CN106939355A - A kind of screening of influenza virus attenuated live vaccines strain and authentication method - Google Patents
A kind of screening of influenza virus attenuated live vaccines strain and authentication method Download PDFInfo
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Abstract
The present invention relates to a kind of screening of influenza virus attenuated live vaccines strain and authentication method, belong to biological pharmacy technical field.The present invention is to have researched and developed a kind of high-density random insertional mutagenesis library by building and screening viral gene is mediated by Mu transposon of phage, the method of attenuated live vaccines candidate's strain is obtained, and is developed a set of to the technical system of the systemic evaluation of attenuated live vaccines strain progress obtained.These methods not only may be directly applied to the screening and evaluation of influenza virus attenuated live vaccines strain, and have extensive reference for the development and exploitation of other virus attenuated live vaccines.
Description
Technical field
The present invention relates to a kind of screening of influenza virus attenuated live vaccines strain and authentication method, belong to bio-pharmaceuticals skill
Art field.
Background technology
Influenza is the respiratory infectious disease of a kind of serious threat human health and public health security.Currently prevent and treat influenza
Main method be patient treated using antiviral drugs and to normal population carry out immunity inoculation.But it is due near
Popular influenza virus all has universal drug resistance over year so that the curative effect of the anti-influenza virus medicament used at present is very
Limited, the treatment of influenza faces the available situation of no medicine.Therefore vaccine inoculation turns into base the most in current flu countermeasure system
Plinth and effective means.The influenza vaccines used at present have two kinds, and one kind is inactivated influenza virus vaccine, another for influenza disease
Malicious attenuated live vaccine.China uses vaccinum influenzae inactivatum.
Traditional inactivated vaccine is all that this make it that its application faces problems by the use of chicken embryo as production matrix.It is first
First, the composition of some remaining chicken embryos of meeting in the influenza vaccines produced by chicken embryo, these compositions often result in vaccine recipient's appearance
Than more serious side reaction, this make it that some crowds can not be inoculated with influenza vaccines at all；In order to which the pair for reducing vaccine inoculation is anti-
Should, be now to remove chicken embryo albumen by complicated purge process, and which again increases the time of production of vaccine and it is economical into
This.Secondly, used inactivated vaccine is not fine for the immune effect of infant and the elderly at present, and it is main former
Because being probably that the heterogenetic antigen in chicken embryo vaccine has disperseed the limited immune response ability of these crowd's bodies, in addition also with
Difference of the birds with human cell in terms of the micro- modification of antigen is relevant.In addition, after a kind of novel influenza occurs, utilizing
The chicken embryo production system of current vaccines, which generally requires the nearly time of 6 months, can just produce new vaccine, and this often causes us
The vaccine of sufficient amount can not be produced and laid in time when facing influenza pandemic or being pandemic.And some stream bird flus
The viral height lethal due to it to chicken embryo, may can not just produce such viral vaccine by chicken embryo at all.Influenza
Attenuated live vaccine is due to its advantageous feature in terms of mediating cellular immune and mucosal immunity, and it can provide very good to human body
Immune protective effect.These acclimatization to cold attenuated vaccines are to be held wild type influenza virus under non-viral physiological condition
Continuous passage, makes virus obtain certain mutation during acclimatization to cold, and then filter out being capable of conduct from the virus of mutation
The strain of attenuated live vaccines.This method very takes, and the obtainable mutated viruses amount of acclimatization to cold is very limited, causes
Alternative very little in candidate's screening process.In addition.The mutated viruses that acclimatization to cold is obtained, often in the genome of virus
Occur one or several point mutation, this mutation is very easy to occur recovery in follow up vaccine production and immunologic process
Mutation, is likely to result in the reversion of vaccine, and then cause disease.Nevertheless, existing acclimatization to cold attenuated vaccine is all by foreign countries
Relevant unit is researched and developed, China's also attenuated vaccine strain without oneself independent intellectual property rights, and this is hindered to a certain extent
Production and application of the attenuated influenza virus live vaccine in China.As can be seen here, develop new Gripovax to screen and comment
Valency system, attenuated vaccine strain of the exploitation with independent intellectual property right, it is reply to overcome traditional attenuated vaccine to develop system defect
The influenza prevention and control situation active demand of current rigorous, it may have very high economic worth.
The content of the invention
The present invention researched and developed it is a kind of by build and screening viral gene mediated by Mu transposon of phage
High-density random insertional mutagenesis library, obtains the method for attenuated live vaccines candidate's strain, and develops a set of weak to what is obtained
Virus live vaccine strain carries out the technical system of systemic evaluation.These methods not only may be directly applied to the weak poison of influenza virus
The screening and evaluation of live vaccine strain, and there is extensive use for reference to anticipate for the development and exploitation of other virus attenuated live vaccines
Justice.
To achieve the above object, the present invention is adopted the following technical scheme that：
A kind of screening of influenza virus attenuated live vaccines strain and authentication method, it is characterised in that comprise the following steps：
(1) the radom insertion technology mediated using Mu transposon of phage sets up the high density mutation library of M genes：
Using the mediation of Finnzymes companies Mu transposon of phage radom insertion mutagenesis kit to influenza virus A/
The oligonucleotide sequence of this 15nt length of 5 '-NNNNNTGCGGCCGCA-3 ' is inserted between each base of WSN/1933M genes, from
And obtain the high density mutation library of influenza virus M genes；
(2) virus mutant storehouse is obtained by influenza virus reverse Genetics Technique：
The plasmid for carrying M gene mutation bodies is transformed into Escherichia coli DH10B receptor cells by the method for electricity consumption conversion,
M gene mutation bodies storehouse plasmid is extracted from restructuring strain, is then reversely lost using the Plasma viral of A/WSN/33H1N1 influenza viruses 8
Pass and learn operating system acquisition virus mutant storehouse；
(3) virus mutant storehouse component is analyzed by second generation sequencing technologies：
The virus mutant storehouse virus for taking step (2) to obtain is passed on mdck cell, is then tried using TRIzol
Viral RNA is extracted in agent, and carries out reverse transcription according to reverse transcription reagent box iScriptTM cDNA Synthesis kit to each RNA
Produce corresponding cDNA, using the cDNA as template, respectively using 3 M genes specific forward primer 5 '-
AGCAAAAGCAGGTAGATATT-3 ', 5 '-GGGGCCAAAGAAATAGCACT-3 ', 5 '-TCCTAGCTCCAGTGCTGGTC-
3 ' do PCR amplifications with the Vic insetion sequence specific reverse primers marked, are set by the PCR fluorescence labeling PCR primers obtained
Once repeat and Liz-500 molecular weight standards are sequenced using 96- capillary 3730xl DNA analysis instrument, produced number
Analyzed according to using ABI softwares according to following standard, removed the non-specific number that PCR processes, primer and sequencing instrument are produced
According to；
(4) screening influenza virus attenuated live vaccines candidate's strain in Mice Body：
Mutant library virus is concentrated by ultracentrifugal method first, determining is used for follow-up mouse after its virus titer
Infection experiment, virus infects the C57/B6 mouse of 6-8 week old by the method for collunarium, respectively after infection second day, the 4th
My god, the 6th day and collect within the 8th day the lung tissue of mouse and carry out homogenized, TRIzol reagents are used from lung homogenate
Total serum IgE is extracted, the method according to above-mentioned steps (3) is sequenced to virus M genes in sample and carries out qualitative and quantitatively divide
Analysis, according to presence situation of the different M gene mutations virus in each sample, determines attenuated live vaccines candidate's strain；
(5) attenuated live vaccines candidate strain genetic stability and safety evaluatio：
(a) separation of attenuated live vaccines and phenotypic evaluation：Dan Ke has been carried out to above-mentioned attenuated live vaccines candidate strain first
Longhua, expands the strain virus of W7-757, W7-791 and W7-797 tri- that wherein can be effectively replicated in mdck cell
Increase, filter out the W7-791 viruses for showing more preferable replication capacity and relatively low cytotoxicity；
(b) attenuated live vaccines genetic stability is detected：W7-791 viruses are passed in mdck cell and Mice Body
In generation, the sequence that the M genes in viral gene order are obtained from cell or mouse lung homogenate is measured, W7- is determined
The mutation of 791 virus M genes can be gone down by stably heredity；
(c) safety evaluation of attenuated vaccine：With the W7-791 virus immunity 6-8 week old mouse of different titers, do not find
Mouse produces Body weight loss and flu-like symptom；Newborn BALB/c mouse collunarium to 15 ages in days is inoculated with the W7-791 of different titers
Or 104TCID50 wild type WSN viruses, detect to Mouse Weight and lung lesion, are not seen on W7-791 Mice Inoculateds
Body weight loss and pulmonary lesion as wild type WSN virus infected mices are observed, it is to flow to thereby determine that W7-791 viruses
Influenza Virus attenuated live vaccines strain.
Further, the condition of the electricity conversion in step (2) is 2.0kV, 200 Ω, 25 μ F.
Further, the specific method in acquisition virus mutant storehouse is in step (2)：The HEK293T cells of culture are gone to
In 6 well culture plates, when cell confluency degree reaches 80~90%, according to transfection reagent operating instruction, insertion mutation M bases will be contained
The plasmid of cause and the plasmid mixed in equal amounts containing other 7 genetic fragments of influenza virus, are mixed, room in proportion with transfection reagent
Temperature is incubated 15min, is added dropwise in HEK293T cell culture fluids, 37 DEG C, and 48h is cultivated in 5%CO2 incubators, collects transfection
Cell conditioned medium, and by virus inoculation in being expanded on mdck cell, infection collects virus after 48 hours, and fractionated viral is frozen
For future use.
Further, PCR uses Novagen PCR enzyme KOD Hot-Start polymerase, PCR in step (3)
Reaction condition be 95 DEG C of pre-degeneration, 10min；95 DEG C of denaturation, 45s；52 DEG C of annealing, 30s；72 DEG C of extension, 90s；Operation 30
Circulation；Last 72 DEG C of extensions 10min.
Further, the method for the non-specific data of removal is in step (3)：(a) all data all meet the silent of standard
Recognize detection level；(b) because the initial 70bp of sequence has stronger non-specific background, so being removed；(c) it is all
Sequence DNA sequence dna all corresponding with influenza virus M genes carries out connection and matched somebody with somebody；(d) sequencing data is done relative to wild type A/ respectively
The normalized of WSN/1933 virus infected cells, uninfecting virus cell and the control of different genes library.
Further, the standard of determination attenuated vaccine candidate's strain is in step (4)：Virus after infection can be effective
Ground is replicated, and is removed at 6-8 days by body.
Further, the titre in step (5) is 106-108TCID50。
Obtained influenza virus attenuated live vaccines strain is screened in Chinese microorganism strain preservation pipe by the inventive method
Reason committee General Microbiological Culture collection has carried out preservation, depositary institution address：BeiChen West Road, Chaoyang District, BeiJing City 1
Number No. 3 Institute of Microorganism, Academia Sinica of institute, deposit number is CGMCC No.13784, Classification And Nomenclature：Influenza A,
Preservation date：2017-02-21.Its M gene has the sequence as shown in SEQ ID No.1.
The advantages of the present invention
The radom insertion technology of Mu transposon of phage mediation can quickly, the high density of any gene of high flux acquisition
Mutant library, with reference to influenza virus reverse genetics operating system, can obtain the influenza virus mutant body storehouse of very big storage capacity, should
Virus mutant storehouse provides material base for screening influenza virus attenuated vaccine strain.This passes through acclimatization to cold better than traditional
The method that the method for mutation builds attenuated vaccine strain.In addition, the emerging second generation high-flux sequence skill of integrated use in the invention
The vaccine triage techniques of attenuated live vaccines candidate's strain is screened and then obtained in art and Mice Body to virus mutant storehouse,
The features such as technology also has fast high flux, speed and reliable results, can greatly shorten the lead time of attenuated vaccine.Most
Afterwards, the present invention has developed the comprehensive vaccine immunity effect evaluation system of set of system, is not only able to scientific evaluation vaccine for body
The immune protective efficiency that can be provided, and the specific mechanism that vaccine provides immunoprotection can be illustrated to a certain extent, it is epidemic disease
The further improvement and optimization of seedling provide help.
Brief description of the drawings
The Establishing process schematic diagram in M genetic fragment mutant influenza virus storehouse transposon-mediated Fig. 1；
The genetic analysis figure in Fig. 2 M gene mutation bodies storehouse；
Fig. 3 genotypic results done after the collunarium infecting mouse of M gene mutation bodies storehouse in different time；
Distribution overview (A) of the different mutant clons that Fig. 4 are separated by being mutated for the first time on M genes；Above-mentioned M bases
Because mutant plasmid is transfected virus titer change schematic diagram (B) in 293T cells；
Influence schematic diagram of Fig. 5 influenza viruses to mdck cell vigor；
Fig. 6 infect the virus titer change schematic diagram of mdck cell；
Influence of the strains of influenza viruses (W7-791) of Fig. 7 present invention with wild strains of influenza viruses (WT-WSN) to mouse is shown
It is intended to；
After the strains of influenza viruses (W7-791) of Fig. 8 mouse inoculations present invention, mouse resists work to same subtype influenza virus
Use schematic diagram；
Immune response schematic diagram of Fig. 9 mouse to the strains of influenza viruses (W7-791) of the present invention.
Wherein, in Fig. 1 (A) in influenza virus (A/WSN/1933) M genetic fragments using transposon-mediated mutation with
The machine transplanting of rice enters 15 bases, obtains the M gene plasmids storehouse of high density mutation.By the M gene mutation plasmid storehouses of acquisition and other seven
Wt influenza genetic reverse genetics operates plasmid co-transfection to set up mutated viruses storehouse.Contain mutated viruses storehouse
Cell conditioned medium is continued for infecting mdck cell or direct infection mouse.Infected mouse is collected in different time points
Lung and lymphoid organ carry out isolated viral.The mutation that viral growth slows down is caused to be identified come and further commented with Genotyping
The internal and external infectious effect of valency；(B) enter performing PCR with the primer of gene-specific primer and identification 15nt Insert Fragments to expand,
And the PCR products of acquisition are sequenced, so that it may obtain the position of all catastrophe points in storehouse.(C) influenza mutation library can in vivo or body
Screen outside.By the storehouse genotypic results for not being screened and being screened just can determine that in genome it is required and
Nonessential region.
In Fig. 2, the genetic analysis result summary (top in influenza virus A WSN (H1N1) M1 and M2 albumen gross mutations storehouse
Point).Column represents insertion point in gene.All mutation libraries are transfected in 293T cells, and are expanded in mdck cell several
Wheel, is marked as 1-4 generations.The fluorescence intensity that peak value is represented has reacted the amount (lower part) of viral RNA.
In Fig. 3,8 6-8 week old C57/B6 mouse are infected in different time points with M genetic fragment mutated viruses storehouse collunarium
Collect lungs and do Genotyping.Each peak value represents the viral RNA amount of insertion position.PBS is handled and wild type WSN infection
Lung homogenate be used as positive control.
In Fig. 4, the single virus clone (A) filtered out in M gene mutations storehouse by being mutated what is separated for the first time
Distribution overview of 67 different mutant clons on M genes.(B) above-mentioned M gene mutations plasmid is transfected in 293T cells,
Cell conditioned medium is collected and determines virus titer.
In Fig. 6, different time is detected with 0.25MOI wild type WSN viruses and W7-791 viruses infection mdck cell
The virus titer of point.
In Fig. 7, (A, C) inoculation 106、107Or 108Mouse Weight after TCID50 W7-791 or wild type WSN virus
Detection；The the 4th and the 6th day virus titer is determined after (B, D) inoculation；(E) W7-791, WSN or PBS inoculation new life BALB/c are small
Mouse Weight is monitored after mouse.
In Fig. 8, (A) mouse immune and virus infected flow journey schematic diagram；(B-C) every group of 5 mouse Nasal immunizations 105PFU
W7-791 or same volume PBS, 4 times of MLD50 of inoculation WSN viruses, are regularly detected after virus infection after being immunized one month
Mouse Weight and survival condition；(D-E) every group of 5 mouse Nasal immunizations 105PFU W7-791 or PBS, is immunized one month
4 times of MLD50 PR8 viruses are inoculated with afterwards, and Mouse Weight and survival condition are regularly detected after virus infection.* * represent P- values<
0.001。
In Fig. 9, virus titer is detected in (A) mouse lung homogenate；(B) immune serum HAI Activity determinations；(C) exempt from
Epidemic disease mice serum antibodies against influenza virus is detected；(D) neutralization in microneutralization measuring W7-791 immune serums resists
Body titre；(E-F) adopt W7-791 immune mouses serum to be not immunized Mice Body in, after 24 hours be inoculated with lethal dose WSN
With HK68/H3 viruses, observe and record each time point mouse survival rate；(G-H) T cell of W7-791 immune mouses of adopting is arrived
It is not immunized in Mice Body, WSN and the HK68/H3 virus of lethal dose is inoculated with after 24 hours, observes and records each time point mouse
Survival rate.
Embodiment
Below in conjunction with the accompanying drawings, the present invention is specifically described and illustrated by embodiment：
The screening of novel influenza attenuated live vaccines and the concrete technical scheme such as figure of evaluation method that the present invention is set up
Shown in 1, it is described in detail below：
1. the radom insertion technology mediated using Mu transposon of phage sets up the high density mutation library of M genes
First, mediated according to Finnzymes companies Mu transposon of phage radom insertion mutagenesis kit (MGS kit,
Finnzymes) the operating procedure of specification, to inserting 5 ' between each base of influenza virus A/WSN/1933M gene-
The oligonucleotide sequence of this 15nt length of NNNNNTGCGGCCGCA-3 ', so as to obtain the high density mutation of influenza virus M genes
Storehouse (such as Figure 1A；Fig. 2).
2. virus mutant storehouse is obtained by influenza virus reverse Genetics Technique
The plasmid for carrying M gene mutation bodies is transformed into Escherichia coli (E.coli) DH10B impressions by the method for electricity consumption conversion
Body cell, the condition of electricity conversion is 2.0kV, 200 Ω, 25 μ F (ElectroMax TM DH10B, Invitrogen).From restructuring
Strain extracts M gene mutation bodies storehouse plasmid, the A/WSN/33 (H1N1) then set up using Hoffmann etc. (PNAS, 2000)
The Plasma viral reverse genetics operating system of influenza virus 8 obtains virus mutant storehouse.Specific method is：The HEK293T of culture
Cell is gone in 6 well culture plates, when cell confluency degree reaches 80~90%, according to transfection reagent operating instruction, will contain insertion
The plasmid of M genes and the plasmid mixed in equal amounts containing other 7 genetic fragments of influenza virus are mutated, with transfection reagent in proportion
Mix, be incubated at room temperature 15min, be added dropwise in HEK293T cell culture fluids, 37 DEG C, 48h is cultivated in 5%CO2 incubators,
Transfectional cell supernatant is collected, and by virus inoculation in being expanded on mdck cell.Infection collects virus after 48 hours, by portion
Partitivirus freezes (such as Figure 1A) for future use.
3. virus mutant storehouse component is analyzed by second generation sequencing technologies
Take above-mentioned obtained virus mutant storehouse virus to be passed on mdck cell, then tried using TRIzol
Viral RNA is extracted in agent (Invitrogen), and to each RNA according to reverse transcription reagent box iScriptTM cDNA Synthesis
The requirement of kit (Bio-Rad) operating instruction carries out reverse transcription and produces corresponding cDNA.Using the cDNA as template, respectively 3 are used
Specific forward primer (the 5 '-AGCAAAAGCAGGTAGATATT-3 ', 5 '-GGGGCCAAAGAAATAGCACT- of individual M genes
3 ', 5 '-TCCTAGCTCCAGTGCTGGTC-3 ') PCR is with the Vic insetion sequence specific reverse primers marked, PCR makes
With Novagen PCR enzyme KOD Hot-Start polymerase.PCR reaction condition is that (1 follows 95 DEG C of 10min of pre-degeneration
Ring)；95 DEG C of denaturation, 45s；52 DEG C of annealing, 30s；72 DEG C of extension, 90s；30 circulations of operation；Last 72 DEG C of extensions 10min (1
Circulation).The fluorescence labeling PCR products (setting is once repeated) and Liz-500 molecular weight standards (Applied obtained by PCR
Biosystem 96- capillary 3730xl DNA analyses instrument (3730xl DNA Analyzer, Applied) are utilized
Biosystems) it is sequenced (such as Figure 1B).Produced data application ABI softwares are analyzed according to following standard, (1)
All data all meet the acquiescence detection level of standard；(2) because the initial 70bp of sequence has the stronger non-specific back of the body
Scape, so being removed；(3) all sequences DNA sequence dna progress connection all corresponding with influenza virus M genes is matched somebody with somebody；(4) to sequencing
Data are done relative to wild type A/WSN/1933 virus infected cells, uninfecting virus cell and different genes library pair respectively
According to normalized, that removes PCR processes, primer and sequencing instrument produce non-specific data.
4. screening influenza virus attenuated live vaccines candidate's strain in Mice Body
In the present invention, our Application mouse models and above-mentioned second generation sequencing technologies identification virus mutant storehouse component
Method influenza virus attenuated live vaccines candidate strain (such as Figure 1A) is screened from M gene mutation virus bases.Pass through hypervelocity first
The method concentration mutant library virus of centrifugation, determines and be used for after its virus titer the experiment of follow-up mouse infection.Virus passes through collunarium
Method infect the C57/B6 mouse of 6-8 week old, every group 8, respectively after infection second day, the 4th day, the 6th day and the 8th
It is collected the lung tissue of mouse and carries out homogenized, and total serum IgE is extracted with TRIzol reagents from lung homogenate, according to
Above-mentioned 3 method is sequenced to virus M genes in sample and is carried out quantitative analysis.In an experiment, handled and wild with PBS
The total serum IgE extracted in type WSN virus infected mice lung tissues is as control.Using above-mentioned 3 method to M bases in extracted RNA
The sequence of cause carries out qualitative and quantitative analysis, to determine presence situation of the different M gene mutations viruses in each sample.We
It was observed that the virus (such as Fig. 3) of three kinds of different duplicating dynamics.As shown in figure 3, A clusters are viral, this virus has effective multiple
Ability processed, they may result in disease as wild-type virus；B clusters mutated viruses may have impact on virus due to mutation
The structure of gene and albumen is with function or causes the forfeiture of viral escape host immune response function and is seriously caused weak, growth
Slowly, these viruses in body due to that can hardly survive, so being unable to effective stimulus body produces immune response, so
It is not preferable vaccine candidate strain.By contrast, although C clusters virus can be replicated effectively in the first six day after infection,
Then removed at 6-8 days by body, be now nearly no detectable these viral presence.Such virus is with regard to that can stimulate body
Stronger immune response is produced, but because it can not continue to replicate and can not cause disease, so it is living to can serve as weak poison
Vaccine candidate strain.
5. candidate's attenuated live vaccines strain genetic stability and safety evaluatio
Good attenuated live vaccines are needed with absolute security, and its phenotype and genotype were required in generation
Stable heredity between border.So, we carry out system comprehensively safety to the above-mentioned attenuated vaccine candidate strain obtained that screens
Property and genetic stability evaluation are the very important parts of this technology system.Therefore, We conducted following experiment：(1)
The separation of weak poison poison and phenotypic evaluation：We have carried out monoclonal to above-mentioned C clusters virus first, and 67 diseases are obtained altogether
Poison clone.And the strain virus of W7-757, W7-791 and W7-797 tri- that wherein can be effectively replicated in mdck cell is carried out
Amplification (Fig. 4).And wherein W7-791 shows more preferable replication capacity and relatively low cytotoxicity (Fig. 5, Fig. 6).So I
It was initially believed that W7-791 be probably more satisfactory attenuated vaccine candidate's strain；(2) attenuated vaccine genetic stability is detected：
In order to ensure back mutation will not occur for vaccine, the phenomenon for occurring attenuated vaccine reversion, we are thin in MDCK by W7-791 viruses
A series of passage is carried out in born of the same parents and Mice Body, the gene order spy to obtaining virus from cell or mouse lung homogenate
It is not that the sequences of M genes is determined, it has been found that the mutation of W7-791 virus M genes can be gone down by stably heredity,
Can't occur the phenomenon of deletion or the back mutation of insertion mutation.And increasing with passage number, W7-791 viruses
Titre is gradually lowered.The heredity that the mutation and phenotype that this explanation w7-791 viruses have can stablize down.(3) vaccine
Safety evaluation：With the W7-791 virus immunity 6-8 week old mouse of different titers, even when every Murine Virus inoculation
Amount up to 107TCID50, we do not have found that mouse produces Body weight loss and flu-like symptom yet.Compared with, 103TCID50 open country
Then there is obvious flu-like symptom and Body weight loss occurs in the mouse of raw type WSN virus infection.6 days diseases of w7-791 infecting mouses
Malicious carrying capacity is lower 100 times (Fig. 7 A, B, C, D) than wild type WSN viruses and H3 subtype viral infection mouse lung inner virus titres.
If the lungs of 4 days mouse after observation infection, it has been found that the lungs of PBS groups and W7-791 infecting mouses do not occur substantially
Lesion, and serious lung injury is then presented in the mouse of wild type WSN virus infection.In order to further confirm that W7-791's
Security, we are inoculated with not same amount (10 to the newborn BALB/c mouse collunarium of 15 ages in days6,107or 108TCID50 W7-)
791 or 104TCID50 wild type WSN is viral, and Mouse Weight and lung lesion testing result show, W7-791 Mice Inoculateds
On do not observe the Body weight loss as wild type WSN virus infected mices and pulmonary lesion (Fig. 7 E).These results all tables
It is bright, we screen acquisition influenza virus mutant strain W7-791 be can only in vitro and in vivo in it is restricted replicate, to adult and
Newborn mice all has the low virulent strain new compared with high safety.
6. the systematicness evaluation of candidate vaccine strain protecting effect
Determine after candidate vaccine strain, it is necessary to which it is evaluated for the immune protective efficiency that body can be provided.Wherein lead
It is related to content：(1) immune protective is detected：W7-791 immune mouses are used, the January after immune, with 4 times of MLD50's
Wild type WSN viruses or PR8 viruses are infected mouse.We have found that non-immune group mouse body weight in experimentation is tight
Decline again and dead, and W7-791 immune mouses have been always maintained at normal body weight, and any flu-like symptom is not shown
(such as Fig. 8 A-E).(2) humoral immunity level is detected：Neutralizing experiment by hirst's hemagglutination Inhibition test or virus can survey
Determine influenza-specific antibody or virucidin in immune serum.Immune mouse antibody test result shows, W7-791
Immune mouse only produces the antibody of WSN virus-specifics, without for PR8 viruses, HK68 (H3N1), Wis (H3N2)
The antibody (such as Fig. 9 A-C) of virus.And non-immune mouse is given by the serum adoptive transfer of W7-791 immune mouses, using various diseases
When poison is to these mouse infections, immune serum decapacitation provide part for WSN in itself protect outer, can not protect
Other infection (Fig. 9 D-F) of virus to mouse.This just illustrates that humoral immunity is not that W7-791 Strain provides immunity
Exclusive source.(3) cellullar immunologic response level is detected：By the T lymphocytes adoptive transfer of W7-791 immune mouses to rather
Epidemic disease mouse, then with different wild type influenza virus infecting mouses, it may be mouse institute energy to observe T lymphocytes of adopting
The immunity of offer, so that it is determined that the effect played in vaccine protection is immunized in T cell.It was found that when W7-791 is immune
The T cells adoptive transfer of mouse is given after non-immune mouse, mouse can be made to obtain the protection of part wide spectrum, so that certain
Occurring degree and disease symptomses (Fig. 9 D-F) of the mouse when by various influenza infections are reduced in degree.Thus illustrate
W7-791 can effectively induce body to produce protectiveness T cell immune response, and this also complies with what influenza virus attenuated live vaccines were immunized
Feature.
SEQUENCE LISTING
<110>Suzhou system medicine research institute
<120>A kind of screening of influenza virus attenuated live vaccines strain and authentication method
<130> 2017
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1027
<212> DNA
<213> A/WSN/1933
<400> 1
ctgcagccct agatattgga aagatgagtc ttctaaccga ggtcgaaacg tacgttctct 60
ctatcgtccc gtcaggcccc ctcaaagccg agatcgcaca gagacttgaa gatgtctttg 120
cagggaagaa caccgatctt gaggttctca tggaatggct aaagacaaga ccaatcctgt 180
cacctctgac taaggggatt ttaggatttg tgttcacgct caccgtgccc agtgagcggg 240
gactgcagcg tagacgcttt gtccaaaatg ctcttaatgg gaacggagat ccaaataaca 300
tggacaaagc agttaaactg tataggaagc ttaagaggga gataacattc catggggcca 360
aagaaatagc actcagttat tctgctggtg cacttgcctg ttgtatgggc ctcatataca 420
acaggatggg ggctgtgacc actgaagtgg catttggcct ggtatgcgca acctgtgaac 480
agattgctga ctcccagcat cggtctcata ggcaaatggt gacaacaacc aatccactaa 540
tcagacatga gaacagaatg gttctagcca gcactacagc taaggctatg gagcaaatgg 600
ctggatcgag tgagcaagca gcagaggcca tggatattgc tagtcaggcc aggcaaatgg 660
tgcaggcgat gagaaccgtt gggactcatc ctagctccag tgctggtcta aaagatgatc 720
ttcttgaaaa tttacaggcc tatcagaaac gaatgggggt gcagatgcaa cgattcaagt 780
gatcctctcg tcattgcggc cgcagtcatt gcagcaaata tcattggaat cttgcacttg 840
atattgtgga ttcttgatcg tctttttttc aaatgcattt atcgtcgctt taaatacggt 900
ttgaaaagag ggccttctac cgaaggagtg ccagagtcta tgagggaaga atatcgaaag 960
gaacagcaga gtgctgtgga tgttgacgat ggtcattttg tcaacataga gctggagtaa 1020
aaaacta 1027
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
agcaaaagca ggtagatatt 20
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
ggggccaaag aaatagcact 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
tcctagctcc agtgctggtc 20
Claims (5)
1. screening and the authentication method of a kind of influenza virus attenuated live vaccines strain, it is characterised in that comprise the following steps：
(1) the radom insertion technology mediated using Mu transposon of phage sets up the high density mutation library of M genes：
Using the radom insertion mutagenesis kit of Finnzymes companies Mu transposon of phage mediation to influenza virus A/WSN/
The oligonucleotide sequence of this 15nt length of 5 '-NNNNNTGCGGCCGCA-3 ' is inserted between each base of 1933 M genes, so as to obtain
Obtain the high density mutation library of influenza virus M genes；
(2) virus mutant storehouse is obtained by influenza virus reverse Genetics Technique：
The plasmid for carrying M gene mutation bodies is transformed into Escherichia coli DH10B receptor cells by the method for electricity consumption conversion, from restructuring
Strain extracts M gene mutation bodies storehouse plasmid, then utilizes the Plasma viral reverse genetics behaviour of A/WSN/33 H1N1 influenza viruses 8
Make system and obtain virus mutant storehouse；
(3) virus mutant storehouse component is analyzed by second generation sequencing technologies：
The virus mutant storehouse virus for taking step (2) to obtain is passed on mdck cell, is then carried using TRIzol reagents
Viral RNA is taken, and reverse transcription generation is carried out according to reverse transcription reagent box iScriptTM cDNA Synthesis kit to each RNA
Corresponding cDNA, using the cDNA as template, respectively using the specific forward primer of 3 M genes, its sequence is respectively SEQ ID
No.2, SEQ ID No.3, SEQ ID No.4, do PCR amplifications, by PCR with the Vic insetion sequence specific reverse primers marked
The fluorescence labeling PCR primer of acquisition is set once repeatedly is divided with Liz-500 molecular weight standards using 96- capillary 3730xl DNA
Analyzer is sequenced, and produced data application ABI softwares are analyzed, and removes what PCR processes, primer and sequencing instrument were produced
Non-specific data；
(4) screening influenza virus attenuated live vaccines candidate's strain in Mice Body：
Mutant library virus is concentrated by ultracentrifugal method first, determines and be used for after its virus titer follow-up mouse infection reality
Test, virus infects the C57/B6 mouse of 6-8 week old, difference second day, the 4th day, the 6th day after infection by the method for collunarium
With the lung tissue for collecting mouse on the 8th day and carry out homogenized, extract total serum IgE with TRIzol reagents from lung homogenate,
According to above-mentioned steps（3）Middle method is sequenced to virus M gene in sample and is carried out qualitative and quantitative analysis, according to different M
Presence situation of the gene mutation virus in each sample, determines attenuated live vaccines candidate's strain；
(5) attenuated live vaccines candidate strain genetic stability and safety evaluatio：
（a）The separation of attenuated live vaccines and phenotypic evaluation：Monoclonal has been carried out to above-mentioned attenuated live vaccines candidate strain first,
The strain virus of W7-757, W7-791 and W7-797 tri- that wherein can be effectively replicated in mdck cell is expanded, tentatively
Filter out the W7-791 viruses for showing more preferable replication capacity and relatively low cytotoxicity；
（b）Attenuated live vaccines genetic stability is detected：By W7-791 virus passed in mdck cell and Mice Body, to from
The sequence that the M genes in the gene order of virus are obtained in cell or mouse lung homogenate is measured, and determines W7-791 viruses
The mutation of M genes can be gone down by stably heredity；
（c）The safety evaluation of attenuated vaccine：With the W7-791 virus immunity 6-8 week old mouse of different titers, mouse is not found
Produce Body weight loss and flu-like symptom；Newborn BALB/c mouse collunarium to 15 ages in days is inoculated with the W7-791 or 10 of different titers4
TCID50 wild type WSN viruses, detect to Mouse Weight and lung lesion, are not observed on W7-791 Mice Inoculateds
Body weight loss and pulmonary lesion as wild type WSN virus infected mices, it is influenza disease to thereby determine that W7-791 viruses
Malicious attenuated live vaccines strain.
2. screening and the authentication method of influenza virus attenuated live vaccines strain according to claim 1, it is characterised in that：Step
Suddenly（2）In electricity conversion condition be 2.0kV, 200 Ω, 25 μ F.
3. screening and the authentication method of influenza virus attenuated live vaccines strain according to claim 1, it is characterised in that：Step
Suddenly（2）It is middle obtain virus mutant storehouse specific method be：The HEK293T cells of culture are gone in 6 well culture plates, treat that cell converges
It is right when reaching 80~90%, according to transfection reagent operating instruction, by the plasmid of the genes of M containing insertion mutation and contain influenza virus
The plasmid mixed in equal amounts of other 7 genetic fragments, is mixed in proportion with transfection reagent, is incubated at room temperature 15 min, is added dropwise
In HEK293T cell culture fluids, 37 DEG C, 48 h are cultivated in 5%CO2 incubators, transfectional cell supernatant is collected, and by virus inoculation
In being expanded on mdck cell, infection collects virus after 48 hours, and fractionated viral is frozen for future use.
4. screening and the authentication method of influenza virus attenuated live vaccines strain according to claim 1, it is characterised in that：Step
Suddenly（3）Middle PCR is pre-degeneration 95 using Novagen PCR enzyme KOD Hot-Start polymerase, PCR reaction condition
DEG C, 10 min；95 DEG C of denaturation, 45s；52 DEG C of annealing, 30s；72 DEG C of extension, 90s；30 circulations of operation；Last 72 DEG C of extensions 10
min。
5. screening and the authentication method of influenza virus attenuated live vaccines strain according to claim 1, it is characterised in that：Step
Suddenly（4）It is middle determine attenuated live vaccines candidate's strain standard be：Virus can effectively be replicated in mouse lung after infection, but
After infection 6-8 days when removed by body.
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| PCT/CN2017/080560 WO2018157454A1 (en) | 2017-03-01 | 2017-04-14 | Method for screening and identifying live attenuated influenza vaccine strains |
| US15/908,723 US20180251769A1 (en) | 2017-03-01 | 2018-02-28 | Recombinant Influenza Virus |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592276A (en) * | 2019-07-08 | 2019-12-20 | 北京世纪元亨动物防疫技术有限公司 | Specific primer and kit for detecting canine influenza virus |
| CN112313748A (en) * | 2018-06-20 | 2021-02-02 | 香港中文大学 | Measurement and prediction of viral gene mutation patterns |
| CN114381440A (en) * | 2022-01-27 | 2022-04-22 | 浙江迪福润丝生物科技有限公司 | A group of influenza A virus attenuated strains based on synonymous mutation and/or deletion mutation and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025583B (en) | 2021-05-24 | 2021-09-24 | 华南农业大学 | Whole-avian-source H5N2 subtype avian influenza recombinant strain, vaccine and application thereof |
| CN113136372B (en) * | 2021-05-28 | 2023-08-01 | 广西大学 | Construction method of recombinant phage |
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| CN1082606A (en) * | 1992-04-14 | 1994-02-23 | 纽约市立大学西奈山医学院 | Use genetically engineered attenuated viruses |
| CN1644686A (en) * | 2003-10-08 | 2005-07-27 | 中国疾病预防控制中心病毒病预防控制所 | High yielded strain of mammalia influenza virus, its recommbined strains and preparation and use thereof |
| CN104582725A (en) * | 2012-07-17 | 2015-04-29 | 梅里亚有限公司 | Attenuated swine influenza vaccines and methods of making and use thereof |
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| ES2393492T3 (en) * | 2004-05-25 | 2012-12-21 | Medimmune, Llc | Variants of hemagglutinin and influenza neuraminidase |
| WO2011125054A2 (en) * | 2010-04-09 | 2011-10-13 | The Catholic University Of America | Protein and nucleic acid delivery vehicles, components and mechanisms thereof |
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2017
- 2017-03-01 CN CN201710118851.XA patent/CN106939355A/en active Pending
- 2017-04-14 WO PCT/CN2017/080560 patent/WO2018157454A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1082606A (en) * | 1992-04-14 | 1994-02-23 | 纽约市立大学西奈山医学院 | Use genetically engineered attenuated viruses |
| CN1644686A (en) * | 2003-10-08 | 2005-07-27 | 中国疾病预防控制中心病毒病预防控制所 | High yielded strain of mammalia influenza virus, its recommbined strains and preparation and use thereof |
| CN104582725A (en) * | 2012-07-17 | 2015-04-29 | 梅里亚有限公司 | Attenuated swine influenza vaccines and methods of making and use thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112313748A (en) * | 2018-06-20 | 2021-02-02 | 香港中文大学 | Measurement and prediction of viral gene mutation patterns |
| CN110592276A (en) * | 2019-07-08 | 2019-12-20 | 北京世纪元亨动物防疫技术有限公司 | Specific primer and kit for detecting canine influenza virus |
| CN114381440A (en) * | 2022-01-27 | 2022-04-22 | 浙江迪福润丝生物科技有限公司 | A group of influenza A virus attenuated strains based on synonymous mutation and/or deletion mutation and preparation method and application thereof |
| CN114381440B (en) * | 2022-01-27 | 2023-12-15 | 浙江迪福润丝生物科技有限公司 | Group of influenza A virus attenuated strains based on synonymous mutation and/or deletion mutation, and preparation method and application thereof |
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