CN112480215A - Virus-like particle of Coxsackie virus CV-A2 - Google Patents

Virus-like particle of Coxsackie virus CV-A2 Download PDF

Info

Publication number
CN112480215A
CN112480215A CN202011403822.6A CN202011403822A CN112480215A CN 112480215 A CN112480215 A CN 112480215A CN 202011403822 A CN202011403822 A CN 202011403822A CN 112480215 A CN112480215 A CN 112480215A
Authority
CN
China
Prior art keywords
thr
leu
ala
vlps
val
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202011403822.6A
Other languages
Chinese (zh)
Other versions
CN112480215B (en
Inventor
申硕
禹玉婷
胡岗
钱莎莎
卢佳
王泽鋆
孟胜利
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
WUHAN INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
Original Assignee
WUHAN INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by WUHAN INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD filed Critical WUHAN INSTITUTE OF BIOLOGICAL PRODUCTS CO LTD
Priority to CN202011403822.6A priority Critical patent/CN112480215B/en
Publication of CN112480215A publication Critical patent/CN112480215A/en
Application granted granted Critical
Publication of CN112480215B publication Critical patent/CN112480215B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86Viral vectors
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525Virus
    • A61K2039/5258Virus-like particles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/54Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011Details
    • C12N2710/14011Baculoviridae
    • C12N2710/14041Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011Details
    • C12N2770/32011Picornaviridae
    • C12N2770/32023Virus like particles [VLP]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011Details
    • C12N2770/32011Picornaviridae
    • C12N2770/32034Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Communicable Diseases (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

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]
There are at least 20 types of enteroviruses that cause HFMD, including Coxsackie virus group a (CVA) types 2, 4, 5, 6, 10, 12 and 16, Coxsackie virus group B (CVB) type 5, echovirus (Echo virus, Echo) and Enterovirus type 71 (Enterovirus 71, EV-a 71). 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, and epidemic chest painRespiratory tract infection, hand-foot-mouth disease, infantile diarrhea and the like to cause 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 was also found that CV-A2 can cause severe cases. 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
Figure BDA0002817981460000041
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
Figure BDA0002817981460000042
Expression System
Figure BDA0002817981460000043
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
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 2577
<212> DNA
<213> Coxsackievirus
<400> 1
atgggggcgc agttttcaac tcagcgctca ggttcccacg agacgagcaa tgtagctcgt 60
gatggatcaa ccattaactt cacgaacatc aactactata aggactctta cgcggcctct 120
gcagctaaac atgacttcac tcaagatcct ggcaagttca cacaacctgt gctggatgct 180
ctacgcgaag cggtgccacc attacagtca ccgagtgctg aggcatgtgg ttacagtgac 240
agagtggcgc agttgacagt tggcaactcg actatcacca ctcaagaagc tgccaatata 300
atagtgtctt atgctgagtg gccagaatat tgtccagaca cggacgccac tgcagtcgac 360
aaaccaaccc gcccagatgt gtcagtgaac agattctaca ctatgccagc aactttgtgg 420
gagacagaat cgaaaggatg gtattggaag tttcctgatt tactcaatga gataggcgtt 480
ttcggacaga acgcccaatt tcactattta tatagatcag ggttttgcat tcacgtgcag 540
tgtaacgcca gtaagttcca tcaaggggct ctgcttgtag cggtaattcc tgagtttgtt 600
gtggccgagc aggacgccac ccagaagccc aatacagcaa agcacccttc gttggaagcc 660
acgcaacccg gcaagaaagg taaggcgttc agacaccctt acgtccttga ttgtggcgtg 720
ccattaagcc aagcccttgt cttcccccat cagtggatca acttacgcac taacaactgt 780
gctaccatag tagtcccata tattaatgcc ttaccttttg attctgcaat aaaccattcc 840
aatttctcac tggcagtgat accagtttgc ccattgaaat acaacacagg tgccacccct 900
tcaataccca tcacgattac agtcgcaccc ttgtgctcgg agtttgccgg gctacgccaa 960
gcagtcaagc aaggagtccc ggtagagatg aaacctggaa caaaccaatt tttaacaact 1020
gatgatggga cctcagcacc aatactacct ggctttcacc ccacaccaac catacacatc 1080
cccggtgaag tccatagttt gttagagttg tgtcagattg agactatgct tgaggtgaac 1140
aatacatcta aggcaatgga gctcgaccgg ctgaggatac ctgtgtctgc acagagcgcc 1200
gtagacaccc tgtgcgcggc ttttagagtg gaccccggta gggacggtcc ctggcagtcc 1260
actatggtag gacaactatg caggtactac acccagtggt caggctccct aaagattaca 1320
ttcatgttca ctggctcatt tatggccact gggaagatgc tggtcgccta cacaccgccg 1380
ggaggcgcgc aacctgccac acgggagcta gctatgttgg gcacacatgt aatctgggat 1440
tttggcctgc agtcgtcggt tacactagtg gtcccatgga taagcaacac tcactatagg 1500
gctgtggaga ctgggggtgt gtttgattac tacaccactg gcgtggtgac catatggtac 1560
cagactaact ttgtagtgcc tcctgacact cctaccagcg cttacatctt agcatttgga 1620
gctggtcaaa agaacttcac tctcaagctg tgcaaagaca cggatgaaat tacccagcaa 1680
gcaacactgc agggtgatgg tatagaagat gccataacca acacagtgaa tgctacgata 1740
gacagggtgt tgagtcggcc agtgtctcat tcaccaacag ctgctaacac ccaggtgagt 1800
cagcactcca ttgagactgg gcgtgtacct gcactacaag ctgctgaaac aggtgctact 1860
tccaatgcta ctgatgaaaa cctgatagaa acaagatgtg tagttaacaa aaatagtgtg 1920
gaagaggcta gtataaacca cttcttctcc cgagcagctt tggttggtaa ggtggagtta 1980
aatgacacag gtacgagtgc cacagggttt accaactggg atatagatat aatggggtat 2040
gctcaactgc gtaggaaact agagatgttc acctacatgc gcttcaatgc tgagtttaca 2100
tttgtggcta ccaccagagc tggacgggta ccatctagag tgctccagta catgtatgtc 2160
ccacctggag cacccaaacc ggacggcagg gaagcttttc aatggcaatc ctcgactaac 2220
ccatcagtgt tcagcaaaat gacggaccct ccaccacagg tctcagtccc attcatgtcg 2280
cctgctagtg cgtaccaatg gttctatgac ggatacccca cctttggaga gcacagtggc 2340
gaagatagcc tgcgctacgg tcaatgcccc aacaacgcat tgggcacctt ttcagtgaga 2400
ttcgttagtg aagagataac taacgagcga atcaccatca ggatatatat gaggcttaaa 2460
cacgtgcggg cttgggtccc gcgcccactc aggtctgagc cttatgtatt gaagaatttc 2520
ccaaattata ctgcagtgac tcacgtcacc gccaaccgtc ccgccattac aaacaca 2577
<210> 2
<211> 1935
<212> DNA
<213> Coxsackievirus
<400> 2
ggcccgagtc tcgactttgc tttatcacta ctgagaagaa atgtcaggca agtccaaaca 60
gaccaagggc actttaccat gctgggagtc agagaccgct tagctgttct tccacgacat 120
tcacaccctg ggaagacaat ctggatagag cacaaactcg tgaacgtcct agacgctgtc 180
gaattagtgg atgaacaagg ggtcaatttg gagttaacct tagtcacact agacaccaat 240
gaaaagttca gggatattac caaattcatt ccagaaagta tcagcgccgc tagtgacgct 300
actttagtga tcaacacaga gcatatgcct tcaatgtttg tgccagtagg tgacgtcgtg 360
cagtatggct ttctaaatct cagtggaaag ccaactcacc gcactatgat gtacaatttt 420
cccactaagg caggtcagtg tggaggggta gtcacatcag ttgggaaggt cattggcatt 480
cacatagggg gtaatggcag gcaaggcttt tgtgcgggac tcaagagaag ctattttgct 540
agtgaacaag gagagatcca gtgggtcaaa cctaataaag aaacaggaag gctaaacatt 600
aatgggccaa ctcgcactaa actcgagccc agtgtgttcc atgacgtctt taagggcaac 660
aaagagccag cagtcttaca cagtaaagat ccccgcctcg aggtggactt tgagcaggca 720
ttgttttcta agtacgtggg aaacacgcta catgagcctg atgagtacgt cagagaggca 780
gctcttcact atgcgaatca gttgaagcag ctagacatag acaccaccca gatgagcatg 840
gaggaagcat gctacggcac ggacaacctt gaggccattg acctccacac tagcgcaggc 900
tacccctaca gtgctctggg aataaaaaag agagatatcc tagaccctac cactagagac 960
gtgagcaaga tgaagtttta catggataag tatggccttg acctccctta ctccacctat 1020
gtcaaagatg agcttcgctc gatagacaag atcaagaagg ggaaatctcg actgattgaa 1080
gccagtagtt taaatgactc agtctacctc agaatggcct ttgggcacct ctatgaaacc 1140
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 (8)

1. Virus-like particle VLPs of coxsackievirus CV-A2, wherein the VLPs are expressed using an insect vector, comprising an optimized CV-A2P1 protein and an optimized CV-a 23 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
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;
the strain CV-A2 of the coxsackievirus is CV-A2-1580V4/CHN XY/2017.
2. The method of producing VLPs of the virus-like particles of coxsackievirus CV-a2 of claim 1, comprising the steps of:
(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;
(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:
the obtained recombinant baculovirus was inoculated into insect cells of sf-9 at an MOI of 0.5, the supernatant was harvested 5 to 6 days later, and coxsackie virus VLPs were isolated and extracted.
3. The method of claim 2, further comprising:
(4) VLPs were identified using SDS-PAGE, Western-Blot, indirect immunofluorescence and transmission electron microscopy.
4. The method of claim 2 or 3, wherein the step of isolating the coxsackievirus VLPs in step (3) comprises:
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.
5. The amino acid sequence of the protein is shown as SEQ ID NO.5 of coxsackievirus CV-A2P 1.
6. The amino acid sequence of the protein of the coxsackievirus CV-A23 CD shown in SEQ ID NO. 6.
7. A nucleotide fragment encoding the coxsackievirus protein of claim 5 or 6.
8. Use of the virus-like particle VLPs of coxsackievirus CV-A2 of claim 1, the coxsackievirus CV-A2P1 protein of claim 5, the coxsackievirus CV-a 23 CD protein of claim 6 and the nucleotide fragment of claim 7 in the preparation of a vaccine or medicament, preferably a vaccine or medicament for the treatment or prevention of hand-foot-and-mouth disease.
CN202011403822.6A 2020-12-04 2020-12-04 Virus-like particle of Coxsackie virus CV-A2 Active CN112480215B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202011403822.6A CN112480215B (en) 2020-12-04 2020-12-04 Virus-like particle of Coxsackie virus CV-A2

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202011403822.6A CN112480215B (en) 2020-12-04 2020-12-04 Virus-like particle of Coxsackie virus CV-A2

Publications (2)

Publication Number Publication Date
CN112480215A true CN112480215A (en) 2021-03-12
CN112480215B CN112480215B (en) 2021-09-17

Family

ID=74939259

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202011403822.6A Active CN112480215B (en) 2020-12-04 2020-12-04 Virus-like particle of Coxsackie virus CV-A2

Country Status (1)

Country Link
CN (1) CN112480215B (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114540315A (en) * 2021-10-18 2022-05-27 武汉生物制品研究所有限责任公司 Thermostable coxsackievirus A group 2 type virus strain
CN115010813A (en) * 2022-05-19 2022-09-06 桂林医学院 Enterovirus 71 type virus-like particle, preparation method and application thereof

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102465144A (en) * 2010-11-19 2012-05-23 中国科学院上海巴斯德研究所 Coxsackie virus A16 type virus-like particle vaccine
CN102533797A (en) * 2012-01-12 2012-07-04 北京工业大学 Preparation method for recombinant coxsackie virus A16 like particle and applications thereof
CN102899336A (en) * 2012-09-19 2013-01-30 北京工业大学 Preparation method and application of Coxsackie virus B3 virus-like particles
CN103436553A (en) * 2013-08-22 2013-12-11 上海博唯生物科技有限公司 Method for preparing recombinant coxsackievirus A16 type virus-like particles
CN104745606A (en) * 2013-12-26 2015-07-01 上海泽润生物科技有限公司 Coxsackie A16 type virus-like particles
CN107384944A (en) * 2016-05-17 2017-11-24 中国科学院上海巴斯德研究所 The virus-like particle of Coxsackie virus A 6 of Yeast expression and its application
CN107384943A (en) * 2016-05-17 2017-11-24 中国科学院上海巴斯德研究所 Preparation and its application of the virus-like particle of Coxsackie virus A 6 in insect cell
CN107746832A (en) * 2017-10-12 2018-03-02 泰山医学院 The Coxsackie virus A 10 of one plant height titre tames strain and its application
CN108624601A (en) * 2017-03-17 2018-10-09 中国科学院上海巴斯德研究所 10 virus-like particle of Coxsackie virus A of Yeast expression and its application
CN108624609A (en) * 2017-03-24 2018-10-09 斯澳生物科技(苏州)有限公司 It is used to prepare the nucleic acid construct and method of coxsackie virus A 16-type virus-like particle
CN109234241A (en) * 2018-08-22 2019-01-18 武汉生物制品研究所有限责任公司 A kind of Coxsackie virus CVA16 type velogen strain CVA16-B6-714 and its application
CN109680026A (en) * 2019-02-28 2019-04-26 深圳鑫泰康生物科技有限公司 The purifying of recombinant C A16 virus-like particle, the application in vaccine and vaccine
CN110184242A (en) * 2019-06-11 2019-08-30 武汉生物制品研究所有限责任公司 The mouse strong virus force of 6 type of Coxsackie virus A group (CVA6) attacks strain and its application
CN111411086A (en) * 2020-02-17 2020-07-14 武汉生物制品研究所有限责任公司 Coxsackie group A5 virus strain capable of effectively infecting old young mice
CN111778168A (en) * 2020-06-19 2020-10-16 北京民海生物科技有限公司 Hansenula polymorpha engineering bacteria for efficiently expressing CA10 virus-like particles and application thereof
CN112011572A (en) * 2020-07-17 2020-12-01 北京科兴生物制品有限公司 Virus-like particle of Coxsackie virus A7 and preparation method and application thereof

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102465144A (en) * 2010-11-19 2012-05-23 中国科学院上海巴斯德研究所 Coxsackie virus A16 type virus-like particle vaccine
CN102533797A (en) * 2012-01-12 2012-07-04 北京工业大学 Preparation method for recombinant coxsackie virus A16 like particle and applications thereof
CN102899336A (en) * 2012-09-19 2013-01-30 北京工业大学 Preparation method and application of Coxsackie virus B3 virus-like particles
CN103436553A (en) * 2013-08-22 2013-12-11 上海博唯生物科技有限公司 Method for preparing recombinant coxsackievirus A16 type virus-like particles
CN104745606A (en) * 2013-12-26 2015-07-01 上海泽润生物科技有限公司 Coxsackie A16 type virus-like particles
CN107384944A (en) * 2016-05-17 2017-11-24 中国科学院上海巴斯德研究所 The virus-like particle of Coxsackie virus A 6 of Yeast expression and its application
CN107384943A (en) * 2016-05-17 2017-11-24 中国科学院上海巴斯德研究所 Preparation and its application of the virus-like particle of Coxsackie virus A 6 in insect cell
CN108624601A (en) * 2017-03-17 2018-10-09 中国科学院上海巴斯德研究所 10 virus-like particle of Coxsackie virus A of Yeast expression and its application
CN108624609A (en) * 2017-03-24 2018-10-09 斯澳生物科技(苏州)有限公司 It is used to prepare the nucleic acid construct and method of coxsackie virus A 16-type virus-like particle
CN107746832A (en) * 2017-10-12 2018-03-02 泰山医学院 The Coxsackie virus A 10 of one plant height titre tames strain and its application
CN109234241A (en) * 2018-08-22 2019-01-18 武汉生物制品研究所有限责任公司 A kind of Coxsackie virus CVA16 type velogen strain CVA16-B6-714 and its application
CN109680026A (en) * 2019-02-28 2019-04-26 深圳鑫泰康生物科技有限公司 The purifying of recombinant C A16 virus-like particle, the application in vaccine and vaccine
CN110184242A (en) * 2019-06-11 2019-08-30 武汉生物制品研究所有限责任公司 The mouse strong virus force of 6 type of Coxsackie virus A group (CVA6) attacks strain and its application
CN111411086A (en) * 2020-02-17 2020-07-14 武汉生物制品研究所有限责任公司 Coxsackie group A5 virus strain capable of effectively infecting old young mice
CN111778168A (en) * 2020-06-19 2020-10-16 北京民海生物科技有限公司 Hansenula polymorpha engineering bacteria for efficiently expressing CA10 virus-like particles and application thereof
CN112011572A (en) * 2020-07-17 2020-12-01 北京科兴生物制品有限公司 Virus-like particle of Coxsackie virus A7 and preparation method and application thereof

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
GUO WP, ET AL.: "ALB72925.1", 《GENBANK》 *
OBERSTE MS, ET AL.: "AY421760.1", 《GENBANK》 *
YANG Q, ET AL.: "ANQ47259.1", 《GENBANK》 *
刘子薇,等: "柯萨奇病毒A组2型的分离鉴定与遗传进化分析", 《中华实验和临床病毒学杂志》 *
姚学君,等: "柯萨奇病毒A组2型分离毒株序列及遗传进化分析", 《江苏预防医学》 *
尉秀霞,等: "一起聚集性发热疫情中柯萨奇病毒A组2型的鉴定及基因特征分析", 《病毒学报》 *
王永芳,等: "《动物生物制品技术》", 31 August 2011, 中国农业大学出版社 *
祖伟,等: "北京市朝阳区2012-2015年柯萨奇A2型肠道病毒的基因特征分析", 《中国病毒病杂志》 *
禹玉婷,等: "柯萨奇病毒A组2型及5型的研究进展", 《中国生物制品学杂志》 *
秦露伟: "柯萨奇病毒A2型的鉴定与手足口病乳鼠模型的构建", 《中国优秀硕士学位论文全文数据库(电子期刊)医药卫生科技辑》 *
罗志宇,等: "2017年湖北省襄阳市手足口病患者柯萨奇病毒A2型分离株的突变和重组分析", 《微生物学免疫学进展》 *
胡岗,等: "柯萨奇病毒A组2型VP1和VP3蛋白原核表达及多克隆抗体的制备", 《微生物学免疫学进展》 *
陈静,等: "柯萨奇病毒A6病毒样颗粒的制备及免疫原性初步研究", 《病毒学报》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114540315A (en) * 2021-10-18 2022-05-27 武汉生物制品研究所有限责任公司 Thermostable coxsackievirus A group 2 type virus strain
CN114540315B (en) * 2021-10-18 2023-05-16 武汉生物制品研究所有限责任公司 Thermostable coxsackievirus A group 2 virus strain
CN115010813A (en) * 2022-05-19 2022-09-06 桂林医学院 Enterovirus 71 type virus-like particle, preparation method and application thereof
CN115010813B (en) * 2022-05-19 2024-04-05 桂林医学院 Enterovirus 71 virus-like particle, and preparation method and application thereof

Also Published As

Publication number Publication date
CN112480215B (en) 2021-09-17

Similar Documents

Publication Publication Date Title
AU661479B2 (en) Modified plant viruses as vectors
CN112480215B (en) Virus-like particle of Coxsackie virus CV-A2
CN101831410B (en) Preparation method of recombinant enterovirus 71 type virus-like particles
Sukhumsirichart et al. Complete nucleotide sequence and genomic organization of hepatopancreatic parvovirus (HPV) of Penaeus monodon
CN110078802B (en) Cat parvovirus VP2 protein and prepared virus-like particle
CN113461788B (en) Cat coronavirus recombinant antigen, genetic engineering subunit vaccine thereof and application
CN104404074A (en) Foot-and-mouth disease virus capsid protein tandem coexpressions and virus-like particle preparation method
CN101695569A (en) Univalent and bivalent gene engineered subunit vaccine for hand-foot-and-mouth disease and preparation method thereof
WO2018170603A1 (en) Norovirus fusion proteins and vlps comprising norovirus fusion proteins
WO2016086576A1 (en) Vector expressing poliomyelitis virus-like granule protein and method for preparing poliomyelitis virus-like granules
US20230293663A1 (en) Modified norovirus vp1 proteins and vlps comprising modified norovirus vp1 proteins
Li et al. High yield expression of duck hepatitis A virus VP1 protein in Escherichia coli, and production and characterization of polyclonal antibody
CN103555680A (en) PRRSV (porcine reproductive and respiratory syndrome virus) virus-like particles with immunogenicity as well as preparation and application thereof
CN113896773B (en) Recombinant FCV antigen and feline calicivirus genetic engineering subunit vaccine
CN114507277B (en) Beet armyworm gene with antiviral effect and application thereof
CN113061167B (en) Rabbit hemorrhagic disease virus recombinant antigen and application thereof
CN110295197B (en) Recombinant expression vector, prepared III type duck hepatitis A virus-like particle, preparation method and application
CN114058524A (en) Bursal disease subviral particle vaccine and preparation method thereof
WO2011136506A2 (en) Method for producing recombinant complex antigens using nanoparticles of human rotavirus
CN111575315A (en) Rabbit viral hemorrhagic disease virus type II VLP vaccine
CN111235114A (en) EV71 replication-defective virus and preparation method and application thereof
WO2024131960A1 (en) Vector for expressing poliovirus-like particles, preparation method therefor, and use thereof
CN100404549C (en) Method for preparing capsid protein (VP)of enteric cytopathogenic human orphan virus 30
JP7429684B6 (en) Modified Norovirus VP1 protein and VLP comprising modified Norovirus VP1 protein
CN111778220A (en) Preparation and application of porcine parvovirus-like particle for displaying B cell epitope and T cell epitope of south Africa type 2 foot-and-mouth disease virus

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant