CN109536464B

CN109536464B - Chikungunya virus infectious clone with deletion of capsid protein gene, construction method and application in preparation of attenuated vaccine

Info

Publication number: CN109536464B
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: 2022-06-10
Anticipated expiration: 2038-12-10
Also published as: CN109536464A

Abstract

The invention belongs to the field of biotechnology, and particularly discloses chikungunya virus infectious clone with a deleted capsid protein gene, a construction method and application in preparation of attenuated vaccines. It proved to be sufficiently attenuated in the A129 and C57BL/6 mouse models, highly safe and capable of producing immune protection against the CHIKV virus. The virus can be used as a safe and effective attenuated vaccine to prevent chikungunya virus infection, and simultaneously can be used as an expression vector for the expression of foreign genes, thereby having good application prospect.

Description

Chikungunya virus infectious clone with deletion of capsid protein gene, construction method and application in preparation of attenuated vaccine

Technical Field

The invention belongs to the field of biotechnology. Specifically, the invention relates to a chikungunya virus infectious clone with a deleted capsid protein gene, a construction method and application in preparation of attenuated vaccines.

Background

Chikungunya virus (CHIKV) belongs to the genus Alphavirus (Alphavirus) of the family Togaviridae (Togaviridae). The virus particles are spherical, have the diameter of about 60-70 nm and are enveloped. The genome of CHIKV is a single positive-stranded RNA of approximately 11800bp in length. The entire genome includes a 5 'non-coding region, 2 independent Open Reading Frames (ORFs), and a 3' non-coding region. The 5 'end contains a cap structure, the 3' end contains a Poly (A) tail, 2 ORFs respectively encode non-structural proteins and structural proteins of the virus, 4 non-structural proteins (nsP1, nsP2, nsP3 and nsP4) and 5 structural proteins (capsid protein C, envelope protein E3, E2, 6K and E1 proteins) are sequentially arranged from the 5 'end to the 3' end, and a 66bp connecting region is arranged between the non-structural genes and the structural genes and is a non-translation region. Meanwhile, the minus strand of the virus can replicate to form a subgenomic structure which occupies 1/3 about the entire genome length and is identical to the 3' end, namely 26S RNA, and the subgenomic structure is used as a template for translation of structural proteins in the later period of virus replication.

CHIKV is a causative agent of Chikungunya fever (Chikungunya feber, CHIK), transmitted primarily by the bite of aedes mosquitoes. Chikungunya fever, which is clinically manifested by fever, rash and severe arthralgia, is a common disease of both humans and animals and is mainly prevalent in africa and southeast asia. People are infected with CHIKV, and the incubation period is generally 3-7 d. Although the disease is low in lethality, it is likely to cause large-scale outbreaks and epidemics in areas with high mosquito vector density. CHIKV was of africa origin, the first known outbreak was in 1952 in african tanzania, state of nevala. Chikungunya virus was isolated from the serum of sick women in this area in 1953, and also from aedes mosquitoes. After the 60's of the 20 th century, the east of CHIK moved to the southeast asia. A pandemic occurred in madras in india in 1965 with about 30 million people infected. Since 2005 CHIKV was prevalent in islands in indian ocean and continental india in tandem, more than 139 million suspected cases of chikungunya were reported in india in 2006, with incidence rates in some regions exceeding 45%. With the increasing mobility of population, the ecological environment changes and other factors, the prevalence frequency and the prevalence range of the population tend to be increasingly expanded globally. According to the world health organization reports, there are 37 countries and regions around the world that are endemic or at risk of potential endemic. In 2008, the first input chikungunya fever case in China was detected in Guangdong. In 9 months 2010, the Dongguan city in China is exposed to chikungunya fever and rapidly causes hundreds of people to get ill, which is the first chikungunya fever epidemic in China and has attracted wide attention as a new infectious disease. The aedes is widely distributed in south China, southwest and the like, and becomes a potential cause of epidemic outbreak of the disease. This suggests that with the increasing frequency of people and economic exchanges around the world, our country has a high possibility of re-input and outbreak.

At present, chikungunya fever still has no effective antiviral drug treatment, and because the infection, the pathogenic mechanism and the like of the CHIKV are less understood, the research and development of the vaccine are still in the laboratory stage. However, the CHIKV is mainly transmitted in people through mosquito bites, the transmission speed is high, the harm is large, the people are easy to panic, and the problem of harming the public health of the people is a great problem. Therefore, the deep development of the research on the CHIKV vaccine is of great significance.

The CHIKV Capsid protein Capsid comprises two functional domains, namely an N-terminal RNA binding domain and a C-terminal serine protease domain. Wherein the C-terminal serine protease mediates the cleavage of the polyprotein Capsid-E2-E1-6k, releasing the mature Capsid protein. The mature capsid protein is specifically combined with the CHIKV genome 49S RNA through an N-terminal RNA binding domain to form a virus nucleocapsid, and then the nucleocapsid is transported to a cell membrane to interact with envelope protein to promote virus particles to bud out to form virus particles with infectivity. Thus, capsids are indispensable proteins that make up the viral nucleocapsid, facilitating the assembly of viral particles to complete the life cycle. In previous studies on flaviviruses, deletion of the Capsid gene maintained the ability of replication of genomic RNA, and viral particles could not be packaged.

The traditional attenuated live vaccine is prepared by changing a strain into an attenuated strain or an avirulent strain through a virulence variation or artificial selection culture method, has better immune effect than an inactivated vaccine, is difficult to store and has the risk of virulence reversion. Therefore, the development of a new safer chikungunya virus attenuated live vaccine has important public health significance and application prospect.

Disclosure of Invention

The invention aims to provide a chikungunya virus infectious clone CHIKV-delta C with a deleted capsid protein gene, and the sequence of the chikungunya virus infectious clone CHIKV-delta C is shown in SEQ ID NO. 1.

Another objective of the invention is to provide an application of the infectious clone CHIKV-delta C, wherein the infectious clone can be used as a target protein expression vector, and the rescued recombinant virus can also be used as an attenuated vaccine.

In order to achieve the purpose, the invention adopts the following technical measures:

a chikungunya virus infectious clone CHIKV-delta C with a deleted Capsid protein gene is obtained by taking a full-length infectious clone CHIKV-WT as a framework and deleting all Capsid genes, and the sequence of the chikungunya virus infectious clone CHIKV-delta C is shown in SEQ ID NO. 1.

The application of the infectious clone CHIKV-delta C comprises the application of the infectious clone as an expression vector, or the rescue of recombinant virus to prepare an attenuated vaccine of chikungunya virus.

Compared with the prior art, the invention has the following advantages and effects:

1. the reverse genetics technology adopted by rescuing the chikungunya virus lacking the capsid protein gene has the advantages of advanced maturity, convenience, simplicity, controllable positioning and the like.

2. The rescued chikungunya virus CHIKV-delta C lacking capsid protein genes can be amplified in large quantity by a cell culture method, and the obtaining mode is simpler.

3. After the CHIKV-delta C virus provided by the invention passes through 5 generations of passages, plaques and growth curves are not changed, and the Capsid gene is still deleted by structural protein gene sequencing. The CHIKV-delta C virus is shown to have good genetic stability, the possibility of reverting to the wild type virus is low, and the safety of the virus serving as a vaccine is improved.

4. The CHIKV-delta C attenuated virus provided by the invention is proved to be fully attenuated in A129 and C57BL/6 mouse models, has high safety and can generate immune protection against the CHIKV virus.

5. The CHIKV-delta C infectious clone provided by the invention can be used as an expression vector, and the eGFP-CHIKV-delta C reporter virus infectious clone constructed on the basis can be conveniently and quickly used for replacing an eGFP reporter gene with other exogenous genes such as interleukin IL-15 and the like for expression.

In conclusion, the chikungunya virus CHIKV-delta C with the deletion of the capsid protein gene can be used as a safe and effective attenuated vaccine to prevent chikungunya virus infection, and has good application prospect and very important theoretical and practical significance.

Drawings

FIG. 1 is a schematic diagram of the construction of infectious clones of chikungunya virus lacking capsid protein genes and the indirect immunofluorescence of recombinant viruses rescued by the infectious clones;

a: constructing infectious clones of the capsid protein RNA binding domain (CHIKV-delta C-115aa) and all capsid protein genes (CHIKV-delta C);

b: indirect immunofluorescence pictures at different time points after CHIKV-WT, CHIKV-delta C-115aa and CHIKV-delta C transfect BHK-21 cells respectively;

c: CHIKV-WT, CHIKV-delta C-115aa and CHIKV-delta C are used for infecting BHK-21 cells respectively and then performing indirect immunofluorescence mapping for 72 h.

FIG. 2 is a graphical representation of CHIKV-WT and CHIKV- Δ C virus plaque morphology, growth curves, and a comparison of infectious virus particle counts;

a: comparing the morphology of CHIKV-WT and CHIKV-delta C plaques;

b: comparing the growth curves of CHIKV-WT and CHIKV-delta C;

c: CHIKV-WT and CHIKV- Δ C infectious virus particle number comparisons.

FIG. 3 is a schematic diagram of the detection of genetic stability of CHIKV-. DELTA.C virus;

A: RT-PCR compares the target band sizes of P0 and P5 generation CHIKV-delta C cells and supernatant samples;

b: comparing the plaque morphology of the CHIKV-delta C viruses of the P0 and P5 generations;

c: comparing growth curves of the CHIKV-delta C viruses of the P0 and P5 generations;

d: E2/Capsid protein expression conditions of the CHIKV-delta C viruses of P0 and P5 generations;

e: structural protein sequencing peak diagrams of three strains of A/B/C CHIKV-delta C virus at P5 generation.

FIG. 4 is a schematic representation of the use of CHIKV- Δ C attenuated vaccines in the assessment of virulence in A129 mouse models;

a: survival of mice immunized with the CHIKV- Δ C attenuated vaccine;

b: swelling of footpads of mice immunized with the CHIKV- Δ C attenuated vaccine;

c: the weight change of mice immunized with the CHIKV-Delta C attenuated vaccine;

d: immunizing mice with CHIKV- Δ C attenuated vaccines for toxemia;

e: photograph of swelling of footpad of mice immunized with CHIKV-. DELTA.C attenuated vaccine.

FIG. 5 is a schematic representation of the use of CHIKV-. DELTA.C attenuated vaccines for immune protection in A129 mouse model;

a: a flow chart of immunization and virus attack of the CHIKV-delta C attenuated vaccine in an A129 mouse model;

b: the neutralizing experiment detects the titer of neutralizing antibodies generated by mice immunized with the CHIKV-delta C attenuated vaccine;

c: survival of mice immunized with the CHIKV- Δ C attenuated vaccine after challenge;

D: swelling of footpads of mice immunized with the CHIKV- Δ C attenuated vaccine after challenge;

e: the weight change of mice immunized with the CHIKV- Δ C attenuated vaccine after challenge;

f: the condition of the virus blood of mice immunized with the CHIKV-delta C attenuated vaccine after challenge.

FIG. 6 is a schematic diagram of the use of CHIKV-. DELTA.C attenuated vaccine for immune protection in a C57BL/6 mouse model;

a: the neutralizing experiment detects the titer of neutralizing antibodies generated by mice immunized with the CHIKV-delta C attenuated vaccine;

b: swelling of footpads of mice immunized with the CHIKV- Δ C attenuated vaccine after challenge;

c: the condition of the virus blood of mice immunized with the CHIKV-delta C attenuated vaccine after challenge.

FIG. 7 is a schematic diagram of the construction and application of an eGFP-CHIKV- Δ C reporter virus infectious clone lacking capsid protein gene and inserted with exogenous gene eGFP;

a: constructing schematic diagrams of eGFP-CHIKV-delta C report virus infectious clones with deletion of capsid protein genes and insertion of exogenous gene eGFP;

b: eGFP-CHIKV-delta C report virus in BHK-21 cell passage after eGFP expression.

Detailed Description

The PCR, enzyme digestion, ligation, transformation, RNA extraction, RT-PCR and other experimental methods involved in this section all adopt conventional methods in the field if no special description is provided. The following are merely illustrative of several embodiments of the present invention. It is obvious that the present invention is not limited to the following embodiments, but many variations are possible. Therefore, modifications and improvements based on the disclosure of the present invention should be made by those skilled in the art within the scope of the claims of the present invention.

Example 1:

construction of a capsid gene-deleted infectious clone of chikungunya virus and rescued virus:

this example explores the nature of the infectious clones it constructs by exploring different regions of the deleted capsid protein:

1. constructing infectious clones of the RNA binding domain (CHIKV-delta C-115aa) of the deleted capsid protein and all genes (CHIKV-delta C) of the deleted capsid protein: six primers were synthesized according to the sequence of CHIKV-WT (GenBank accession No. KC488650), the sequences of which are F1 and R1 in Table 1; f2 and R2; f3 and R3.

The fragment CHIKV-. DELTA.C-115 aa was PCR-amplified with CHIKV-WT as a template and F1 and R2, F2 and R1 as primers respectively using PrimeSTAR HS enzyme (purchased from Takara), and PCR products were recovered, and 2-stage PCR-recovered products obtained above were used as templates for fusion PCR amplification with primers F1 and R1, and PCR products were recovered.

The CHIKV-delta C fragment is subjected to PCR amplification by PrimeSTAR HS enzyme by using CHIKV-WT as a template and F1, R3, F3 and R1 as primers respectively, a PCR product is recovered, the obtained 2-segment PCR recovery product is used as a template, fusion PCR amplification is carried out, the primers are F1 and R1, and the PCR product is recovered.

The two amplified PCR reaction systems are as follows: 94 ℃ for 2min, 94 ℃ for 20s, 55 ℃ for 10s, 68 ℃ for 3min, 68 ℃ for 10min, and 30 cycles.

The two recovered fusion fragments are subjected to double enzyme digestion by RsrII and NdeI respectively, and are transformed into escherichia coli competent HB101 after being connected with full-length infectious clone CHIKV-WT treated by the same enzyme; the plasmids are identified to be correct by DNA sequencing and are respectively named as infectious clone CHIKV-delta C-115aa and infectious clone CHIKV-delta C. The capsid protein RNA binding domain is deleted from CHIKV-delta C-115aa, all genes of the capsid protein are deleted from CHIKV-delta C, and the cloning construction schematic diagram is shown as A in figure 1.

2. Linearization and phenol chloroform extraction of plasmids: respectively carrying out enzyme digestion on 10 mu g of plasmids CHIKV-delta C-115aa and CHIKV-delta C by using BamHI, carrying out enzyme digestion for two hours at 37 ℃, identifying the enzyme digestion completion by 0.8% agarose gel electrophoresis, adding 100 mu l of saturated phenol (purchased from national drug group chemical reagent company) into an enzyme digestion product, oscillating and mixing uniformly, centrifuging for 5min at 17000g, sucking supernatant into a new centrifuge tube, adding 100 mu l of sterile water into the original centrifuge tube, oscillating and mixing uniformly, and centrifuging for 5min at 17000 g; sucking the supernatant, mixing with the supernatant obtained in the previous step (the total volume is about 200 μ l), adding 200 μ l chloroform (purchased from chemical reagent of national drug group), mixing, and centrifuging at 17000g for 5 min; sucking the supernatant into a centrifugal tube (about 150-200 mu l) without RNAase, adding one tenth of the volume of sodium acetate (purchased from national drug group chemical reagent company) and 2.5 times of the volume of absolute ethyl alcohol (purchased from national drug group chemical reagent company) to mix evenly, standing at-20 ℃ for 30min, and centrifuging at 17000g for 5 min; absorbing and discarding the supernatant, adding 1ml of 70% ethanol for washing, centrifuging at 17000g for 5min, and absorbing and discarding the supernatant; standing at room temperature for 15min, adding 11 μ l of RNAase-free water, determining DNA concentration by Thermo Scientific NanoDrop 2000, detecting DNA quality by 0.8% agarose gel electrophoresis, and storing at-20 deg.C for use.

3. In vitro transcription of RNA: mu.g of the phenol chloroform-extracted linearized product was used as a template, and recombinant CHIKV-. DELTA.C-115 aa and CHIKV-. DELTA.C RNAs were obtained using an in vitro transcription kit T7mMESSAGE mMACHINE kit (purchased from Ambion, USA) according to the kit instructions. RNA concentration was determined using Thermo Scientific NanoDrop 2000, RNA quality was checked using 0.8% freshly prepared agarose gel electrophoresis and stored at-80 ℃ until use.

4. The RNA of the recombinant CHIKV-delta C-115aa and CHIKV-delta C obtained by in vitro transcription is transfected into BHK-21 cells by a liposome transfection method, and the RNA of the transfected wild-type virus CHIKV-WT is used as a positive control: one day before transfection, 2X 10 inoculation⁵BHK-21 cells were plated in 35mm cell culture dishes with three 10mm by 10mm coverslips per dish to achieve approximately 80% cells on the day of transfection; when transfection is carried out, the medium in the culture dish is discarded, 1ml of Opti-MEM is used for washing once, and then 1ml of Opti-MEM is added (cells are in an infiltration state); adding 1ml of Opti-MEM into a 1.5ml EP tube, adding 4ul of DMRIE-C (mixing DMRIE-C before use), mixing by reversing the top and the bottom, adding 1ug of RNA of the recombinant CHIKV obtained by in vitro transcription (no RNA is added in a control group), and mixing by reversing the top and the bottom; quickly abandoning the Opti-MEM in the culture dish, and adding the mixture into the dish (the action is light, and the mixture is not blown against the cells); after 4 hours of incubation at 37 ℃ in a carbon dioxide incubator, the cultures were discarded and 2mL of DMEM medium containing 2% FBS was added. Observing the cell state of the experimental group and the control group under a microscope, respectively taking a

slide

24, 48 and 72 hours after transfection, fixing the cells by using 5% acetone fixing solution (purchased from national drug group chemical reagent company), fixing for 15 minutes at room temperature, washing for three times by using PBS, Storing at 4 deg.C, collecting virus supernatant when cytopathic effect is obvious, and storing at-80 deg.C to obtain CHIKV- Δ C-115aa virus and CHIKV- Δ C virus.

5. Detecting the virus protein expression conditions of the recombinant CHIKV-delta C-115aa and the CHIKV-delta C by indirect Immunofluorescence (IFA): and (3) incubating the slide glass stored at 4 ℃ in the step (4) at room temperature to obtain a primary antibody, wherein the primary antibody is a polyclonal antibody of the CHIKV E2 protein of rabbit origin diluted 1:250 times. Incubating the goat anti-rabbit antibody for 1-2 h at room temperature, washing the goat anti-rabbit antibody with PBS for 10 times, and incubating the goat anti-rabbit antibody with the coupling Fluorescein Isothiocyanate (FITC) diluted by 1:125 times at room temperature in a dark place. After 1h incubation with secondary antibody, the slides were washed 10 times with PBS, taken out and labeled, with a small dot of 95% glycerol at each label, the cover slip was placed cell side down on the glycerol drop and observed under a fluorescence microscope using 200 x magnification (fig. 1, B).

The result shows that the positive rate of the recombinant CHIKV-delta C virus at each time point is lower than that of the CHIKV-WT, but the number of positive cells of the CHIKV-delta C virus gradually increases along with the prolonging of the culture time after transfection, and the trend is consistent with that of the CHIKV-WT virus; and the number of positive cells of the recombinant CHIKV-delta C-115a is not increased along with the prolonging of the culture time after transfection, and the positive cells are scattered, which shows that the CHIKV-delta C can successfully save infectious virus particles like the CHIKV-WT, and the CHIKV-delta C-115a can normally replicate but cannot generate the infectious virus particles.

And 6, IFA (IFA) is used for detecting the infection conditions of the rescued recombinant CHIKV-delta C-115aa and CHIKV-delta C viruses: and (5) respectively infecting the CHIKV-WT, the CHIKV-delta C-115aa and the CHIKV-delta C viruses collected in the step (4) with the BHK-21 cells for 72h, and carrying out IFA detection (C in the figure 1) by the same method. The results indicate that the recombinant CHIKV- Δ C-115aa is not infectious, whereas the recombinant CHIKV- Δ C can produce infectious virus particles, similar to the recombinant CHIKV-WT.

TABLE 1 primers used for construction of infectious clones of chikungunya virus lacking capsid protein genes

Example 2:

comparing the morphology, growth curve and number of infectious virus particles of the CHIKV-delta C virus and the CHIKV-WT plaque:

1. the CHIKV-delta C virus rescued in example 1 is subjected to plaque experiment with CHIKV-WT to determine virus titer and morphology comparison: each well of a 24-well cell culture plate was seeded with 1X 10⁵And (3) removing the culture medium in the BHK-21 cells when the cell confluency reaches 90%, adding 100 mu l of the virus collected in the step (3) diluted by 10 times in a DMEM culture medium containing 2% FBS, adsorbing for 1h at 37 ℃, and fully shaking once every 15 minutes. After completion of the adsorption, the virus solution in each well was aspirated, a DMEM medium containing 2% FBS and a 2% methylcellulose cover were added, the mixture was cultured at 37 ℃ in an incubator containing 5% CO2 for 3 days, staining was performed with a staining solution containing 1% crystal violet and 3.7% formaldehyde after plaque formation, the staining solution in the well was taken out after 30min treatment at room temperature, the bottom of the well was washed with running water, and after drying, the virus titer was counted and converted, and the plaque morphology was observed (A in FIG. 2). As can be seen, the plaque of CHIKV-. DELTA.C virus is smaller than that of CHIKV-WT.

2. Inoculating 2X 10 cells in 35mm cell culture dish⁵BHK-21 cells, 5% CO at 37 ℃₂Under the culture condition, when the confluence degree reaches 60%, 400 μ l of diluted CHIKV- Δ C and CHIKV-WT virus are added into each dish respectively according to the MOI of 0.01, and the temperature is 37 ℃ and the CO content is 5%₂Adsorbing in incubator for 2 hr, discarding virus solution, adding 2ml DMEM medium containing 2% fetal calf serum into each well, and culturing at 37 deg.C with 5% CO₂Culturing under culture condition, collecting 400 μ l virus supernatant every 12h after infection, adding 400 μ l DMEM medium containing 2% fetal calf serum, collecting virus as virus sample of CHIKV- Δ C with the same infection complex number as CHIKV-WT virus at different time points, and storing at-80 deg.C. The virus titers collected at different time points were determined according to the plaque assay described above, and growth curves were plotted (FIG. 2, panel B). The results show that the growth trend of the CHIKV-. DELTA.C virus is similar to that of the CHIKV-WT.

3. And (3) preparing protein samples from the CHIKV-delta C and the CHIKV-WT viruses of different PFUs respectively, carrying out Western blotting detection by using a virus specific antibody E1, and carrying out gray scale analysis on a detected E1 protein band (C in figure 2). The results showed that the number of infectious virus particles of CHIKV-. DELTA.C was about 1% of that of CHIKV-WT.

Example 3:

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

1. the CHIKV-. DELTA.C virus rescued in example 1 (P0 generation) was infected into BHK-21 cells at a multiplicity of infection MOI of 0.01, cell supernatants (P1 generation) were significantly collected by CPE after 5 days, three virus strains were transmitted in parallel for each generation, and this procedure was repeated 5 times; the cells and supernatants collected from P0 and P5 passages were subjected to Trizol and QIAamp Viral RNA Mini Kit instructions to extract Viral RNA and stored at-80 ℃ for use. RT-PCR detection was performed with the PrimeScript One Step RT-PCR Kit, primers CHIKV-6791-F: 5'-gatgattcacttgcgcttac-3' and CHIKV-8498-R: 5'-gatgcttgtagcagctgat-3'.

2. The supernatant virus liquid collected from the P0 and P5 generations was compared for plaque morphology (the same method as in example 2); the two were compared with the growth curve in BHK-21 cells at a multiplicity of infection MOI of 0.01 (same procedure as in example 2); IFA (method same as example 1) detection of protein expression is carried out on cells of P0 and P5 generations by using virus-specific antibodies E2 and Capsid; simultaneously, virus RNA is extracted from the P5 generation A/B/C three strains of viruses, and the primers are used for carrying out RT-PCR on CHIKV-6791-F and CHIKV-8498-R, and the amplified products are sequenced. The RT-PCR results of the cells of the P0 and P5 generations and the supernatant sample show that the sizes of the target bands obtained by the RT-PCR of the P0 and P5 generations are consistent and are smaller than the target band obtained by the RT-PCR of the CHIKV-WT (A in figure 3), which indicates that the Capsid gene of the CHIKV-delta C virus is still deleted after passage of 5 generations; the plaque and growth curves show that the sizes of the P0 and P5 virus plaques are not obviously different and are relatively uniform (B in figure 3), and the growth curves of the two are similar in trend (figure 3C); IFA results show that the viruses of P0 and P5 generations can detect the expression of E2 protein, but cannot detect the expression of Capsid (D in figure 3), further indicating that the Capsid gene of the virus after passage is deleted; sequencing results show that the structural protein sequences of the three strains of the P5 generation virus are consistent and all lack the Capsid gene (3E in the figure), and further show that the CHIKV-delta C virus has good genetic stability.

Example 4:

the application of the CHIKV-delta C virus provided by the invention in preparing attenuated vaccines comprises the following steps:

this example examined the virulence assessment of the CHIKV-ac virus in an a129 mouse model.

1. Three groups of six-week-old A129 mice (six mice per group) were separately immunized subcutaneously at 1X 10²CHIKV-WT, 1X 10 of PFU⁴CHIKV-. DELTA.C virus from PFU and Mock group without any treatment. After immunization, mice were recorded daily for survival, swelling of foot pads and weighed for a total of 14 days, and blood was collected from the orbit on

days

1, 3 and 5 after challenge, and the toxic blood was detected by fluorescence quantitative PCR: 5 drops of blood were taken from each mouse in 1.5ml EP tubes, 1ml Trizol was added to each well for lysis, and RNA extraction was performed according to the instructions. The Real-Time RT-PCR assay was performed with One Step SYBR PrimeScript PLUS RT-PCR Kit (Perfect Real Time) (available from TAKARA) with primers CHIKV-nsP 1-F: 5'-ACGTGGATATAGACGCTGACAG-3' and CHIKV-nsP 1-R: 5'-GCATGGTCATTTGATGTGACC-3' are provided. The reaction systems are as follows: reverse transcription at 42 ℃ for 5min, pre-denaturation at 95 ℃ for 10s, PCR for 40 cycles including denaturation at 95 ℃ for 10s, annealing and extension at 60 ℃ for 34 s.

The results showed that the mice immunized with the CHIKV- Δ C virus survived (a in fig. 4), the footpad did not swell (E in B/4 in fig. 4), the body weight gradually increased (C in fig. 4) and no toxemia was detected (D in fig. 4), consistent with the Mock group mice that did not receive any treatment. On the other hand, mice immunized with CHIKV-WT all died at day 8, and the phenomena of pad swelling appeared, and the body weight gradually decreased and higher levels of toxic blood could be detected. Proves that the constructed chikungunya virus with the deletion of capsid protein gene is determined to be attenuated in an A129 mouse model and is immunized by 1 multiplied by 10 ⁴CHIKV-. DELTA.C of PFU did not cause any clinical symptoms in mice, but 1X 10 mice were immunized²CHIKV-WT from PFU was lethal to mice.

Example 5:

this example investigated the use of CHIKV-ac virus in immune protection in a129 mouse model:

1. three groups of six-week-old A129 mice (six mice per group) were separately immunized subcutaneously at 1X 10⁴CHIKV-. DELTA.C virus from PFU, an equal volume of DMEM medium with 2% FBS, and Mock group without any treatment. Antibody neutralization experiments were performed on orbital bleeds at 14, 21 and 28 days post-immunization, respectively: taking 5 drops of blood from each mouse, placing the blood drops in a 1.5ml EP tube, standing the tube at 4 ℃ for 3h, centrifuging the tube, collecting serum, inactivating the serum at 56 ℃ for 30 minutes, and freezing the serum at-20 ℃ for later use; serially diluting serum by Using DMEM medium containing 2% FBS according to the ratio of 1:50, 1:200, 1:800 and 1:3200, taking 100 mu l of diluted serum (the control group is 100 mu l of DMEM medium containing 2% FBS) and mixing with equal volume of eGFP-CHIKV reporter virus containing complex infection MOI of 0.01 (the virus is obtained by rescuing eGFP-CHIKV infectious clone constructed in the laboratory, Deng CL, Liu SQ, Zhou DG, Xu LL, Li XD, Zhang PT, Li PH, Ye HQ, Wei HP, Yuan, Qin CF, Zhang B.development of Neutrallion Assay Using eGFP Chikungunya Virus. 2016 Jun 28; 8 (7)) and incubating for 1h at 37 ℃; the 24-well cell culture plate was seeded with 8X 10 cells per well one day in advance ⁴BHK-21 cells, 5% CO at 37 ℃₂Under the culture condition, when the confluence reaches 60%, 200 μ l of the incubated serum and virus mixture is added into each well, and the mixture is incubated at 37 deg.C and 5% CO₂Adsorbing for 1 hr in incubator, discarding virus solution, washing with PBS once, adding 1ml DMEM medium containing 2% fetal calf serum into each well, 37 deg.C, and 5% CO₂Continuously culturing for 48h in the incubator; the serum was diluted to a dilution at which the eGFP fluorescence intensity was reduced by 50% from that of the control group, as the neutralizing antibody titer (A in FIG. 5), by observing the expression of eGFP under a fluorescent microscope and detecting the eGFP fluorescence intensity with a multifunctional microplate reader.

2. 30 days after immunization, three groups of mice were each foot-padded to challenge 1X 10³Mice were recorded daily for survival, footpad swelling and weighed by PFU CHIKV-WT for 14 days, while blood was drawn from the orbit daily for 5 days after challenge and blood toxicity was detected by fluorescent quantitative PCR (same as example 4). The results showed that mice immunized with the CHIKV- Δ C virus produced neutralizing antibodies (B in fig. 5) and survived challenge (C in fig. 5), footpadSwelling did not appear (fig. 5D), body weight gradually increased (E in fig. 5) and no toxemia was detected (F in fig. 5), consistent with Mock group mice without any treatment. While the control mice immunized with DMEM medium containing 2% FBS did not detect neutralizing antibodies, all died at day 8 after challenge, the swelling of the footpad appeared, the body weight gradually decreased and higher levels of toxic blood could be detected. The capsid protein gene-deleted chikungunya virus constructed by the invention is proved to be attenuated in an A129 mouse model, and can be used as a vaccine to provide good immune protection for the A129 mouse.

Example 6:

this example examined the use of CHIKV-ac virus in immune protection in a C57BL/6 mouse model:

1. three groups of six-week-old C57BL/6 mice (six mice per group) were separately immunized subcutaneously at 1X 10⁴CHIKV-WT, CHIKV- Δ C virus from PFU and an equal volume of DMEM medium with 2% FBS. Orbital bleeds were taken at 14, 21 and 28 days after immunization for antibody neutralization experiments (same as example 5), and three groups of mice were each foot-filled for 2.5X 10 of challenge 30 days after immunization⁵The swelling of the footpad of the mice was measured daily for 14 days with wild type ECSA CHIKV from PFU, and the venoclysis was detected by fluorescent quantitative PCR method (same as example 4) after challenge at

orbital bleeding days

1, 3 and 5. The results showed that both mice immunized with the CHIKV-WT and CHIKV- Δ C viruses produced neutralizing antibodies (a in fig. 6), and the mice had no swelling of the footpad and no detection of toxic blood after the challenge (C in B/6 in fig. 6), while the control mice immunized with DMEM medium containing 2% FBS did not detect neutralizing antibodies, and had swelling of the footpad and a higher level of toxic blood after the challenge. The capsid protein gene-deleted chikungunya virus constructed by the invention is proved to be attenuated in a C57BL/6 mouse model, and can be used as a vaccine to provide good immune protection for a C57BL/6 mouse.

Example 7:

the application of chikungunya virus infectious clone CHIKV-delta C with capsid protein gene deletion as an expression vector comprises the following steps:

1. constructing infectious clone of eGFP-CHIKV-delta C reporter virus with capsid protein gene deletion and exogenous gene eGFP insertion: six primers were synthesized based on the sequence of CHIKV-WT and eGFP-CHIKV, and the sequences are F4 and R4 in Table 2; f5 and R5; f6 and R6. Using CHIKV-WT as a template, using F4 and R5 primers to amplify to obtain a fragment A, using F6 and R4 primers to amplify to obtain a fragment C, using eGFP-CHIKV as a template, using F5 and R6 primers to amplify to obtain a fragment B, respectively recovering three fragments, using recovered A/B as a template, performing fusion PCR amplification, using F4 and R6 primers to obtain a fragment AB, using recovered fragment AB and fragment C as templates, performing fusion PCR amplification, using F4 and R4 primers to obtain a fragment ABC, and recovering the fragment. The PCR reaction systems are as follows: 94 ℃ for 2min, 94 ℃ for 20s, 55 ℃ for 10s, 68 ℃ for 3min, 68 ℃ for 10min, and 30 cycles.

The recovered fragment ABC is subjected to double enzyme digestion by NdeI and BamHI, and is transformed into escherichia coli competent HB101 after being connected with full-length infectious clone CHIKV-delta C treated by the same enzyme; the plasmids were all identified as correct by DNA sequencing, and the resulting infectious clone was named eGFP-CHIKV- Δ C. The cloning construction scheme is shown as A in FIG. 7.

2. The above-mentioned eGFP-CHIKV- Δ C reporter virus infectious clone was linearized, phenol chloroform extracted, RNA transcribed in vitro, transfected into BHK-21 cells, and rescued to obtain the eGFP-CHIKV- Δ C reporter virus, which was blind-transmitted three times on BHK-21 cells, and it was found by observing the expression of eGFP under a fluorescence microscope that the eGFP-CHIKV- Δ C could be stably amplified on BHK-21 cells, similar to the wild-type eGFP-CHIKV reporter virus (B in FIG. 7).

TABLE 2 primers used for eGFP-CHIKV- Δ C reporter virus infectious clone construction

Sequence listing

<110> Wuhan Virus institute of Chinese academy of sciences

<120> infectious clone of chikungunya virus with deletion of capsid protein gene, construction method and application in preparation of attenuated 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 chikungunya virus infectious clone with a deleted capsid protein gene has a sequence shown in SEQ ID NO. 1.

2. A method for preparing chikungunya virus infectious clone with a deleted Capsid protein gene comprises the steps of taking a full-length infectious clone CHIKV-WT as a framework and obtaining the chikungunya virus infectious clone after all Capsid genes are deleted.

3. Use of the infectious clone of claim 1 as an expression vector.

4. The infectious clone of claim 1 rescued recombinant virus.

5. Use of an infectious clone according to claim 1 or a rescued recombinant virus thereof in the manufacture of an attenuated vaccine of chikungunya virus.