CN109609467A - A kind of CV-A6 virus seed culture of viruses and its inactivated vaccine for human - Google Patents
A kind of CV-A6 virus seed culture of viruses and its inactivated vaccine for human Download PDFInfo
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- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
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- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
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- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32311—Enterovirus
- C12N2770/32351—Methods of production or purification of viral material
Abstract
The present invention discloses a kind of CV-A6 virus seed culture of viruses and its inactivated vaccine for human；The classification naming of seed culture of viruses is 6 type of Coxsackie virus A group, and deposit number is CGMCC No.16217, the composition of made people CV-A6 inactivated vaccine are as follows: CV-A6 inactivates 100 μ g/ml of purifying antigen, aluminium hydroxide 1mg/ml.There is good immunogene and safety through the zoopery vaccine product.
Description
Technical field
The present invention relates to a kind of CV-A6 virus seed culture of viruses and its inactivated vaccine for human.
Background technique
Hand-foot-and-mouth disease (hand foot mouth disease, HFMD) is in the whole world, the especially Asian-Pacific area, popular scale
It influences increasingly to increase with threatening, and it is worth noting that, the process that this disease increasingly aggravates the harm of children population.So far
Until the present, the epidemiology statistics in China have shown the disease since 2008 are listed in Class C infectious disease, in 2009
It becomes first of Class C infectious disease incidence number, overall case fatality rate is about 0.05%.The prevention and control of the infectious disease, especially in children group
Prevention and control in body, it has also become an important public health problem.
6 type of CV-A6(Coxsackie virus A group in recent years) infection cause HFMD break out or prevalence trend be continuously increased, have
A little areas have become the pathogen of the main HFMD in addition to enterovirns type 71 (EV-A71) and CV-A16, and with it is a variety of its
Its enterovirus includes that EV-A71 and CV-A16 the phenomenon that alternate cycles, mixed infection occur.It is popular that this has highlighted control HFMD
Arduousness.
At present both at home and abroad in addition to EV-A71 vaccine, it there is no other special effect medicine therapeutic HFMD, and vaccine is that it is main pre-
Anti- means.After being very popular from China HFMD in 2008, HFMD has become the serious Disease Spectrum in China, and vaccine becomes control disease
The urgent need kind of disease, after R&D works in 7 years, EV71 vaccine is approved for the prevention and treatment of hand-foot-and-mouth disease.To solve
EV71 vaccine is difficult to control other enteroviruses such as CV-A16 and CV-A6 and causes the prevalence of HFMD, and may thus cause
The EV-A71 vaccine public receives and queries problem, proposes research and development using EV-A71, CV-A16 as core, increases the enteron aisles such as CV-A6 disease
The necessity of poison joint HFMD vaccine as main component.CV-A16 vaccine is being researched and developed at present, the research and development to CV-A6 vaccine
Still lack.
Summary of the invention
The technical problem to be solved by the present invention is to, overcome defect described above, provide a kind of CV-A6 virus seed culture of viruses and its
Screening technique, and the safely and effectively people CV-A6 inactivated vaccine prepared with CV-A6 virus seed culture of viruses.
In order to solve the problems, such as that techniques described above, CV-A6 virus seed culture of viruses of the present invention, classification naming are Coxsack disease
Malicious 6 type of A group, deposit number are CGMCC No.16217, which is named as YNKA1205；The poison of the CV-A6 virus
Kind preparation method the following steps are included:
(1) CV-A6 Strain is separated
Hand-foot-and-mouth disease severe childhood Feces of Patients 1g is taken to add 5ml physiological saline, suspension 3000xg is centrifuged 30min, and supernatant is through nothing
It after bacterium filtering, is inoculated in RD cell (i.e. the pernicious embryo's rhabdomyoma cell of people), after 3-5 days lesions, continues to pass on 3 in RD cell
Generation, and through RT-PCR and sequencing acquisition virus whole genome sequence and the amino acid sequence of supposition, the genotype of the Strain
It is consistent with the genotype of the CV-A6 of current Chinese epidemic strain for D3；
(2) adaptation of the strain virus on KMB17 cell
The P28 for having grown up to fine and close single layer is selected for KMB17 cell, old culture solution (referring to MEM) is abandoned, cleans cell table with PBS solution
0.125% trypsin digestion and cell, cell bottle to be patted is added in face, and cell is slided from bottle wall drift sand sample, and cell culture is added
Liquid, by 2 × 105Cell/bottle is inoculated with small square vase, and after 37 DEG C are cultivated 3~4 days, CV-A6 200 μ l of virus obtained by step (1) are inoculated with
In cell face, 37 DEG C are adsorbed 1 hour, the MEM cell maintenance medium for being free of calf serum are then added, 37 DEG C are cultivated and observe cell
Lesion situation, virus freeze spare up to 90% harvest virus；
(3) clone purification of virus stain
The CV-A6 virus harvest liquid passed on to the 5th generation has been adapted on KMB17 cell, is diluted to 10 with viral dilution-3、
10-4、10-5Three dilutions are inoculated in six well culture plate of KMB17 cell for having formed single layer, and 37 DEG C adsorb 1 hour, are added not
Viral maintaining liquid containing calf serum removes culture solution after 48 hours, be added and contain 0.8% agarose-MEM, after agar solidified,
It is inverted in 37 DEG C of cultures and draws out plaque with pipette tips in microscopic observation plaque after 3 days, be placed in 1.5ml centrifuge tube, be added
0.5mlMEM maintaining liquid, vortex oscillation are inoculated in single layer KMB17 cell immediately, and 37 DEG C are cultivated 3~4 days, after cytopathy
Harvest virus, same method carry out plaque purification twice again, obtain the purified colonies strain for being identified as CV-A6；The purifying gram
Grand strain is passed in KMB17 cell i.e. as primordial seed after 4 generations, and after resuming the 3 generations i.e. main seed of conduct, then passing for 3 generations again is
Work seed；This primordial seed, main seed and work seed are CV-A6 virus seed culture of viruses；
(4) seed culture of viruses of primordial seed obtained by step (3), main seed and the seed that works is identified respectively, shows the seed culture of viruses
All have good immunogenicity, genetic stability and the good conformity proliferative capacity on KMB17 cell.
The identification method of YNKA1205 seed culture of viruses of the present invention:
(1) YNKA1205 strain virus infection titer measures
Seed culture of viruses carries out 10 times and is serially diluted (i.e. 10-1, 10-2…….10-8), each dilution adds 8 holes, every 100 μ l of hole, by containing
2×105A/ml RD cell, every 100 μ l cell suspension of hole add to each 8 hole of dilution, and 37 DEG C are cultivated 5~7 days, observation knot
Fruit.
(2) RT-PCR and sequencing
Virus genomic nucleic acid extraction presses Axygen Body Fluid viral DNA/RNA Miniprep Kit manipulator
Volume carries out, and viral gene RNA reverse transcription uses One-step RNA PCR kit (AMV) reagent of TaKaRa company production
Box is carried out by operation manual, and is sequenced according to " walk method ", by being compared in NCBI BLAST after sequencing, the nucleotide
Sequence and the homology of CV-A6 are 75%, according to parting standard, can determine whether that the virus is CV-A6.
(3) different passages strain viral genome stabilities and sequence compare
Virus is virus genomic by harvesting the virus harvest liquid of P1 generation to P15 generation in KMB17 cell secondary culture
Nucleic acid extraction is carried out by Axygen Body Fluid viral DNA/RNA Miniprep Kit extracts kit operation manual,
The primer and VP1 base of 5 ' non-coding region genes and 3 ' Noncoding genes are directed to according to the design synthesis of CV-A6 virus gene sequence
Because of two pairs of primers, viral gene RNA reverse transcription is tried using the One-step RNA PCR kit (AMV) that TaKaRa company produces
Agent box is carried out by operation manual, after being sequenced according to " walk method ", using MEGA software to the full base group sequence of different generations
Column are compared, and consensus sequence is using this laboratory to complete acquired in the 2nd generation that the isolated strain is inoculated with KMB17 and adapts to
Gene order, and compare the gene order of 2,5,10 and 15 generation viruses finds nucleotide between them and amino acid sequence
Homologous to be up to 99.6% and 100% respectively, this prompts the strain genetic stability preferable.
The people of CV-A6 virus seed culture of viruses of the present invention preparation is with CV-A6 inactivated vaccine, including following component: CV-A6 antigen
100 μ g/ml, aluminium hydroxide 1mg/ml；People CV-A6 inactivated vaccine the preparation method is as follows:
(1) Strain is inoculated in human diploid cell strain KMB17, after adapting to, and passes through plaque three times on KMB17 cell
Purifying obtains the clone strain for being identified as CV-A6.The clone strain is used as primordial seed after passing for 4 generations in KMB17 cell, after
Resumed for 3 generations i.e. as main seed, then passing for 3 generations again is the seed that works.
(2) identification experiment, one step growth experiment, drop are carried out respectively to the seed culture of viruses of primordial seed, main seed and the seed that works
The detection such as degree, sterile, mycoplasma and immunogenicity, shows to have good immunogenicity and have on KMB17 cell
Good conformity proliferative capacity.
(3) vertification regulation is produced according to KMB17 cell, selects the 28th fine and close generation KMB17 cell of growth, abandons culture solution,
Cell face is cleaned with PBS solution, abandons washing lotion.
(4) it is inoculated with the Working viral seed of 0.01MOI in the cell face of KMB17 cell, 37 DEG C are placed after mixing,
The a certain amount of viral maintaining liquid (added amount is determined according to the size of culture bottle) without serum is added, in 37 DEG C of 3-5 in 30min
It, viral CPE(cytopathic effect) up to 90% or more harvest virus liquid.
(5) formaldehyde is added by 1:4000 in the virus harvest liquid and presses 1:2000 in 37 DEG C of 3 days or virus harvest liquids
Beta-propiolactone is added in 4 DEG C of 2 days inactivation of viruses, is concentrated by ultrafiltration to the 5% of original volume, it is purified to obtain refined solution, identified institute
The purity for stating refined solution is higher than 96%, measures its protein concentration not less than 95% using Lowry method；
(6) by after above-mentioned refined solution filtration sterilization, 100 μ g/ml are adjusted to PBS solution, aluminium hydroxide 1mg/ml is to get CV-A6
Inactivated vaccine finished product.
At present vaccine principal mode be attenuated vaccine and two kinds of inactivated vaccine, but attenuated vaccine there are virulence reply reversion and
Vaccine related disease can be caused, and inactivated vaccine can then overcome this disadvantage.The present invention is in addition to filtering out one plant of immunogenicity and peace
Outside the preferable strain of full property, used cellular matrix --- KMB17 is also very crucial as viral vaccine production carrier.At present
Cellular matrix for vaccine for man is mainly two training body cells of African green monkey kidney cell (Vero cell) and human embryo lung (HEL), such as
2BS and KMB17.As vaccine for man, cellular matrix of the cell from people as its production is selected, can not only be provided good
Good quality, and there is better safety.Therefore, CV-A6 of the present invention is to be located away from CV-A6 grave infection children
Separation strains, not only there is preferably proliferation and adaptability in human archeocyte KMB17, but also can also generate on mouse good
Immunogenicity.KMB17 is the distinctive source of people source of applicant's unit and approved vaccine for man production cellular matrix.In addition it selects
D3 hypotype strain is vaccine strain in the genotype of China's Major Epidemic CV-A6 virus at present, keeps vaccine more targeted, exempts from
Epidemic disease better effect.
In CV-A6 virus purification of the present invention and cultural method, pass through improvement tradition CV-A6 virus purification and culture
Method, using the MEM cell maintenance medium for being free of calf serum, 37 DEG C are cultivated and are observed cytopathy, increase the culture effect of CV- A 6
Rate.
CV-A6 seed culture of viruses strain YNKA1205 of the present invention was protected on 08 20th, 2018 to Chinese microorganism strain
It hides administration committee's common micro-organisms center (CGMCC) and submits preservation, number: CGMCC No.16217, depositary institution address
Are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Compared with the prior art, the invention has the characteristics that:
1. providing a kind of seed culture of viruses that can be used for people's CV-A6 inactivated vaccine, vaccine there is no to list at present；
CV-A6 inactivated vaccine method is prepared as cellular matrix 2. providing one kind and training body cell using KMB17 source of people two, experiment shows this
Vaccine has good immunity and safety；
3. in CV-A6 virus purification of the invention and cultural method, by improvement tradition CV-A6 virus purification using the training that suspends
The method of supporting, and the viral maintaining liquid without calf serum is used in Virus culture, increase CV-A6 separation and inactivated vaccine production effect
Rate.
Detailed description of the invention
Fig. 1 a is CV-A6 strain YNKA1205 at KMB17 cell generation pathological change form (after kind poison for 24 hours)；
Fig. 1 b is CV-A6 strain YNKA1205 at KMB17 cell generation pathological change form (48h after kind poison)；
Fig. 1 c is CV-A6 strain YNKA1205 at KMB17 cell generation pathological change form (72h after kind poison)；
Fig. 1 d is CV-A6 strain YNKA1205 at KMB17 cell generation pathological change form (96h after kind poison)；
Fig. 2 is the plaque that CV-A6 seed culture of viruses YNKA1205 is formed on KMB17 cell (arrow meaning is the plaque to be formed)；
Fig. 3 is CV-A6 strain YNKA1205 in KMB17 cell previous step growth curve；
Fig. 4 is the neutralize antibody titers of the primordial seed, main seed and the seed that works.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and examples.
Embodiment one: separation, adaptation and the purification process of strain involved in the present invention
(1) Virus Sample is obtained
The hand-foot-and-mouth disease that collection pediatric hospital is accredited as CV-A6 infection suffers from excreta sample, and 1g excrement is taken to be resuspended in 5mL physiology salt
In water, 3000g, which is centrifuged, takes supernatant for 30 minutes, and uses 0.45 μm of filter (being purchased from Millipore company) filtering, -20 DEG C of guarantors
It deposits spare.
(2) adaptation of the virus on RD cell
The RD cell for having grown up to fine and close single layer is selected, old culture solution is abandoned, cleans cell surface with appropriate PBS solution, is added appropriate
0.125% trypsin digestion and cell, cell bottle to be patted, cell are slided from bottle wall drift sand sample, and cell culture fluid is added, presses
The every hole 2ml is inoculated with 24 orifice plates, and 200 μ l of filtered sample is inoculated in cell face, after 37 DEG C are cultivated 3~4 days, observes cytopathy
Situation virus CPE freezes spare up to 90% or more harvest virus.The CV-A6 separation method passes through improvement tradition CV-A virus purification
Method is centrifuged using 3000g and takes within 30 minutes supernatant, and higher virus recycling yield and impurity-eliminating effect, and the training that suspends are obtained
It supports, to increase virus purification efficiency.
(3) adaptation of the virus on KMB17 cell
The P23 for having grown up to fine and close single layer is selected for KMB17 cell, old culture solution is abandoned, cleans cell surface with appropriate PBS solution,
Appropriate 0.125% trypsin digestion and cell, cell bottle to be patted is added, cell is slided from bottle wall drift sand sample, and cell training is added
Nutrient solution, by 2 × 105Cell/bottle is inoculated with small square vase and 200 μ l of CV-A6 virus is inoculated in cell face after 37 DEG C are cultivated 3~4 days,
37 DEG C adsorb 1 hour, the MEM cell maintenance medium for being free of calf serum are then added, 37 DEG C are cultivated and observe cytopathy situation
Viral CPE freezes spare up to 90% harvest virus.CV-A6 culture and adaptive method pass through the cultural method of improvement, in KMB17
Cell adapted middle use is free of the viral maintaining liquid of calf serum, and 37 DEG C are cultivated and observed cytopathy, to improve CV-A6 training
Support efficiency.
(4) clone purification of virus stain
The CV-A6 virus harvest liquid passed on to the 5th generation has been adapted on KMB17 cell, is diluted to 10-3、10-4、10-5Three
Dilution is inoculated in six well culture plate of KMB17 cell for having formed single layer, and 37 DEG C adsorb 1 hour, is added without calf serum
Viral maintaining liquid removes culture solution after 48 hours, be added and be inverted in 37 DEG C of trainings after agar solidified containing 0.8% agarose-MEM
It supports, in microscopic observation plaque after 3 days, draws out plaque with pipette tips, be placed in 1.5ml centrifuge tube, 0.5mlMEM maintaining liquid is added,
Vortex oscillation is inoculated in KMB17 cell monolayer immediately, and 37 DEG C are cultivated 3~4 days, and virus, harvest virus are harvested after cytopathy
Continue to be inoculated in the growth passage of KMB17 cell.
(5) prepared by seed culture of viruses
The clone strain for being identified as CV-A6 is passed in KMB17 cell after 4 generations i.e. as primordial seed, after resuming 3 generations i.e. conduct
Main seed, then passing for 2 generations again is the seed that works.
The culture of two: CV-A6 strain YNKA1205 of embodiment and vaccine preparation method
(1) virus is in KMB17 cell culture
The P28 for having grown up to fine and close single layer is selected for KMB17 cell, old culture solution is abandoned, cleans cell surface with appropriate PBS solution,
Appropriate 0.125% trypsin digestion and cell, cell bottle to be patted is added, cell is slided from bottle wall drift sand sample, and cell training is added
Nutrient solution, by 2 × 105Cell/bottle is inoculated with small square vase and 200 μ l of CV-A6 virus is inoculated in cell face after 37 DEG C are cultivated 3~4 days,
37 DEG C adsorb 1 hour, the MEM cell maintenance medium for being free of calf serum are then added, 37 DEG C are cultivated and observe cytopathy situation
Viral CPE freezes spare up to 90% harvest virus.Attached drawing 1a-1d is CV-A6 strain in KMB17 cell generation pathological change form
Cytopathy, third day cytopathic effect i.e. reachable 90% takes place for inoculation second day in (culture 4 days).
(2) virus titer measures
Seed culture of viruses carries out 10 times and is serially diluted, and each dilution adds 8 holes, every 100 μ l of hole, by containing 2 × 105A/mlRD cell, often
100 μ l cell suspension of hole adds to each 8 hole of dilution, and 37 DEG C are cultivated 5~7 days, and observation presses Reed- as a result, result calculates
Muench Method formula calculates；The infection titer of the seed culture of viruses can reach 7.25-75CCID50/L.Fig. 3 is that CV-A6 exists
KMB17 cell previous step growth curve, in third day, i.e., arrival logarithmic growth phase, infection titer reach highest
7.25CCID50/L。
(3) viral purification
Formaldehyde is added by 1:4000 in virus harvest liquid, and 37 DEG C of 3 days inactivation of viruses are concentrated by ultrafiltration to the 5% of original volume, chromatographic column
Purifying, the purity through electrophoresis, HPLC identification viral purification liquid are higher than 96%, measure its protein concentration using Lowry method.
(4) prepared by finished product
After above-mentioned refined solution filtration sterilization, 100 μ g/ml, aluminium hydroxide 1mg/ml are adjusted to PBS solution, which is institute
The CV-A6 inactivated vaccine finished product needed.
Embodiment three: the identification method of seed culture of viruses
(1) sterility test and detection of mycoplasma experiment
THIOGLYCOLLIC ACID salt broth 4 is taken, pancreas junket soya peptone fluid nutrient medium 2, every 0.5 ml of inoculation is viral.Inoculation
2 30~35 DEG C of cultures of THIOGLYCOLLIC ACID salt broth afterwards；THIOGLYCOLLIC ACID salt broth 2 and pancreas junket soya peptone liquid
Body culture medium 2 are set 20~25 DEG C of cultures；It is operated simultaneously using virus-culturing fluid with method as negative control.Culture determines after 14 days
As a result.Mycoplasma inspection semisolid culturemedium and broth bouillon each 4 are taken, semisolid culturemedium boils thawing, is cooled to
56℃.Go out energy calf serum and the dual anti-mixed liquor 2.5ml with the mixing of 4:1 ratio in advance is added in two kinds of culture mediums every, fast
Speed shakes up, and then every culture medium is added sample to be examined 0.5ml and makees originally culture, observes in 35 DEG C of cultures.When to the 7th day,
Two kinds of primary culture mediums respectively take out 2 transferred speciess, each transferred species of every culture medium culture medium of the same race 2, and 0.5ml/ branch is cultivated in 35 DEG C
Observation was to 21 days；It is primary to continue culture observation to 21 days.
(2) pathogenicity is observed
Mouse intracranial inoculation 10 in 1-2 days after taking-up is raw5CCID50Virus quantity.Observation 2 times daily, and record the fiber crops of clinical appearance
Numbness death condition.It observes 14 days altogether.What is occurred in 24 hours causes paralysis or died to give as null mice because injection damages
It deletes.According to clinical observation result, calculates 4 indexs and evaluated: (1) half paralyzing dose (PD50)；(2) median lethal
Dosage (LD50), it is calculated by Spearman-karber method；(3) average Clinical score (MCS).It is calculated by 3 score values: 0 point of expression
Without paralysis sample symptom during observation；1 point of expression was once benumbed, but remained to survival to expiring；2 points of expressions are dead or seriously benumb
Midway is needed to put to death.(4) there is day (MFT) in average clinical symptoms.There is clinical paralysis symptom to the 1st time after pointed injection sample
Day.
(3) identification experiment
Virus genomic nucleic acid extraction presses Axygen Body Fluid viral DNA/RNA Miniprep Kit manipulator
Volume carries out, and viral gene RNA reverse transcription uses One-step RNA PCR kit (AMV) reagent of TaKaRa company production
Box is carried out by operation manual, and by comparing in NCBI BLAST after sequencing, the homology of the nucleotide sequence and CV-A6 are
75%, according to parting standard, it can determine whether that the virus is CV-A6.
(4) different passages strain viral genome stabilities and sequence compare.
Virus is by harvesting virus harvest liquid of the P1 generation to P15 generation, viral gene in KMB17 cell secondary culture
The nucleic acid extraction of group presses Axygen Body Fluid viral DNA/RNA Miniprep Kit extracts kit operation manual
Carry out, according to CV-A6 virus gene sequence design synthesis for the primer of 5 ' non-coding region genes and 3 ' Noncoding genes and
The One-step RNA PCR kit that VP1 gene two produces primer, viral gene RNA reverse transcription using TaKaRa company
(AMV) kit is carried out by operation manual, after being sequenced according to " walk method ", using MEGA software to the complete of different generations
Base group sequence is compared, and consensus sequence is inoculated with KMB17 to the isolated strain using this laboratory and the 2nd generation adapted to is obtained
The complete genome sequence taken, and compare the gene order of 2,5,10 and 15 generation viruses.
Example IV: the immunology detection of inactivated vaccine
(1) after the viral purification liquid for obtaining purification, after selection kit is to the detection of its viral antigen content quantitative or protein quantification,
The immunizing dose group being serially diluted is set, is injected intraperitoneally, immunized mice, in 2,4,6,8 weeks detection neutralizing antibodies, compare,
Evaluate immunogenicity and antigenicity.
(2) detection of neutralize antibody titers: by viral dilution to 100CCID50The 1:4,1:8,1:16,1 of/ml and equivalent:
After 32,1:64 and 1:128 immune serum mixed in equal amounts, 37 DEG C of water-baths 60 minutes are set, are inoculated with RD cell, 35 DEG C are cultivated 5~7 days
Determine result.Cell-free lesion is the positive；It sets serum simultaneously and cell controls is feminine gender.
(3) observation group is immunized in experiment seedling
Two weeks after vaccine inoculation, surrounding detect its antibody level, booster immunization is primary after four weeks if necessary, periodic detection antibody
Potency evaluates the dynamic change characterization of antibody response.
(4) female to pass antibody suckling mouse protection test group
Need to design dosage grouping according to experiment, grouping is immune, and every group of immune adult mice female mice 4, male mouse 2 separates feeding
It supports, after just exempting from four weeks, carries out booster immunization, two exempt from after a week, and female mice 4, male mouse 2 are raised with nest, give birth to suckling mouse to female mice
Afterwards, using one age in days suckling mouse of lethal viral dosage LD50 intracerebral injection, observation, the clinical paralysis death condition occurred of record are commented
Valence mother passes antibody suckling mouse protective effect.The result shows that female antibody that passes can protect suckling mouse from infection CV-A6 virus.
SEQUENCE LISTING
<110>China Medical Sciences Academy Medical Biology Institute
<120>CV-A6 virus stain YNKA1205 whole genome sequence
<130> PCT/CN2018/111833
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 7442
<212> DNA
<213> coxsackievirus A6
<400> 1
TTAAAACAGCCCTGTGGGTTGTACCCACCCACAGGGCCCACTGGGCGCTAGCACACTGATTCTATGGAATCT
TTGTGCGCCTGTTTTATAACCCCTTCCCCAAAACTGTAACTTAGAAGAATATCACACTACCGATCAATAGCAGGCA
TGGCGCGCCAGTCATGTCTAGATCAAGCACTTCTGTCTCCCCGGATTGAGTATCAATAGACTGCTAGCGCGGTTGA
AGGAGAAAACGTCCGTTACCCGGCTAACTACTTCGAGAAACTTAGTAGCACCATTGAAGCTGCGGAGTGTTTCGCT
CAGCACTCTCCCAGTGTAGATCAGGTCGATGAGTCACTGCACTCCCCACGGGCGACCGTGGCAGTGGCTGCGTTGG
CGGCCTGCCTATGGGGCAACCCATAGGACGCTCTAAAGTGGACATGGTGCGAAGAGTCTATTGAGCTAGTTAGTAG
TCCTCCGGCCCCTGAATGCGGCTAATCCTAACTGCGGAGCACATGCCCTCAATCCAGGGGGTGGTGTGTCGTAACG
GGCAACTCTGCAGCGGAACCGACTACTTTGGGTGTCCGTGTTTCCTTTTTTTCTTATATTGGCTGCTTATGGTGAC
AATTGAGAGATTGTTACCATATAGCTATTGGATTGGCCATCCAGTGACAAACAGAGCTTTGATATACTTGTTCGTG
GGTTTCGTTCCACTCATCAGTCGTACAGTTCATACTTTAAAGTACATTCTGATTCTGAACAATAGAAAATGGGCGC
CCAAGTTTCAACAGAAAAATCTGGGTCGCACGAGACAAAGAATGTAGCGACCGAAGGGTCTACTATCAACTTCACC
AACATTAATTACTATAAGGATTCTTATGCAGCGTCAGCTAGTAAACAGGACTTTGCACAAGATCCTGCAAAGTTCA
CACGCCCTGTCTTGGATACCATCAGGGAGGTTGCAGCCCCCTTGCAATCCCCTTCTGTTGAGGCGTGCGGTTATAG
TGACCGAGTCGCACAGTTGACTGTGGGCAACTCAACCATTACTACCCAAGAGGCAGCCAACATTGTGTTGAGTTAC
GGAGAGTGGCCAGAATATTGTCCCTCCACGGACGCTACAGCTGTGGACAAACCTACTCGCCCTGACGTGTCAGTAA
ATAGGTTCTACACACTGTCAACTAAGAGTTGGAAGACAGAATCTACTGGCTGGTACTGGAAATTCCCTGATGTGCT
AAACGACACAGGAGTGTTCGGTCAAAACGCCCAATTCCACTACTTGTACCGCTCGGGTTTCTGCATGCACGTTCAG
TGCAATGCAAGCAAGTTCCATCAGGGGGCCCTCTTAGTGGCTGCAATCCCCGAGTTTGTGATTGCTGCAAGCAGCC
CTCCCGCGAAGCCTAATGGACGAGGGTTATACCCAGATTTCACTCACACTAACCCAGGTAAAAATGGCCAAGAGTT
TCGAGATCCTTATGTCTTGGATGCTGGTGTCCCCCTAAGTCAAGCACTGGTTTACCCCCATCAATGGATCAATCTA
CGAACTAATAACTGCGCGACCATCATCATGCCCTATGTCAATGCGCTTCCATTTGATTCGGCGCTTAACCACTCAA
ATTTTGGATTGGTTGTGATCCCTATTAGCCCTTTAAAATATTGTAATGGAGCTACCACAGAGGTGCCAATCACACT
AACTATTGCCCCACTTAACTCGGAGTTTAGCGGCCTCCGACAAGCAATAAAACAAGGGTTTCCCACAGAGCTTAAG
CCTGGGACCAATCAATTTCTTACAACTGACGACGGGACATCCCCACCAATACTGCCCGGTTTTGAACCAACTCCAT
TGATTCACATTCCTGGCGAGTTCACTTCTTTGTTAGATTTGTGCCAAATAGAAACCATACTAGAAGTCAATAATAC
CACTGGCACCACCGGGGTCAATAGATTACTAATCCCCGTTCGAGCACAGAACAATGTGGACCAGTTGTGCGCATCA
TTCCAAGTAGACCCTGGGCGCAATGGCCCGTGGCAATCCACAATGGTCGGTCAGATCTGCAGGTATTACACTCAAT
GGTCAGGTTCTCTTAAGGTAACCTTTATGTTCACGGGTTCTTTTATGGCCACAGGGAAAATGCTGATAGCCTACAC
ACCACCTGGTAGTGCTCAGCCCGCTACAAGGGAAGCAGCAATGCTTGGGACTCATATAGTGTGGGATTTTGGTTTG
CAATCATCGGTTACCCTAGTTATACCTTGGATTAGTAATACTCATTTTAGAGCAGTTAAGACTGGAGGGGTATATG
ACTACTACGCAACCGGGATTGTCACCATTTGGTACCAAACCAACTTTGTAGTGCCACCAGACACCCCCACTGAGGC
TAATATTATAGCTCTTGGAGCAGCACAGAAAAACTTTACTCTAAAGTTGTGCAAGGACACTGACGAGATCCAGCAA
ACAGCAGAGTACCAAAATGATCCCATTACAAATGCAGTGGAAAGCGCTGTGAGCGCGCTTGCTGACACCACAATAT
CCCGGGTGACCGCAGCTAACACTGCAGCTAGCACCCACTCCCTGGGAACAGGGCGTGTACCAGCATTGCAAGCCGC
AGAAACGGGAGCAAGCTCTAATGCTAGTGATGAGAACCTTATTGAGACTCGCTGTGTGATGAATCGAAACGGGGTT
AATGAGGCGAGTGTGGAACACTTTTACTCTCGTGCAGGGCTGGTAGGAGTTGTGGAGGTGAAGGACTCGGGCACTA
GCCCGGATGGGTACACAGTCTGGCCCATAGATGTGATGGGCTTCGTGCAACAGCGGCGCAAGCTAGAGCTGTCAAC
ATACATGCGCTTTGATGCCGAGTTCACTTTTGTGTCCAACCTCAGTGACAGTACGACGCCCGGGATGCTGCTGCAG
TATATGTATGTACCACCAGGGGCTCCTAAGCCGGATAGCAGGAAATCATATCAATGGCAGACTGCTACTAACCCGT
CGGTATTTGCAAAATTGAGAGATCCACCCCCCCAGGTATCTGTTCCGTTCATGTCGCCAGCAACAGCTTATCAGTG
GTTTTATGATGGTTACCCTACATTTGGTGAGCACAAACAAGCTACCAATTTGCAATATGGGCAGTGTCCTAATAAC
ATGATGGGCCATTTTGCCATCCGAACAGTTAGTGAATCTACCACCGGGAGAAACGTCCACGTTCGGGTGTACATGA
GAATTAAGCACGTGAGAGCTTGGGTACCTAGACCCCTTCGATCCCAAGCTTATATGGTCAAGAACTACCCGACATA
CAGCCAAACAATAACTAACACTGCAACTGACCGTGCAAGCATAACCACCACGGATTATGAAGGCGGGATACCAGCA
AACCCACAAAGGACATTTGGTAGGTTTGGCCAACAGTCCGGGGCTATCTATGTAGGTAACTTCAGAGTGGTGAACC
GACACCTCGCCACTCATAATGATTGGGCAAATCTAGTATGGGAAAGTAGCTCACGAGATCTTCTGGTGTCCTCCAC
CACTGCTCAGGGATGTGATACCATTGCCCGATGTGATTGTCAAACAGGAGTGTATTACTGCAACTCTAGAAGGAAA
CACTACCCGGTTAGTTTTTCTAAGCCTAGCCTCGTCTTCGTGGAAGCTAGTGAGTATTACCCTGCCAGGTATCAGT
CGCACCTCATGCTTGCGAAGGGACATTCTGAACCCGGGGACTGTGGTGGCATTCTTAGGTGCCAACATGGCGTGAT
TGGTATCGTGTCCACTGGTGGTAATGGACTTGTCGGATTTGCAGATGTCAGAGATCTTTTGTGGCTGGATGAAGAA
GCTATGGAACAGGGTGTGTCAGATTACATCAAAGGGCTTGGTGACGCATTCGGAACTGGTTTTACTGATGCAGTGT
CTAGGGAGGTGGAGGCTCTTAAGAACTACCTTATAGGATCTGAAGGGGCTGTTGAAAAGATCTTGAAGAATTTAAT
TAAATTGATCTCAGCATTAGTCATAGTGATCAGAAGTGATTATGACATGGTAACCCTCACAGCAACCTTGGCACTC
ATAGGGTGTCATGGCAGCCCCTGGGCGTGGATCAAGGCTAAGACAGCATCCATCTTAGGCATCCCTATCGCCCAGA
AGCAGAGTGCGTCATGGCTTAAGAAGTTTAACGACATGGCCAATGCTGCCAAGGGATTTGAGTGGATTTCCAATAA
GATTAGCAAATTTATTGATTGGCTTAAGGAGAAAATTATACCAGCAGCTAGAGAGAAGGTTGAGTTTTTGAACAAC
CTAAAACAACTGCCATTGTTGGAGAACCAAATCTCAAACCTGGAGCAGTCCGCCGCTTCGCAAGAAGACCTTGAAG
CAATGTTTGGGAATGTATCGTATCTCGCTCACTTCTGCCGTAAATACCAACCACTTTATGCTACAGAAGCCAAAAG
AGTTTATGCTTTGGAAAAGAGGATGAACAATTACATGCAGTTCAAGAGCAAACACCGTATTGAACCTGTATGTCTT
ATCATCAGAGGCTCCCCAGGTACCGGAAAGTCCTTGGCAACCGGTATAATTGCCCGAGCAATAGCTGACAAGTACC
ACTCTAGTGTGTACTCACTTCCGCCAGATCCAGACCACTTTGATGGTTACAAACAGCAAGTGGTCACAGTTATGGA
CGATCTATGCCAAAATCCTGATGGCAAGGATATGTCACTCTTTTGCCAGATGGTATCCACCGTAGATTTTATCCCA
CCAATGGCTTCTTTAGAAGAGAAAGGGGTCTCATTCACATCTAAATTTGTTATTGCATCCACTAATGCTAGCAATA
TCATAGTGCCAACAGTGTCTGATTCTGATGCTATCCGCCGCAGGTTCTATATGGACTGCGACATCGAGGTGACGGA
CTCATATAAAACAGATTTGGGTAGGTTAGACGCTGGAAGAGCTGCCAAATTATGCTCTGAAAATAACACAGCAAAC
TTCAAACGCTGCAGCCCACTAGTGTGTGGGAAGGCCATCCAATTAAGAGATAGGAAGTCCAAAGTTAGATACAGTG
TGGATACGGTGGTTTCAGAGCTCATAAGGGAGTACAATAACAGATCTGCCATTGGAAACACAATTGAAGCGTTGTT
CCAGGGGCCACCCAAGTTTAGACCTATTAGGATTAGTCTTGAGGAGGCGCCAGCACCAGATGTTATTAGTGATCTT
CTTGCCAGTGTGGATAGTGAAGAGGTGCGCCAATACTGTAGAGACCAAGGTTGGATCATACCAGAAACCCCTACCA
ACGTTGAGCGACATCTAAGTAGGGCTGTGCTAATTATGCAATCCATCGCCACGGTCGTTGCAGTAGTCTCACTAGT
GTATGTTATCTACAAACTTTTTGCTGGATTTCAGGGTGCATATTCTGGCGCTCCTAAGCAAGTGCTCAAGAAACCT
ATCCTCCGTACGGCAACAGTGCAGGGGCCTAGCCTTGATTTTGCCCTATCCCTACTGAGAAGGAACATCAGGCAGG
TTCAGACAGATCAAGGGCACTTCACTATGTTGGGTGTCAGGGATCGCTTAGCAGTTCTCCCGCGCCACTCACAGCC
CGGAAAAACAATCTGGGTGGAGCACAAACTCGTGAACATCCTGGATGCTGTCGAGTTGGTGGATGAGCAAGGGGTC
AATCTAGAGCTCACTCTAATCACTCTTGATACCAATGAGAAATTCAGAGATATCACCAAGTTCATTCCAGAAAACA
TCAGCGCTGCTAGTGACGCCACCCTAGTGATTAATACAGAACACATGCCCTCAATGTTTGTACCTGTGGGAGATGT
CGTACAATACGGTTTCCTGAATCTCAGTGGAAAACCCACCCACCGCACCATGATGTACAACTTCCCTACTAAGGCA
GGACAGTGTGGAGGAGTGGTGACATCAGTTGGAAAGGTTATTGGAATTCACATAGGAGGCAATGGTAGGCAAGGTT
TCTGTGCGGGACTTAAGAGGAGCTACTTTGCTAGTGAGCAAGGAGAGATCCAATGGGTAAAGCCTAACAAAGAAAC
TGGGAGACTTAACATCAACGGGCCAACTCGCACCAAGCTCGAACCTAGTGTGTTCCATGATGTCTTTGAGGGTAAC
AAGGAGCCAGCGGTCTTACATAGTAAGGACCCTCGCCTTGAGGTGGACTTCGAGCAGGCGTTGTTCTCTAAGTATG
TAGGGAACACACTCCATGAACCTGATGAGTATATCAGAGAAGCAGCCCTTCACTATGCAAATCAGTTGAAACAGCT
GGACATAGACACCACCCAGATGAGCATGGAGGAGGCTTGTTATGGGACAGACAATCTTGAAGCTATCGACCTTCAA
ACTAGTGCAGGTTATCCCTACAGTGCTCTGGGAATAAAGAAAAGAGATATTTTAGACCCTACCACCAGAGACGTCA
GTAGGATGAAGTTCTACATGGACAAGTATGGTCTCGACCTTCCATATTCTACCTATGTCAAAGACGAGCTTCGCTC
GATAGACAAGATCAAGAAAGGGAAGTCTCGTCTGATTGAGGCCAGCAGTTTGAATGACTCGGTCTATCTCAGAATG
GCCTTCGGGCACCTCTATGAAACTTTCCATGCAAATCCTGGGACAGTAACTGGCTCGGCTGTGGGGTGTAACCCAG
ATGTGTTTTGGAGTAAGCTACCAATTCTGCTCCCTGGGTCCCTCTTTGCCTTTGACTATTCAGGCTACGATGCTAG
CCTTAGCCCAGTTTGGTTTAGAGCACTGGAATTAGTCCTTAGAGAGATAGGCTATAGTGATGAGGCAGTCTCGCTC
ATTGAAGGAATCAACCACACGCACCATGTGTACCGTAACAAAACCTACTGCGTACTTGGTGGAATGCCCTCAGGTT
GCTCAGGAACATCCATCTTTAACTCAATGATTAATAACGTCATTATCAGAGCATTACTCATTAAAACATTCAAGGG
CATTGATCTGGATGAACTCAACATGGTTGCCTACGGGGACGATGTGCTCGCTAGTTACCCCTTTCCAATTGACTGC
CTAGAGCTAGCAAAAACAGGTAAGGAGTACGGTCTAACCATGACTCCTGCAGACAAGTCCCCTTGCTTCAATGAAG
TTAATTGGGAAAACGCAACCTTCCTCAAGAGAGGCTTCTTGCCTGATGAGCAATTTCCGTTTTTGATCCACCCCAC
CATGCCAATGAAGGAAATTCATGAATCCATTCGGTGGACCAAGGATGCACGCAATACTCAAGATCACGTGCGGTCC
CTATGCCTATTGGCGTGGCACAATGGTAAGCAAGAATACGAAAAATTTGTGAACTCAATTAGATCCGTCCCAGTAG
GAAGAGCATTGGCAATCCCCAATTATGAAAATCTGAGACGTAAGTGGCTCGAATTGTTTTAGAGGTTGAACAAACC
TCAACCCCACCAGAAATCTGGTCGTGAATATAACTGGTGGGGGTAAATTTGTTATACCCAGAATAGCAAAATCA 18
Claims (2)
1. a kind of CV-A6 virus seed culture of viruses, which is characterized in that the classification naming of the seed culture of viruses is 6 type of Coxsackie virus A group, preservation
Number is CGMCC No.16217；The preparation method of the seed culture of viruses of the CV-A6 virus the following steps are included:
(1) CV-A6 Strain is separated
Hand-foot-and-mouth disease severe childhood Feces of Patients 1g is taken to add 5ml physiological saline, suspension 3000xg is centrifuged 30min, and supernatant is through nothing
After bacterium filtering, it is inoculated in RD cell, after 3-5 days lesions, continues to pass on for 3 generations in RD cell, and is somebody's turn to do through RT-PCR and being sequenced
The CV-A6's of viral whole genome sequence and the amino acid sequence of supposition, the genotype of the Strain and current Chinese epidemic strain
Genotype is consistent；
(2) adaptation of the strain virus on KMB17 cell
The P28 for having grown up to fine and close single layer is selected for KMB17 cell, old culture solution is abandoned, cleans cell surface with PBS solution, is added
0.125% trypsin digestion and cell, cell bottle to be patted, cell are slided from bottle wall drift sand sample, cell culture fluid are added, by 2
×105Cell/bottle is inoculated with small square vase, and after 37 DEG C are cultivated 3~4 days, 200 μ l of CV-A10 virus obtained by step (1) are inoculated in carefully
Born of the same parents face, 37 DEG C adsorb 1 hour, the MEM cell maintenance medium for being free of calf serum are then added, 37 DEG C are cultivated and observe cytopathy
Situation, virus freeze spare up to 90% harvest virus；
(3) clone purification of virus stain
The CV-A6 virus harvest liquid passed on to the 5th generation has been adapted on KMB17 cell, is diluted to 10 with viral dilution-3、
10-4、10-5Three dilutions are inoculated in six well culture plate of KMB17 cell for having formed single layer, and 37 DEG C adsorb 1 hour, are added not
Viral maintaining liquid containing calf serum removes culture solution after 48 hours, be added and contain 0.8% agarose-MEM, after agar solidified,
It is inverted in 37 DEG C of cultures and draws out plaque with pipette tips in microscopic observation plaque after 3 days, be placed in 1.5ml centrifuge tube, be added
0.5mlMEM maintaining liquid, vortex oscillation are inoculated in single layer KMB17 cell immediately, and 37 DEG C are cultivated 3~4 days, after cytopathy
Harvest virus, same method carry out plaque purification twice again, obtain the purified colonies strain for being identified as CV-A6；The purifying gram
Grand strain is passed in KMB17 cell i.e. as primordial seed after 4 generations, and after resuming the 3 generations i.e. main seed of conduct, then passing for 3 generations again is
Work seed；This primordial seed, main seed and work seed are CV-A6 virus seed culture of viruses；
(4) seed culture of viruses of primordial seed obtained by step (3), main seed and the seed that works is identified respectively, shows the seed culture of viruses
All have good immunogenicity, genetic stability and the good conformity proliferative capacity on KMB17 cell.
2. a kind of people's CV-A6 inactivated vaccine of the preparation of the CV-A6 virus seed culture of viruses as described in claim 1, which is characterized in that described
Vaccine includes following component: CV-A6 antigen 1 00 μ g/ml, aluminium hydroxide 1mg/ml；The preparation of people CV-A6 inactivated vaccine
Method is as follows:
(1) vertification regulation is produced according to KMB17 cell, selects the 28th fine and close generation KMB17 cell of growth, abandons culture solution, use PBS
Solution cleans cell face, abandons washing lotion；
(2) it is inoculated with the Working viral seed of 0.01MOI in the cell face of the KMB17 cell, is placed in 37 after mixing
The viral maintaining liquid for being free of serum is added in DEG C 30min, in 37 DEG C 3-5 days, viral cytopathic effect is fallen ill up to 90% or more
Malicious harvest liquid；
(3) formaldehyde is added by 1:4000 in step (2) virus harvest liquid and presses 1 in 37 DEG C of 3 days or virus harvest liquids:
2000 be added beta-propiolactones in 4 DEG C 2 days, be concentrated by ultrafiltration to the 5% of original volume, it is purified that refined solution, purity are higher than 96%,
Its protein concentration is measured not less than 95% using Lowry method；
(4) by after step (3) the refined solution filtration sterilization, it is adjusted to 100 μ g/ml, aluminium hydroxide 1mg/ml with PBS solution, i.e.,
Obtain CV-A6 inactivated vaccine finished product.
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