CN107384944A - The virus-like particle of Coxsackie virus A 6 of Yeast expression and its application - Google Patents
The virus-like particle of Coxsackie virus A 6 of Yeast expression and its application Download PDFInfo
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Abstract
The invention provides the virus-like particle of Coxsackie virus A 6 of Yeast expression and its application, specifically, the present invention provides a kind of method for preparing COxsackie A6 virus-like particles, the COxsackie A6 virus P1 albumen and 3CD albumen coded sequences used in this method through codon optimization, expressed in yeast cells, can assemble to form VLP automatically, and expression quantity is high, it is easy to purify, purifying, which obtains VLP, has stronger immunogenicity.
Description
Technical field
The invention belongs to biomedicine field, and specifically, the present invention relates to the Coxsackie virus of Yeast expression
A6 virus-like particles and its application.
Background technology
Hand-foot-and-mouth disease is a kind of contact sexually transmitted disease, is widely current in the sub- Pacific region.Main infection five years old
Following children cause heating, hand, foot, mouth and buttocks bleb, and symptom, a small number of infants such as cough can be short-term
Inside there are serious neurological symptoms and cardiorespiratory system complication, in addition it is dead.But there is presently no for brothers' mouth
The available vaccine of disease.
From 2008, enterovirns type 71 (EV71) and the type of Coxsackie virus 16 (CA16) were extensive because of its
Spread and epidemic turns into two main pathogens of hand-foot-and-mouth disease, and the research and development of vaccine also more is directed to two kinds of viruses.So
And recent years, on hand-foot-and-mouth disease Global prevalence caused by COxsackie CA6, especially Asia and Europe ground
The popular report in area is on the increase, and has attracted extensive concern.COxsackie CA6 virus infection can cause heating,
The typical hand-foot-and-mouth disease symptom such as bleb, while with atypia brothers such as ulcer, incrustation and piptonychias at bleb outburst
Stomatosis symptom, and can not only infect young children and also adult is caused serious injury.Recently studies have reported that
The capsid protein composition of COxsackie CA6 virions is disclosed, and proves non-structural protein white region (2A-3D) change
Change is the major reason for causing virus infection to cause atypia hand-foot-and-mouth disease symptom.
By taking the Clinical Report of some provinces and cities of China as an example, 2013, the hand-foot-and-mouth disease in Guangdong Province 60.3% was quick-fried
Hair is due to that COxsackie CA6 virus infection causes, and 66.9% also by this in the hand-foot-and-mouth disease case of Jilin Changchun outburst
Virus causes.These Clinical Reports show that COxsackie CA6's is popular more and more extensive, are increasingly becoming hand
One of main pathogen of sufficient stomatosis outburst, still can be by moreover, the crowds of EV71 or CA16 vaccines has been immunized
COxsackie CA6 virus infection, thus it is very necessary for COxsackie CA6 vaccine development, also can be later double
Place mat is made in the research and development of valency or polyvaccine.
Therefore, in order to effectively, targetedly prevent COxsackie CA6 infection, there is an urgent need to open for this area
Vaccine and its application of the hairpin to COxsackie CA6.
The content of the invention
It is an object of the invention to provide a kind of virus-like particle of Coxsackie virus A 6 using Yeast expression,
Its preparation method and its application.
The first aspect of the present invention, there is provided a kind of polynucleotides through codon optimization of separation, the multinuclear
Thuja acid encodes COxsackie A6 virus P1 albumen；And the polynucleotides are selected from the group：
(a) polynucleotides of the sequence as shown in SEQ ID NO.3；
(b) multinuclear of nucleotide sequence and homology >=95% (preferably >=98%) of sequence shown in SEQ ID NO.3
Thuja acid；
(c) polynucleotides complementary with any described polynucleotides of (a)-(c).
The second aspect of the present invention, there is provided a kind of expression vector, the expression vector contain first party of the present invention
Polynucleotides described in face.
In another preference, the expression vector also includes the more of coding COxsackie A6 virus 3CD albumen
Nucleotide sequence.
In another preference, the expression vector includes the first expression cassette and the second expression cassette, first table
Include the polynucleotides or its complementary series shown in SEQ ID NO.3 up to box；Second expression cassette includes SEQ ID
Polynucleotides or its complementary series shown in NO.8.
In another preference, the expression vector is recombinant baculovirus.
In another preference, first expression cassette also includes promoter, and the promoter is located at SEQ ID NO.3
The upstream of shown polynucleotides, preferably described promoter are PAOX1 promoters.
In another preference, second expression cassette also includes promoter, and the promoter is located at SEQ ID NO.8
The upstream of shown polynucleotides, preferably described promoter are PAOX1 promoters.
The third aspect of the present invention, there is provided a kind of host cell, described host cell contain the present invention second
Expression vector described in aspect, or it is integrated with genome the polynucleotides described in first aspect present invention.
In another preference, the host cell is yeast cells.
The fourth aspect of the present invention, there is provided a kind of virus-like particle of Coxsackie virus A 6 (VLP), the virus
Sample particle is as the host cell expression described in third aspect present invention.
In another preference, the virus-like particle includes SEQ ID NO.4, SEQ ID NO.5 and SEQ ID
Polypeptide shown in NO.6.
In another preference, the virus-like particle is by SEQ ID NO.4, SEQ ID NO.5 and SEQ ID
Polypeptide composition shown in NO.6.
The fifth aspect of the present invention, there is provided a kind of method for preparing Coxsackie virus A 6VLP, including step：
Under conditions suitable for the expression, the cell described in third aspect present invention is cultivated, so as to give expression to the present invention
Virus-like particle (VLP) described in fourth aspect；With the separation virus-like particle (VLP).
The sixth aspect of the present invention, there is provided a kind of pharmaceutical composition, described composition contain the present invention the 4th
Polynucleotides described in virus-like particle (VLP), first aspect present invention or second party of the present invention described in aspect
The host cell described in expression vector or third aspect present invention described in face, and pharmaceutically acceptable carrier
And/or auxiliary material.
In another preference, described pharmaceutical composition includes vaccine combination.
In another preference, described vaccine combination also contains adjuvant.
In another preference, described adjuvant includes aluminum oxide, saponin(e, quil A, muramyl dipeptide, ore deposit
Thing oil or vegetable oil, the adjuvant based on vesica, non-ionic block copolymer or deae dextran, cell factor (bag
Include IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12 and CpG).
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and below (such as implementation
Example) in specifically describe each technical characteristic between can be combined with each other, so as to form new or preferable skill
Art scheme.As space is limited, no longer tire out one by one herein and state.
Brief description of the drawings
Fig. 1 Pichia pastoris co-expresses CA6 P1 and 3CD albumen (A) plasmid YCA6-003 schematic diagrames.
TRP2-L and TRP2-R, TRP upstream and downstream region；PAOX1, AOX1 promoter；CYC1 TT,
CYC1 transcription termination regions；ADE2, amino-imidazole ribotide carboxylase is encoded, as selection markers.(B)
Height expression strain screening.Anti- CA6VP0 is as detection antibody.The yeast thalline lysate conduct of empty carrier conversion
Negative control (ctr).(C) Western blot analyze yeast clone expression status.Swimming lane M：Marker,
Swimming lane C：The yeast thalline lysate of empty carrier conversion, swimming lane 1-3：Plasmid YCA6-003 conversions bacterial strain 7,
12nd, 15 lysate.
Fig. 2 .CA6 VLP assembling.The yeast thalline lysate of plasmid YCA6-003 conversions crosses 10%-50%,
Take 12 layers successively from top to bottom.(A)SDS-PAGE.(B) Western blotting, primary antibody：It is anti-
CA6 VP0.(C) Western blotting, primary antibody：Anti- CA6 VP1.(D) Western blotting,
Primary antibody：Anti- CA6 VP3.(E) electron microscope.Bar=100nm.
Fig. 3 mouse immunes and antibody response.(A) SDS-PAGE analyzes CA6 VLP with compareing antigen.
(B) CA6VP0, VP1, VP3 equal proportion mixing wrapper sheet make ELISA detections.Three exempt from surrounding serum 1：20
Dilution.(C) CA6 VLP wrapper sheets make ELISA detections.Three exempt from surrounding serum 1：1000 dilutions.(D)
CA6 Specific antibody titres detect.CA6 VLP are as envelope antigen.Each point represents a mouse, horizontal
Line represents geometrical mean.Significant difference：* *, P<0.001.
The anti-VLP serum of Fig. 4 assigns mouse and protected completely.The μ l of 7 age in days ICR mouse peritoneal injections 50
Anti- VLP serum or control serum.After 24 hours, mouse peritoneal injection (A, B) CA6/Gdula or (C,
D)CA6/S0087b.Continuous 15 days (the A, C) survival rates for observing and recording mouse and (B, D) clinical condition
Shape.Symptom scores grade is as follows：0, health；1, it is slow in action；2, incoordination；3, paralysis；
4, it is dead.
Embodiment
The present inventor is by extensive and in-depth study, a kind of side for preparing COxsackie A6 virus-like particles of acquisition
Method, using COxsackie A6 virus P1 albumen and 3CD albumen coded sequences through codon optimization in this method,
Expressed in yeast cells, can assemble to form VLP automatically, and expression quantity is high, is easy to purify, purifying obtains VLP
With stronger immunogenicity.On this basis, the present invention is completed.
Before describing the present invention, it should be understood that the invention is not restricted to described specific method and experiment condition,
Because this kind of method and condition can change.It should also be understood that its purpose of term used herein is only that description
Specific embodiment, and it is not intended to be restricted, the scope of the present invention is by only by appended claim
Book limits.
Unless otherwise defined, otherwise whole technologies used herein are respectively provided with such as institute of the present invention with scientific terminology
The identical meanings that the those of ordinary skill in category field is generally understood that.As used herein, mentioning what is specifically enumerated
In use, term " about ", which means that the value can change from the value enumerated, is not more than 1% in numerical value.For example,
As used herein, statement " about 100 " include 99 and 101 and between whole values (for example, 99.1,99.2,
99.3rd, 99.4 etc.).
Appoint although can be used in the implementation or test of the present invention to heretofore described similar or of equal value
Where method and material, herein place enumerate preferable method and material.
A kind of safe and efficient CA6 viruses (COxsackie A6 viruses) vaccine of purpose of the present invention research and development, energy
Enough protect body from the viral invasion and attack.Using Yeast expression CA6 P1 and 3CD albumen, with specificity
More anti-detection finds that P1 albumen can be cut by 3CD and obtain capsid protein VP0, VP1 and VP3, this three kinds
The spontaneous assembling of structural proteins energy forms the virus-like particle for lacking nucleic acid.Importantly, use with the VLP epidemic diseases
Seedling have successfully been obtained anti-CA6-VLP serum after mouse is immunized, and the CA6-VLP vaccines are in itself, can not only
Protect newborn mice from homologous viral CA6/Gdula attack, can also protect mice against heterologous disease well
Malicious CA6/S0087b infection.The CA6-VLP vaccines that the present invention researchs and develops can protect body from CA6 diseases
Poison infringement, provides safeguard for the research and development of hand-foot-and-mouth disease polyvaccine.
Hand-foot-and-mouth disease (Hand, foot and mouth disease, HFMD)
Hand-foot-and-mouth disease is the contagious infection disease of children below a kind of main infection five years old, with being popular in Asia-Pacific
Area.The type of Coxsackie virus 6 (Coxsackievirus A6, CA6) is one of the sick principal causative original,
The report on the viral infected children and adult was on the increase in recent years.However, it currently there are no pin
To the available vaccine of the virus.
In our current research, CA6 P1 and 3CD are co-expressed using yeast cells, obtained just definite
The virus-like particle (Virus Like Particle, VLP) for cutting and assembling.Result of study shows, P1 eggs
Can correctly be cut by 3CD in vain obtain can spontaneous assembling formed complete VLP capsid protein VP0, VP1 and
VP3.Mouse through VLP vaccine immunities can produce very strong serum antibody response, in default of suitable
CA6 Virus culture cell lines, hinder the external implementation for neutralizing experiment, but passive protection and active in vivo
Protecting effect is fine.After seven age in days newborn mices are injected intraperitoneally with anti-CA6-VLP serum, respectively with original
Strain CA6/Gdula or clinical separation strain CA6/S0087b viruses are attacked, and are as a result shown, anti-VLP blood
Mouse can be protected from the infection of homologous virus and heterologus virus clearly.Meanwhile Neonatal Mouse is chosen on 1st
It is immune that age and 7 ages in days carry out CA6-VLP intraperitoneal injections, during 14 age in days respectively with prime strain CA6/Gdula or
Clinical separation strain CA6/S0087b viruses are attacked, and are as a result shown, CA6-VLP vaccines can also be protected
Mouse is from two kinds of viral infection.
The type of Coxsackie virus 6 (Coxsackievirus A6, CA6)
The type of Coxsackie virus A 6 (Coxsackievirus A6, CA6) is hand-foot-and-mouth disease (Hand, foot
Andmouth disease, HFMD) one of main pathogen, the incidence of disease in children and adult is just gradual
Rise, ever-increasing CA6, which infects, brings serious threat to publilc health, but there is presently no pin
To CA6 vaccine.
Traditionally, inactivated vaccine and attenuated live vaccine are the most frequently used vaccine development strategies.Both approaches will
Virus is asked to breed in cell, however, CA6 is a kind of it is difficult to the Coxsackie virus of culture.Also, CA6 is not
Efficient replication in cell that can be the most frequently used in Vero and MRC-5 both production of vaccine.Therefore, tradition is passed through
Method exploitation CA6 vaccines will face huge challenge.In this research, inventors investigated based on VLP plans
Slightly develop the possibility of CA6 vaccines.
Because CA6 viruses are difficult to expand in cell, the exploitation of inactivated vaccine and attenuated live vaccine is limited.
In our current research, inventor developed one kind based on virus-like particle (virus-like particle,
VLP CA6 vaccines).CA6 P1 and 3CD albumen is co-expressed in Pichia pastoris, produces CA6-VLP,
After mouse is immunized with the VLP of acquisition, the specific serum antibodies of CA6 can be induced in Mice Body.Passively
In protection test, antiserum can protect mouse to be survived after the CA6 attacks by lethal dose.This
Invention successfully develops CA6 vaccines based on CA6 VLP strategies.
COxsackie A6 viruses CA6 and other principal causative originals of hand-foot-and-mouth disease CA16, CA10 and EV71 virus
Compare, there is significant difference, nucleotide homology is respectively 76.4%, 81.5%, 73.8%, its amino
Acid homology is respectively 48.0%, 22.8%, 42.2%, thus can be assumed that CA6 for be totally different from CA16,
CA10 and EV71 virus.
The amino acid sequence of COxsackie A6 virus P1 albumen is as follows：
MGAQVSTEKSGSHETKNVATEGSTINFTNINYYKDSYAASASRQDFAQDPAK
FTRPVLDTIREVAAPLQSPSVEACGYSDRVAQLTVGNSTITTQEAANIVLSYGEWPE
YCPSTDATAVDKPTRPDVSVNRFYTLSTKSWKTESTGWYWKFPDVLNDTGVFGQN
AQFHYLYRSGFCMHVQCNASKFHQGALLVAAIPEFVVAASSPATKPNGQGLYPDF
AHTNPGKNGQEFRDPYVLDAGVPLSQALVYPHQWINLRTNNCATIIMPYVNALPFD
SALNHSNFGLVVIPISPLKYCNGATTEVPITLTIAPLNSEFSGLRQAIKQGFPTELKPG
TNQFLTTDDGTSPPILPGFEPTPLIHIPGEFTSLLDLCQIETILEVNNTTGTTGVSRLLIP
VRAQNNVDQLCASFQVDPGRNGPWQSTMVGQICRYYTQWSGSLKVTFMFTGSFM
ATGKMLIAYTPPGSAQPATREAAMLGTHIVWDFGLQSSVTLVIPWISNTHFRAVKT
GGVYDYYATGIVTIWYQTNFVVPPDTPTEANIIALGAAQKNFTLKLCKDTDEIQQT
AEYQNDPITNAVESAVSALADTTISRVTAANTAASTHSLGTGRVPALQAAETGASS
NASDENLIETRCVMNRNGVNEASVEHFYSRAGLVGVVEVKDSGTNLDGYTVWPV
DVMGFVQQRRKLELSTYMRFDAEFTFVSNLNDSTTPGMLLQYMYVPPGAPKPDSR
KSYQWQTATNPSVFAKLSDPPPQVSVPFMSPATAYQWFYDGYPTFGEHKQATNLQ
YGQCPNNMMGHFAIRTVSESTTGKNVHVRVYMRIKHVRAWVPRPLRSQAYMVKN
YPTYSQTITNTATDRASITTTDYEGGVPANPQRTS(SEQ ID NO.1)。
The wild-type polynucleotide sequence for encoding COxsackie A6 virus P1 albumen is as follows：
ATGGGTGCCCAAGTTTCAACAGAAAAATCTGGGTCGCACGAGACAAAGAATGTAGCGACCGAA
GGGTCTACTATCAACTTCACCAACATCAATTACTATAAGGATTCTTATGCAGCGTCAGCTAGTAGAC
AGGATTTTGCACAAGATCCCGCAAAGTTCACACGCCCTGTCTTGGATACCATCAGGGAGGTTGCAGC
CCCGTTGCAATCCCCTTCTGTTGAGGCGTGCGGTTATAGTGACCGAGTCGCACAGTTGACTGTGGGC
AACTCAACCATTACTACCCAAGAGGCAGCCAACATTGTGTTGAGCTACGGAGAGTGGCCAGAATATT
GTCCCTCCACGGACGCTACAGCTGTGGACAAGCCTACTCGCCCTGACGTGTCAGTGAATAGGTTCTA
CACACTGTCAACTAAGAGTTGGAAGACAGAATCTACTGGCTGGTACTGGAAATTCCCTGATGTGCTA
AATGACACAGGAGTATTCGGTCAAAACGCCCAATTCCACTACTTGTACCGCTCGGGTTTCTGCATGC
ACGTTCAGTGCAATGCAAGCAAGTTCCATCAGGGGGCCCTCTTAGTGGCTGCAATCCCCGAATTTGT
GGTTGCTGCAAGCAGTCCTGCCACGAAGCCTAATGGACAAGGGTTGTACCCAGATTTCGCTCACACT
AACCCGGGAAAAAATGGCCAAGAGTTTCGAGATCCTTATGTCTTGGATGCTGGTGTCCCCCTAAGTC
AAGCACTGGTTTACCCCCATCAATGGATCAATCTACGAACTAATAACTGCGCGACCATTATCATGCC
ATATGTCAATGCGCTTCCATTTGATTCAGCGCTTAACCACTCAAATTTTGGATTGGTTGTGATCCCT
ATTAGCCCATTAAAATATTGTAATGGAGCTACCACAGAGGTGCCAATCACACTAACTATTGCCCCAC
TTAACTCGGAGTTTAGCGGCCTCCGACAAGCAATAAAACAAGGGTTTCCCACAGAGCTCAAGCCTGG
GACCAATCAATTTCTTACAACTGATGACGGGACATCCCCACCAATACTGCCCGGTTTTGAACCAACT
CCATTGATTCACATTCCTGGTGAGTTCACCTCTTTGTTAGATTTGTGTCAAATAGAAACCATACTAG
AAGTCAATAATACCACTGGCACCACCGGGGTCAGTAGATTACTAATCCCCGTTCGAGCACAGAACAA
TGTGGACCAGTTGTGCGCATCATTCCAAGTAGACCCTGGGCGCAATGGCCCGTGGCAATCCACAATG
GTCGGTCAGATCTGCAGGTATTACACTCAATGGTCAGGTTCCCTTAAGGTAACCTTTATGTTCACGG
GTTCTTTTATGGCCACAGGGAAAATGCTGATAGCCTACACACCACCTGGTAGTGCTCAACCCGCTAC
AAGGGAAGCAGCAATGCTTGGGACTCATATAGTGTGGGATTTTGGTTTGCAATCATCGGTTACCCTA
GTTATACCTTGGATTAGTAACACCCATTTTAGAGCAGTTAAGACTGGAGGGGTATACGACTACTACG
CAACCGGGATCGTCACCATTTGGTACCAAACCAATTTCGTAGTGCCACCAGATACCCCCACTGAGGC
TAATATTATAGCTCTTGGAGCAGCACAGAAAAACTTTACCCTAAAGTTGTGCAAGGACACTGACGAG
ATCCAGCAAACAGCAGAGTACCAAAATGATCCCATTACAAATGCAGTGGAAAGCGCTGTGAGCGCGC
TTGCTGACACCACAATATCCCGGGTGACCGCAGCCAACACTGCAGCTAGCACTCACTCCCTGGGAAC
AGGGCGTGTACCAGCATTGCAAGCCGCAGAAACGGGAGCAAGCTCTAATGCTAGTGATGAGAACCTT
ATTGAGACTCGCTGTGTGATGAATCGAAACGGGGTTAATGAGGCGAGTGTGGAACACTTTTACTCTC
GTGCAGGGCTGGTAGGAGTTGTGGAGGTGAAGGACTCGGGCACTAACCTGGATGGGTACACAGTTTG
GCCTGTAGATGTGATGGGCTTCGTGCAACAGCGGCGCAAGCTAGAGCTGTCAACATACATGCGCTTT
GATGCCGAGTTCACTTTTGTGTCCAACCTCAATGATAGCACGACGCCCGGGATGCTGCTGCAGTATA
TGTATGTACCACCAGGGGCTCCTAAGCCGGATAGCAGGAAATCATATCAATGGCAGACTGCTACTAA
CCCGTCGGTATTCGCAAAATTGAGTGATCCACCCCCCCAGGTATCTGTCCCGTTCATGTCGCCAGCA
ACAGCTTATCAGTGGTTTTATGATGGTTACCCTACATTTGGTGAGCACAAACAAGCTACCAATTTGC
AATATGGGCAGTGTCCTAATAACATGATGGGCCATTTTGCCATCCGAACAGTCAGTGAATCTACCAC
CGGGAAAAATGTCCACGTTCGGGTGTACATGAGAATTAAGCACGTGAGAGCTTGGGTACCTAGACCC
CTTCGATCCCAAGCTTATATGGTCAAAAACTACCCGACATACAGCCAAACAATAACTAACACTGCAA
CTGATCGTGCAAGTATAACCACCACGGATTATGAAGGCGGGGTACCAGCAAACCCACAAAGGACATC
T(SEQ ID NO.2)。
One in the present invention is preferably carried out in mode, and the COxsackie A6 virus P1 albumen by optimization is compiled
Code polynucleotide sequence is as follows：
ATGGGTGCTCAGGTGTCCACCGAGAAGTCCGGTTCCCACGAGACTAAGAACGTGGCCACCGAG
GGTTCCACCATCAACTTCACCAACATCAACTACTACAAGGACTCCTACGCTGCTTCCGCTTCCCGTC
AGGACTTCGCTCAGGACCCCGCTAAGTTCACCCGTCCCGTGCTGGACACTATCCGCGAAGTGGCTGC
TCCCCTGCAGTCCCCATCTGTCGAGGCTTGCGGTTACTCCGACCGTGTGGCTCAGCTGACCGTGGGC
AACTCTACCATCACCACCCAAGAGGCTGCTAACATCGTGCTGTCCTACGGCGAGTGGCCCGAGTACT
GCCCTTCTACCGACGCTACCGCTGTGGACAAGCCCACCAGACCTGACGTGTCCGTGAACCGTTTCTA
CACCCTGTCCACCAAGTCCTGGAAGACCGAGTCCACCGGCTGGTACTGGAAGTTCCCCGACGTGCTG
AACGACACCGGCGTGTTCGGACAGAACGCTCAGTTCCACTACCTGTACCGTTCCGGTTTCTGCATGC
ACGTCCAGTGCAACGCTTCCAAGTTCCACCAGGGTGCTCTGCTGGTGGCTGCTATCCCCGAGTTCGT
CGTGGCTGCTTCATCCCCCGCTACCAAGCCTAACGGCCAGGGCCTGTACCCTGACTTCGCCCACACC
AACCCTGGCAAGAACGGTCAAGAGTTCCGTGACCCCTACGTCCTGGACGCTGGTGTCCCTCTGTCTC
AGGCTCTGGTGTACCCCCACCAGTGGATCAACCTGCGTACCAACAACTGCGCTACCATCATCATGCC
CTACGTGAACGCTCTGCCCTTCGACTCCGCTCTGAACCACTCCAACTTCGGCCTGGTGGTCATCCCC
ATCTCCCCACTGAAGTACTGCAACGGTGCTACCACCGAGGTGCCCATCACCCTGACAATCGCTCCTC
TGAACTCCGAGTTCTCCGGACTGCGTCAGGCTATCAAGCAGGGTTTCCCCACCGAGCTGAAGCCCGG
TACTAACCAGTTCCTGACCACCGACGACGGCACCTCCCCACCTATCCTGCCTGGTTTCGAGCCCACC
CCCCTGATCCACATCCCTGGCGAGTTCACCTCTCTGCTGGACCTGTGCCAGATCGAGACTATCCTGG
AAGTGAACAACACCACCGGAACCACCGGTGTCTCCCGTCTGCTGATCCCTGTGCGTGCTCAGAACAA
CGTGGACCAGCTGTGCGCTAGCTTCCAGGTGGACCCCGGTCGTAACGGTCCTTGGCAGTCCACTATG
GTCGGACAAATCTGCCGCTACTACACCCAGTGGAGCGGTTCCCTGAAAGTGACCTTCATGTTCACCG
GTTCCTTCATGGCTACCGGCAAGATGCTGATCGCTTACACCCCCCCTGGTTCCGCTCAGCCCGCTAC
TCGTGAAGCTGCTATGCTGGGCACCCACATCGTGTGGGACTTCGGCTTGCAGTCCTCTGTGACCCTC
GTGATCCCCTGGATCTCCAACACCCACTTCCGTGCTGTCAAGACCGGTGGCGTGTACGACTACTACG
CTACCGGTATCGTGACCATCTGGTATCAGACCAACTTCGTGGTGCCCCCCGACACCCCTACCGAGGC
TAACATCATCGCTCTGGGCGCTGCTCAGAAGAACTTCACCCTGAAGCTGTGCAAGGACACCGACGAG
ATCCAGCAGACCGCTGAGTACCAGAACGACCCCATCACCAACGCTGTCGAGTCCGCTGTGTCCGCTC
TGGCTGACACCACCATCTCCCGTGTGACCGCTGCCAACACCGCTGCTTCTACCCACTCCCTGGGTAC
TGGTCGTGTGCCCGCTCTGCAGGCTGCTGAGACTGGTGCTTCCTCCAACGCCTCCGACGAGAACCTG
ATCGAAACCCGTTGCGTGATGAACCGTAACGGTGTCAACGAGGCTTCCGTCGAGCACTTCTACTCCC
GCGCTGGACTCGTGGGCGTGGTGGAAGTTAAGGACTCCGGCACCAACCTGGACGGTTACACCGTCTG
GCCCGTGGACGTGATGGGTTTCGTGCAGCAGCGTCGCAAGCTGGAACTGTCCACCTACATGCGTTTC
GACGCTGAGTTCACTTTCGTGTCCAACCTGAACGACTCCACCACCCCCGGCATGCTGCTGCAGTACA
TGTACGTGCCCCCTGGTGCTCCCAAGCCCGACTCCAGAAAGTCCTACCAGTGGCAGACCGCCACCAA
CCCCTCCGTGTTCGCTAAGCTGTCCGACCCCCCACCACAGGTGTCCGTGCCTTTCATGTCCCCTGCT
ACCGCTTACCAGTGGTTCTACGACGGTTACCCCACCTTCGGCGAGCACAAGCAGGCTACCAACCTGC
AGTACGGCCAGTGCCCCAACAACATGATGGGACACTTCGCTATCCGTACCGTGTCCGAGTCTACCAC
TGGAAAGAACGTCCACGTGCGTGTGTACATGCGTATCAAGCACGTGCGCGCTTGGGTGCCCCGTCCT
CTGCGTTCCCAAGCTTACATGGTCAAGAACTACCCTACCTACTCCCAGACCATCACTAACACCGCTA
CCGACCGTGCTTCTATCACCACCACCGACTACGAGGGTGGTGTCCCCGCTAACCCTCAGAGGACCTC
TTAA(SEQ ID NO.3)。
COxsackie A6 virus P1 albumen forms VP0 albumen, VP1 albumen and VP3 eggs after 3CD Protein cleavages
In vain, the amino acid sequence of VP0 albumen is as follows：
MGAQVSTEKSGSHETKNVATEGSTINFTNINYYKDSYAASASRQDFAQDPAKFTRPVLDTIREVAAP
LQSPSVEACGYSDRVAQLTVGNSTITTQEAANIVLSYGEWPEYCPSTDATAVDKPTRPDVSVNRFYT
LSTKSWKTESTGWYWKFPDVLNDTGVFGQNAQFHYLYRSGFCMHVQCNASKFHQGALLVAAIPEFVV
AASSPATKPNGQGLYPDFAHTNPGKNGQEFRDPYVLDAGVPLSQALVYPHQWINLRTNNCATIIMPY
VNALPFDSALNHSNFGLVVIPISPLKYCNGATTEVPITLTIAPLNSEFSGLRQAIKQ(SEQ ID
NO.4)；
The amino acid sequence of VP1 albumen is as follows：
NDPITNAVESAVSALADTTISRVTAANTAASTHSLGTGRVPALQAAETGASSNASDENLIETR
CVMNRNGVNEASVEHFYSRAGLVGVVEVKDSGTNLDGYTVWPVDVMGFVQQRRKLELSTYMRFDAEF
TFVSNLNDSTTPGMLLQYMYVPPGAPKPDSRKSYQWQTATNPSVFAKLSDPPPQVSVPFMSPATAYQ
WFYDGYPTFGEHKQATNLQYGQCPNNMMGHFAIRTVSESTTGKNVHVRVYMRIKHVRAWVPRPLRSQ
AYMVKNYPTYSQTITNTATDRASITTTDYEGGVPANPQRTS(SEQ ID NO.5)；
The amino acid sequence of VP3 albumen is as follows：
GFPTELKPGTNQFLTTDDGTSPPILPGFEPTPLIHIPGEFTSLLDLCQIETILEVNNTTGTTG
VSRLLIPVRAQNNVDQLCASFQVDPGRNGPWQSTMVGQICRYYTQWSGSLKVTFMFTGSFMATGKML
IAYTPPGSAQPATREAAMLGTHIVWDFGLQSSVTLVIPWISNTHFRAVKTGGVYDYYATGIVTIWYQ
TNFVVPPDTPTEANIIALGAAQKNFTLKLCKDTDEIQQTAEYQ(SEQ ID NO.6)。
The amino acid sequence of COxsackie A6 virus 3CD albumen is as follows：(prime strain CA6-Gdula-3CD)
GPSLDFALSLLRRNIRQAQTDQGHFTMLGVRDRLAILPRHSQPGKTIWIEHKLVNVLDAV
ELVDEQGVNLELTLLTLDTNEKFRDITKFIPEAITGASDATLVINTEHMPSMFVPVGDVV
QYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKIIGIHIGGNGRQGFCAGLKRSYFA
SEQGEIQWIKPNKETGRLNINGPTRTKLEPSVFHDVFEGNKEPAVLTSKDPRLEVNFEQA
LFSKYVGNTLHEPDEYVTQAALHYANQLKQLDINTSKMSMEEACYGTEYLEAIDLHTSAG
YPYSALGIKKRDILDPATRDTSKMKLYMDKYGLDLPYSTYVKDELRSLDKIKKGKSRLIE
ASSLNDSVYLRMTFGHLYEVFHANPGTITGSAVGCNPDVFWSKLPILLPGSLFAFDYSGY
DASLSPVWFRALELVLREIGYTEEAVSLIEGINHTHHVYRNKTYCVLGGMPSGCSGTSIF
NSMINNI IIRTLLIKTFKGIDLDELNMVAYGDDVLASYPFPIDCLELAKTGKEYGLTMTP
ADKSSCFNEVTWENATFLKRGFLPDHQFPFLIHPTMPMREIHESIRWTKDARNTQDHVRS
LCLLAWHNGKEEYEKFVSTIRSVPIGKALAIPNFENLRRNWLELF(SEQ ID NO.7)。
The wild-type polynucleotide sequence for encoding COxsackie A6 virus 3CD albumen is as follows：(prime strain
CA6-Gdula-3CD)
GGACCTAGCTTGGACTTTGCTTTGTCTCTCCTGAGGCGTAACATCAGACAGGCGCAGACCGACCAGG
GTCACTTCACCATGCTGGGCGTACGGGACCGCTTAGCTATCCTGCCACGCCACTCGCAACCAGGGAA
AACCATCTGGATAGAACACAAATTGGTCAATGTATTAGATGCAGTTGAATTGGTGGATGAACAAGGT
GTTAATTTAGAACTCACACTGCTGACCTTGGACACTAATGAGAAGTTTAGGGACATCACTAAGTTCA
TTCCAGAGGCAATCACTGGAGCGAGTGATGCAACTCTAGTTATCAACACTGAGCACATGCCCTCGAT
GTTTGTACCAGTAGGTGACGTTGTACAGTATGGGTTCTTGAATCTCAGTGGTAAACCTACTCACAGA
ACCATGATGTACAATTTCCCTACAAAGGCAGGACAATGTGGAGGGGTGGTCACCTCAGTTGGCAAGA
TCATTGGAATCCACATTGGCGGAAATGGACGTCAGGGCTTCTGCGCCGGCTTAAAGAGGAGCTACTT
CGCCAGTGAACAAGGAGAAATCCAGTGGATAAAACCTAACAAAGAAACTGGGAGGCTGAATATTAAT
GGTCCAACTCGGACCAAATTGGAGCCCAGTGTATTCCATGATGTGTTCGAGGGCAACAAAGAGCCGG
CGGTTTTGACCAGCAAGGATCCTAGGTTAGAGGTTAATTTTGAGCAAGCTCTGTTCTCTAAGTACGT
GGGCAACACTCTACATGAACCTGATGAGTATGTGACACAAGCTGCCCTCCACTATGCAAATCAGCTG
AAACAACTAGACATAAACACCAGCAAGATGAGCATGGAGGAGGCGTGCTATGGTACAGAATATTTGG
AAGCAATAGACCTGCATACTAGTGCTGGGTACCCTTATAGCGCCCTGGGTATTAAGAAGAGAGACAT
TCTCGATCCAGCTACCAGAGACACTTCCAAGATGAAATTATACATGGACAAGTATGGACTGGATTTG
CCCTACTCCACTTATGTAAAGGATGAGCTTAGATCTCTAGACAAAATTAAGAAAGGAAAGTCTCGCT
TAATTGAGGCCAGCAGCCTAAATGACTCTGTCTACCTTAGAATGACTTTTGGTCATCTATATGAGGT
GTTTCACGCCAACCCGGGAACCATAACTGGATCTGCAGTCGGGTGTAATCCTGATGTGTTCTGGAGT
AAGCTGCCAATCTTACTGCCGGGCTCGCTCTTTGCATTTGACTACTCAGGATATGATGCAAGCCTTA
GTCCTGTATGGTTTAGAGCTCTAGAGTTGGTTCTGCGGGAGATCGGTTACACGGAGGAGGCTGTGTC
ACTCATAGAAGGAATTAACCACACTCACCACGTGTACCGGAACAAGACATACTGTGTTCTTGGTGGG
ATGCCCTCAGGTTGCTCTGGTACTTCCATTTTCAATTCCATGATTAACAACATAATCATCAGAACCC
TCTTGATTAAAACGTTCAAAGGTATAGACTTAGATGAATTGAACATGGTGGCCTACGGGGATGATGT
GTTGGCTAGCTACCCATTTCCCATTGATTGCTTGGAATTGGCAAAAACTGGCAAGGAGTACGGATTG
ACCATGACTCCTGCCGACAAATCATCCTGTTTCAATGAAGTCACCTGGGAGAATGCAACTTTCTTAA
AACGGGGTTTCTTACCAGATCATCAGTTTCCTTTTCTGATCCATCCCACCATGCCCATGAGGGAAAT
CCACGAGTCCATTCGCTGGACCAAGGATGCTCGTAATACTCAGGACCACGTGCGCTCCCTTTGTTTG
CTGGCATGGCACAATGGAAAAGAGGAATATGAGAAATTTGTGAGCACAATTAGATCAGTTCCCATTG
GAAAAGCTTTGGCAATACCAAATTTTGAGAACTTGAGAAGAAATTGGCTCGAACTATTTTAA(SEQ
ID NO.8)。
Genetically engineered cell
The invention provides a kind of genetically engineered cell (host cell), the genetically engineered cell is eucaryon
Cell, and the expression cassette and 3CD of COxsackie A6 virus P1 albumen are integrated with the genome of the cell
The expression cassette of albumen；Or containing expression vector in the cell, the expression vector contains COxsackie A6 diseases
The expression cassette of malicious P1 albumen and the expression cassette of 3CD albumen；
And the genetically engineered cell is in intracellular expression COxsackie A6 virus P1 albumen and 3CD albumen, and
The P1 albumen is formed capsid protein VP0 albumen, VP1 albumen and and VP3 after the 3CD Protein cleavages
Albumen, the VP0 albumen, VP1 albumen and and VP3 albumen be internally formed virus in the genetically engineered cell
Sample particle (VLP).
In another preference, the cell is yeast cells.
In another preference, the expression cassette of described COxsackie A6 virus P1 albumen can including 5' to 3'
The elements below being operatively connected：Promoter, initiation codon, the ORF of COxsackie A6 virus P1 albumen
Sequence and terminator codon.
In another preference, the expression cassette of described COxsackie A6 virus 3CD albumen can including 5' to 3'
The elements below being operatively connected：Promoter, initiation codon, the ORF of COxsackie A6 virus 3CD albumen
Sequence and terminator codon.
In the present invention, term " being operably connected " means such configuration, wherein regulating and controlling sequence is placed in
Relative to the appropriate location of the coded sequence of polynucleotides so that regulating and controlling sequence instructs the expression of coded sequence.
Composition and application process
Present invention also offers a kind of composition, and it contains：(i) recombinant virus sample particle (VLP) of the invention
Or the polynucleotides of the codified recombinant virus sample particle of the present invention, and (i i) pharmaceutically or in immunology can
The excipient or adjuvant of receiving.
In the present invention, term " containing " represents that various composition can be applied to or be present in together the group of the present invention
In compound.Therefore, term " mainly by ... form " and " consist of " are included in term " containing "
In.
The composition of the present invention includes pharmaceutical composition and vaccine combination.
The composition of the present invention can be monovalent (only containing a kind of recombinant virus sample particle or polynucleotides),
Can also be multivalence (containing a variety of recombinant virus sample particles or polynucleotides).
The pharmaceutical composition or vaccine combination of the present invention can be prepared into various regular dosage forms, including (but
It is not limited to)：Injection, granula, tablet, pill, suppository, capsule, suspension, spray etc..
(1) pharmaceutical composition
The pharmaceutical composition of the present invention includes the recombinant virus sample particle of the present invention of (or containing) therapeutically effective amount
Or polynucleotides.
Term " therapeutically effective amount " used herein refers to therapeutic agent treatment, alleviates or prevents target disease or shape
The amount of condition, or show the amount of detectable treatment or prevention effect.The effect can be for example, by antigen water
Put down to detect.Therapeutic effect also includes the reduction of physical symptoms.Accurate for a certain object effectively measures
Certainly the build and health status in the object, the nature and extent of illness and the therapeutic agent given of selection and
/ or therapeutic agent combination.Therefore, it is useless to preassign accurate effective dose.However, given for certain
For fixed situation, the effective dose can be determined with normal experiment.
For the purposes of the present invention, effective dosage is to give individual about 0.001 mg/kg to 1000 millis
G kg, it is preferably about the recombinant virus sample particle of 0.01 mg/kg to 100 mg/kg body weight.
Pharmaceutical composition can also contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier "
Refer to the carrier for therapeutic agent (recombinant virus sample particle of the invention) administration.The term refers to more such
Medicament carrier：The antibody for producing and being harmful to the individual for receiving said composition themselves is not induced, and after administration
There is no undue toxicity.Suitable carrier can be big, the slow macromolecular of metabolism, such as protein, more
Sugar, PLA (polylactic acid), polyglycolic acid etc..These carriers are ordinary skill people
Known to member.In Remington ' s Pharmaceutical Sciences (Mack Pub.Co., N.J.
1991) discussing fully on pharmaceutically acceptable carrier or excipient can be found in.
The upper acceptable carrier of combination of traditional Chinese medicine may include liquid, such as water, salt solution, glycerine and ethanol.Separately
Outside, complementary material, such as wetting agent or emulsifying agent, pH buffer substance are there is likely to be in these carriers.
Generally, composition can be made to injectable agent, such as liquid solution or suspension；It may also be fabricated which and fit before the injection
Close supplying solution or suspension, the solid form of liquid excipient.Liposome is also included within pharmaceutically acceptable
Carrier definition in.
(ii) vaccine combination
The vaccine (composition) of the present invention can be preventative (i.e. prevention disease) or curative (suffer from
Disease is treated after being ill).
These vaccines include immunising antigen (including recombinant virus sample particle of the present invention), and generally with " medicine
Acceptable carrier on " combines, and these carriers include itself not inducing produced to receiving said composition
Any carrier of the harmful antibody of body.Suitable carrier is typically big, is metabolized slow macromolecular, such as egg
White matter, polysaccharide, PLA, polyglycolic acid, amino acid polymer, amino acid copolymer, lipid aggregates
(such as oil droplet or liposome).These carriers are well known to those of ordinary skill in the art.In addition, these
Carrier can play immunostimulant (" adjuvant ").In addition, antigen can also be with bacterial toxoid (as in vain
The toxoid of the pathogen such as larynx, lockjaw, cholera, helicobacter pylori) coupling.
The preferred adjuvant of enhancing immune composition effect includes but is not limited to：(1) aluminium salt (alum), such as hydrogen-oxygen
Change aluminium, aluminum phosphate, aluminum sulfate etc.；(2) oil-in-water emulsion formula, for example, (a) MF59 is (referring to WO
90/14837), (b) SAF, and (c) Ribi adjuvant systems (RAS) (Ribi Immunochem, Hamilton,
MT), (3) saponin adjuvant；(4) Freund Freund's complete adjuvants (CFA) and Freund Freund's incomplete adjuvants (IFA)；(5)
Cell factor, such as interleukin (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12),
Interferon (such as interferon), macrophage colony stimulatory factor (M-CFS), TNF (TNF)；
(6) the detoxification variant of bacterial ADPribosylating toxin (such as E.coli LT LT)；And
(7) other materials of composition effect are strengthened as immunostimulant.
Including immunogenic composition vaccine combination (such as, it may include antigen, can pharmaceutically connect
The carrier and adjuvant received), usually contain diluent, such as water, salt solution, glycerine, ethanol etc..In addition,
Auxiliary substances, such as wetting agent or emulsifying agent, pH buffer substance may be present in this kind of carrier.
More specifically, the vaccine including immunogenic composition, the immunogene comprising immunological effective amount
Property polypeptide, and above-mentioned other required components." immunological effective amount " refers to single dose or continuous agent one
It is effective to treating or preventing to give and give individual amount.The dosage can be according to the health status for treating individual
With physiological status, treat the classification (such as people), the ability of individual immunity system synthesis antibody, required of individual
Degree of protection, vaccine preparation, treating physician assessment and other correlative factors to medical conditions and
It is fixed.It is expected that the dosage can be determined in relatively wide scope by normal experiment.
Generally, vaccine combination or immunogenic composition can be made to injectable agent, for example, liquid solution or
Suspension；It may also be fabricated which and be adapted to supplying solution or suspension, the solid form of liquid excipient before the injection.The system
Agent is also emulsifiable or is encapsulated in liposome, to strengthen adjuvant effect.
In addition, the vaccine combination of the present invention can be unit price or polyvaccine.
(iii) method of administration and dosage
Once being made into the composition of the present invention, it can be directly given to object.Object to be treated can be fed
Newborn animal, especially people.
When as vaccine, the recombinant virus sample particle of the present invention can be directly applied to known method
Body.Generally use applies these with conventional vaccine identical route of administration and/or simulation pathogenic infection path
Vaccine.
Giving the approach of pharmaceutical composition of the present invention or vaccine combination includes (but being not limited to)：Intramuscular, skin
Under, intracutaneous, intrapulmonary, intravenous, intranasal, by oral administration or other parenteral route of administration.If desired,
Can be with combination medicine-feeding approach, or be adjusted according to disease event.Vaccine combination can be with single dose or multi-agent
Amount is given, and can include giving booster to trigger and/or maintain immunity.
Recombinant virus sample particle vaccines should be given with " effective dose ", i.e., the amount of recombinant virus sample particle is selected
Immune response is adequate to bring about in administration routes, can effectively promote to protect host to resist related disease.
Representational disease includes (but being not limited to)：Rabbit hemorrhagic disease virus infection etc..
The amount of selected recombinant virus sample particle in each vaccine dose part, it is by can trigger the immune protective should
Answer and depending on the amount without obvious side effect.Generally, after host cells infected, each dose of vaccine is enough to contain
There are about 1 μ g-1000mg, preferably 1 μ g-100mg, more preferably 10 μ g-50mg protein.It can use
Standard research techniques including the IgG titers in the object of observation and other reactions determine specific vaccine
Optimum amount.It can determine the need for strengthening dosage by monitoring the immunity level of vaccine offer.Commenting
After having estimated the IgG titers in serum, it may be necessary to from enhancing dose immunizations.Using adjuvant and/
Or immunostimulant can improve the immune response of the protein to the present invention.
Method for optimizing is to give immunogenic composition from parenteral (subcutaneously or intramuscularly) approach by injection.
In addition, the vaccine of the present invention can be given together with reference to other immunomodulators, or and other treatment
Agent is given together.
Main advantages of the present invention are：
(1) a kind of new generation vaccine based on COxsackie A6 virus vlps is disclosed first, can be safer higher
Effect ground protection body from CA6 virus infection, while hand-foot-and-mouth disease principal causative original CA16, CA10 and
EV71 etc. does not have cross protection, and CA6 harmfulness is further serious, and exploitation CA6 vaccines are by for hand-foot-and-mouth disease
Wide spectrum polyvaccine lays the foundation, and expands the protection domain of vaccine for hand-foot-mouth disease.
(2) present invention build containing COxsackie A6 viruses P1 protein expressions box and 3CD protein expression boxes
Carrier, it can succeed and COxsackie A6 virus P1 albumen and 3CD albumen are expressed in genetically engineered cell, and
The P1 albumen is formed capsid protein VP0 albumen, VP1 albumen and and VP3 after the 3CD Protein cleavages
Albumen, the VP0 albumen, VP1 albumen and and VP3 albumen can be autonomous inside the genetically engineered cell
Form virus-like particle (VLP).
(3) present invention is built using the COxsackie A6 virus P1 albumen Codon sequences Jing Guo more suboptimization
VLP expression vectors, high efficient expression, VLP yield it can be significantly improved in yeast cells.3CD albumen exists
Important function is equally served in VLP forming processes, the present invention carries out excellent for 3CD albumen Codon sequences
Change, as a result display can not significantly improve VLP expression yield, therefore, wild type still be used in the present invention
3CD protein coding genes.
(4) expanding effect of the CA6 viruses on the cells such as Vero is bad at present, nearest one research hair
Existing CA6 viruses expand titre on RD cells and only reach 107TCID50/mL, viral yield is relatively low, right
The dilution factor of this plant of CA6 viruses is relatively low, and these factors greatly limit opening for CA6 inactivated vaccine
Hair, the application are based on gene engineering strategy exploitation CA6 new generation vaccines, in viral yield and immunogenicity etc.
Aspect has significant advantage, breaches the limitation bottleneck of exploitation CA6 inactivated vaccines.
With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are only used for
The bright present invention rather than limitation the scope of the present invention.The experiment side of unreceipted detailed conditions in the following example
Method, generally write according to normal condition such as U.S. Sambrook.J etc.《Molecular Cloning: A Laboratory room guide》It is (yellow
Training hall etc. is translated, Beijing：Science Press, 2002) described in condition, or built according to manufacturer
The condition of view.Unless otherwise indicated, otherwise percentage and number are calculated by weight.It is used in following examples
Experiment material and reagent can be obtained unless otherwise instructed from commercially available channel.
Material and method
1.1 bacterial strain and antibody
The two plants of CA6 viruses used in this experiment, CA6/Gdula (GenBank ID:AY421764.1) purchase
From American Type Culture Collecti (ATCC#VR-165), clinical strain CA6/S0087b (GenBank ID:
KT183533.1) given by Chinese Academy of Sciences's Shanghai Pasteur institute's control and prevention of disease with diagnostic center.In large intestine bar
CA6 VP0 albumen is expressed in bacterium, it is more anti-with anti-CA6 VP0 are obtained after the protein immunization rabbit, with same
Sample method expression produce CA6 VP1 and VP3 albumen, respectively be immunized mouse after obtain anti-CA6 VP1 and
Anti- CA6 VP3 are more anti-.
1.2 vector construction
CA6/Gdula cDNA genes are obtained by reverse transcription).Then, obtained by template amplification of cDNA
To 3CD fragments and cloned pPink-HC (Invitrogen) generation plasmids YCA6-3CD.Optimization
Clinical strain SZc173/13 (GenBank ID:KF682362.1 P1 genes (SEQ ID NO.1)) are in gold
Si Rui companies (Shanghai, China) synthesis, clones into pPink-HC (Invitrogen), produces plasmid YCA6
‐P1.3CD expression cassettes are inserted into by plasmid YCA6-P1 in Bgl II sites by the method for homologous recombination
In, produce plasmid YCA6-003.
1.3 yeast conversions and screening
Single endonuclease digestion EcoNI sites linearize plasmid YCA6-003, and electricity conversion enters PichiaPinkTM
Strain 1(Invitrogen).Yeast conversion and the screening of subsequent transformant are grasped according to product description
Make.The yeast clone of conversion is first cultivated on a small scale and methanol induction.After induction terminates, bacterium solution is centrifuged,
Crack thalline.Lysate supernatant is collected to analyze for ELISA and Western blotting.
1.4 enzyme-linked immunosorbent assays and immunoblot experiment
Lysate supernatant obtained above is taken to carry out enzyme-linked immunosorbent assay.Contain 5 μ l lysates per hole
The μ l PBS solutions of supernatant 45 are coated with 96 hole Elisa plates, and 4 DEG C overnight, then use and contain 5% degreasing ox
37 DEG C of the PBST solution incubation 1h of milk, then 37 DEG C of primary antibody is used as by the use of anti-CA6 VLP polyclonal antibody
2h is incubated, the secondary antibody of corresponding horseradish peroxidase (HRP) mark carries out incubation 1h, finally uses TMB
Colour developing reads light absorption value OD450 after terminating.Immunoblot experiment is as it was previously stated, by specific polyclonal
Antibody (anti-CA6 VP0, anti-CA6VP1 and anti-CA6VP3) and its corresponding HRP mark secondary antibodies are carried out
Detection and analysis.
1.5 VLP expression and purification
Bibliography (Zhang C, et al. (2015) High-yield production of
recombinant virus-like particles of enterovirus 71in Pichia pastoris
and their protective efficacy against oral viral challenge in mice.
Vaccine 33:2335-2341.) method of report carries out CA6 VLP expression and purifying.Empty carrier
As a control group, the albumen of purifying is as control antigen for pPink-HC transformed yeasts bacterium.The CA6 finally given
VLP determines concentration by Bradford methods.
1.6 Electronic Speculum detect
After CA6 virus-like particles are dyed by 0.5% water acetic acid uranyl, in Tecnai G2
Spirit observed under electron microscope, further determine that the sample of preparation has assembled to form complete virus-like
Particle.
1.7 mouse immunes and antibody test
CA6-VLP after purification is diluted with 0.15M PBS, then by 6 μ g CA6 VLP or control antigen
(300 μ l) is isometric with isometric aluminium adjuvant Alhydrogel (3mg) (Invivogen, USA)
1:1 is sufficiently mixed.Control antigen is injected intraperitoneally respectively from two groups (6/group) 6 week old ICR female mices
Or CA6 VLP, 1 μ g/ are only.It is immune at the 0th, 2,5 week, it is immunized altogether three times.Taken a blood sample at the 7th, 9 week,
Separate serum and be used for antibody test and the experiment of internal passive protection.Tested by ELISA and determine CA6 in serum
Specific IgG antibody.In brief, 96 orifice plates (10ng/ holes) are coated with CA6 VLP, or
With CA6VP0, VP1, VP3 mixing wrapper sheet (1 of Bacillus coli expression:1:1,200ng/ hole), 4 DEG C
Overnight, 1h are closed with 37 DEG C of the PBST solution containing 5% skim milk, then respectively with anti-pPink HC
Serum with anti-CA6 VLP is as 37 DEG C of incubation 2h of primary antibody, after the secondary antibody incubation 1h that corresponding HRP is marked
Read light absorption value OD450.
1.8 external cross neutralization tests
Verified through test of many times, CA6 viruses can not be normal in RD, Vero, 293T and Neuro-2A etc.
Expand for a long time in rule cell line, can not in vitro culture obtain enough infectious titer, so unused acquisition
Antiserum carries out the evaluation of external neutralization to CA6 viruses.Serum only be have detected to CA16, CA10
And the neutralization of EV71 viruses, method are as follows：Take acquisition immune serum make 16 ×, 32 ×, 64 ×,
128 ×, 256 ×, 512 ×, 1024 × and 2048 × doubling dilution after 50 μ l/ holes add in 96 orifice plates, after
Tri- kinds of viruses of 100TCID50/ holes CA16, CA10 and EV71 are separately added into, 37 DEG C of incubation 1h, are added
Enter 104The RD cells in/hole, detect the anti-CA6-VLP serum external neutralization viral to this three kinds.
Passive protection is tested in 1.9 bodies
Select four groups of ICR mouse, the anti-VLP serum of 50 μ l of intraperitoneal injection or control group serum, after 24h,
Intraperitoneal injection 1.469 × 106The CA6/Gdula viruses of individual copy or 1.3383 × 104Individual copy
CA6/S0087b viruses.Then, the continuous 15 days clinical conditions and survival rate for observing and recording mouse.It is clinical
Scoring criterion is as follows：0, health；1, it is slow in action；2, incoordination；3, paralysis；4, it is dead.
The analysis of 2.9 data statistics significant differences completes double tails using the softwares of GraphPad Prism version 5
T- is detected.
The VLP of embodiment 1 expression and purifying
In order to express CA6 VLP, 3CD and P1 genes are together inserted pPink-HC carriers by the present inventor
Upper (Figure 1A), is built into plasmid YCA6-003, then converts Pichia pastoris competence with the plasmid, small
Amount expression obtains cellular lysate liquid centrifugation supernatant and analyzed for ELISA and Western blotting.Conversion
Empty carrier pPink-HC yeast clone operation repetitive is as negative control.Compared with control group, plasmid
The saccharomycete lysate supernatant of YCA6-003 conversions shows obvious antigen-antibody reaction (Figure 1B).
Choose three most strong progress Western blotting analyses of reaction in yeast clone, experimental result is such as
(Fig. 1 C) shown in figure, respectively by the use of anti-CA6VP1, anti-CA6VP0, anti-CA6VP3 as detection antibody,
All there is corresponding band in three yeast lysate samples, and size is respectively 35kDa, 27kD, 39kDa,
There is not corresponding band in control group.The yeast strains for having crossed the YCA6-003 conversions of explanation plasmid above can
P1 albumen is expressed, and P1 can be cut into VP0, VP1, VP3 by 3CD.
The yeast thalline lysate of YCA6-003 conversions is used subsequently to sucrose density gradient centrifugation.In #7, #8 and
#9 layers can be clearly seen that three bands, and size is respectively 39kDa, 35kD, 27kDa (Fig. 2A).
Take saccharose gradient sample carry out Western blotting analyses, respectively with anti-CA6 VP1, anti-CA6 VP0,
Anti- CA6 VP3 are as detection antibody, in #7, #8 and #9 layers can see size be respectively 39kDa, 35kD,
27kDa three bands (Fig. 2 B-D).VP0, VP1, VP3 are in #7, the co-precipitation of #8 and #9 layers
Phenomenon illustrates that assembling forms VLP structures altogether for they.Then #7, #8 and #9 layers are mixed under Electronic Speculum
Observation, it can be seen that the spheric granules structure (Fig. 2 E) that diameter is about 30nm.It these results suggest that complete
Red yeast can express complete CA6 virus-like particles structure.
The mouse immune of embodiment 2
Before immune mouse, prepare CA6 VLP and control antigen and quantitative (Fig. 3 A), then helped with aluminium
Agent mixes.Take two groups of (6/group) ICR mouse that CA6 VLP were injected intraperitoneally respectively at the 0th, 2,5 week
Vaccine and control antigen.Taken a blood sample at the 7th, 9 week for elisa assay.Mixed with VP0, VP1, VP3
Compound equal proportion wrapper sheet, ELISA results as shown in Figure 3 B, from vaccine group mouse collection 6 parts of serum in,
There are 5 parts obvious antigen-antibody reaction all occur, and control group is without significant reaction.It is worth noting that,
When with CA6 VLP wrapper sheets, all there is obvious antigen-antibody reaction (Fig. 3 C) in all vaccine group mouse,
And geometric mean titer has reached 17959 (Fig. 3 D).Result above, which not only demonstrates CA6 VLP, to be had
Very high immunogenicity, and illustrate that CA6VLP induces antibody primarily directed to conformational epitope's.
The external cross-neutralization experiment of the CA6-VLP serum of embodiment 3
The anti-CA6 serum 1 obtained:16 2 doubling dilutions of starting, to tri- kinds of CA16, CA10 and EV71
The cross-neutralization experimental result of virus confirms CA6 serum in minimum dilution factor is no to three strain virus and handed over
Pitch effect.
Passive protection is tested in the CA6 VLP vaccine antiserum bodies of embodiment 4
The present inventor goes to evaluate the protecting effect of anti-VLP antibody by the experiment of serum passive protection.7 ages in days are new
The raw μ l of mouse peritoneal injection 50 anti-VLP serum or control group serum, and be injected intraperitoneally after 24 hours
CA6/Gdula the or CA6/S0087b viruses of lethal dose are attacked, experimental result such as Fig. 4 A and 4B
It is shown, the mouse of control serum is injected, with engendering serious clinical condition after CA6/Gdula virus attacks
Shape, including be slow in action, incoordination and paralysis, and it is all dead in 6d after poison is attacked.Phase therewith
Instead, the mouse of anti-VLP serum is injected, does not occur obvious clinical symptoms within the observation period of 15 days.
Likewise, during with CA6/S0087b virus attacks, control group mice is all dead in 9 days, and injection is anti-
The mouse of VLP serum all survives and does not occur obvious clinical symptoms (Fig. 4 C-D).Experiment knot above
Fruit confirm the antibody that CA6 VLP induce can assign mouse it is complete inside protect.
Discuss
Shown in P1 albumen optimization codon and SEQ ID NO.9 shown in the SEQ ID NO.1 of the present invention
3CD albumen codons, can not only in Pichia pastoris can high efficient expression, moreover, expressed
P1 albumen can be formed after 3CD Protein cleavages capsid protein VP0 albumen, VP1 albumen and and VP3 albumen,
And portion forms virus-like particle (VLP) in the cell.Mouse is immunized using the virus-like particle (VLP) of purifying,
There is a very high immunogenicity, and the antibody that induces of the CA6 VLP of the present invention is primarily directed to conformational epitope,
High specificity, mouse can be protected from virus attack.Therefore, the CA6 VLP of yeast sources are a kind of pole
Potential CA6 candidate vaccines.
In the last few years, the main pathogenic microbes for causing HFMD are EV71, CA16 and CA6.It is presently believed that
Develop the key that a kind of polyvaccine with broad spectrum protection effect is HFMD preventions.Recently, EV71 VLP
Expressed with CA16 VLP successes in Pichia pastoris, and zoopery has been verified that their protectiveness
Effect.Therefore, the CA6 VLP of Pichia anomala expression, EV71 VLP and CA16 VLP can form one kind
Trivalent vaccine, prevention of this polyvaccine for HMFD will produce very active influence.
All it is incorporated as referring in this application in all documents that the present invention refers to, just as each document
It is individually recited as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read,
Those skilled in the art can make various changes or modifications to the present invention, and these equivalent form of values equally fall within this Shen
Please appended claims limited range.
Claims (10)
- A kind of 1. polynucleotides through codon optimization of separation, it is characterised in that polynucleotide encoding Ke Sa Qi A6 virus P1 albumen；And the polynucleotides are selected from the group：(a) polynucleotides of the sequence as shown in SEQ ID NO.3；(b) multinuclear of nucleotide sequence and homology >=95% (preferably >=98%) of sequence shown in SEQ ID NO.3 Thuja acid；(c) polynucleotides complementary with any described polynucleotides of (a)-(c).
- 2. a kind of expression vector, it is characterised in that the expression vector contains more nucleosides described in claim 1 Acid.
- 3. expression vector as claimed in claim 2, it is characterised in that the expression vector also includes compiling The polynucleotide sequence of code COxsackie A6 virus 3CD albumen.
- 4. expression vector as claimed in claim 2, it is characterised in that the expression vector includes the first table Up to box and the second expression cassette, first expression cassette includes the polynucleotides or its complementary sequence shown in SEQ ID NO.3 Row；Second expression cassette includes the polynucleotides or its complementary series shown in SEQ ID NO.8.
- 5. expression vector as claimed in claim 2, it is characterised in that the expression vector is shaft-like to recombinate Virus.
- 6. expression vector as claimed in claim 4, it is characterised in that first expression cassette also includes opening Mover, the promoter are located at the upstream of the polynucleotides shown in SEQ ID NO.3, and preferably described promoter is PAOX1 promoters；And/orSecond expression cassette also includes promoter, and the promoter is located at more nucleosides shown in SEQ ID NO.8 The upstream of acid, preferably described promoter is PAOX1 promoters.
- 7. a kind of host cell, it is characterised in that described host cell contains the expression described in claim 2 Carrier, or it is integrated with genome the polynucleotides described in claim 1；Preferably, the host cell is yeast cells.
- 8. a kind of virus-like particle of Coxsackie virus A 6 (VLP), it is characterised in that the virus-like particle is by weighing Profit requires the host cell expression described in 7.
- 9. a kind of method for preparing Coxsackie virus A 6VLP, including step：Under conditions suitable for the expression, the cell described in claim 7 is cultivated, so as to give expression to claim 8 Described virus-like particle (VLP)；With the separation virus-like particle (VLP).
- 10. a kind of pharmaceutical composition, it is characterised in that described composition contains the disease described in claim 8 Polynucleotides described in malicious sample particle (VLP), claim 1 either the expression vector described in claim 2 or Host cell described in claim 7, and pharmaceutically acceptable carrier and/or auxiliary material；Preferably, described pharmaceutical composition includes vaccine combination.
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| CN111000991A (en) * | 2019-12-20 | 2020-04-14 | 浙江普康生物技术股份有限公司 | Novel coxsackievirus A group 6 type recombinant subunit protein vaccine and preparation method thereof |
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| 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 |
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| 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 |
| CN112480215A (en) * | 2020-12-04 | 2021-03-12 | 武汉生物制品研究所有限责任公司 | Virus-like particle of Coxsackie virus CV-A2 |
| CN115707776A (en) * | 2021-08-20 | 2023-02-21 | 华淞(上海)生物医药科技有限公司 | Recombinant Coxsackie virus A6 virus-like particle and application thereof |
| CN115707776B (en) * | 2021-08-20 | 2024-03-19 | 华淞(上海)生物医药科技有限公司 | Recombinant coxsackievirus A6 virus-like particles and uses thereof |
| CN114807060A (en) * | 2022-06-23 | 2022-07-29 | 北京民海生物科技有限公司 | Coxsackie virus A6 type strain and immunogenic composition and application thereof |
| CN114807060B (en) * | 2022-06-23 | 2022-09-30 | 北京民海生物科技有限公司 | Coxsackie virus A6 type strain and immunogenic composition and application thereof |
| WO2023246639A1 (en) * | 2022-06-23 | 2023-12-28 | 北京民海生物科技有限公司 | Coxsackievirus a6 type strain, and immunogenic composition and use thereof |
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