CN109536461A - A kind of O-shaped foot and mouth disease virus mutant strain and its preparation method and application - Google Patents
A kind of O-shaped foot and mouth disease virus mutant strain and its preparation method and application Download PDFInfo
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- CN109536461A CN109536461A CN201811406871.8A CN201811406871A CN109536461A CN 109536461 A CN109536461 A CN 109536461A CN 201811406871 A CN201811406871 A CN 201811406871A CN 109536461 A CN109536461 A CN 109536461A
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Abstract
The present invention provides a kind of O-shaped foot and mouth disease virus mutant strains and its preparation method and application, belong to vaccine candidate strain technical field.O-shaped foot and mouth disease virus mutant strain is using rHN Strain as maternal Strain, amino acid following in the G-H ring of VP3 albumen is mutated: the 173rd Aspartic acid mutations are asparagine, and the 174th valine mutation is glutamic acid and the 179th asparagine mutation is cysteine.The virus mutation strain, which has genetic stability and obtains caveolin, mediates the ability for infecting CHO-K1 cell.By the cross-neutralization ability for detecting immuno positive serum, the results showed that compared with maternal Strain, the cross-protection ability for the foot and mouth disease virus neutralizing antibody that induction body generates is obviously improved for virus mutation strain, shows excellent antigen broad spectrum activity.The virus mutation strain should can be used for preventing the infection of O-shaped foot and mouth disease virus through the vaccine of inactivation preparation.
Description
Technical field
The invention belongs to vaccine candidate strain technical fields, and in particular to a kind of O-shaped foot and mouth disease virus mutant strain and its preparation
Methods and applications.
Background technique
Aftosa (foot-and-mouth disease, FMD) is by foot and mouth disease virus (foot-and-mouth
Disease virus, FMDV) a kind of urgency for causing the major livestocks such as pig, ox, sheep and other domestic, wild artiodactyls to be suffered from altogether
Property, hot, high degree in contact and can quickly long-distance communications deadly infectious disease.The disease is World Organization for Animal Health
One of the zoonosis of (office international des é pizooties, OIE) statutory report, China is also arranged
First of a kind of animal epidemic disease list [Ministry of Agriculture announce No. 1125,2008].Have from first time in 1546 more definite
Aftosa literature record since, the disease is existing to be still worldwide widely current for a long time, endangers huge.Although aftosa at
The lethality of year susceptible animal is not high (< 5%), but the disease infectiousness is extremely strong, and disease incidence may be up to 100%, due to the life of affected animal
Performance decline is produced, animal product is imported and exported international trade and is limited, and will cause heavy direct economic loss and indirect economy damage
Lose, and the latter be often the former decades of times so that hundred times (Rowlands DJ.Foot-and-Mouth Disease.2003).
For example, 2001, all therefore Britain surpasses parliament's general election of 8,000,000,000 pounds or even current year because of economic loss caused by aftosa epidemic situation
(Knowles NJ, et al.Outbreak of Foot-and-Mouth Disease Virus Serotype is postponed the time
O in the UK Caused by a Pandemic Strain.Vet Rec.2001,148:258–259).Therefore the disease is not only
The sustainable and healthy development of farming and animal husbandry or even entire national economy is seriously threatened, and will cause serious society's negative effect, element
There is the title of " political economy disease ".
China is that aquaculture big country and aftosa endanger more serious one of country.According to Ministry of Agriculture's statistical data,
Calculate that the loss of industrial chain year is exceeded 100 billion, (the economics research summary agricultural of Pu China Animal diseases prevention and control that the situation is tense for prevention and control
Economic problems .2006,6:61-64).It comes and goes increasingly frequent today in international trade, prevention and control, purification and the root of China's aftosa
Except not only to developing agricultural economy, ensure food safety, health plays a crucial role with health and maintenance country reputation, and to pushing away
The control of dynamic whole world aftosa is of great significance.
Foot and mouth disease virus is the Zoonotic virus that first case is found, and belongs to small (micro-) RNA virus section aftosa (sore) disease
Poison belongs to, and has O, A, C, Asia1, SAT1-3 totally seven serotype, rarely has immunological cross protection between each serotype, same serotype
There is also difference, mutual cross-protection (the Brown F.The History of such as or not antigenicity between different subtype
Research in Foot-and-Mouth Disease.Virus Res.2003,91:3–7).History generation in China's is current
Popular has O, A and Asia1 type foot and mouth disease virus, wherein the threat of O-shaped foot and mouth disease virus is maximum, A type takes second place, in May, 2009
Later again without Asia1 type aftosa clinical case (Bai X, et al.Evolution and Molecular
Epidemiology of Foot-and-Mouth Disease Virus in China.Chinese Sci Bull.2011,
56:2191–2201).Currently, the separated O-shaped foot and mouth disease virus in China further includes in addition to history representative strains Aksu of Xinjiang poison
Chinese classical type (Cathay), Pan Asia -1 (PanAsia-1) pedigree, Burma 98 (Mya98) pedigree, India 2001d
(IND2001d) branch (the aftosa Analysis on epidimic situation such as Liu Xiangtao China veterinary science, 2017,47 (S1): 45-49).
Vaccine immunity is the important means for preventing aftosa.The immune efficacy of aftosa vaccine mainly depends on vaccine seed culture of viruses
Ability (the Barchrach HL.Immune and of the humoral immunity level of excitation, i.e. B cell epitope induction neutralizing antibody
Antibody Responses to an Isolated Capsid Protein of Foot-and-Mouth Disease
Virus.Immunol.1975,115:1636–1641).In addition, the cellular immunity mediated by t cell epitope is in prevention aftosa
The later period of virus infection also plays a very important role the (Zhejiang the foot and mouth disease virus t cell epitope research overview such as Liu new life
Agricultural sciences, 2010, (5): 1113-1117).
The aftosa vaccine of the existing mature production technology process in China includes inactivated vaccine and synthetic peptide vaccine.Although synthesis
A possibility that peptide vaccine can evade inactivated vaccine there are viral escapes and malicious risk is dissipated, but it is inoculated with ox and can not obtain completely
Immunoprotection, be only test on pig body be successfully [Ministry of Agriculture announce No. 437,2004].Moreover, synthetic peptide is anti-
Original spectrum is narrow, immunity is limited, then when having new strain invasion, it is possible that immuning failure.Now from the point of view of (city), there are no energy
Compare favourably with " quality-high and inexpensive " of inactivated vaccine foot-and-mouth disease gene engineering vaccine (whole world Liu Xin aftosa Control Technology and
Pathogenic characteristic general research Scientia Agricultura Sinica .2015,48:3547-3564).European Union and some countries in South America pass through tens
The immunity inoculation of the foot and mouth disease virus inactivated vaccine in year, effectively controls aftosa, this is considered anti-with inactivated vaccine
Control mark post (Sobrino F, the et al.Foot-and-Mouth Disease Virus:a Long Known of aftosa
Virus,but a Current Threat.Vet Res.2001,32:1–30).Although foot and mouth disease virus has significant heredity
Variation property, but compared with A type foot and mouth disease virus, the antigenic variation of O-shaped foot and mouth disease virus is smaller, and vaccine strain can be resisted
Most of Field epidemic strains.For example, O-shaped aftosa vaccine strain used in the American-European and some countries and regions in Asia be only limitted to and
- 1 pedigree of Pan Asia belongs to the Middle East-South Asia topological type O1Manisa and IND R2/75 (Asia) and Europe-South America topological type
O1K, O1BFS (Europe) and O1Campos, O1Caseros (South America), however seed culture of viruses is used in the production of the O-shaped aftosa vaccine in China
And its component is many and diverse various (Beijing Xie Qingge aftosa: Chinese agriculture publishing house .2004), this innate immune pressure and outer
The folded rush effect of boundary's environmental factor accelerates quasispecies advantage positive selection of foot and mouth disease virus under the conditions of being resistant to naturally, very unfavorable
Purification and elimination (Woodbury EL, et al.Analysis of Mixed Foot-and-Mouth in aftosa
Disease Virus Infections in Saudi Arabia:Prolonged Circulation of an Exotic
Serotype.Epidemiol Infect.1994,112:201–211)。
Complete 146S virion needs such as dendron when invading body as most effective antigen in inactivated foot-and-mouth disease vaccine
The assistance of the antigen presenting cells such as shape cell (dendritic cells, DC) (antigen presenting cells, APC),
And (leaching is delivered to by major histocompatibility complex (major histocompatibility complex, MHC) molecule
Bar) cell surface, important target cell of the Dendritic Cells as foot and mouth disease virus inactivated vaccine, it is anti-for adjusting immunity of organism
It is necessary (Bayry J, et al.Interaction of Foot-and-Mouth Disease Virus with for imperial
Dendritic Cells.Trends Microbiol.2006,14:346–347).The foot and mouth disease virus of inactivation passes through VP1G-H
Arginine-glycine-aspartic acid (the Arginine-Glycine-Aspartic acid, RGD guarded in ring；145th~
147, for O-shaped) integrin (integrins, Ins) of three peptide motifs and cell surface combines, starts and activate not
Mature ox bone marrow Dendritic Cells apoptosis series of signals access, can be detrimental to immune response (Jin H, et
al.Induction of Immature Dendritic Cell Apoptosis by Foot and Mouth Disease
Virus is an Integrin Receptor Mediated Event before Viral Infection,J Cell
Biochem.2007,102:980–991).But the foot and mouth disease virus that mature monocytic dendritic shape cell combines integrin
It infects and insensitive, the immune complex of neutralization or the foot and mouth disease virus of inactivation still have the ability for identifying immunocyte
(Robinson L,et al.Foot-and-Mouth Disease Virus Exhibits an Altered Tropism in
the Presence of Specific Immunoglobulins,Enabling Productive Infection and
Killing of Dendritic Cells.J Virol.2011,85:2212–2223).Therefore, using Dendritic Cells as target
Mark cell helps to develop effective foot-and-mouth disease virus resistant vaccine.Although mutual with Dendritic Cells about foot and mouth disease virus at present
The molecular mechanism of effect is also not very clear, still, the endocytosis side that Heparan sulfate (heparan sulfate, HS) mediates
Formula is maximally efficient for internalization of the foot and mouth disease virus in Dendritic Cells acts on, and Dendritic Cells present antigen is to leaching
Bar cell, the special IgG reaction of induction foot and mouth disease virus.The virus and its infectious RNA that non-sulfuric acid heparan combines are to tree
Prominent shape cell combination also can induce specific immune response (Harwood LJ, the et al.Dendritic Cell of reduced levels
Internalization of Foot-and-Mouth Disease Virus:Influence of Heparan Sulfate
Binding on Virus Uptake and Induction of the Immune Response.J Virol.2008,82:
6379–6394)。
Foot and mouth disease virus utilizes the external capsid protein that the molecular basis of cell surface receptor is positioned at virion surface
Amino acids characteristic sequence or structural domain in VP1-3.Wild type foot and mouth disease virus identifies integrin, warp using RGD motif
Clathrin-mediated endocytosis action path infects sensitive organization's cell (O'Donnell V, et al.Analysis of
Foot-and-Mouth Disease Virus Internalization Events in Cultured Cells.J
Virol.2005,79:8506–8518).And cell toxicant can be gradually adsorbed onto carefully in adaptation process by Heparan sulfate
Cellular surface (Bai X, et al.Effects of Two Amino Acid Substitutions in the Capsid
Proteins on the Interaction of Two Cell-adapted PanAsia-1 Strains of Foot-
and-Mouth Disease Virus Serotype O with Heparan Sulfate Receptor.Virol
J.2014,11:132), certain foot and mouth disease virus cell adapted strains can also obtain the ability using Heparan sulfate as receptor
(Jackson T,et al.Efficient Infection of Cells in Culture by Type O Foot-and-
Mouth Disease Virus Requires Binding to Cell Surface Heparan Sulfate.J
Virol.1996,70:5282–5287).The cell adapted poison of Heparin-sensitive type mediates it to complete cell internalization by caveolin
Process (O'Donnell V, et al.Heparan Sulfate-Binding Foot-and-Mouth Disease Virus
Enters Cells via Caveola-mediated Endocytosis.J Virol.2008,82:9075–9085).Cause
Most of the amino acid variation of Receptors of Foot-and-mouth type conversion is located at five solid axles, triad close to foot and mouth disease virus particle
With (Sobrino F, et al.Foot-and-Mouth Disease Virus:Current on pentamer (two solid axles) interface
Research and Emerging Trends.Norfolk:Caister Academic Press.2017)。
China can utilize Heparan sulfate receptor pathway for the vaccine strain OZK/93 of O-shaped aftosa most worthy
Infecting host cell BHK-21 and CHO-K1, (the O-shaped Pan Asia 1 in the China Bai Xingwen is the molecule of foot and mouth disease virus biological phenotype difference
Basic Beijing: Chinese Academy of Agricultural Sciences .2012), but it the O-shaped foot and mouth disease virus of a small number of hypotypes is not achieved it is complete intersection
The immune effect of protection.It is certain also on host cell using the foot and mouth disease virus cell adapted strain of Heparan sulfate receptor
It can show to cause weak virulence phenotype (but except OZK/93 etc.).Using reverse genetic manipulation to foot and mouth disease virus B cell table
Position, i.e., several amino acid progress rite-directed mutagenesis in antigen site can expand aftosa vaccine strain spectrotype, and [Liu is in new equal with instead
Aftosa vaccine strain spectrotype and vaccine preparation method .ZL201010256781.22013.01.09 are expanded to genetic manipulation].But
The genetic engineering poison rHN and its vaccine candidate seed culture of viruses lose ability (the Li P, et for infecting CHO-K1 cell
al.Evaluation of a Genetically Modified Foot-and-Mouth Disease Virus Vaccine
Candidate Generated by Reverse Genetics.BMC Vet Res.2012,8:57), and foot and mouth disease virus
Certain (a) key amino acid variations in T/B cell epitope are severely limited to the double action of antibody response and cell absorption
(Mateu MG,et al.A Single Amino Acid Substitution Affects Multiple Overlapping
Epitopes in the Major Antigenic Site of Foot-and-Mouth Disease Virus of
Serotype C.J Gen Virol.1990,71:629-637), and then kind of poison may be significantly affected and produced in aftosa vaccine
With the cross-protection ability of replication performance and virus titer and its neutralizing antibody in BHK-21 cell.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of O-shaped foot and mouth disease virus mutant strain and preparation method thereof and answering
With the amino acid being mutated in the O-shaped foot and mouth disease virus mutant strain is located on non-T/B cell epitope (such as VP3G-H ring), the O
Type foot and mouth disease virus mutant strain has the ability for inducing the stronger horizontal neutralizing antibody of cross protection.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179C, using rHN Strain as mother
This Strain, following amino acid sites mutate in the G-H ring including VP3 albumen: the 173rd Aspartic acid mutations are asparagus fern
Amide, the 174th valine mutation is glutamic acid and the 179th asparagine mutation is cysteine.
The present invention provides the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179CPreparation method, including
Following steps:
(1) using pOFS plasmid as template, PCR is carried out with OSEP3+ and NEC- primer pair, A amplified production is obtained, uses NEC+
PCR is carried out with ONNP3'- primer pair, obtains B amplified production；
The nucleotide sequence of the OSEP3+ is as shown in SEQ ID NO.1 in sequence table；
The nucleotide sequence of the NEC- is as shown in SEQ ID NO.2 in sequence table；
The nucleotide sequence of the NEC+ is as shown in SEQ ID NO.3 in sequence table；
The nucleotide sequence of the ONNP3'- is as shown in SEQ ID NO.4 in sequence table；
(2) it using the A amplified production and B amplified production as template, is merged with OSEP3+ and ONNP3'- primer pair
PCR obtains VP3D173N+V174Y+N179CDNA fragmentation；
(3) by the VP3D173N+V174Y+N179CDNA fragmentation and pSK-HDV carrier Spe I/Not I double digestion, connection,
Obtain pSK-VP3D173N+V174E+N179CPlasmid；
(4) by VP0 genetic fragment, VP1+P2 genetic fragment, 5'UTR+L genetic fragment, P3+3'UTR gene fragment clone
To the pSK-VP3D173N+V174E+N179CIn plasmid, pOFS is obtainedD3173N+V3174E+N3179CPlasmid；
(5) by the pOFSD3173N+V3174E+N3179CObtained linear plasmid is transfected BSR/T7-5 by plasmid linearization processing
Cell obtains O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179C。
Preferably, the PCR system of A amplified production or B amplified production is as follows in step (1): 10 × LA Taq buffer 5
μ l, 2.5mmol/L dNTP 5 μ l, 0.5 μ l of upstream primer, 0.5 μ l of downstream primer, 0.5 μ l, LA Taq enzyme of template, 0.5 μ l,
ddH2O 38μl。
Preferably, the fusion DNA vaccine program in step (1) in the PCR program or step (2) of A amplified production or B amplified production
It is independent are as follows: 94 DEG C of 5min；94 DEG C of 1min, 61 DEG C of 1min 20s, 72 DEG C of 3min, 30 circulations；72℃10min.
Preferably, the reaction system of Spe I/Not I double digestion is as follows in step (3): 10 × Basal buffer, 5 μ l,
25 μ l, Not I of template, 5 μ l, Spe I 5 μ l, ddH2O 10μl；The reaction condition of the Spe I/Not I double digestion is 37 DEG C
Warm bath 2h.
Preferably, primer pair used in amplification VP0 genetic fragment is OSBP4+ and OEP2- in step (4)；The OSBP4+
Nucleotide sequence as shown in SEQ ID NO.5 in sequence table；SEQ ID in the nucleotide sequence of the OEP2- such as sequence table
Shown in NO.6；
Expanding primer pair used in VP1+P2 genetic fragment is OS2A+/OBN-；The nucleotide sequence of the OS2A+ such as sequence
In list shown in SEQ ID NO.7；The nucleotide sequence of the OBN- is as shown in SEQ ID NO.8 in sequence table；
Expanding primer pair used in 5'UTR+L genetic fragment is OST7+/OBL-；The nucleotide sequence of the OST7+ such as sequence
In list shown in SEQ ID NO.9；The nucleotide sequence of the OBL- is as shown in SEQ ID NO.10 in sequence table.
Expanding primer pair used in P3+3'UTR genetic fragment is 3A'+/DNS-；The nucleotide sequence of the 3A'+ such as sequence
In list shown in SEQ ID NO.11；The nucleotide sequence of the DNS- is as shown in SEQ ID NO.12 in sequence table.
The present invention provides the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179COr the preparation method system
Standby obtained O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179CImproving the application in neutralizing antibody cross protection.
The present invention provides the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179COr the preparation method system
Standby obtained O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179CApplication in infection CHO series of cell system.
The present invention provides the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179COr the preparation method system
Standby obtained O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179CApplication in Immune Cell Antigens submission.
The present invention provides a kind of based on the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179COr the system
The O-shaped foot and mouth disease virus mutant strain rHN that Preparation Method is preparedD3173N+V3174E+N3179CThe vaccine strain of inactivation.
The present invention provides a kind of O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179C, using rHN Strain as mother
This Strain mutates amino acid following in the G-H ring of VP3 albumen: the 173rd Aspartic acid mutations are asparagine,
174th valine mutation is glutamic acid and the 179th asparagine mutation is cysteine.The present invention is by foot and mouth disease virus
Reverse genetics manipulation technology platform successfully saves out O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179C, tested through RT-PCR
Card, the virus mutation strain reach the 10th generation Successful amplification and go out purpose band, and sequencing result shows that the virus mutation strain has
Preferable genetic stability；One step growth curve is as the result is shown: pinpointing with maternal Strain rHN and single foot and mouth disease virus
Mutant strain rHNV3174Y(comparative example 1) compares, rHND3173N+V3174E+N3179CProliferation trend on BHK-21 cell is similar, three
Duplicating dynamics do not show apparent difference, illustrate the mutation of specific site and have not been changed virus in BHK-21 cell
Replication capacity；Use TCID50It is thin to BHK-21 that measurement result shows that it is not significantly changed in virus mutation strain described in vitro test
The infectivity of born of the same parents；Meanwhile using LD50Measurement result shows that virus mutation strain described in vivo studies is obvious to the pathogenicity of suckling mouse
Weaken, with rHNV174YIt compares, rHND3173N+V3174E+N3179CThe weak effect of cause become apparent；Plaque assay and exempt from indirectly
Epidemic disease fluorescence and laser confocal microscope testing result explanation, the virus mutation strain obtain caveolin mediation and infect CHO
The ability of series of cell system.By detect immuno positive serum cross-neutralization ability the result shows that, the virus mutation strain
It induces the cross-protection ability of the neutralizing antibody generated to be obviously improved, illustrates that the virus mutation strain has excellent antigen wide spectrum
Property.
Detailed description of the invention
Fig. 1 is that the foot-and-mouth disease virus gene group full length cDNA clone of VP3 rite-directed mutagenesis constructs schematic diagram
Fig. 2 is pOFSV3174YAnd pOFSD3173N+V3174E+N3179CPlasmid and its qualification result of virus mutation strain；Fig. 2A: M,
DNA molecular quality standard (1Kb)；1~3, it is followed successively by pOFS, pOFSV3174YAnd pOFSD3173N+V3174E+N3179CThe Spe I/ of plasmid
Not I digestion products；Fig. 2 B:M, DNA molecular quality standard (DL5000)；1~3, it is followed successively by rHN, rHNV3174YAnd rHND3173N +V3174E+N3179CThe RT-PCR product of amplification；Fig. 2 C: being from left to right rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CIn VP3
Encode the sequencing peak figure of the 171st~181 amino acids；
Fig. 3 is rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CPlaque form on BHK-21 and CHO-K1 cell；
Fig. 4 is rHN, rHNV3174YAnd rHND3173N+V3174E+N3179COne step growth curve；
Fig. 5 is rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CWhether there is or not aftosas after inoculation BHK-21 and CHO-K1 cell
The indirect immunofluorescene assay result of virus nonstructural protein 3A expression；
Fig. 6 is rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CInfect the laser co-focusing of BHK-21 and CHO-K1 cell
Microscopy Results；
Fig. 7 be virus neutralization tests measure immune swine after the 21st day acquisition serum neutralizing antibody r value variance analysis.
Specific embodiment
The present invention provides a kind of O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179C, using rHN Strain as mother
This Strain, following amino acid sites mutate in the G-H ring including VP3 albumen: the 173rd Aspartic acid mutations are asparagus fern
Amide, the 174th valine mutation is glutamic acid and the 179th asparagine mutation is cysteine.
In the present invention, the rHN Strain is the O/ saved using foot and mouth disease virus reverse genetics manipulation technology platform
HN/CHA/93 (i.e. OZK/93, classical vaccine strain) genetic engineering poison.The rHN Strain has been published, and article is specifically shown in
(Li P,et al.Evaluation of a Genetically Modified Foot-and-Mouth Disease Virus
Vaccine Candidate Generated by Reverse Genetics.BMC Vet Res.2012,8:57)。
In the present invention, the mentality of designing of the mutation is as follows:
By on influenced on capsid protein outside foot and mouth disease virus in substance, three-dimensional conformation stability it is relatively conservative
Non- T, B cell epitope area (section of mutation neither foot and mouth disease virus t cell epitope area, nor foot and mouth disease virus B cell
Epitope area.It is transformed from three-dimensional conformation, different from other people is selected by t cell epitope and/or B cell table
Position transformation promotes the cross-neutralization ability of foot and mouth disease virus) in potential target spot carry out pointed decoration, target is sexually revised with feature
Mark the intermolecular forces such as the space occupy-place (position) of amino acid and its interaction amino acid, positive and negative charge, the disulfide bond of institute's band
Mode, the flexibility of Lai Youhua foot and mouth disease virus main neutrality epitope area (VP1 G-H ring, i.e. antigen site 1-1)
And improve its efficiency for targeting submission；The non-T, B cell epitope area are specially but are not limited to VP3 G-H ring, it is tied in space
Close to antigen site 1-1,1-2 (end VP1 C-) on structure；Pointed decoration is preferably mutated, such as is replaced；The characteristic changes
Becoming is specially the change of properties such as amino acid chemical structure, polarity, acid-base property that pointed decoration causes；The targeting submission is mouth hoof
Epidemic disease poison identifies antigen presenting cell and its depends on receptor mediated endocytosis path；The present antigens such as Dendritic Cells are given
Lymphocyte, the humoral immunity and cell immune response of inducing specific.
The present invention provides the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179CPreparation method, including
Following steps:
(1) using pOFS plasmid as template, PCR is carried out with OSEP3+ and NEC- primer pair, A amplified production is obtained, uses NEC+
PCR is carried out with ONNP3'- primer pair, obtains B amplified production；
The nucleotide sequence of the OSEP3+ is as shown in SEQ ID NO.1 in sequence table；
The nucleotide sequence of the NEC- is as shown in SEQ ID NO.2 in sequence table；
The nucleotide sequence of the NEC+ is as shown in SEQ ID NO.3 in sequence table；
The nucleotide sequence of the ONNP3'- is as shown in SEQ ID NO.4 in sequence table；
(2) it using the A amplified production and B amplified production as template, is merged with OSEP3+ and ONNP3'- primer pair
PCR amplification obtains VP3D173N+V174Y+N179CDNA fragmentation；
(3) by the VP3D173N+V174Y+N179CDNA fragmentation and pSK-HDV carrier Spe I/Not I double digestion, connection,
Obtain pSK-VP3D173N+V174E+N179CPlasmid；
(4) by VP0 genetic fragment, VP1+P2 genetic fragment, 5'UTR+L genetic fragment, P3+3'UTR gene fragment clone
To the pSK-VP3D173N+V174E+N179CIn plasmid, pOFS is obtainedD3173N+V3174E+N3179CPlasmid；
(5) by the pOFSD3173N+V3174E+N3179CObtained linear plasmid is transfected cell, obtained by plasmid linearization processing
To O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179C。
Building schematic diagram such as Fig. 1 of the foot-and-mouth disease virus gene group full length cDNA clone of the VP3 rite-directed mutagenesis.
The present invention carries out PCR with OSEP3+ and NEC-, NEC+ and ONNP3'- primer pair respectively using pOFS plasmid as template,
Obtain A amplified production and B amplified production；
The nucleotide sequence of the OSEP3+ is as shown in SEQ ID NO.1 in sequence table；
The nucleotide sequence of the NEC- is as shown in SEQ ID NO.2 in sequence table；
The nucleotide sequence of the NEC+ is as shown in SEQ ID NO.3 in sequence table；
The nucleotide sequence of the ONNP3'- is as shown in SEQ ID NO.4 in sequence table；
In the present invention, the pOFS plasmid is using pSK-HDV as carrier, includes the rHN female parent Strain (base of OZK/93
Because engineering poison) Genomic full_length cDNA recombinant plasmid.The pOFS plasmid has been reported that (Li P, et in the prior art
al.Evaluation of a Genetically Modified Foot-and-Mouth Disease Virus Vaccine
Candidate Generated by Reverse Genetics.BMC Vet Res.2012,8:57)。
In the present invention, the PCR system of the A amplified production or B amplified production is preferably as follows: 10 × LA Taq
5 μ l, 2.5mmol/L dNTP of buffer 5 μ l, 0.5 μ l of upstream primer, 0.5 μ l of downstream primer, 0.5 μ l, LA Taq enzyme of template
0.5 μ l, ddH2O 38μl。
In the present invention, the PCR program of A amplified production or B amplified production is independently preferred are as follows: 94 DEG C of 5min；94℃1min,
61 DEG C of 1min 20s, 72 DEG C of 3min, 30 circulations；72℃10min.
After obtaining A amplified production and B amplified production, the present invention is used using the A amplified production and B amplified production as template
OSEP3+ and ONNP3'- primer pair carries out fusion DNA vaccine, obtains VP3D173N+V174Y+N179CDNA fragmentation.
In the present invention, the amplification program of the fusion DNA vaccine is preferred are as follows: 94 DEG C of 5min；94℃1min,61℃1min
20s, 72 DEG C of 3min, 30 circulations；72℃10min.The amplification system of the fusion DNA vaccine is preferably 10 × LA Taq buffer
5 μ l, 2.5mmol/L dNTP 5 μ l, 0.5 μ l of upstream primer, 0.5 μ l, A amplified production of downstream primer, 0.5 μ l, B amplified production
0.5 μ l, LA Taq enzyme 0.5 μ l, ddH2O37.5μl.In the present invention, the fusion DNA vaccine primer include OSEP3+ and
ONNP3'-。
Obtain VP3D173N+V174Y+N179CAfter DNA fragmentation, the present invention is by the VP3D173N+V174Y+N179CDNA fragmentation and
PSK-HDV carrier Spe I/Not I double digestion, double enzyme digestion product recycling, connection, obtains pSK-VP3D173N+V174E+N179CMatter
Grain.
In the present invention, the pSK-HDV carrier has been reported that (the foot and mouth disease virus such as Cao Weijun O/HN/ in the prior art
The rescue of 93 vaccine strains and virus activity identify North China Agricultural Journal, 2010,25 (3): 32-37).
In the present invention, the reaction system of Spe I/Not I double digestion is preferably as follows: 10 × Basal buffer, 5 μ l,
25 μ l, Not I of template, 5 μ l, Spe I 5 μ l, ddH2O 10μl.The reaction condition of the Spe I/NotI double digestion is preferably
37 DEG C of warm bath 2h.The present invention is not particularly limited the method for the connection, using connection scheme known in the art.
Obtain pSK-VP3D173N+V174E+N179CAfter plasmid, by VP0 genetic fragment, VP1+P2 genetic fragment, 5'UTR+L gene
Segment, P3+3'UTR gene fragment clone to the pSK-VP3D173N+V174E+N179CIn plasmid, pOFS is obtainedD3173N +V3174E+N3179CPlasmid.
In the present invention, the template for expanding VP0 genetic fragment is pOFS plasmid.Expand primer used in VP0 genetic fragment
To preferably OSBP4+ and OEP2-；The nucleotide sequence of the OSBP4+ is preferably as shown in SEQ ID NO.5 in sequence table；Institute
The nucleotide sequence of OEP2- is stated preferably as shown in SEQ ID NO.6 in sequence table.Expand the same A of PCR system of VP0 genetic fragment
The PCR system of amplified production, the difference is that: 10 × LA Taq buffer (5 μ l) is changed to 5 × PrimeSTARTTM
HS DNA polymerase Buffer (10 μ l), LA Taq (0.5 μ l) are changed to PrimeSTARTTM HS DNA
polymerase(0.5μl)；Correspondingly, ddH2O is reduced to 33 μ l.The PCR program of the amplification VP0 genetic fragment is expanded with A
The PCR program of product.The VP0 genetic fragment and pSK-VP3 that will be obtainedD173N+V174E+N179CPlasmid carries out double digestion, and double digestion produces
Object recycling, connection, obtain pSK-VP0+VP3D173N+V174E+N179CPlasmid.The same step of the reaction system of the double digestion (3).It is described
Restriction enzyme in reaction system is Spe I/EcoR I.
In the present invention, the template for expanding VP1+P2 genetic fragment is pOFS plasmid.Expand the genetic fragment institute of VP1+P2
Primer pair is preferably OS2A+/OBN-.The nucleotide sequence of the OS2A+ is preferably such as SEQ ID NO.7 institute in sequence table
Show；The nucleotide sequence of the OBN- is preferably as shown in SEQ ID NO.8 in sequence table.The amplification VP1+P2 genetic fragment
PCR system of the PCR system with VP0 genetic fragment.The PCR program of the amplification VP1+P2 genetic fragment is the same as VP0 genetic fragment
PCR program.The VP1+P2 genetic fragment and pSK-VP0+VP3 that will be obtainedD173N+V174E+N179CPlasmid carries out double digestion, double enzymes respectively
Product recycling, connection are cut, pSK-VP0+VP3 is obtainedD173N+V174E+N179C+ VP1+P2 plasmid.The reaction system of the double digestion is same
Step (3).Restriction enzyme in the VP1+P2 genetic fragment double enzyme digestion reaction system is Nhe I/Not I.It is described
pSK-VP0+VP3D173N+V174E+N179CRestriction enzyme in plasmid double enzyme digestion reaction system is that Spe I/Not I (needs
Bright, Nhe I and Spe I are isocaudarner).
In the present invention, the template for expanding 5'UTR+L genetic fragment is pOFS plasmid.Expand 5'UTR+L genetic fragment institute
Primer pair is preferably OST7+/OBL-；The nucleotide sequence of the OST7+ is preferably such as SEQ ID NO.9 institute in sequence table
Show；The nucleotide sequence of the OBL- is preferably as shown in SEQ ID NO.10 in sequence table.The amplification 5'UTR+L genetic fragment
PCR system with VP0 genetic fragment PCR system.The PCR program of the amplification 5'UTR+L genetic fragment is the same as VP0 genetic fragment
PCR program.The 5'UTR+L genetic fragment and pSK-VP0+VP3 that will be obtainedD173N+V174E+N179C+ VP1+P2 plasmid carries out double enzymes
It cuts, double enzyme digestion product recycling, connection obtain pSK-5'UTR+L+VP0+VP3D173N+V174E+N179C+ VP1+P2 plasmid.Double enzymes
The same step of the reaction system cut (3).Restriction enzyme in the reaction system is Spe I/BamH I.
In the present invention, the template for expanding P3+3'UTR genetic fragment is pOFS plasmid.Expand P3+3'UTR genetic fragment
Primer pair used is preferably 3A'+/DNS-；The nucleotide sequence of the 3A'+ is preferably such as SEQ ID NO.11 institute in sequence table
Show；The nucleotide sequence of the DNS- is preferably as shown in SEQ ID NO.12 in sequence table.The amplification P3+3'UTR gene piece
PCR system of the PCR system of section with VP0 genetic fragment.The PCR program of the amplification P3+3'UTR genetic fragment is the same as VP0 gene
The PCR program of segment.The P3+3'UTR genetic fragment and pSK-5'UTR+L+VP0+VP3 that will be obtainedD173N+V174E+N179C+VP1+P2
Plasmid carries out double digestion, and double enzyme digestion product recycling, connection obtain pSK-5'UTR+L+VP0+VP3D173N+V174E+N179C+VP1+P2+
P3+3'UTR, i.e. pOFSD3173N+V3174E+N3179CPlasmid.The same step of the reaction system of the double digestion (3).In the reaction system
Restriction enzyme be BglII/NotI.
Obtain pOFSD3173N+V3174E+N3179CAfter plasmid, by the pOFSD3173N+V3174E+N3179CPlasmid linearization processing, will
Obtained linear plasmid transfection cell, obtains O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179C。
In the present invention, the linearization process is preferably NotI with restriction enzyme.The digestion system of the Not I
Reaction system described in same step (3), difference are: restriction enzyme only has Not I (5 μ l), correspondingly, ddH2The body of O
Product increases to 15 μ l.Digestion condition described in the digestion conditional synchronization of the NotI rapid (3).
After obtaining digestion products, preferably the digestion products are recycled, obtain linear plasmid.The recycling is preferably adopted
It is carried out with kit.In embodiments of the present invention, the kit is purchased from the agarose of precious bioengineering (Dalian) Co., Ltd
Gel recycling and/or DNA fragmentation purification kit.
The present invention is not particularly limited linear plasmid transfection cell, is using transfection method known in the art
It can.The cell is preferably BSR/T7-5 cell.Stablize the BSR/T7-5 cell line of expression T7 RNA polymerase by German Karl-
Klaus professor Conzelmann give.
In order to determine whether virus mutation strain heredity prepared by the present invention is stable, the present invention is by the pOFSD3173N +V3174E+N3179CPlasmid carries out linearization process and transfects BSR/T7-5 cell, the cell and suspension of harvest transfection in 72h.Freeze repeatedly
Melt the continuous passage on BHK-21 cell three times, until typical induced cytopathic effect occurs in 90% or more cell
The time of (cytopathic effect, CPE) tends towards stability.The 10th generation genetic engineering poison is selected, by RNeasy Mini Kit
Specification operation, extracts total serum IgE, and RT-PCR expands P1 gene [upstream primer 204:5'-acctccgacgggtggtacgc-3'
(SEQ ID No.15), downstream primer NK61:5'-gacatgtcctcctgcatctg-3'(SEQ ID No.16)], agarose
After gel electrophoresis, recycling target fragment, the sequencing of the Beijing Liuhe Huada Genomics Technology Co., Ltd Song Ji.The result shows that this
The virus mutation strain succeeding generations of invention preparation do not mutate, have preferable genetic stability.
The present invention also provides another schemes of the specific amino acid mutation in O-shaped foot and mouth disease virus VP3 G-H ring, i.e.,
rHNV3174Y, it is maternal Strain with rHN, is tyrosine by the 174th valine mutation in VP3 G-H ring.It is described
rHNV3174YThe preparation method of plasmid is building pOFS firstV3174YPlasmid.The pOFSV3174YThe construction method of plasmid is substantially same
pOFSD3173N+V3174E+N3179CThe construction method of plasmid, difference are: amplification VP3V174YPrimer pair be OSEP3+/3174Y-,
3174Y+/ONNP3'- and OSEP3+/ONNP3'-.The nucleotide sequence of the 3174Y- such as SEQ ID No.13 institute in sequence table
Show；The nucleotide sequence of the 3174Y+ is as shown in SEQ ID No.14 in sequence table.The amplification system and amplification journey of the PCR
Orderisomorphism builds pOFSD3173N+V3174E+N3179CAmplification system and amplification program when plasmid.The rHNV3174Y, as positive control
Illustrate the rHN that the present invention protectsD3173N+V3174E+N3179CBiological characteristics.
Whether the rescue in order to identify the virus mutation strain successful and its biological characteristics, has carried out following test: erosion
Spot forms test, the drafting of one step growth curve, TCID50And LD50Measurement, indirect immunofluorescence and laser co-focusing it is micro-
Microscopy is surveyed.
Wherein plaque assay can identify the receptor of present invention genetic engineering poison obtained utilize phenotype whether because
It changes for mutation.Test result is as follows: compared with rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CIt is infected
The ability of CHO-K1 cell.
One step growth curve is as the result is shown: with maternal Strain rHN and single foot and mouth disease virus rite-directed mutagenesis strain
rHNV3174YCompare, virus mutation strain rHND3173N+V3174E+N3179CProliferation trend on BHK-21 cell is similar, the duplication of three
Dynamics does not show apparent difference, illustrates that the mutation of specific site does not change virus replication capacity in cell.
TCID50Measurement, TCID50It is equivalent in vitro test, in order to identify that genetic engineering poison obtained is right
Whether the pathogenic change effect ability of BHK-21 cell changes；TCID50Measurement result show, maternal Strain rHN and aftosa
Virus mutation strain rHNV3174YAnd rHND3173N+V3174E+N3179CTCID50Between 10-6.500~10-7.125Between, between three
Difference is not significant, and it is really unknown that this result has further proved specific amino acid mutation in O-shaped foot and mouth disease virus VP3 G-H ring
It is aobvious to change its infectivity to BHK-21 cell.
LD50Measurement, LD50It is equivalent in vivo studies, in order to identify genetic engineering poison obtained to suckling mouse
Whether pathogenicity changes.LD50Measurement result show, compared with rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CTo suckling mouse
Pathogenicity obviously weakens, and the latter is particularly evident.
Meanwhile indirect immunofluorescene assay result is shown: rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CIt is thin in BHK-21
In born of the same parents can normal replication, translation, confirming specific amino acid mutation in O-shaped foot and mouth disease virus VP3G-H ring makes which give senses
Contaminate the ability of CHO-K1 cell.
Laser co-focusing testing result is shown: rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CInfect BHK-21 cell according to
Rely in two kinds of endocytosis paths of clathrin and caveolin mediation；It infects on CHO-K1 cell, really only rHNV3174Y
And rHND3173N+V3174E+N3179CWith caveolin common location, illustrate that CHO-K1 cell is infected in two kinds of foot and mouth disease virus rite-directed mutagenesis strains
The endocytosis path of caveolin mediation should be to rely on.
In order to detect the antigen broad spectrum activity of virus mutation strain provided by the invention, detection virus mutation strain inactivated vaccine is immune
The cross-neutralization ability of positive serum, as a result, it has been found that, the neutralization that virus mutation strain induction body (pig) provided by the invention generates
The cross-protection ability of antibody is obviously improved.
The present invention provides the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179COr the preparation method system
Standby obtained O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179CApplication in increasing antibody and in cross protection.
The present invention provides the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179COr the preparation method system
Standby obtained O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179CApplication in infection CHO series of cell system.
The present invention provides the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179COr the preparation method system
Standby obtained O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179CApplication in Immune Cell Antigens submission.
The present invention provides a kind of based on the O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179COr the system
The O-shaped foot and mouth disease virus mutant strain rHN that Preparation Method is preparedD3173N+V3174E+N3179CThe vaccine strain of inactivation.
The present invention is not particularly limited the method for the inactivation, using inactivation scheme known in the art.?
In the embodiment of the present invention, Montanide is added in 1:1 (V/V) ratio in Xiang Suoshu mutant strainTMISA201 VG emulsification
Foot and mouth disease virus inactivated vaccine is made in 20h, laboratory.
Application of the foot and mouth disease virus inactivated vaccine in the prevention and treatment of O-shaped aftosa.
Below with reference to embodiment to a kind of O-shaped foot and mouth disease virus mutant strain provided by the invention and its preparation method and application
It is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
The introduction of raw material sources
The source of 1.1 plasmids or cell
The BSR/T7-5 cell line for stablizing expression t7 rna polymerase teaches favour by German Karl-Klaus Conzelmann
It gives.
BHK-21 cell (Ins+, HS+) and CHO-K1 cell (Ins-, HS+) are respectively quoted from China typical culture collection
Center and Unite States Standard biology product collecting center.
1.2 main agents
LA Taq、PrimeSTARTTMHS DNA polymerase, restriction enzyme, T4 DNA ligase, DNA point
Protonatomic mass standard, Ago-Gel recycling and DNA purification kit, plasmid extraction kit are purchased from precious bioengineering (Dalian)
Co., Ltd.
E.coli JM109 competent cell is purchased from Transgene company.
Opti-MEM I、LipofectamineTM2000, GMEM, DMEM, 2 × MEM are purchased from Invitrogen company.
Ampicillin-streptomysin and 0.25%Trypsin-EDTA are purchased from Gibco company.
Fetal calf serum (fetal bovine serum, FBS) is purchased from Hyclone company.
Phosphate buffer (phosphate buffered saline, PBS；PH=7.4) it is purchased from Biological
Industries company.
RNeasy Mini Kit is purchased from Qiagen company.
Bassora gum is purchased from MP Biomedicals company.
O-shaped aftosa guinea pig antiserum, foot and mouth disease virus non-structural protein (non-structural proteins,
NSPs) 3A monoclonal antibody (monoclonal antibody, McAb) is mentioned by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
For.
Clathrin (clathrin) heavy chain McAb and rabbit source caveolin (caveolin) polyclonal antibody
(polyclonal antibody, PcAb) is purchased from ThermoFisher company.
Goat Anti-cavy IgG H&L (647) it is purchased from Abcam company.
The mountain sheep anti-mouse igg (H+L) and goat anti-rabbit igg (H+L) of FITC label are century biotechnology purchased from Beijing health
Co., Ltd.
DAPI is purchased from the green skies Bioisystech Co., Ltd in Shanghai.
Binary ethylenimine, sodium thiosulfate are given by middle peasant Witter biotech inc.
MontanideTMISA201 VG is purchased from Seppic company (Qingpu Shanghai factory).
O-shaped foot and mouth disease virus Liquid-phase blocking ELISA antibody assay kit and foot and mouth disease virus 3ABC blocking ELISA
Antibody assay kit is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's diagnostic center.
O/rV-1 plants and O-shaped O/BY/CHA/2010 plants of foot and mouth disease virus inactivated vaccine immuno positive serum (being shown in Table 4), by
OIE/ China national aftosa reference laboratory provides.
1.3 experimental animal
1~3 age in days suckling mouse (Kunming system), is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's Experimental Animal Center.
18~22g mouse (Kunming system), is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's Experimental Animal Center.
Cavy (250~350g), is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's Experimental Animal Center.
The susceptible pig (25~40kg) of health pinpoints pig farm purchased from Gansu Province.
Embodiment 1
The foot-and-mouth disease virus gene group full length cDNA clone (pOFS of VP3 rite-directed mutagenesisD3173N+V3174E+N3179CPlasmid) building
The first step, firstly, PCR amplification is carried out with OSEP3+/NEC- and NEC+/ONNP3'- using pOFS plasmid as template,
Reaction system is (50 μ l of total volume): 10 × LA Taq buffer, 5 μ l, 2.5mM dNTP 5 μ l, 0.5 μ l of upstream primer,
0.5 μ l of downstream primer, 0.5 μ l, LA Taq enzyme of template 0.5 μ l, ddH2O 38μl.Response procedures are equal are as follows: 94 DEG C of 5min；94℃
1min, 61 DEG C of 1min20s, 72 DEG C of 3min, 30 circulations；72℃10min.Amplification obtains two segments of A and B.
Then, purpose product is obtained using the Ago-Gel QIAquick Gel Extraction Kit of precious bioengineering (Dalian) Co., Ltd.
Table 1 constructs primer used in foot and mouth disease virus VP3 rite-directed mutagenesis full length cDNA clone
To recycle obtained two segments as template (OSEP3+/NEC- and NEC+/ONNP3'- amplified production), using melting
It closes PCR and obtains VP3 using OSEP3+/ONNP3'- as primer pair amplifies purpose productD173N+V174Y+N179CDNA fragmentation.
Step 2: digestion connects
With Spe I/Not I double digestion pSK-HDV carrier and VP3D173N+V174Y+N179CDNA fragmentation.The reaction system of digestion
It is (50 μ l of total volume): 10 × Basal buffer, 5 μ l, 25 μ l of template, 15 μ l of restriction enzyme, restriction enzyme
25 μ l, ddH2O 10μl.Warm bath 2h under the conditions of 37 DEG C obtains two digestion products.Digestion products are separately recovered, connect, even
The reaction system connect is (10 μ l of total volume): 2 × ligase buffer, 5 μ l, the 1 μ l of pSK-HDV carrier of digestion recycling, digestion
The VP3 of recyclingD173N+V174Y+N179C3 μ l, T4 DNA ligase1 μ l of DNA fragmentation.16 DEG C overnight.Conversion, finally obtains pSK-
VP3D173N+V174E+N179CPlasmid.
Step 3:, using OSBP4+/OEP2- as primer pair, expanding VP0 genetic fragment using pOFS plasmid as template.Reactant
It is the same first step, difference is: 10 × LA Taq buffer (5 μ l) is changed to 5 × PrimeSTARTTM HS DNA
Polymerase Buffer (10 μ l), LA Taq (0.5 μ l) are changed to PrimeSTARTTM HS DNA polymerase(0.5μ
l)；Correspondingly, ddH2O is reduced to 33 μ l.Response procedures are same as above.Recycling condition is same as above.
Then, with the VP0 genetic fragment of Spe I/EcoR I double digestion amplification, pSK-VP3D173N+V174E+N179CPlasmid.Instead
Answer system and the same second step of reaction condition.Recycling, connection.Reaction system is (10 μ l of total volume): 2 × ligase buffer, 5 μ
L, the 3 μ l of VP0 genetic fragment of digestion recycling, the pSK-VP3 of digestion recyclingD173N+V174E+N179C1 μ l, T4 DNA ligase, 1 μ
l.16 DEG C overnight.Conversion, finally obtains pSK-VP0+VP3D173N+V174E+N179CPlasmid.
Step 4: OS2A+/OBN- is primer pair using pOFS plasmid as template, VP1+P2 purpose product is expanded.Reactant
System and the same third step of response procedures.Recycle VP1+P2 genetic fragment.
Then, the VP1+P2 genetic fragment expanded with Nhe I/Not I double digestion, with Spe I/Not I double digestion pSK-
VP0+VP3D173N+V174E+N179CPlasmid (Nhe I and Spe I are isocaudarner).The same third step of reaction system and reaction condition.Recycling
Digestion products, connection, the reaction system of the connection are (10 μ l of total volume): 2 × ligase buffer, 5 μ l, digestion recycling
3 μ l of VP1+P2 genetic fragment, digestion recycling pSK-VP0+VP3D173N+V174E+N179C1 μ l, T4 DNA ligase, 1 μ l.16
DEG C overnight.Conversion, finally obtains pSK-VP0+VP3D173N+V174E+N179C+ VP1+P2 plasmid.
Step 5: OST7+/OBL- is primer pair using pOFS plasmid as template, 5'UTR+L genetic fragment is expanded.Reactant
The same third step of the response procedures of system.Recycle amplified production.
Then, with the 5'UTR+L genetic fragment and pSK-VP0+VP3 of the amplification of Spe I/BamH I double digestionD173N +V174E+N179C+ VP1+P2 plasmid.The same third step of reaction system and reaction condition.Recycle digestion products, connection.The reactant of connection
It is for (10 μ l of total volume): 2 × ligase buffer, 5 μ l, the 3 μ l of 5'UTR+L genetic fragment of digestion recycling, digestion recycling
pSK-VP0+VP3D173N+V174E+N179C1 μ l, T4DNA ligase of+VP1+P2,1 μ l.16 DEG C overnight.Conversion, finally obtain and
pSK-5'UTR+L+VP0+VP3D173N+V174E+N179C+ VP1+P2 plasmid.
Step 6: 3A'+/DNS- is primer pair using pOFS plasmid as template, P3+3'UTR genetic fragment is expanded.Reactant
System and the same third step of response procedures.Recycle amplified production.
Then, with the P3+3'UTR genetic fragment and pSK-5'UTR+L+VP0+ of the amplification of Bgl II/Not I double digestion
VP3D173N+V174E+N179C+ VP1+P2 plasmid.The same third step of reaction system and reaction condition.Digestion products are recycled, connection, connection is instead
Answering system is (10 μ l of total volume): 2 × ligase buffer, 5 μ l, the 3 μ l of P3+3'UTR genetic fragment of digestion recycling, digestion
The pSK-5'UTR+L+VP0+VP3 of recyclingD173N+V174E+N179C1 μ l of+VP1+P21 μ l, T4DNA ligase.16 DEG C overnight.Turn
Change, finally obtains pSK-5'UTR+L+VP0+VP3D173N+V174E+N179C+ VP1+P2+P3+3'UTR plasmid, i.e. pOFSD3173N +V3174E+N3179CPlasmid.
It finally, will be through the correct pOFS of Spe I/Not I double digestion qualification resultD3173N+V3174E+N3179CPlasmid, which is sent, posts Beijing
Six directions Hua Da Gene science limited liability company carries out sequencing.
Comparative example 1
Respectively with OSEP3+/3174Y- and 3174+/ONNP3'- for two pairs of primer pairs, using pOFS plasmid as template, PCR is obtained
To C amplified fragments and D amplified fragments, the scheme of the amplification system and amplification program with the first step in embodiment 1.According to implementation
The scheme of the first step carries out fusion DNA vaccine in example 1, obtains VP3V174Y, real according to the scheme of second step in embodiment 1 to the 6th step
It applies, obtains pOFSV3174YPlasmid.
Embodiment 2
By the correct pOFS of sequencing result in embodiment 1 and comparative example 1D3173N+V3174E+N3179CPlasmid and pOFSV3174YWith
NotI carries out digestion and segment recycling respectively, obtains two kinds of plasmids of linearization process.
By LipofectamineTMIt is thin to be transfected BSR/T7-5 by 2000 operational manuals respectively for the 2.5 μ g plasmid linearized
Born of the same parents, the cell and suspension of the interior harvest transfection of 72h.Multigelation three times, the continuous passage on BHK-21 cell, until 90% or more
The time that typical induced cytopathic effect (cytopathic effect, CPE) occurs in cell tends towards stability.Select the 10th generation base
It because of engineering poison, is operated by RNeasy Mini Kit specification, extracts total serum IgE.Using total serum IgE as template, carries out RT-PCR and expand P1
Gene [upstream primer 204:5'-acctccgacgggtggtacgc-3'(SEQ ID No.15), downstream primer NK61:5'-
Gacatgtcctcctgcatctg-3', (SEQ ID No.16)], amplified production agarose gel electrophoresis, recycling target fragment
Afterwards, the Beijing Liuhe Huada Genomics Technology Co., Ltd Song Ji is sequenced.
Using pOFS plasmid as positive control, to the pOFS of buildingV3174YAnd pOFSD3173N+V3174E+N3179CPlasmid respectively into
As a result the identification of row Spe I/Not I double digestion obtains two purpose bands (Fig. 2A) being consistent with expected size.Sequencing knot
Fruit sufficiently confirms that the foot-and-mouth disease virus gene group full length cDNA clone of two kinds of VP3 rite-directed mutagenesis constructs successfully.
pOFSV3174YAnd pOFSD3173N+V3174E+N3179CPlasmid linearizes respectively through Not I, after transfection BSR/T7-5 cell,
It observed apparent CPE in 48h.By the continuous passage on BHK-21 cell of the transfection sample of harvest, the two is opened from the 4th generation
The time for beginning that 90% or more cell is caused CPE occur tends towards stability (about 8~10h).10th generation of selection inoculation BHK-21 cell
Genetic engineering poison, carries out RT-PCR identification, as a result obtains the purpose band of about 2474bp (base pairs, base-pair),
The sequencing result of gel recovery product is consistent with original plasmid sequence (Fig. 2 B, Fig. 2 C).Two kinds of virus mutation strains are respectively designated as
rHNV3174YAnd rHND3173N+V3174E+N3179C。
Embodiment 3
Plaque assay
By rHN and rHNV3174YAnd rHND3173N+V3174E+N3179CVirus mutation strain carries out 10 times and is serially diluted, and is inoculated in respectively
Single layer BHK-21 and the CHO-K1 cell (200 hole μ l/) cultivated in 6 orifice plates, 37 DEG C, 1h, during which 10~15min of interval gently shakes
It is 1 time dynamic, so that cell surface is kept infiltration.Then, inhale and abandon inoculation liquid, every hole covering 2ml MEM culture medium (1ml containing 2%FBS 2
The bassora gum aqueous solution of × MEM+1ml 1.2%) continue culture to 48h (BHK-21) or 72h (CHO-K1).Training is abandoned finally, inhaling
Base is supported, fixed (+50% acetone of 50% methanol), 0.2% violet staining are observed and calculates plaque forming unit (plaque
Forming unit, PFU).
Plaque assay the result shows that, on BHK-21 cell, compared with maternal Strain rHN, single aftosa
Viral rite-directed mutagenesis strain rHNV3174YPlaque form slightly become larger, levels of replication slightly improves, and virus mutation strain rHND3173N +V3174E+N3179CPlaque form tend to become smaller, replication capacity weakens, but statistical analysis difference is not significant (Fig. 3, table 2).
RHN cannot form plaque on CHO-K1 cell, and rHNV3174YAnd rHND3173N+V3174E+N3179CCan on CHO-K1 cell
It is formed plaque (Fig. 3, table 2).
2 plaque assay result of table
Embodiment 4
The drafting of one step growth curve
By 1.0MOI (multiplicity of infection, infection multiplicity) by rHN and rHNV3174YAnd rHND3173N +V3174E+N3179CVirus mutation strain is seeded to BHK-21 cell respectively, after 4 DEG C of absorption 1h, inhales and abandons inoculation liquid, then rejoin and be free of
The DMEM culture medium of FBS, 37 DEG C be incubated for the 2nd, 4,6,8,9,10,12,16,20, for 24 hours when harvest virus respectively.Multigelation 3
After secondary, according to 1.6 measurement PFU, one step growth curve is drawn.
As a result see Fig. 4: rHN, rHNV3174YAnd rHND3173N+V3174E+N3179COne step growth curve.As shown in Figure 4, rHN,
rHNV3174YAnd rHND3173N+V3174E+N3179COne step growth curve it is almost the same, this illustrates rHNV3174YAnd rHND3173N +V3174E+N3179CThe mutation of specific site do not influence the of self-replication capacity of the Strain.
Embodiment 5
1、TCID50Measurement
BHK-21 cell is made 2 × 106The suspension of/ml, rHN, the rHN being serially diluted with 10 timesV3174YAnd rHND3173N +V3174E+N3179CIt respectively takes 50 μ l to be added in 96 orifice plates (8 holes/dilution), selects 10-4~10-6Three gradients, then set 37 DEG C,
CO2Incubator is to 72h.It inhales and abandons culture medium, fix 30min with 10% formalin solution, 0.05% methylene blue solution dyes
It is rinsed after 30min, after spontaneously drying, counts lesion hole count, according toMethod calculates TCID50。
2、LD50Measurement
Respectively take 10-4~10-8Diluted rHN and two kinds of foot and mouth disease virus mutant strains inoculate 1~3 age in days suckling mouse (200 μ
L/, 5/dilution), the incidence of suckling mouse is observed, until the 7th day, the death toll of suckling mouse is counted, according to Reed-Muench
Method calculates LD50。
Measure TCID50It is to understand the genetic engineering poison of acquisition to the pathogenic change effect ability of BHK-21 cell and whether change
Become, is equivalent in vitro test.Measure LD50Be in order to understand genetic engineering poison obtained to the pathogenicity of suckling mouse and whether change,
It is equivalent in vivo studies.
TCID50Measurement result show, maternal Strain rHN and two kinds of virus mutation strain rHNV3174YAnd rHND3173N +V3174E+N3179CTCID50Between 10-6.500~10-7.125Between, the difference between three is not significant, this result is further
It has proved specific amino acid mutation in O-shaped foot and mouth disease virus VP3G-H ring and its infectivity to BHK-21 cell is really not significantly changed
(table 2).LD50Measurement result show, compared with rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CIt is bright to the pathogenicity of suckling mouse
Aobvious to weaken, the latter is particularly evident, and (2) 10 times of decline or more, is shown in Table.
Embodiment 6
Indirect immunofluorescence assay
By rHN and rHNV3174YAnd rHND3173N+V3174E+N3179CIt is inoculated on BHK-21 or CHO-K1 cell respectively, sets 37
℃、CO2In incubator.It after 4~8h, inhales and abandons inoculation liquid, 4% paraformaldehyde fixed 30min, 0.2%TritonX-100 is penetrating
15min, 5%BSA close 1h, and foot and mouth disease virus 3AMcAb (3A24,1:400), 37 DEG C of incubation 1h is added, and add FITC label
Mountain sheep anti-mouse igg (1:50) react 1h.DAPI (1:10000) is added and contaminates core 5min.It is washed with PBS after the completion of each step
3~5 times.It is observed under fluorescence inverted microscope (EVOS FL) finally, setting.
Indirect immunofluorescene assay result shows, after maternal Strain and virus mutation strain inoculation BHK-21 cell 4h,
It is generated due to it is observed that reacting after 3A24 (McAb) identifies foot and mouth disease virus 3A with the host specificity IgG of FITC label
Green fluorescence, show rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CIt can normal replication, translation in BHK-21 cell
(Fig. 5).But rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CAfter being inoculated with 4~8h of CHO-K1 cell, only the former fails to examine
The expression of FMDV 3A is measured, so that confirming specific amino acid mutation in O-shaped foot and mouth disease virus VP3G-H ring makes
Which give the abilities (Fig. 5) of infection CHO-K1 cell.
Embodiment 7
Laser confocal microscope detection
RHN and its two kinds of virus mutation strains are inoculated in BHK-21 or CHO-K1 cell respectively by 100MOI.37 DEG C of incubations
It after 15min, inhales and abandons inoculation liquid, PBS is washed 3 times.Sequentially add O-shaped aftosa guinea pig antiserum (1:500), Clathrin heavy chain
McAb (1:500) or rabbit source caveolin PcAb (1:200),The goat Anti-cavy IgG H&L of 647 labels
The mountain sheep anti mouse or anti-rabbit IgG (1:50) of (1:400), FITC label, respectively act on 1h under the conditions of 37 DEG C.DAPI contaminates core 5min, PBS
Washing 5~8 times after 50% glycerol mounting, is set and is observed under laser confocal microscope (TCS SP8).
Laser co-focusing testing result shows that maternal Strain is inoculated with BHK-21 cell 15min with two kinds of virus mutation strains
Afterwards, respectively it is observed that foot and mouth disease virus and clathrin and caveolin common location, illustrate rHN, rHNV3174YWith
rHND3173N+V3174E+N3179CInfect two kinds of endocytosis paths that BHK-21 cell is mediated dependent on clathrin and caveolin
(Fig. 6).But on CHO-K1 cell, only rHN reallyV3174YAnd rHND3173N+V3174E+N3179CWith caveolin common location, explanation
The endocytosis path that CHO-K1 cell should be to rely on caveolin mediation is infected in two kinds of virus mutation strains.
Embodiment 8
Foot and mouth disease virus representative strains
Mya98 pedigree: O/BY/CHA/2010 (and current O-shaped aftosa vaccine production one of representative seed culture of viruses),
GenBank accession number JN998095 (Zheng H, et al.Genetic Characterization of a New
Pandemic Southeast Asia Topotype Strain of Serotype O Foot-and-Mouth Disease
Virus Isolated in China during 2010.Virus Genes.2012,44(1):80–88)。
- 1 pedigree representative strains of Pan Asia: O/CHA/7/2011, GenBank accession number JF837375.
Cathay topological type representative strains: O/SCGH/CHA/2016, GenBank accession number KX161429.
IND2001d branch representative strains: O/XJ/CHA/2017, GenBank accession number: MF461724 (Zhu Z, et
al.First Detection of Foot-and-Mouth Disease Virus O/ME-SA/Ind2001in
China.Transbound Emerg Dis.2018May 9.doi:10.1111/tbed.12895)。
O/rV-1 strain is that applicant newly applies in the national inventing patent obtained and a kind of novel chiral synthon certificate of registry
Related one plant of foot-and-mouth disease gene engineering virus, is disclosed in the form of national inventing patent and novel chiral synthon certificate of registry.Country's hair
The grant number of bright patent is ZL201210035986.7.The entitled Schweineseuche O-shaped virus 3A3B epitope missing inactivation of novel chiral synthon
Vaccine (O/rV-1 plants), card number demonstrate,prove word 50 for (2017) novel chiral synthon, and research institute is that Chinese Academy of Agricultural Sciences Lanzhou animal doctor grinds
Study carefully institute, Zhongmu Industry Co., Ltd, middle peasant Witter biotech inc, data of issue: October 16 in 2017
Day.
By rHN, rHN of above-mentioned representative hoof-and-mouth disease strain and the 10th generationV3174YAnd rHND3173N+V3174E+N3179C(totally 8
Strain) it is used as research material, the TCID of each Strain is measured according to the method for embodiment 550, the results are shown in Table 4.
1) preparation of foot-and-mouth disease virus antigen and safety examination
By the 10th generation rHN, rHNV3174YAnd rHND3173N+V3174E+N3179CIt is inactivated, is separately added into binary ethylenimine to end
During which concentration 3mmol/L, 30 DEG C of effect 48h are uninterruptedly stirred or are shaken.Be added sodium thiosulfate to final concentration 1.2%~
2.0%, it is sufficiently stirred, is blocked excessive inactivator.
Then, to above 3 kinds O-shaped foot-and-mouth disease virus antigen samplings, for safety examination, (remaining sample is rapidly cooled to 4
DEG C or less and save): respectively with mouse 5, subcutaneous injection inactivation antigen 0.5ml/ is only；Cavy 2 are respectively used, subcutaneous injection inactivation is anti-
Former 2ml/ is only.It is observed continuously 7, the typical clinical symptom caused by not occurring because of mouth disease virus infection.
2) preparation of foot and mouth disease virus inactivated vaccine
After the safety examination of inactivation antigen is qualified, Montanide is added in 1:1 (V/V) ratioTMISA201VG cream
Change 20h, obtains 3 kinds of O-shaped foot and mouth disease virus inactivated vaccines.
3) immuno positive serum
With above-mentioned 3 kinds O-shaped each 2 of immune swines of foot and mouth disease virus inactivated vaccine difference, (O-shaped foot and mouth disease virus liquid phase is blocked
ELISA antibody titer is feminine gender not higher than 1:8 and 3ABC antibody test), it takes a blood sample within 7,14,21 days after immune, 37 DEG C put
After setting 1h, overnight in 4 DEG C.8000rpm, centrifugation 15min, collect serum.
4) O-shaped foot and mouth disease virus Liquid-phase blocking ELISA antibody test
It is proceeded as follows by O-shaped foot and mouth disease virus Liquid-phase blocking ELISA antibody assay kit specification:
Each 100 μ l of Swine serum to be checked is taken, 2 is carried out with PBST × is serially diluted that (positive and negative control serum carries out identical behaviour
Make).
The serum to be checked of 1:4~1:512 is added in 96 orifice plates by column, 50 holes μ l/；It is (negative right to set up positive and negative control
According to serum, 1:1~1:8；Positive control serum, dilution are 1:2~1:256) and viral antigen control wells (4 hole).
Viral antigen is diluted to working concentration (1:25) with PBST (containing Tween-20), serum dilution holes (50 μ l/ are added
Hole) and viral antigen control wells (100 hole μ l/) in；Sealing plate, oscillation, 4 DEG C stand overnight or 37 DEG C of incubation 90min.
Antigen-antibody complex is transferred in order from 96 orifice plates and has been coated with O-shaped foot and mouth disease virus rabbit-anti (body)
On elisa plate, 50 holes μ l/；Sealing plate, 37 DEG C of incubation 60min.
With the continuous board-washing of PBST 3~5 times, pat dry；O-shaped foot and mouth disease virus guinea pig antibodies working solution, 50 holes μ l/ are added；Envelope
Plate, 37 DEG C of incubation 30min.
With the continuous board-washing of PBST 3~5 times, pat dry；Rabbit-anti cavy IgG-HRP working solution, 50 holes μ l/ are added；Sealing plate, 37 DEG C
It is incubated for 30min.
With the continuous board-washing of PBST 3~5 times, pat dry；Tmb substrate solution A and B solution are mixed into (1:1, V/V), shaken up, is pressed
The substrate solution after the mixing is added in the amount in 50 holes μ l/, sets 37 DEG C of incubation 15min (chromogenic reaction).
After chromogenic reaction, terminate liquid (50 hole μ l/) is added and terminates reaction；OD450nm value is read in microplate reader.
With the OD of viral antigen control wells450Nm value is critical value, determines the antibody titer of serum to be checked.
The ELISA antibody titer of Swine serum to be checked the results are shown in Table 3.
Table 3O type foot and mouth disease virus Liquid-phase blocking ELISA antibody test result
3 kinds of O-shaped foot and mouth disease virus inactivated vaccines are immunized animal the 7th day, have O-shaped foot and mouth disease virus specific ELISA anti-
The generation of body.To the 14th day, the O-shaped foot and mouth disease virus specific ELISA antibody level of aftosa vaccine immune swine all on
It rises；After 21 days, the antibody levels of all immune swines >=1:90 (table 3).
5) virus neutralization experiment (Virus Neutralization Test, VNT)
The immuno positive serum of O-shaped foot and mouth disease virus Liquid-phase blocking ELISA antibody titer >=1:128 is selected, virus is carried out
Experiment is neutralized, to study and judge immuno positive serum to the cross-neutralization ability of O-shaped different genotype foot and mouth disease virus.Specific steps are such as
Under (in triplicate):
Test strain is diluted to 1000TCID respectively50、100TCID50、10TCID50And 0.1TCID50；It chooses
100TCID50Dilution is separately added into the 1st~10 column of 96 orifice plates (50 hole μ l/), and the 11st, 12 column are separately added into 4 holes
1000TCID50、100TCID50、10TCID50And 0.1TCID50Virus liquid.
By selected aftosa vaccine immuno positive serum in 56 DEG C of effect 30min, 2 are carried out with DMEM basic culture solution ×
It is serially diluted；Each to be added in above-mentioned 96 orifice plate of a line (1:2~1:1024,50 holes μ l/), the training of the basis DMEM is added in the 11st, 12 column
Nutrient solution (50 hole μ l/)；Set CO2In cell incubator, 37 DEG C of effect 1h.
By BHK-21 cell suspension (2 × 106A cell/ml, about 10ml) it is added in above-mentioned 96 orifice plate, 50 holes μ l/, set 37
DEG C, CO2In cell incubator, it is incubated for 72h.
The liquid abandoned in 96 orifice plates is inhaled, fixed, dyeing determines result (Reed-Muench method).
It the results are shown in Table 4.
The antibody titer result of the Swine serum to be checked of table 4
5 kinds of immuno positive serum count display, the O-shaped 3A3B table of swine foot-and-mouth disease virus to the VNT result of 8 kinds of foot and mouth disease viruses
Position missing inactivated vaccine production with O/rV-1 plants of seed culture of viruses of immuno positive serum to O-shaped Cathay topological type, Mya98,
The neutralising capacity of PanAsia-1 pedigree and IND2001d branch foot and mouth disease virus is preferable (value > 0.3 r), O/BY/CHA/2010
Immuno positive serum stablizes between 0.31~0.85 (r value) neutralising capacity of other genotype foot and mouth disease viruses；But
RHN immuno positive serum is poor to the neutralising capacity of PanAsia-1 pedigree foot and mouth disease virus (r value is between 0.08~0.14), VP3
Upper V174Y leads to its neutralising capacity point to Mya98 pedigree and PanAsia-1 pedigree and IND2001d branch foot and mouth disease virus
Is not declined and risen；It is worth noting that rHND3173N+V3174E+N3179CImmuno positive serum is to O-shaped four genotype mouth hoof
The neutralising capacity of epidemic disease poison is obviously improved, and its r value is all larger than 0.50 (table 4).
OIE terrestrial animal diagnostic test and vaccine handbook are recommended, and ideal aftosa vaccine preparation kind poison will not only have good
Good production performance (lesion time short, inheritance stability on BHK-21 cell etc.), and it is different to same serotype in vitro
The neutralization levels and immune efficacy of genotype foot and mouth disease virus should meet the requirement (OIE, 2012) that r value is not less than 0.3 respectively.It is right
VNT result carries out variance analysis, and D173N+V174E+N179C can significantly improve foot and mouth disease virus immuno positive on rHN VP3
The cross-neutralization ability of serum implies rHND3173N+V3174E+N3179CInheritance stability strain may have to O-shaped Cathay topological type,
Mya98 and PanAsia-1 pedigree and IND2001d branch foot and mouth disease virus are intersected well attacks poison protection (Fig. 7).
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences
<120>a kind of O-shaped foot and mouth disease virus mutant strain and its preparation method and application
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Claims (10)
1. a kind of O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179C, which is characterized in that using rHN Strain as female parent
Strain, following amino acid sites mutate in the G-H ring including VP3 albumen: the 173rd Aspartic acid mutations are asparagus fern acyl
Amine, the 174th valine mutation is glutamic acid and the 179th asparagine mutation is cysteine.
2. O-shaped foot and mouth disease virus mutant strain rHN described in claim 1D3173N+V3174E+N3179CPreparation method, feature exists
In, comprising the following steps:
(1) using pOFS plasmid as template, carry out PCR with OSEP3+ and NEC- primer pair, obtain A amplified production, with NEC+ and
ONNP3'- primer pair carries out PCR, obtains B amplified production；
The nucleotide sequence of the OSEP3+ is as shown in SEQ ID NO.1；
The nucleotide sequence of the NEC- is as shown in SEQ ID NO.2；
The nucleotide sequence of the NEC+ is as shown in SEQ ID NO.3；
The nucleotide sequence of the ONNP3'- is as shown in SEQ ID NO.4；
(2) using the A amplified production and B amplified production as template, fusion DNA vaccine is carried out with OSEP3+ and ONNP3'- primer pair, is obtained
To VP3D173N+V174Y+N179CDNA fragmentation；
(3) by the VP3D173N+V174Y+N179CDNA fragmentation and pSK-HDV carrier use Spe I/NotI double digestion respectively, and connection obtains
To pSK-VP3D173N+V174E+N179CPlasmid；
(4) by VP0 genetic fragment, VP1+P2 genetic fragment, 5'UTR+L genetic fragment, P3+3'UTR gene fragment clone to institute
State pSK-VP3D173N+V174E+N179CIn plasmid, pOFS is obtainedD3173N+V3174E+N3179CPlasmid；
(5) by the pOFSD3173N+V3174E+N3179CPlasmid linearization processing is thin by obtained linear plasmid transfection BSR/T7-5
Born of the same parents obtain O-shaped foot and mouth disease virus mutant strain rHND3173N+V3174E+N3179C。
3. preparation method according to claim 2, which is characterized in that A amplified production or B amplified production in step (1)
PCR system is as follows: 10 × LATaqbuffer5 μ l, 2.5mmol/LdNTP5 μ l, 0.5 μ l of upstream primer, 0.5 μ l of downstream primer,
0.5 μ l, LATaq enzyme of template 0.5 μ l, ddH2O38μl。
4. preparation method according to claim 2 or 3, which is characterized in that A amplified production or B amplified production in step (1)
PCR program or step (2) in fusion DNA vaccine program it is independent are as follows: 94 DEG C of 5min；94℃1min,61℃1min20s,72℃
3min, 30 circulations；72℃10min.
5. preparation method according to claim 2, which is characterized in that the reaction of SpeI/Not I double digestion in step (3)
System is as follows: 10 × Basalbuffer5 μ l, template 25 μ l, NotI5 μ l, SpeI5 μ l, ddH2O10μl；The SpeI/NotI
The reaction condition of double digestion is 37 DEG C of warm bath 2h.
6. preparation method according to claim 2, which is characterized in that draw used in amplification VP0 genetic fragment in step (4)
Object is to for OSBP4+ and OEP2-；The nucleotide sequence of the OSBP4+ is as shown in SEQ ID NO.5；The OEP2-'s
Nucleotide sequence is as shown in SEQ IDNO.6 in sequence table；
Expanding primer pair used in VP1+P2 genetic fragment is OS2A+/OBN-；Such as sequence table of the nucleotide sequence of the OS2A+
Shown in middle SEQIDNO.7；The nucleotide sequence of the OBN- is as shown in SEQ ID NO.8；
Expanding primer pair used in 5'UTR+L genetic fragment is OST7+/OBL-；Such as sequence table of the nucleotide sequence of the OST7+
Shown in middle SEQIDNO.9；The nucleotide sequence of the OBL- is as shown in SEQ ID NO.10；
Expanding primer pair used in P3+3'UTR genetic fragment is 3A'+/DNS-；Such as sequence table of the nucleotide sequence of the 3A'+
Shown in middle SEQIDNO.11；The nucleotide sequence of the DNS- is as shown in SEQ ID NO.12.
7. O-shaped foot and mouth disease virus mutant strain rHN described in claim 1D3173N+V3174E+N3179COr claim 2~6 is any one
The O-shaped foot and mouth disease virus mutant strain rHN that the item preparation method is preparedD3173N+V3174E+N3179CIntersect improving neutralizing antibody
Application in protection.
8. O-shaped foot and mouth disease virus mutant strain rHN described in claim 1D3173N+V3174E+N3179COr claim 2~6 is any one
The O-shaped foot and mouth disease virus mutant strain rHN that the item preparation method is preparedD3173N+V3174E+N3179CIn infection CHO series of cell
Application in system.
9. O-shaped foot and mouth disease virus mutant strain rHN described in claim 1D3173N+V3174E+N3179COr claim 2~6 is any one
The O-shaped foot and mouth disease virus mutant strain rHN that the item preparation method is preparedD3173N+V3174E+N3179CIn Immune Cell Antigens submission
In application.
10. one kind is based on O-shaped foot and mouth disease virus mutant strain rHN described in claim 1D3173N+V3174E+N3179COr claim 2
The O-shaped foot and mouth disease virus mutant strain rHN that preparation method described in~6 any one is preparedD3173N+V3174E+N3179CThe epidemic disease of inactivation
Miao Zhu.
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| CN112029735A (en) * | 2020-08-31 | 2020-12-04 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease virus non-structural protein 3B dominant epitope deletion marker strain and preparation method and application thereof |
| CN112094821A (en) * | 2020-09-28 | 2020-12-18 | 中国农业科学院兰州兽医研究所 | O-type foot-and-mouth disease virus strain and application thereof |
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| CN117210502A (en) * | 2023-09-13 | 2023-12-12 | 中国农业科学院兰州兽医研究所 | Construction of recombinant foot-and-mouth disease virus strain with foot-and-mouth disease virus VP1 protein immunosuppression site mutation |
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| CN111303251A (en) * | 2020-02-25 | 2020-06-19 | 中国农业科学院兰州兽医研究所 | Method for in-vitro assembly of foot-and-mouth disease virus-like particles and application thereof |
| CN111303251B (en) * | 2020-02-25 | 2021-07-30 | 中国农业科学院兰州兽医研究所 | Method for in-vitro assembly of foot-and-mouth disease virus-like particles and application thereof |
| CN111944770A (en) * | 2020-08-27 | 2020-11-17 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease virus attenuated mutant strain and preparation method and application thereof |
| CN111944770B (en) * | 2020-08-27 | 2022-04-15 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease virus attenuated mutant strain and preparation method and application thereof |
| CN112029735A (en) * | 2020-08-31 | 2020-12-04 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease virus non-structural protein 3B dominant epitope deletion marker strain and preparation method and application thereof |
| CN112094821A (en) * | 2020-09-28 | 2020-12-18 | 中国农业科学院兰州兽医研究所 | O-type foot-and-mouth disease virus strain and application thereof |
| CN113061587A (en) * | 2021-04-30 | 2021-07-02 | 中国农业科学院兰州兽医研究所 | Antigen spectrum expanded O-type foot-and-mouth disease virus strain and construction method and application thereof |
| CN113061587B (en) * | 2021-04-30 | 2023-03-31 | 中国农业科学院兰州兽医研究所 | Antigen spectrum expanded O-type foot-and-mouth disease virus strain and construction method and application thereof |
| CN113337476A (en) * | 2021-05-28 | 2021-09-03 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease O type Panasia-2 lineage reserve vaccine strain and construction method and application thereof |
| CN113337476B (en) * | 2021-05-28 | 2023-06-20 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease O-type PanASia-2 pedigree reserve vaccine strain, construction method and application thereof |
| CN117210502A (en) * | 2023-09-13 | 2023-12-12 | 中国农业科学院兰州兽医研究所 | Construction of recombinant foot-and-mouth disease virus strain with foot-and-mouth disease virus VP1 protein immunosuppression site mutation |
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