ C virus and CHIKV-WT carries out plaque assay measurement virus titer And form compares: being inoculated with 1 × 10 respectively in each hole of 24 porocyte culture plates⁵A BHK-21 cell, reaches to cell confluency degree When to 90%, the culture medium in hole is abandoned, the above-mentioned steps 3 of DMEM culture medium 10 doubling dilution of the 100 μ l containing 2%FBS are added The virus of collection, 37 DEG C of absorption 1h, sufficiently shakes primary for every 15 minutes.The virus liquid in each hole is inhaled after absorption and is abandoned, is added The covering for entering the DMEM culture medium containing 2%FBS and 2% methylcellulose cultivates 3 in 37 DEG C, the incubator of 5%CO2 It, is dyed after plaque formation with containing the dyeing liquor of 1% crystal violet and 3.7% formaldehyde, after room temperature handles 30min, takes out hole In dyeing liquor, and with flowing water flushing hole bottom, after drying be it is countable be converted into virus titer, while observing plaque form (Fig. 2 Middle A).From the figure, it can be seen that the plaque of CHIKV- Δ C virus is small compared with CHIKV-WT.

2. being inoculated with 2 × 10 in 35mm Tissue Culture Dish⁵BHK-21 cell, at 37 DEG C, 5%CO₂Under condition of culture, wait converge Right is 0.01 according to infection multiplicity MOI when reaching 60%, the CHIKV- Δ C after 400 μ l dilution is added into each ware respectively With CHIKV-WT virus, 37 DEG C, 5%CO₂After adsorbing 2h in incubator, virus liquid is abandoned, 2ml is added in every hole and contains 2% tire ox blood Clear DMEM culture medium, in 37 DEG C, 5%CO₂It is cultivated under condition of culture, every 12h receives 400 μ l viral supernatants simultaneously after infection The DMEM culture medium that 400 μ l contain 2% fetal calf serum is added, the virus of collection is identical as CHIKV-WT virus as CHIKV- Δ C The virus-like of different time points under infection multiplicity is saved in -80 DEG C.It is checked and accepted according to above-mentioned plaque assay method measurement different time The virus titer of collection is drawn growth curve (B in Fig. 2).The result shows that the growth tendency of CHIKV- Δ C virus and CHIKV-WT It is similar.

3. taking the CHIKV- Δ C of different PFU and CHIKV-WT virus to prepare albumen sample respectively, with special viral antibody E1 The E1 protein band that carries out Western blotting detection, and will test carries out gray analysis (C in Fig. 2).The result shows that The infectious viral particle number of CHIKV- Δ C is about the 1% of CHIKV-WT.

Embodiment 3:

CHIKV- Δ C virus provided by the invention has genetic stability:

1. it is 0.01 infection BHK-21 that embodiment 1 is saved to obtained CHIKV- Δ C viral (P0 generation) by infection multiplicity MOI Cell, CPE obviously collects cell conditioned medium (P1 generation) after 5 days, and per generation passes three strain virus in parallel, repeats 5 this steps；It will P0, P5 withhold cell and supernatant the saying according to Trizol and QIAamp Viral RNA Mini Kit kit respectively of collection Bright book extracts the RNA of virus, saves backup in -80 DEG C.RT-PCR is carried out with PrimeScript One Step RT-PCR Kit Detection, primer CHIKV-6791-F:5 '-gatgattcacttgcgcttac-3 ' and CHIKV-8498-R:5 '- gatgcttgtagcagctgat-3’。

2. the viral supernatant liquid progress plaque form for taking P0, P5 to withhold collection compares (method is with embodiment 2)；By the two by sense Contaminating plural number MOI is 0.01 progress growth curve comparison in BHK-21 cell (method is with embodiment 2)；And it is thin by P0, P5 generation Born of the same parents carry out IFA (method is with embodiment 1) detection protein expression situation with special viral antibody E2 and Capsid；Simultaneously by P5 generation Tri- strain virus of A/B/C extract viral RNA after, carry out RT- with primer pair CHIKV-6791-F and CHIKV-8498-R above-mentioned Amplified production is sequenced PCR.The cell in P0, P5 generation and the RT-PCR of supernatant sample are the result shows that the RT-PCR in P0, P5 generation Obtained purpose band is in the same size, (A in Fig. 3) smaller than the purpose band that the RT-PCR of CHIKV-WT is obtained, and illustrates to pass by 5 generations What the Capsid gene of Dai Hou, CHIKV- Δ C virus was still missing from；Plaque and growth curve show that the virus in P0, P5 generation is bitten Spot size no significant difference, and it is more uniform (B in Fig. 3), the growth curve trend of the two is also similar (Fig. 3 C)；IFA result table The virus in bright P0, P5 generation can detect E2 protein expression, but Capsid expression (D in Fig. 3) is not detected, and further illustrate What the Capsid gene of the virus after passage was missing from；Sequencing result shows that the marismortui structural protein sequence of P5 three strain virus of generation is consistent, Capsid gene (3E in figure) is lacked, further demonstrates that CHIKV- Δ C virus has good genetic stability.

Embodiment 4:

Application of the CHIKV- Δ C virus provided by the invention in preparation attenuated vaccine:

What the present embodiment was investigated is virulence evaluation of the CHIKV- Δ C virus in A129 mouse model.

1. three group of six week old A129 mouse (every group six), difference subcutaneous inoculation 1 × 10²The CHIKV-WT of PFU, 1 × 10⁴The CHIKV- Δ C virus of PFU and Mock group without any processing.The Survival of mouse, foot are recorded after immune daily It pads swelling and weighs in, measure 14 days altogether, while the 1st, the 3 and 5 day eye socket after attacking poison takes blood, uses quantitative fluorescent PCR Method detects malicious blood: every mouse takes 5 to bleed in 1.5ml EP pipe, is separately added into 1ml Trizol and is sufficiently cracked, is pressed Book carries out RNA extraction as directed.With One Step SYBR PrimeScript PLUS RT-PCR Kit (Perfect Real Time) (being purchased from TAKARA company) carries out Real-time RT-PCR detection, primer CHIKV-nsP1-F:5 '- ACGTGGATATAGACGCTGACAG-3 ' and CHIKV-nsP1-R:5 '-GCATGGTCATTTGATGTGACC-3 '.Reaction system Are as follows: 42 DEG C of 5min of reverse transcription, 95 DEG C of 10s of initial denaturation, PCR 40 circulations include 60 DEG C of 95 DEG C of 10s of denaturation, annealing and extension 34s。

The result shows that the mouse that CHIKV- Δ C virus has been immunized survives (A in Fig. 4), foot pad does not occur swelling (in Fig. 4 E in B/4), weight gradually increases (C in Fig. 4) and is not detected malicious blood (D in Fig. 4), small with the Mock group without any processing Mouse phenomenon is consistent.And the mouse of immune CHIKV-WT is all dead in the 8th day, footpad swelling phenomenon occurs, weight gradually under It drops and higher malicious blood level can be detected.Prove the chikungunya virus of missing capsid protein gene constructed by the present invention Determination is attenuation in A129 mouse model, is immunized 1 × 10⁴The CHIKV- Δ C of PFU will not make mouse generate any clinical condition Shape, and immune 1 × 10²The CHIKV-WT of PFU can make mouse lethal.

Embodiment 5:

What the present embodiment was investigated is application of the CHIKV- Δ C virus in the protection of A129 mouse model immunization:

1. three group of six week old A129 mouse (every group six), difference subcutaneous inoculation 1 × 10⁴The CHIKV- Δ C virus of PFU, Isometric DMEM culture medium containing 2%FBS and Mock group without any processing.14,21 and 28 days after immune Eye socket takes blood to carry out in antibody and test: every mouse takes 5 to bleed in 1.5ml EP pipe, is centrifuged and collects after 4 DEG C of standing 3h Serum, 56 DEG C inactivate 30 minutes, -20 DEG C freeze it is spare；DMEM culture medium of the serum containing 2%FBS is pressed into 1:50,1:200,1: 800,1:3200 is serially diluted, the serum after taking 100 μ l to dilute (control group is DMEM culture medium of the 100 μ l containing 2%FBS) (virus is the eGFP- of this laboratory building to the eGFP-CHIKV reporter virus for being 0.01 with isometric MOI containing infection multiplicity The rescue of CHIKV infection clones obtains, Deng CL, Liu SQ, Zhou DG, Xu LL, Li XD, Zhang PT, Li PH, Ye HQ,Wei HP,Yuan ZM,Qin CF,Zhang B.Development of Neutralization Assay Using an eGFP Chikungunya Virus.Viruses.2016 Jun 28；8 (7)) mix after in 37 DEG C of incubation 1h；Mention the previous day 8 × 10 are inoculated in the every hole of 24 porocyte culture plates⁴BHK-21 cell, at 37 DEG C, 5%CO₂Under condition of culture, reached to convergence degree When to 60%, the serum after 200 μ l are incubated for and virus mixture, 37 DEG C, 5%CO are added into every hole respectively₂It is inhaled in incubator After attached 1h, virus liquid is abandoned, PBS is washed once, and the DMEM culture medium that 1ml contains 2% fetal calf serum, 37 DEG C, 5%CO are added in every hole₂ Continue to cultivate 48h in incubator；It is examined by the expression in fluorescence microscopy microscopic observation eGFP and with multi-function microplate reader Survey eGFP fluorescence intensity, using eGFP fluorescence intensity compared with control group reduce by 50% when dilution as the serum neutralizing antibody Titre (A in Fig. 5).

2. three groups of mouse insoles are attacked poison 1 × 10 respectively 30 days after immune³The CHIKV-WT of PFU, records mouse daily Survival, footpad swelling and weigh in, measure 14 days altogether, while 5 days after attacking poison, daily eye socket takes blood, uses Fluorescence quantifying PCR method (with embodiment 4) detects malicious blood.The result shows that during the mouse that CHIKV- Δ C virus has been immunized can generate With antibody (B in Fig. 5), and mouse survives (C in Fig. 5) after carrying out attacking poison, and foot pad does not occur swelling (Fig. 5 D), weight by It cumulative length (E in Fig. 5) and is not detected malicious blood (F in Fig. 5), it is consistent with the Mock group mouse phenomenon without any processing.And Neutralizing antibody is not detected in the control group mice of the immune DMEM culture medium containing 2%FBS, after carrying out attacking poison all in the 8th day , there is footpad swelling phenomenon in death, and weight is gradually reduced and higher malicious blood level can be detected.Prove institute's structure of the present invention The determination in A129 mouse model of the chikungunya virus for the missing capsid protein gene built is attenuation, and can be used as epidemic disease Seedling provides good immunoprotection to A129 mouse.

Embodiment 6:

What the present embodiment was investigated is application of the CHIKV- Δ C virus in the protection of C57BL/6 mouse model immunization:

1. three group of six week old C57BL/6 mouse (every group six), difference subcutaneous inoculation 1 × 10⁴The CHIKV-WT of PFU, CHIKV- Δ C virus and isometric DMEM culture medium containing 2%FBS.After immune 14,21 and 28 days eye sockets take blood into In row antibody and (with embodiment 5) is tested, and 30 days after immune, three groups of mouse insoles are attacked into poison 2.5 × 10 respectively⁵PFU's Wild type ECSA CHIKV measures the footpad swelling situation of mouse daily, measures 14 days altogether, while the 1st, 3 and 5 after attacking poison Its eye socket takes blood, detects malicious blood with fluorescence quantifying PCR method (with embodiment 4).The result shows that be immunized CHIKV-WT and The mouse of CHIKV- Δ C virus can generate neutralizing antibody (A in Fig. 6), and mouse insole does not occur swelling simultaneously after carrying out attacking poison And malicious blood (C in B/6 in Fig. 6) is not detected, and during the control group mice of the immune DMEM culture medium containing 2%FBS is not detected And antibody, occur footpad swelling phenomenon after carrying out attacking poison and higher malicious blood level can be detected.Prove institute of the present invention The determination in C57BL/6 mouse model of the chikungunya virus of the missing capsid protein gene of building is attenuation, and can be with Good immunoprotection is provided to C57BL/6 mouse as vaccine.

Embodiment 7:

The chikungunya virus infection clones CHIKV- Δ C of capsid protein gene is lacked as answering in expression vector With:

1. lacking capsid protein gene, it is inserted into the eGFP-CHIKV- Δ C reporter virus infection clones of foreign gene eGFP Building: according to CHIKV-WT and eGFP-CHIKV sequence, six primers, F4 and R4 in sequence such as table 2 are synthesized；F5 and R5；F6 With shown in R6.Using CHIKV-WT as template, segment A is obtained with F4 and R5 primer amplification, F6 and R4 primer amplification obtains segment C, Using eGFP-CHIKV as template, segment B is obtained with F5 and R6 primer amplification, three segments are separately recovered, is with A/B after the recovery Template, fusion DNA vaccine amplification, primer is F4 and R6, obtains segment AB, then using segment AB after the recovery and segment C as template, melt PCR amplification is closed, primer is F4 and R4, obtains segment ABC, recycles segment.PCR reaction system is equal are as follows: 94 DEG C of 2min, 94 DEG C of 20s, 55 DEG C of 10s, 68 DEG C of 3min, 68 DEG C of 10min, 30 circulations.

Segment ABC after the recovery carries out double digestion with NdeI and BamHI, with the full-length infectious clone with identical enzymatic treatment CHIKV- Δ C is ligated and transformed into E. coli competent HB101；Plasmid passes through DNA sequencing and is accredited as the sense correctly obtained Metachromia clone designation is eGFP-CHIKV- Δ C.Clone constructs A in schematic diagram such as Fig. 7.

2. above-mentioned eGFP-CHIKV- Δ C reporter virus infection clones are according to the method linearisation in embodiment 1, phenol chlorine RNA, transfection BHK-21 cell is transcribed in vitro in imitative extracting, and rescue obtains eGFP-CHIKV- Δ C reporter virus, by the virus in Blind passage three generations on BHK-21 cell passes through the expression discovery in fluorescence microscopy microscopic observation eGFP, eGFP-CHIKV- Δ C Amplification can be stablized on BHK-21 cell, it is similar to the eGFP-CHIKV reporter virus of wild type (B in Fig. 7).

Table 2.eGFP-CHIKV- Δ C reporter virus infection clones construct the primer

Sequence table

<110>Wuhan Virology Institute,Chinan academy of Sciences

<120>it is a kind of lack capsid protein gene chikungunya virus infection clones and construction method and preparation be attenuated Application in vaccine

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 11283

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atggctgcgt gagacacacg tagcctatca gtttcttact gctctactct gcaaagcaag 60

agattaataa cccatcatgg attctgtgta cgtggatata gacgctgaca gcgccttttt 120

gaaggccctg caacgtgcgt accccatgtt tgaggtggaa cctaggcagg tcacatcaaa 180

tgaccatgct aatgctagag cgttctcgca tctagccata aaactaatag agcaggaaat 240

tgatcccgac tcaaccatcc tggatatagg tagtgcgcca gcaaggagga tgatgtcgga 300

caggaagtac cactgcgttt gcccgatgcg cagcgcagaa gatcccgaga gactcgctaa 360

ttatgcgaga aagctcgcat ctgccgcagg aaaagtcctg gacagaaaca tttctggaaa 420

gatcggggac ttacaagagg tgatggccgt gccagacacg gagacgccaa cattttgctt 480

acacacagat gtctcatgta gacagagagc agacgtcgcg atataccaag acgtctatgc 540

tgtacatgca cccacgtcgc tatatcacca ggcgattaaa ggagtccgag tggcgtactg 600

ggtagggttc gacacaaccc cgttcatgta caacgctatg gcgggtgcct acccctcata 660

ctcgacaaat tgggcggatg agcaggtact gaaggctaag aacataggat tatgttcaac 720

agacctgaca gaaggtagac gaggcaaatt gtctatcatg agagggaaaa agctaaaacc 780

gtgcgaccgt gtgctgttct cagtagggtc aacgctttac ccggaaagcc gcatgctact 840

taagagctgg cacctaccat cggtgttcca tctaaagggc aagcttagct tcacatgccg 900

ctgtgacaca gtggtttcgt gtgagggcta cgtcgttaag agaataacga tgagcccagg 960

cctttatgga aaaaccacgg ggtatgcggt aacccaccac gcagacggat tcttgatgtg 1020

caagactacc gacacggtag acggtgaaag agtgtcattc ccggtgtgca cgtacgtgcc 1080

ggcgaccatt tgtgatcaaa tgaccggcat ccttgctaca gaagtcacgc cggaggatgc 1140

acagaagctg ttggtggggc tgaaccagag aatagtggtt aacggcagaa cgcaacggaa 1200

cacgaacacc atgaagaact acctgcttcc cgtggtcgcc caggccttca gtaagtgggc 1260

gaaggagtgc cggaaggaca tggaagatga gaagcttctg ggggtcagag aaagaacact 1320

gacctgctgc tgtctgtggg catttaagaa gcagaaaaca cacacggtct acaagaggcc 1380

tgatacccag tcaatccaga aggttcaggc cgaattcgac agctttgtag taccaggcct 1440

gtggtcgtcc gggttgtcaa tcccgttgag gactagaatc aagtggctgt tacgcaaggt 1500

gccgaaagca gacctgatcc catacagcgg aaatgcccaa gaagcccagg atgctgaaaa 1560

agaagcagag gaagaacgag aagcagaact gactcatgag gctctaccac ccctacaggc 1620

agcacaggaa gatgtccagg tcgaaatcga cgtggaacaa cttgaggata gagctggtgc 1680

tggaataata gagactccga gaggcgctat taaagttact gcccaactaa cggaccacgt 1740

cgtgggggag tacctggtac tttccccgca gaccgtatta cgcagccaga agctcagcct 1800

gatccacgct ttagcggagc aagtgaagac gtgtacgcat agcggacgag tagggaggta 1860

tgcggtcgaa gcgtacgatg gccgagtcct agtgccctca ggctatgcaa tttcgcccga 1920

agacttccag agtctaagcg aaagcgcaac gatggtgtac aacgaaagag agttcgtaaa 1980

cagaaagtta caccacattg cgatgcacgg accagctctg aacactgacg aagagtcgta 2040

tgagcttgtg agggcagaga ggacagaaca cgagtacgtc tacgacgtgg accagagaag 2100

atgctgcaag aaggaagaag ctgcaggatt ggtactggtg ggcgacttga ctaatccgcc 2160

ctaccacgaa ttcgcatacg aagggctaaa aattcgcccc gcttgtccat ataaaattgc 2220

agtcatagga gtcttcgggg taccaggatc tggtaagtca gccattatca agaacctagt 2280

taccaggcaa gacctggtga ctagcggaaa gaaagaaaac tgccaagaaa tcagcaccga 2340

cgtgatgaga cagagaggtc tagaaatatc tgcacgtacg gttgattcgc tgctcttgaa 2400

tggatgcaat agaccagtcg acgtgttgta cgtagacgag gcgtttgcgt gccactctgg 2460

aacgttactt gccttgatcg ccttggtgag accaagactg aaagttgtac tttgtggtga 2520

cccgaagcag tgcggcttct tcaatatgat gcagatgaaa gtcaactaca atcataacat 2580

ctgcactcaa gtgtaccaca aaagtatctc caggcggtgc acactgcctg tgactgccat 2640

tgtgtcgtcg ttgcattacg aaggcaaaat gcgcactacg aatgagtaca acatgccgat 2700

tgtagtggac actacgggct caacaaaacc tgaccctgga gacctcgtgt taacgtgctt 2760

cagagggtgg gttaaacaac tgcaaattga ctatcgtgga cacgaggtca tgacagcagc 2820

cgcatcccaa gggttaacca gaaaaggagt ttacgcagtt aggcaaaaag ttaacgaaaa 2880

cccactctat gcatcaacat cagagcacgt caacgtactc ctaacgcgta cggaaggtaa 2940

actggtatgg aagacactct ctggtgaccc gtggataaag acgctgcaga acccaccgaa 3000

aggaaacttc aaggcaacta ttaaggagtg ggaggtggag cacgcatcga taatggcggg 3060

catctgcagt caccaagtga cctttgacac gttccaaaac aaagccaacg tttgctgggc 3120

taagagcttg gtccctatcc tcgaaacagc ggggataaaa ctaaatgata ggcagtggtc 3180

ccagataatt caagctttca aagaagacaa agcatactca cccgaagtag ccctgaatga 3240

aatatgcacg cgcatgtatg gggtggatct agacagcggg ctattctcta aaccgttggt 3300

atctgtgtat tacgcggata accactggga taataggccg ggaggaaaga tgttcggatt 3360

caaccctgag gcagcgtcca ttctagaaag aaagtacccg tttacaaaag gaaagtggaa 3420

catcaacaag cagatctgcg tgactaccag gaggatagaa gacttcaacc ctaccaccaa 3480

cattataccg gccaacagga gactaccaca ctcattagtg gccgaacacc gcccagtaaa 3540

aggggaaaga atggaatggc tggttaacaa gataaacgga catcatgtgc tcctggttag 3600

cggctataac cttgcactgc ctactaagag agtcacctgg gtagcgccac taggtgtccg 3660

cggagcggac tatacataca acctagagat gggtctaccg gcaacacttg gtaggtatga 3720

cctagtggtc ataaacatcc acacaccttt tcgcatacac cattaccaac agtgcgtaga 3780

tcacgcaatg aaactgcaaa tgctaggagg tgactcactg agactgctca aaccgggtgg 3840

ctctctattg atcagagcat acggttacgc agatagaacc agtgaacgag taatctgcgt 3900

actgggacgt aagtttagat cgtccagagc attgaaacca ccatgtatca ccagtaatac 3960

tgagatgttc ttcctattta gcagttttga caatggcaga aggaatttta caacgcatgt 4020

tatgaacaat caactgaacg cagcctttgt aggacaggcc acccgagcag gatgtgcacc 4080

atcgtaccgg gtaaaacgca tggatatcgc gaagaacgat gaagagtgcg tggtcaacgc 4140

cgccaaccct cgcgggttac cgggtgacgg tgtttgcaag gcagtatata aaaaatggcc 4200

ggagtccttt aaaaatagtg caacaccagt aggaaccgca aaaacagtta tgtgcggtac 4260

atatccagta atccatgccg taggaccaaa cttctcaaat tacacggagt ccgaagggga 4320

ccgggaattg gcggctgcct atcgagaagt cgcaaaggaa gtaactagac tgggagtaaa 4380

tagcgtagct atacctctcc tctccacagg tgtatactca ggagggaaag acaggctaac 4440

ccagtcactg aaccacctct ttacagccat ggactcgacg gatgcagacg tggtcatcta 4500

ctgccgagac aaggaatggg agaagaaaat atctgaggcc atacagatgc ggacccaagt 4560

ggagctgctg gatgagcaca tctccataga ctgcgatgtc attcgcgtgc accctgacag 4620

tagtttggca ggtagaaaag gatacagcac cacggaaggc gcactgtact catatctaga 4680

agggacacgt tttcaccaga cggcagtgga tgtggcagag atacatacta tgtggccaaa 4740

gcaaatagag gccaatgagc aagtctgcct atatgccctg ggggaaagta ttgagtcaat 4800

caggcagaaa tgcccggtgg atgatgcaga tgcatcatct cccccgaaaa ccgtcccgtg 4860

cctttgccgt tatgccatga ctcctgaacg cgtcacccga cttcgcatga accatgtcac 4920

aaatataatt gtgtgttctt catttcccct tccaaagtac aagatagaag gagtgcaaaa 4980

agtcaaatgc tccaaggtaa tgttatttga tcacaatgtg ccatcgcgcg taagtccaag 5040

ggaatacaga tcttcccagg agtctgtacg ggaagtgagt atgacaacgt cattgacgca 5100

tagtcagttt gatctaagcg ccgatggcga gacactgccc gtcccgtcag acctggatgc 5160

tgacgcccca gccctagaac cggccctaga cgacggggcg atacatacga ccggaaacct 5220

tgcggccgtg tctgactggg taatgagcac cgtacccgtc gcgccgccta gaagaaggag 5280

agggagaaac ctgaccgtga tatgtgacga gagagaaggg aatataacac ccatggctag 5340

cgtccgattc tttagagcag agcagtgtcc gaccgtacaa gaaacagcgg agacgcgtga 5400

cacagctatt tcctttcggg caccgccaag tatcaccgtg gaactgagcc atccaccgat 5460

ctccttcgga gcaccaagcg agacgttccc catcacattt ggggacttca acgatggaga 5520

aatcgaaagc ttgtcttctg agctactaac tttcggagac ttcctacccg gtgaagtgga 5580

tgatttgaca gatagcgact ggtccacgtg ctcagacacg gacgacgagt tatgactaga 5640

cagggcaggt gggtatatat tctcgtcgga cactggtcca ggccatttac aacagaagtc 5700

ggtacgccag tcagtgctgc cggtaaacac cctggaggaa gttcacgagg agaagtgtta 5760

cccacctaag ctggatgaat taaaggagca actactactt aagaaactcc aggagagtgc 5820

gtccacggcc aatagaagca ggtatcaatc acgcaaagtg gaaaatatga aagcaacaat 5880

catccagaga ctaaagagag gctgtaaact gtatttaatg gcagagaccc cgaaagtccc 5940

gacttatcgg accgtatacc cggcgcctgt gtactcgcct ccgatcaacg tccgattgtc 6000

caatcccgag tccgcagtgg cagcatgtaa cgagttctta gctagaaact acccaactgt 6060

ttcatcatac caaatcaccg atgagtatga tgcatatcta gacatggtgg acgggtcgga 6120

gagttgcttg gaccgagcga cattcaatcc gtcaaaactt aggagctacc cgaaacaaca 6180

tgcttatcac gcgccctcta tcagaagcgc tgtaccttcc ccattccaga acacactaca 6240

gaatgtactg gcagcagcca cgaaaaggaa ctgcaacgtc acacagatga gggaattacc 6300

cactttggac tcagcagtat tcaacgtgga gtgttttaaa aaattcgcat gcaaccgaga 6360

atactgggaa gaatttgctg ccagccctat caggataacg actgagaatc taacaaccta 6420

tgtcactaaa ttaaaggggc caaaagcagc agcgttgctt gcaagaaccc ataatctgct 6480

gccgctgcag gatgtaccaa tggataggtt cacagtagat atgaaaaggg acgtgaaggt 6540

aactcctggc acaaagcata cagaggaaag gcctaaggtg caggttatac aggcggctga 6600

acccttggca acagcgtacc tatgtggaat tcacagagaa ttggttagga gattgaacgc 6660

cgccctccta cccaacgtgc atacactatt tgacatgtct gccgaggact tcgatgccat 6720

tatagccgca cactttaagc caggagacgc cgttttagaa acggacatag cctcctttga 6780

taagagccag gatgattcac ttgcgcttac cgccttaatg ctgttagaag atttgggagt 6840

ggatcactcc ttgttggacc tgatagaggc tgcttttgga gagatttcca gctgtcacct 6900

gccgacaggt acgcgcttca agttcggcgc tatgatgaaa tccggtatgt tcctaactct 6960

gttcgtcaac acattgttaa atatcaccat cgctagccgg gtgttggaag atcgtctgac 7020

aaaatctgca tgcgcggcct tcatcggcga cgacaacata atacatggtg tcgtctccga 7080

tgaattgatg gcagccagat gcgctacttg gatgaacatg gaagtgaaga tcatagatgc 7140

agttgtatcc cagaaagctc cctacttttg tggagggttt atactgcatg atactgtgac 7200

aggaacagct tgcagggtgg cggacccgct aaaaaggtta tttaaactgg gcaaaccgtt 7260

agcggcaggt gacgaacaag acgaagacag aaggcgggcg ctggctgatg aagtaatcag 7320

atggcaacga acagggctaa tagatgagct ggagaaagcg gtgtactcta ggtacgaagt 7380

gcagggtata tcagttgctg taatgtccat ggccaccttt gcaagctcca gatccaactt 7440

cgagaagctc agaggacccg tcataacctt gtacggcggt cctaaatagg tacgcactac 7500

agctacctat tttgcaaaag ccgacagcag gtacctaaat accaatcagc cataatgagt 7560

ctggccattc cagttatgtg cctgctggca aataccacgt tcccctgctc ccagccccct 7620

tgcacaccct gctgctacga aaaagagccg gagaaaacct tgcgcatgct tgaagacaat 7680

gtcatgagcc ccgggtacta tcagctgcta caagcatcct taacatgttc tccccgacgc 7740

cagcggcgca gtattaagga ccacttcaat gtctataaag ccacaagacc gtacctagct 7800

cactgtcccg actgtggaga agggcactcg tgccatagtc ccgtagcgct agaacgcatc 7860

agaaacgaag cgacagacgg gacgttgaaa atccaggttt ccttgcaaat cggaataaag 7920

acggatgata gccatgattg gaccaagctg cgttatatgg acaatcacat gccagcagac 7980

gcagagcggg ccgggctatt tgtaagaacg tcagcaccgt gcacgattac tggaacaatg 8040

ggacacttca ttctggcccg atgtccgaaa ggagaaactc tgacggtagg gttcactgac 8100

ggtaggaaga tcagtcactc atgtacgcac ccatttcacc atgaccctcc tgtgataggc 8160

cgggaaaaat tccattcccg accgcagcac ggtagggaac taccttgcag cacgtacgcg 8220

cagagcaccg ctgcaactgc cgaggagata gaggtacata tgcccccaga caccccagat 8280

cgcacattaa tgtcacaaca gtccggcaat gtaaagatca cagtcaatag tcagacggtg 8340

cggtacaagt gcaattgtgg tgactcaagt gaaggattaa ccactacaga taaagtgatt 8400

aataactgca aggttgatca atgccatgcc gcggtcacca atcacaaaaa atggcagtat 8460

aattcccctc tggtcccgcg taatgctgaa ttcggggacc ggaaaggaaa agttcacatt 8520

ccatttcctc tggcaaatgt gacatgcagg gtgcctaaag caagaaaccc caccgtgacg 8580

tacggaaaaa accaagtcat catgttgctg tatcctgacc acccaacgct cctgtcctac 8640

aggaatatgg gagaagaacc aaactatcaa gaagagtggg tgacgcataa gaaggagatc 8700

aggttaaccg tgccgactga agggctcgag gtcacgtggg gtaacaatga gccgtacaag 8760

tattggccgc agttatccac aaacggtaca gcccacggcc acccgcatga gataattctg 8820

tattattatg agctgtaccc aactatgact gtggtagttt tgtcagtggc ctcgttcata 8880

ctcctgtcga tggtgggtgt ggcagtgggg atgtgcatgt gtgcacgacg cagatgcatt 8940

acaccgtacg aactgacacc aggagctacc gtccctttcc tgcttagcct aatatgctgc 9000

attagaacag ctaaagcggc cacataccag gaggccgcgg tatacctgtg gaacgagcag 9060

cagcctttat tttggctgca agcccttatt ccgctggcag ccctgattgt cctatgtaac 9120

tgtctgagac tcttaccatg ctgttgtaaa atgttgactt ttttagccgt actgagcgtc 9180

ggtgcccaca ctgtgagtgc gtacgaacac gtaacagtga tcccgaacac ggtgggagta 9240

ccgtataaga ctctagtcaa cagaccgggc tacagcccca tggtattgga gatggagctt 9300

ctgtctgtca ccttggaacc aacgctatcg cttgattaca tcacgtgcga gtataaaacc 9360

gttatcccgt ctccgtacgt gaaatgctgc ggtacagcag agtgtaagga caagagccta 9420

cctgattaca gctgtaaggt cttcaccggc gtctacccat tcatgtgggg cggcgcctac 9480

tgcttctgcg acaccgaaaa tacgcaattg agcgaagcac atgtggagaa gtccgaatca 9540

tgcaaaacag aatttgcatc agcatacagg gctcataccg catccgcgtc agctaagctc 9600

cgcgtccttt accaaggaaa taatatcact gtagctgctt atgcaaacgg cgaccatgcc 9660

gtcacagtta aggacgctaa attcatagtg gggccaatgt cttcagcctg gacacctttc 9720

gacaataaaa tcgtggtgta caaaggcgac gtctacaaca tggactaccc gcccttcggc 9780

gcaggaagac caggacaatt tggcgacatc caaagtcgca cgcctgagag cgaagacgtc 9840

tatgctaata cacaactggt actgcagaga ccgtccgcgg gtacggtgca cgtgccgtac 9900

tctcaggcac catctggctt caagtattgg ctaaaagaac gaggggcgtc gctgcagcac 9960

acagcaccat ttggctgtca aatagcaaca aacccggtaa gagcgatgaa ctgcgccgta 10020

gggaacatgc ctatctccat cgacataccg gacgcggcct ttaccagggt cgtcgacgcg 10080

ccatctttaa cggacatgtc gtgtgaggta tcagcctgca cccattcctc agactttggg 10140

ggcgtagcca tcattaaata tgcagccagt aagaaaggca agtgtgcagt gcactcgatg 10200

actaacgccg tcactattcg ggaagctgaa atagaagtag aagggaactc tcagttgcaa 10260

atctcttttt cgacggccct agccagcgcc gaatttcgcg tacaagtctg ttctacacaa 10320

gtacactgtg cagccgagtg ccatccaccg aaagaccata tagtcaatta cccggcgtca 10380

cacaccaccc tcggggtcca agacatttcc gctacggcga tgtcatgggt gcagaagatc 10440

acgggaggtg tgggactggt tgtcgctgtt gcagcactga tcctaatcgt ggtgctatgc 10500

gtgtcgttta gcaggcacta acttgacaac taggtatgaa ggcatacgcg tccctaaaga 10560

gacacaccgc atatagctag gaatcaatag ataagtatag atctaagggc tgaacaaccc 10620

ctgaatagta acaaaatata aaaatcaaca aaaatcataa aatagaaaac tagaaataga 10680

agtaggtaag aaggtatatg tgtcccctaa gagacacacc atatatagct aagaatcaat 10740

agataagcat agatcaaagg gctgaacaac ccctgaataa taacaaaata taaaaaccaa 10800

taaaaatcat aaaatagaaa accacaaata gaagtagttc aaagggctat aaaacccctg 10860

aatagtaaca aaatataaaa ctaataaaaa tcaaacgaat accataattg gcaatcggaa 10920

gagatgtagg tacttaagct tcttaaaagc agccgaactc gctttgagat gtaggcgtag 10980

cacaccgaac tcttccataa ttctccgaac ccacagggac gtaggagatg ttcaaagtga 11040

ctataaaacc ctgaacagta ataaaatata aaattaataa tgagtaccat aattggcaaa 11100

tggaagagac gtaggtacta agcttcttaa aagcagccga actcactttg agatgtaggc 11160

atagcatacc gaactcttcc acaattctcc gtacccatag ggacgtagga gatgttattt 11220

tgtttttaat atttcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aactagtgga 11280

tcc 11283

<210> 2

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

tatatattct cgtcggacac t 21

<210> 3

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

tgcatgtcac atttgccaga g 21

<210> 4

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

tcagccataa tggtcaagca tgaaggtaag 30

<210> 5

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

ttcatgcttg accattatgg ctgattggta 30

<210> 6

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

tcagccataa tgagtctggc cattccagtt 30

<210> 7

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

aatggccaga ctcattatgg ctgattggta 30

<210> 8

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

gagcaccgta cccgtcgcg 19

<210> 9

<211> 59

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

cgcggatcca ctagtttttt tttttttttt tttttttttt tttttttttt tgaaatatt 59

<210> 10

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

agcaggcact aagtcataac cttgtacggc 30

<210> 11

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

caaggttatg acttagtgcc tgctaaacga 30

<210> 12

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

aagttaatta atcttgacaa ctaggtatga 30

<210> 13

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

ctagttgtca agattaatta acttgtacag 30

Claims

1. a kind of chikungunya virus infection clones for lacking capsid protein gene, the sequence of the infection clones is SEQ Shown in ID NO.1.

2. a kind of preparation method for the chikungunya virus infection clones for lacking capsid protein gene, including with full-length infectious Clone CHIKV-WT is skeleton, will be obtained after Capsid gene all missing.

3. infection clones described in claim 1 are as the application in expression vector.

4. the recombinant virus that infection clones described in claim 1 are saved out.

5. infection clones described in claim 1 or its recombinant virus saved out are in the attenuation epidemic disease for preparing chikungunya virus Application in seedling.