CN112480215B - Virus-like particle of Coxsackie virus CV-A2 - Google Patents

Abstract

The invention relates to a virus-like particle of Coxsackie virus CV-A2, which is characterized in that the virus-like particle is expressed by an insect vector and comprises an optimized CV-A2P1 protein and an optimized CV-A2P 13CD protein; the nucleotide sequence and the amino acid sequence of the optimized CV-A2P1 protein are respectively shown as SEQ ID NO.3 and SEQ ID NO. 5; the nucleotide sequence and the amino acid sequence of the optimized CV-A2P 13CD protein are respectively shown as SEQ ID NO.4 and SEQ ID NO. 6; the strain CV-A2 of the coxsackievirus is CV-A2-1580V4/CHN XY/2017.

Description

Virus-like particle of Coxsackie virus CV-A2

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a virus-like particle of coxsackie virus CV-A2.

Background

Hand-Foot-and-Mouth Disease (HFMD) is a viral Disease caused by infection with various enteroviruses. HFMD is seen in many outbreaks worldwide, common in summer and fall. The patients mainly are preschool children, particularly the patients with the highest incidence rate in the age group below 3 years old, the clinical manifestations are herpes of oral mucosa, hands, feet and other parts, and a small number of the patients can cause serious complications such as encephalitis, pulmonary edema, flaccid paralysis, myocarditis, heart failure and the like, and even death cases^[1]。

The HFMD-causing enteroviruses have at least 20 types, including Coxsackie virus group A (CVA) types 2, 4, 5, 6, 10, 12 and 16, and Coxsackie virus group B (Coxsackie virus B, CVB)Type 5, echovirus (Echo), and Enterovirus type 71 (Enterovirus 71, EV-A71). CV-A2 is a member of the Coxsackie virus A group, the prototype strain, Fleetwood, first isolated in Delaware, USA in 1947^[2]The gene structure is divided into: a5 'non-coding region, a 3' non-coding region, and an open reading frame located between the non-coding regions. The coding region comprises a P1 region encoding a structural protein and a P2, P3 region encoding a non-structural protein. The P1 region encodes 4 capsid proteins VP1, VP2, VP3 and VP4, and 3 structural proteins are exposed on the surface of the virus particle except that VP4 is embedded inside the virus particle shell and is tightly connected with the virus core; the P2 and P3 regions encode 7 non-structural proteins 2A, 2B, 2C, 3A, 3B, 3C and 3D, which are involved in the functions of viral RNA replication, transcription, cleavage of viral polyprotein, assembly of viral particles, etc. The recombinant polyprotein P1 expressed by the cells is cleaved by the simultaneously expressed 3CD enzymes into VP0, VP1, VP2, VP3 and VP4, assembled into virus-like particles with components similar to the pre-particles (Procapsid) after viral infection. CV-A2 mainly causes herpangina, encephalitis and myelitis, myocarditis and pericarditis, epidemic chest pain, respiratory infection, hand-foot-and-mouth disease, infantile diarrhea, etc., and causes epidemic outbreak^[3-7]. Taiwan enterovirus epidemic in 2008, 73% (161/221 cases) of 221 collected samples had herpangina, and 27% (60/221 cases) had hand-foot-and-mouth disease^[8]. In the follow-up visit of early infection of Swedish enterovirus in 2012, the infection proportion of CV-A2 accounts for 9.6 percent, and is second only to CV-A9(13.5 percent) and CV-A16(11.5 percent)^[9]. The most diseases caused by CV-A2 infection are mild symptoms, short course of disease, and less complication or sequelae^[10-12]. However, it has been reported that severe disease of hands, feet and mouth caused by CV-A2 infection resulted in death^[13]: necropsy results of more children died after acute infection in 2012 all showed that CV-A2 infected positive, and the isolated strains were recombinant strains of CV-A2 and EV-71 and CV-A4. Research on the etiology of HFMD in the experiment from 10 months in 2016 to 12 months in 2017 and Xiangyang city in Hubei province shows that CV-A2 can cause outbreak of HFMD and is popular with CV-A5, CV-A6, CV-A10, CV-A16 and EV-A71 and becomes one of the main pathogens of HFMD; it has also been found that CV-A2 can also be usedThe cases of the onset of the disease. Therefore, the monitoring and basic research of the serotype enterovirus have very important significance and practical value.

Currently, the main candidate vaccines for prevention of HFMD are cell matrix whole virus inactivated vaccines and Virus Like Particle (VLP) vaccines. However, in addition to EV-A71 and CV-A16, the other major serotypes of inactivated vaccines are difficult to grow in human vaccine matrix cells. VLPs have the characteristics of large yield, easy enrichment and easy purification, and are also gradually used in the field of vaccines. Therefore, the exploration of a baculovirus Bac-to-Bac expression system and the purification of CV-A2 VLPs have important application significance, and can provide powerful reference for the development of CV-A2 and multivalent hand-foot-and-mouth vaccines.

Reference to the literature

[1]PRAGER P,NOLAN M,ANDREWS IP,et al.Neurogenic Pulmonary Edema in Enterovirus 71 Encephalitis is not Uniformly Fatal but Causes Severe Morbidity in survivors[J].Pediatric critical care medicine, 2003,4(3):377-381.

[2]SICKLES GM,DALLDORF G.Serologic Differences Among Strains of The Coxsackie Group of Viruses [J].Proceedings of the Society for Experimental Biology and Medicine.Society for Experimental Biology and Medicine,1949,72(1):30.

[3]TSAI HP,HUANG SW,WU FL,et al.An Echovirus 18-associated Outbreak of Aseptic Meningitis in Taiwan:Epidemiology and Diagnostic and Genetic Aspects[J].Journal of Medical Microbiology,2011,60(9): 1360-1365.

[4]KIM H,KANG B,HWANG S,et al.Clinical and Enterovirus Findings Associated with Acute Flaccid Paralysis in the Republic of Korea During the Recent Decade[J].Journal of Medical Virology,2014,86(9): 1584-1589.

[5]CISTERNA DM,PALACIOS G,RIVERO K,et al.Epidemiology of Enterovirus Associated with Neurologic Diseases[J].Medicina,2007,67(2):113-119.

[6]LI W,GAO HH,ZHANG Q,et al.Large Outbreak of Herpangina in Children Caused by Enterovirus in Summer of 2015in Hangzhou,China[J].Scientific Reports,6,35388.

[7]KUMAR A,SHUKLAD,KUMAR R,et al.Molecular Identification of Enteroviruses Associated with Aseptic Meningitis in Children from India[J].Archives of Virology,2013,158(1):211-215.

[8]YEN TY,HUANG YP,HSU YL,et al.ACase of Recombinant Coxsackievirus A2 Infection with Neurological Complications in Taiwan[J].Journal of Microbiology Immunology and Infection,2016,50(6): 928-930.

[9]YAMASHITA T,ITO M,TANIGUCHI A,et al.Prevalence of coxsackievirus A5,A6,and A10 in patients with herpangina in Aichi Prefecture,2005[J].Japanese Journal of Infectious,2005,58(6):390-391.

[10]PARK K,LEE BH,BAEK KA,et al.Enteroviruses Isolated from Herpangina and Hand-Foot-and-Mouth Disease in Korean Children[J].Virology Journal,2012,9(1):205.

[11]DAVIAJL,BEL PH,NINET VZ,et al.Onychomadesis Outbreak in Valencia,Spain Associated with Hand,Foot,and Mouth Disease Caused by Enteroviruses[J].Pediatric Dermatology,2011,28(1):1-5.

[12] Zhubing, Stadiumyu, Xia Hui Ming, etc. the etiology research of the hand-foot E1 disease in Guangzhou region in 2008 [ J ]. Zhonghua pediatric journal, 2010,2(48): 127-.

[13]YIP CCY,LAU SKP,WOO PCY,et al.Recombinant Coxsackievirus A2 and Deaths of

Disclosure of Invention

The invention successfully constructs CV-A2P1-3CD recombinant Bacmid plasmid and successfully expresses CV-A2 VLPs, which lays a foundation for the research and development of CV-A2 VLPs vaccines in the future.

The invention relates to virus-like particle VLPs of coxsackie virus CV-A2, which is characterized in that the VLPs are expressed by insect vectors and comprise optimized CV-A2P1 protein and optimized CV-A23 CD protein;

the nucleotide sequence and the amino acid sequence of the optimized CV-A2P1 protein are respectively shown as SEQ ID NO.3 and SEQ ID NO.5, and the nucleotide sequence and the amino acid sequence of the optimized CV-A23 CD protein are respectively shown as SEQ ID NO.4 and SEQ ID NO. 6;

preferably, the strain CV-A2 of the coxsackievirus is CV-A2-1580V4/CHN XY/2017.

The invention also relates to a preparation method of the virus-like particles VLPs of the coxsackie virus CV-A2, which is characterized by comprising the following steps:

(1) construction of recombinant baculovirus Bacmid plasmid: optimized CV-A2P1 and CV-A23 CD genes are respectively cloned behind pPh and P10 promoters of pFastBacdual plasmid, escherichia coli DH10Bac competent cells are transformed, and positive plasmid, namely recombinant baculovirus Bacmid plasmid, is extracted.

(2) Expression of recombinant baculovirus: transfecting insect cells by using the recombinant baculovirus Bacmid plasmid to obtain the recombinant baculovirus.

(3) Preparation of Coxsackie Virus CV-A2 VLPs: the obtained recombinant baculovirus was inoculated with insect cells at MOI of 0.5, and after 5 to 6 days, the supernatant was harvested, and coxsackie virus VLPs were isolated by extraction, preferably, the insect cells were sf-9.

Optionally, the method further includes:

(4) VLPs were identified using SDS-PAGE, Western-Blot, indirect immunofluorescence and transmission electron microscopy.

Preferably, the step of extracting and isolating coxsackie virus VLPs in step (3) is:

1) collecting Sf-9 cell sap infected by CV-A2 VLPs, repeatedly freezing and thawing, centrifuging at 3000rpm and 4 ℃ for 10min to remove cell debris;

2) the supernatant is ultrafiltered and concentrated by 30 times by a membrane with the aperture of 30 kD;

3) the concentrated product was passed through 20% sucrose, 15000g bedding, centrifuged for 4h, followed by centrifugation at 1.31g/ml CsCl density 25000g for 20h, and the target bands were extracted as CV-A2 VLPs (middle band in FIG. 7).

The invention also relates to application of the virus-like particles VLPs of the coxsackievirus CV-A2, the coxsackievirus CV-A2P1 protein and the coxsackievirus CV-A23 CD protein in preparation of vaccines or medicaments, preferably, the vaccines or medicaments are vaccines or medicaments for treating or preventing hand-foot-and-mouth diseases.

The invention has the beneficial effects that:

CV-A2 VLPs are successfully assembled by Bac-to-Bac baculovirus expression systems through CV-A2P1 and 3CD genes optimized by insect codons.

The invention successfully constructs CV-A2P 1/3CD recombinant Bacmid plasmid and successfully expresses CV-A2 VLPs, which lays a foundation for the research and development of CV-A2 VLPs vaccines in the future.

Drawings

FIG. 1 and 1a show the partial sequence PCR results (M: Marker; 1: 1bp-500 bp; 2: 1230bp-3780 bp; 3: 3910bp-6210 bp.) for CV-A2-1580R 4/XY/CHN/2017; 1b is CV-A2-1580R4/XY/CHN/2017 partial sequence PCR result (M: Marker; 4: 870bp-2070 bp; 5: 3700bp-5530 bp);

FIGS. 2 and 2a are CV-A2P1 gene identification diagrams; 2b is CV-A23 CD gene identification diagram; lanes in the figure: m: a protein Marker; 1, after BamHI/SpeI enzyme digestion, P1 fragment; 2, Xho I/Sph I enzyme cutting, 3CD segment.

FIG. 3, CV-A2P 1/3CD recombinant Bacmid DNA identification diagram; lanes in the figure: m: marker; 1: amplifying fragments by using M13F and M13R primers; 2: M13F and CV-A2-P1 primers amplified the P1 fragment; 3: CV-A2-3CD and M13R primers amplified the 3CD fragment.

FIG. 4, recombinant baculovirus rCV-A2 Pl/3CD infected insect cell Sf9 pathogram (. times.200).

FIG. 5, rCV-A2 VLPs DNA electrophoresis identification map; lanes in the figure: m: a protein Marker; 1: the M13F/M13R primer amplifies a band; 2: the M13R/CV-A2-3CD (3CD) primer amplified a band.

FIG. 6, Indirect immunofluorescence assay for insect cell Sf9 expression of rCV-A2 VLPs (X200).

FIG. 7 CsCl Density gradient centrifugation purification rCV-A2 VLPs

FIGS. 8 and 8a are SDS-PAGE analyses of rCV-A2 VLPs; 8b Western-Blot detection of rCV-A2 VLPs

FIG. 9, electron microscopy projection of purified rCV-A2VLs, coordinatometer: 200 nm.; magnification, 100000.

Detailed Description

Example 1: construction of CV-A2P 1/3CD recombinant Bacmid

1. Determination of the P1 and 3CD Gene sequences

A clinical specimen is collected from a patient with hand-foot-and-mouth disease clinically diagnosed in Xiangyang city of Hubei province, and CV-A2-1580R4/CHN XY/2017 strain is obtained by separating human rhabdomyosarcoma cells (RD) by a conventional method. Extracting RNA, reverse transcription, and sequencing the whole genome sequence by biological engineering (Shanghai) GmbH.

The P1 and 3CD sequences were determined by alignment with the CV-A2 prototype strain Fleetwood strain (GenBank: AY 421760.1).

The sequencing result shows that:

the P1 gene nucleotide sequence of Coxsackie virus (CV-A2-1580R4/CHN XY/2017 strain) is shown in SEQ ID NO. 1;

the nucleotide sequence of the coxsackievirus (CV-A2-1580R4/CHN XY/2017 strain) 3CD gene is shown as SEQ ID NO. 2.

2. Construction of recombinant plasmid pFastBacDual-P1/3CD

Optimizing codons according to insect cell preference characteristics and synthesizing P1 and 3CD cDNA fragments, wherein the steps are as follows:

SEQ ID NO. 3: optimized coxsackie virus P1 gene nucleotide sequence

SEQ ID NO. 4: optimized coxsackie virus 3CD gene nucleotide sequence

The amino acid sequences are respectively as follows:

SEQ ID No. 5: optimized Coxsackie virus P1 gene amino acid sequence

SEQ ID NO. 6: optimized Coxsackie virus 3CD gene amino acid sequence

The P1 and 3CD fragments were ligated into the pFastBacDual vector by T-A cloning.

Two gene segments, placed behind polyhedrin promoter pPh and P10 promoter, respectively. This work was done by Biotechnology (Shanghai) Inc.

The recombinant plasmid pFastBacDual-P1/3CD is obtained and then identified by BamHI/SpeI and XhoI/SphI double enzyme digestion respectively.

3. CV-A2P 1/3CD recombinant Bacmid

After the obtained pFastBacDual-P1/3CD recombinant plasmid, DH10 BacTM competent cells are further transformed and cultured for 72 hours at 37 ℃, and CV-A2P 1/3CD recombinant Bacmid is obtained.

The obtained CV-A2P 1/3CD recombinant Bacmid was identified using M13F/M13R and M13F/CV-A2-P1, M13R/CV-A2-3CD primers (synthesized by Biotechnology, Shanghai, Inc.), which are shown in Table 1.

Table 1: M3F, M13R, CV-A2-P1 and CV-A2-3CD primers

PCR reaction system and reaction conditions:

reaction buffer 15.2. mu.l, DNA template 0.4. mu.l, Taq enzyme mix 3.6. mu.l, forward and reverse primers 0.8. mu.l each (1.0 ng/. mu.l), supplemented ddH₂O to 20. mu.l.

94℃ 5min；

30s at 94 ℃; 30s at 55 ℃; 30s at 72 ℃ for 35 cycles.

Wherein forward and reverse primers for amplifying M13F/M13R fragment (7016 bp) are respectively M13F and M13R, forward and reverse primers for amplifying P1 fragment (3112bp) are respectively M13F and CV-A2-P1, and forward and reverse primers for amplifying 3CD (3586bp) fragment are respectively CV-A2-3CD and M13R.

And sending the PCR amplification result to Shanghai biological engineering Co., Ltd for sequencing, and splicing the sequencing result by utilizing SeqMan (7.1.0) software to obtain a CV-A2-1580R4/CHN XY/2017 strain genome complete sequence. The sequence is 7401nt in full length, the nucleotide positions of the non-coding region 5 '-UTR and 3' -UTR in the genome are 1-747nt and 7321-7401nt respectively, the length of the coded Polyprotein (Polyprotein) is 2191 amino acid residues, the P1 and 3CD sequence positions of CV-A2-1580R4/CHN XY/2017 strains, P1: 748-3324nt, 3 CD: 5383 7317 nt.

The pFastBacDual-P1 fragment and pFastBacDual-3CD fragment of the recombinant plasmid were digested simultaneously with BamHI/SpeI and XhoI/SphI, respectively, and the sizes of the fragments were predicted, as shown in FIG. 2, indicating that P1 and 3CD were cloned into the pFastBacDual plasmid.

PCR verification is carried out on universal primers M13F/M13R, M13F/CV-A2-P1(P1) and M13R/CV-A2-3CD (3CD) primers, clear bands can be seen on 1% agarose gel electrophoresis above and below 7016 bp, 3112bp and 3586bp, and the clear bands are consistent with the expected target size, and the clear bands are shown in figure 3, which indicates that CV-A2 Pl/3CD gene is successfully transposed to Bacmid.

Example 2: construction and identification of recombinant baculovirus rBac-CV-A2P 1/3CD

1. Construction and DNA identification of recombinant baculovirus rBac-CV-A2P 1/3CD

According to

Expression System

The Reagent operation shows that recombinant Bacmid CV-A2P 1/3CD is transfected into Sf9 cells and cultured in a biochemical incubator at 27 ℃ until cytopathic effect occurs to obtain recombinant baculovirus rBac-CV-A2P 1/3 CD.

Recombinant Bacmid is used for transfecting sf9 insect cells, cells become round and large under a microscope, cell nuclei become large and fill the whole cells, the cells are transparent and edematous, the refractive index of the cells is changed, meanwhile, more and more vacuoles and more particles appear in the cells, and the observation under a microscope is shown in figure 4. Indicating that CV-A2P 1/3CD recombinant Bacmid successfully transfects Sf9 cells.

Recombinant baculovirus DNA was extracted and its inserted DNA sequence was identified by amplification using M13F/M13R and M13R/CV-A2-3CD primers. The PCR reaction system and reaction conditions were the same as above.

As can be seen in FIG. 5, clear bands were seen on the agarose gel electrophoresis at positions 7016 bp and 3586bp, consistent with the expected target size. This further confirmed the successful transfection of CV-A2 Pl-3CD recombinant Bacmid, and the recombinant virus of interest, rBacCVA2-P1/3CD, was obtained.

2. Indirect immunofluorescence assay (IFA)

After rBac-CV-A2 Pl/3CD recombinant Bacmid transfects insect cells Sf 972 h, fixing the cells by 4% paraformaldehyde, and blocking the cells by 5% BSA + 0.5% TritonX-100+ PBS at room temperature;

adding a primary antibody: rabbit anti-CV-A2 VLP polyclonal antibody (1: 1000), incubated overnight at 4 ℃;

adding a secondary antibody: goat anti-mouse Alexa-488(1: 2000), incubated for 1h at 37 ℃; and (4) observing under a fluorescence microscope.

The fluorescence microscope showed a clear green fluorescence, whereas the control group of normal Sf9 cells showed no fluorescence signal (FIG. 6), indicating that the recombinant rCV-A2 VLPs (infection) protein was successfully expressed.

Example 3: purification and characterization of CV-A2 VLPs

1. Sucrose bedding and Cesium chloride Density gradient centrifugation purification rCV-A2 VLPs

Collecting rCV-A2 VLPs infected Sf9 cells, repeatedly freezing and thawing, centrifuging at 3000rpm and 4 ℃ for 10min to remove cell debris, and ultrafiltering and concentrating the supernatant by a membrane with the aperture of 30 kD. The concentrated product was centrifuged through 20% sucrose, 15000g pad for 4h, 1.31g/ml CsCl density 25000g for 20h and the band of interest (middle band in FIG. 7) was extracted.

2. SDS-PAGE and Western-Blot immunoblotting method for detecting target protein expression

The purified rCV-A2 VLPs were identified by 0.1% SDS-12.5% PAGE and Western-Blot, respectively, and detected by conventional methods. Primary antibody in Western-Blot: anti-CV-A2 VLP polyclonal antibody (1: 2000) was incubated overnight at 4 ℃; secondary antibody: goat anti-mouse polyclonal antibody (1: 5000) is incubated for 1h at 37 ℃, and finally, a DAB color development system is used for color development.

As shown in FIG. 8a, it can be clearly seen that VP0 is about 39kD, VP1 is about 34kD, and VP3 is about 27kD, consistent with the expected results. The specific bands of VP0, VP1 and VP3 were detected by Western-Blot, as shown in FIG. 8 b. The successful expression of the recombinant rCV-A2 VLPs (infection) protein is shown.

3. Observation by transmission electron microscope

A sample of 100. mu.l was taken, contacted with the surface of a copper mesh with a supporting film, left to stand for 3min, negatively stained with 3% phosphotungstic acid for 5min, air-dried overnight at normal temperature, and sent to the Wuhan virus institute of Chinese academy of sciences for electron microscopy observation.

When rCV-A2 VLPs (antigens) are observed under a transmission electron microscope after purification, as can be seen from figure 9, the shapes of the virus-like particles are regularly circular and uniformly distributed, the diameter range of the virus-like particles is determined to be about 23-33nm, and the overall shape and size are consistent with those of enterovirus CV-A2. Indicating that rCV-A2 VLPs (infection) protein is expressed successfully.

Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not used to limit the protection scope of the present invention.

SEQUENCE LISTING

<110> Wuhan Biometrics institute of Biotechnology, Inc

<120> a virus-like particle of Coxsackie virus CV-A2

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<170> PatentIn version 3.3

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ttccatgcaa atcctggaac tgtgactggt tcagctgtgg ggtgcaaccc agatacattt 1200

tggagtaagc taccaatctt gctccctggc tccctctttg cctttgacta ctcaggttat 1260

gatgctagtc tcagcccagt ttggttcagg gcactggagc tagttctcag ggagataggc 1320

tatagtgagg aggcggtttc gcttatcgaa ggaatcaacc acacacacca tgtataccgc 1380

aacaagactt attgcgtgct tggtgggatg ccctcaggtt gctcaggaac atccatcttt 1440

aactcaatga tcaacaacat tatcatcaga acactcctta ttaagacatt caagggtatt 1500

gatttggatg aactcaatat ggttgcttac ggagatgatg tgctcgccag ttatcctttc 1560

ccaattgact gcctagaact agcaagaacg ggtaaggagt atggtctaac catgacccct 1620

gctgacaagt ccccttgctt taatgaagtc aattgggaaa atgcaacctt tctcaagagg 1680

ggcttcttgc ccgatgatca gtttccattc ttgatccacc ctaccatgcc aatgaaggag 1740

attcatgaat ccattcgatg gaccaaggat gcacgcaata ctcaagatca cgtgcgatcc 1800

ttatgtctat tggcatggca caacggcaag caagaatatg aagaatttgt gagtacaatc 1860

aggtctgtcc caataggaaa agcattggca attcccaatt atgaaaattt gagacgtaat 1920

tggctcgaat tattt 1935

<210> 3

<211> 2580

<212> DNA

<213> Artificial sequence

<400> 3

atgggtgccc agttcagcac acagcgtagc ggctcccacg agacatccaa cgtggcccgt 60

gacggctcaa ctatcaactt cacaaacatc aactactaca aggacagcta cgccgccagc 120

gccgccaagc acgacttcac ccaggaccct ggcaagttca cccagcctgt gctggacgct 180

ctgcgcgaag ccgtgcctcc cctgcagtcc ccaagcgctg aagcctgtgg ctactcagac 240

cgtgtcgccc agctgactgt tggtaacagc acaatcacta ctcaggaggc tgctaacatc 300

atcgtgtcct acgctgagtg gcctgagtac tgccctgaca ccgacgccac cgccgttgac 360

aagcccactc gcccagacgt gtcagtgaac cgtttctaca caatgcctgc caccctctgg 420

gaaactgaaa gcaagggctg gtactggaag ttcccagacc tgctcaacga gatcggagtc 480

ttcggtcaga acgcccagtt ccactacctg taccgctctg gtttctgtat ccacgttcag 540

tgtaacgctt ccaagttcca ccaaggcgcc ctgttggtcg ctgtgatccc tgagttcgtg 600

gtggccgaac aggacgctac acaaaagcct aacaccgcta agcacccatc cctggaggcc 660

acccagcctg gcaagaaggg caaggccttc cgtcacccat acgtgctcga ctgtggtgtc 720

cctctgagcc aggccctcgt gttcccacac cagtggatca acctgcgcac taacaactgt 780

gctactatcg tggtgcccta catcaacgcc ctccccttcg attccgccat caaccactcc 840

aacttcagcc tcgctgttat cccagtgtgc cctctgaagt acaacaccgg tgctaccccc 900

agcatcccaa tcactatcac cgtcgctccc ctgtgttctg agttcgccgg actccgtcaa 960

gccgtgaagc agggcgtccc agtggaaatg aagccaggca ccaaccagtt cctcactacc 1020

gacgatggca catccgctcc catcctccca ggtttccacc caacacctac catccacatc 1080

cctggtgaag tccacagcct gctggagctc tgtcagatcg aaactatgct ggaagtcaac 1140

aacacctcca aggccatgga actggacagg ctgcgtatcc ctgtgtctgc ccagtctgct 1200

gtggacaccc tgtgcgccgc tttccgtgtc gaccctggtc gcgacggtcc ctggcagagc 1260

acaatggtgg gccagctgtg ccgctactac actcaatgga gcggtagcct caagatcacc 1320

ttcatgttca ctggcagctt catggctacc ggaaagatgc tggttgccta caccccacct 1380

ggaggtgctc agccagccac ccgtgaactc gccatgctcg gaactcacgt gatctgggac 1440

ttcggtctgc agagctcagt gacactggtc gtcccttgga tctccaacac acactaccgc 1500

gctgttgaga ccggcggtgt gttcgactac tacactactg gcgtggtgac catctggtac 1560

caaacaaact tcgtggtgcc tcctgacaca cccacctccg cctacatcct cgccttcgga 1620

gctggacaga agaacttcac cctgaagctg tgcaaggaca ccgacgagat tactcagcag 1680

gccaccctgc aaggtgacgg catcgaggac gctatcacta acaccgttaa cgctaccatc 1740

gatcgtgtcc tgagccgccc agtttcccac agccccacag ccgccaacac ccaggtgtct 1800

cagcactcta tcgagacagg tcgtgtccct gctctgcagg ctgctgagac cggtgccacc 1860

tcaaacgcta ccgacgaaaa cctgatcgag actaggtgcg tggttaacaa gaactccgtc 1920

gaggaagcta gcatcaacca cttcttctct agggctgccc tggtgggcaa ggtcgagctg 1980

aacgacactg gcactagcgc tacaggattc accaactggg acatcgacat catgggttac 2040

gctcagctgc gccgtaagct ggagatgttc acctacatga ggttcaacgc tgagttcaca 2100

ttcgtggcta caacacgcgc cggtagggtg ccatcccgcg tcctgcagta catgtacgtc 2160

cctcccggtg cccctaagcc agacggccgt gaggctttcc aatggcagag ctccactaac 2220

cctagcgttt tcagcaagat gacagaccca cccccccagg ttagcgtgcc cttcatgtcc 2280

cctgcttccg cttaccagtg gttctacgac ggctacccta ccttcggtga acactccgga 2340

gaagacagcc tgaggtacgg ccagtgcccc aacaacgccc tcggtacttt ctccgtgcgc 2400

ttcgttagcg aagaaatcac taacgagcgc atcacaatcc gtatctacat gcgcctgaag 2460

cacgttcgtg cctgggttcc tcgccccctg cgctcagaac cctacgtgct gaagaacttc 2520

ccaaactaca ccgctgtgac ccacgtgaca gctaaccgcc ctgccatcac taacacttaa 2580

<210> 4

<211> 1935

<212> DNA

<213> Artificial sequence

<400> 4

ggacctagcc tggacttcgc cctgagcctc ctgcgccgta acgttcgtca ggtgcagact 60

gaccaaggcc acttcaccat gctgggcgtg cgcgaccgcc tggccgttct gccccgtcac 120

tctcaccctg gcaagacaat ctggatcgag cacaagctgg tgaacgtgct ggacgctgtg 180

gaactggttg acgaacaggg tgttaacctg gaactgactc tggtgacact cgacaccaac 240

gagaagttcc gcgacatcac aaagttcatc ccagagtcta tcagcgctgc ctcagacgct 300

acactcgtca tcaacactga gcacatgcca tccatgttcg tgcctgtcgg cgacgttgtg 360

cagtacggtt tcctgaacct gagcggtaaa cctacccacc gtaccatgat gtacaacttc 420

cccactaagg ccggccagtg cggcggcgtg gtgacttccg tgggaaaggt catcggtatc 480

cacatcggtg gaaacggacg tcagggattc tgcgccggcc tgaagcgcag ctacttcgct 540

agcgagcaag gtgagatcca gtgggtcaag cccaacaagg aaaccggtcg tctgaatatc 600

aacggtccca ctcgcaccaa gctggagcca tcagtcttcc acgacgtgtt caagggtaac 660

aaggaaccag ctgtgctgca cagcaaggac cctcgtctgg aggttgactt cgaacaggct 720

ctcttcagca agtacgtcgg caacacactg cacgagcctg atgaatacgt ccgcgaggcc 780

gctctgcact acgctaacca gctgaagcaa ctggacatcg acaccacaca gatgagcatg 840

gaggaagctt gttacggcac agacaacctc gaagccatcg acctccacac atccgctggt 900

tacccatact ccgccctcgg tatcaagaag cgcgacatcc tggaccccac aacacgtgac 960

gtctcaaaga tgaagttcta catggataag tacggtctgg acctgccata ctccacctac 1020

gtcaaggacg aactgcgttc catcgacaag atcaagaagg gcaagtctcg cctgatcgaa 1080

gccagcagcc tgaacgactc tgtttacctg aggatggctt tcggtcacct gtacgagact 1140

ttccacgcca accccggaac cgtgaccggt tccgctgtcg gttgtaaccc tgacaccttc 1200

tggtccaagc tcccaatcct gttgcctggc tcactgttcg ccttcgacta ctcaggctac 1260

gacgctagcc tcagccccgt gtggttccgc gctctggaac tcgtgctgcg tgagatcggc 1320

tactccgagg aggctgtgag cctcatcgaa ggaatcaacc acactcacca cgtttaccgt 1380

aacaagacct actgcgtcct gggcggaatg cccagcggtt gtagcggaac tagcatcttc 1440

aactccatga tcaacaacat catcatccgc acactcctga tcaagacttt caagggcatc 1500

gacttggacg agctgaacat ggtggcctac ggcgacgacg tgctcgcttc ctacccattc 1560

ccaatcgact gcctggaact cgctcgcact ggcaaggaat acggtctgac catgacacct 1620

gccgacaagt ccccttgttt caacgaggtc aactgggaga acgctacctt cctgaagcgc 1680

ggcttcctgc ctgacgacca gttcccattc ctgatccacc caactatgcc aatgaaggaa 1740

atccacgaat caatccgctg gactaaggac gcccgtaaca cccaggacca cgtccgctcc 1800

ctctgtctgc tggcctggca caacggcaag caggagtacg aggaattcgt gagcactatc 1860

cgctccgtgc ctatcggcaa ggccctggcc atccctaact acgaaaacct gcgccgtaac 1920

tggctggagc tgttc 1935

<210> 5

<211> 859

<212> PRT

<213> Artificial sequence

<400> 5

Met Gly Ala Gln Phe Ser Thr Gln Arg Ser Gly Ser His Glu Thr Ser

1 5 10 15

Asn Val Ala Arg Asp Gly Ser Thr Ile Asn Phe Thr Asn Ile Asn Tyr

20 25 30

Tyr Lys Asp Ser Tyr Ala Ala Ser Ala Ala Lys His Asp Phe Thr Gln

35 40 45

Asp Pro Gly Lys Phe Thr Gln Pro Val Leu Asp Ala Leu Arg Glu Ala

50 55 60

Val Pro Pro Leu Gln Ser Pro Ser Ala Glu Ala Cys Gly Tyr Ser Asp

65 70 75 80

Arg Val Ala Gln Leu Thr Val Gly Asn Ser Thr Ile Thr Thr Gln Glu

85 90 95

Ala Ala Asn Ile Ile Val Ser Tyr Ala Glu Trp Pro Glu Tyr Cys Pro

100 105 110

Asp Thr Asp Ala Thr Ala Val Asp Lys Pro Thr Arg Pro Asp Val Ser

115 120 125

Val Asn Arg Phe Tyr Thr Met Pro Ala Thr Leu Trp Glu Thr Glu Ser

130 135 140

Lys Gly Trp Tyr Trp Lys Phe Pro Asp Leu Leu Asn Glu Ile Gly Val

145 150 155 160

Phe Gly Gln Asn Ala Gln Phe His Tyr Leu Tyr Arg Ser Gly Phe Cys

165 170 175

Ile His Val Gln Cys Asn Ala Ser Lys Phe His Gln Gly Ala Leu Leu

180 185 190

Val Ala Val Ile Pro Glu Phe Val Val Ala Glu Gln Asp Ala Thr Gln

195 200 205

Lys Pro Asn Thr Ala Lys His Pro Ser Leu Glu Ala Thr Gln Pro Gly

210 215 220

Lys Lys Gly Lys Ala Phe Arg His Pro Tyr Val Leu Asp Cys Gly Val

225 230 235 240

Pro Leu Ser Gln Ala Leu Val Phe Pro His Gln Trp Ile Asn Leu Arg

245 250 255

Thr Asn Asn Cys Ala Thr Ile Val Val Pro Tyr Ile Asn Ala Leu Pro

260 265 270

Phe Asp Ser Ala Ile Asn His Ser Asn Phe Ser Leu Ala Val Ile Pro

275 280 285

Val Cys Pro Leu Lys Tyr Asn Thr Gly Ala Thr Pro Ser Ile Pro Ile

290 295 300

Thr Ile Thr Val Ala Pro Leu Cys Ser Glu Phe Ala Gly Leu Arg Gln

305 310 315 320

Ala Val Lys Gln Gly Val Pro Val Glu Met Lys Pro Gly Thr Asn Gln

325 330 335

Phe Leu Thr Thr Asp Asp Gly Thr Ser Ala Pro Ile Leu Pro Gly Phe

340 345 350

His Pro Thr Pro Thr Ile His Ile Pro Gly Glu Val His Ser Leu Leu

355 360 365

Glu Leu Cys Gln Ile Glu Thr Met Leu Glu Val Asn Asn Thr Ser Lys

370 375 380

Ala Met Glu Leu Asp Arg Leu Arg Ile Pro Val Ser Ala Gln Ser Ala

385 390 395 400

Val Asp Thr Leu Cys Ala Ala Phe Arg Val Asp Pro Gly Arg Asp Gly

405 410 415

Pro Trp Gln Ser Thr Met Val Gly Gln Leu Cys Arg Tyr Tyr Thr Gln

420 425 430

Trp Ser Gly Ser Leu Lys Ile Thr Phe Met Phe Thr Gly Ser Phe Met

435 440 445

Ala Thr Gly Lys Met Leu Val Ala Tyr Thr Pro Pro Gly Gly Ala Gln

450 455 460

Pro Ala Thr Arg Glu Leu Ala Met Leu Gly Thr His Val Ile Trp Asp

465 470 475 480

Phe Gly Leu Gln Ser Ser Val Thr Leu Val Val Pro Trp Ile Ser Asn

485 490 495

Thr His Tyr Arg Ala Val Glu Thr Gly Gly Val Phe Asp Tyr Tyr Thr

500 505 510

Thr Gly Val Val Thr Ile Trp Tyr Gln Thr Asn Phe Val Val Pro Pro

515 520 525

Asp Thr Pro Thr Ser Ala Tyr Ile Leu Ala Phe Gly Ala Gly Gln Lys

530 535 540

Asn Phe Thr Leu Lys Leu Cys Lys Asp Thr Asp Glu Ile Thr Gln Gln

545 550 555 560

Ala Thr Leu Gln Gly Asp Gly Ile Glu Asp Ala Ile Thr Asn Thr Val

565 570 575

Asn Ala Thr Ile Asp Arg Val Leu Ser Arg Pro Val Ser His Ser Pro

580 585 590

Thr Ala Ala Asn Thr Gln Val Ser Gln His Ser Ile Glu Thr Gly Arg

595 600 605

Val Pro Ala Leu Gln Ala Ala Glu Thr Gly Ala Thr Ser Asn Ala Thr

610 615 620

Asp Glu Asn Leu Ile Glu Thr Arg Cys Val Val Asn Lys Asn Ser Val

625 630 635 640

Glu Glu Ala Ser Ile Asn His Phe Phe Ser Arg Ala Ala Leu Val Gly

645 650 655

Lys Val Glu Leu Asn Asp Thr Gly Thr Ser Ala Thr Gly Phe Thr Asn

660 665 670

Trp Asp Ile Asp Ile Met Gly Tyr Ala Gln Leu Arg Arg Lys Leu Glu

675 680 685

Met Phe Thr Tyr Met Arg Phe Asn Ala Glu Phe Thr Phe Val Ala Thr

690 695 700

Thr Arg Ala Gly Arg Val Pro Ser Arg Val Leu Gln Tyr Met Tyr Val

705 710 715 720

Pro Pro Gly Ala Pro Lys Pro Asp Gly Arg Glu Ala Phe Gln Trp Gln

725 730 735

Ser Ser Thr Asn Pro Ser Val Phe Ser Lys Met Thr Asp Pro Pro Pro

740 745 750

Gln Val Ser Val Pro Phe Met Ser Pro Ala Ser Ala Tyr Gln Trp Phe

755 760 765

Tyr Asp Gly Tyr Pro Thr Phe Gly Glu His Ser Gly Glu Asp Ser Leu

770 775 780

Arg Tyr Gly Gln Cys Pro Asn Asn Ala Leu Gly Thr Phe Ser Val Arg

785 790 795 800

Phe Val Ser Glu Glu Ile Thr Asn Glu Arg Ile Thr Ile Arg Ile Tyr

805 810 815

Met Arg Leu Lys His Val Arg Ala Trp Val Pro Arg Pro Leu Arg Ser

820 825 830

Glu Pro Tyr Val Leu Lys Asn Phe Pro Asn Tyr Thr Ala Val Thr His

835 840 845

Val Thr Ala Asn Arg Pro Ala Ile Thr Asn Thr

850 855

<210> 6

<211> 645

<212> PRT

<213> Artificial sequence

<400> 6

Gly Pro Ser Leu Asp Phe Ala Leu Ser Leu Leu Arg Arg Asn Val Arg

1 5 10 15

Gln Val Gln Thr Asp Gln Gly His Phe Thr Met Leu Gly Val Arg Asp

20 25 30

Arg Leu Ala Val Leu Pro Arg His Ser His Pro Gly Lys Thr Ile Trp

35 40 45

Ile Glu His Lys Leu Val Asn Val Leu Asp Ala Val Glu Leu Val Asp

50 55 60

Glu Gln Gly Val Asn Leu Glu Leu Thr Leu Val Thr Leu Asp Thr Asn

65 70 75 80

Glu Lys Phe Arg Asp Ile Thr Lys Phe Ile Pro Glu Ser Ile Ser Ala

85 90 95

Ala Ser Asp Ala Thr Leu Val Ile Asn Thr Glu His Met Pro Ser Met

100 105 110

Phe Val Pro Val Gly Asp Val Val Gln Tyr Gly Phe Leu Asn Leu Ser

115 120 125

Gly Lys Pro Thr His Arg Thr Met Met Tyr Asn Phe Pro Thr Lys Ala

130 135 140

Gly Gln Cys Gly Gly Val Val Thr Ser Val Gly Lys Val Ile Gly Ile

145 150 155 160

His Ile Gly Gly Asn Gly Arg Gln Gly Phe Cys Ala Gly Leu Lys Arg

165 170 175

Ser Tyr Phe Ala Ser Glu Gln Gly Glu Ile Gln Trp Val Lys Pro Asn

180 185 190

Lys Glu Thr Gly Arg Leu Asn Ile Asn Gly Pro Thr Arg Thr Lys Leu

195 200 205

Glu Pro Ser Val Phe His Asp Val Phe Lys Gly Asn Lys Glu Pro Ala

210 215 220

Val Leu His Ser Lys Asp Pro Arg Leu Glu Val Asp Phe Glu Gln Ala

225 230 235 240

Leu Phe Ser Lys Tyr Val Gly Asn Thr Leu His Glu Pro Asp Glu Tyr

245 250 255

Val Arg Glu Ala Ala Leu His Tyr Ala Asn Gln Leu Lys Gln Leu Asp

260 265 270

Ile Asp Thr Thr Gln Met Ser Met Glu Glu Ala Cys Tyr Gly Thr Asp

275 280 285

Asn Leu Glu Ala Ile Asp Leu His Thr Ser Ala Gly Tyr Pro Tyr Ser

290 295 300

Ala Leu Gly Ile Lys Lys Arg Asp Ile Leu Asp Pro Thr Thr Arg Asp

305 310 315 320

Val Ser Lys Met Lys Phe Tyr Met Asp Lys Tyr Gly Leu Asp Leu Pro

325 330 335

Tyr Ser Thr Tyr Val Lys Asp Glu Leu Arg Ser Ile Asp Lys Ile Lys

340 345 350

Lys Gly Lys Ser Arg Leu Ile Glu Ala Ser Ser Leu Asn Asp Ser Val

355 360 365

Tyr Leu Arg Met Ala Phe Gly His Leu Tyr Glu Thr Phe His Ala Asn

370 375 380

Pro Gly Thr Val Thr Gly Ser Ala Val Gly Cys Asn Pro Asp Thr Phe

385 390 395 400

Trp Ser Lys Leu Pro Ile Leu Leu Pro Gly Ser Leu Phe Ala Phe Asp

405 410 415

Tyr Ser Gly Tyr Asp Ala Ser Leu Ser Pro Val Trp Phe Arg Ala Leu

420 425 430

Glu Leu Val Leu Arg Glu Ile Gly Tyr Ser Glu Glu Ala Val Ser Leu

435 440 445

Ile Glu Gly Ile Asn His Thr His His Val Tyr Arg Asn Lys Thr Tyr

450 455 460

Cys Val Leu Gly Gly Met Pro Ser Gly Cys Ser Gly Thr Ser Ile Phe

465 470 475 480

Asn Ser Met Ile Asn Asn Ile Ile Ile Arg Thr Leu Leu Ile Lys Thr

485 490 495

Phe Lys Gly Ile Asp Leu Asp Glu Leu Asn Met Val Ala Tyr Gly Asp

500 505 510

Asp Val Leu Ala Ser Tyr Pro Phe Pro Ile Asp Cys Leu Glu Leu Ala

515 520 525

Arg Thr Gly Lys Glu Tyr Gly Leu Thr Met Thr Pro Ala Asp Lys Ser

530 535 540

Pro Cys Phe Asn Glu Val Asn Trp Glu Asn Ala Thr Phe Leu Lys Arg

545 550 555 560

Gly Phe Leu Pro Asp Asp Gln Phe Pro Phe Leu Ile His Pro Thr Met

565 570 575

Pro Met Lys Glu Ile His Glu Ser Ile Arg Trp Thr Lys Asp Ala Arg

580 585 590

Asn Thr Gln Asp His Val Arg Ser Leu Cys Leu Leu Ala Trp His Asn

595 600 605

Gly Lys Gln Glu Tyr Glu Glu Phe Val Ser Thr Ile Arg Ser Val Pro

610 615 620

Ile Gly Lys Ala Leu Ala Ile Pro Asn Tyr Glu Asn Leu Arg Arg Asn

625 630 635 640

Trp Leu Glu Leu Phe

645

Claims (4)

1. Virus-like particles VLPs of Coxsackie virus CV-A2,

the strain CV-A2 of the coxsackievirus is CV-A2-1580V4/CHN XY/2017;

the VLPs are expressed by insect vectors, and the preparation method comprises the following steps:

(1) construction of recombinant baculovirus Bacmid plasmid:

cloning optimized CV-A2P1 and CV-A23 CD genes respectively behind pPh and P10 promoters of pFastBacdual plasmid, transforming escherichia coli DH10Bac competent cells, and extracting positive plasmid, namely recombinant baculovirus Bacmid plasmid;

the nucleotide sequence of the optimized CV-A2P1 gene is shown as SEQ ID NO.3

The nucleotide sequence of the optimized CV-A23 CD gene is shown in SEQ ID NO. 4;

(2) expression of recombinant baculovirus:

transfecting an insect cell by using a recombinant baculovirus Bacmid plasmid to obtain a recombinant baculovirus;

(3) preparation of Coxsackie Virus CV-A2 VLPs:

inoculating insect cells into the obtained recombinant baculovirus according to MOI =0.5, wherein the insect cells are sf-9, and after 5-6 days, harvesting supernatant, and extracting and separating coxsackie virus VLPs; the method for extracting and separating coxsackie virus VLPs comprises the following steps:

1) collecting Sf-9 cell sap infected by CV-A2 VLPs, repeatedly freezing and thawing, centrifuging at 3000rpm and 4 ℃ for 10min to remove cell debris;

2) the supernatant is ultrafiltered and concentrated by 30 times by a membrane with the aperture of 30 kD;

3) the concentrated product was passed through 20% sucrose, 15000g bedding, centrifuged for 4h, followed by centrifugation at 1.31g/ml CsCl density 25000g for 20h, and the target bands were extracted as CV-A2 VLPs.

2. The VLPs of coxsackie virus CV-a2 of claim 1, wherein the method for preparing the VLPs of coxsackie virus CV-a2 further comprises:

(4) VLPs were identified using SDS-PAGE, Western-Blot, indirect immunofluorescence and transmission electron microscopy.

3. The application of the protein of the coxsackievirus CV-A2P1 shown in the amino acid sequence SEQ ID NO.5 and the protein of the coxsackievirus CV-A23 CD shown in the amino acid sequence SEQ ID NO.6 in the preparation of virus-like particles VLPs of the coxsackievirus.

4. Use of the virus-like particle VLPs of coxsackie virus CV-a2 of claim 1 in the preparation of a vaccine or medicament for the treatment or prevention of hand-foot-and-mouth disease.

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