CN104056276B - Influenza virus attenuated live vaccine and preparation method thereof - Google Patents
Influenza virus attenuated live vaccine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an influenza virus attenuated live vaccine with a preservation number of CCTCCNO: V201302, which is obtained based on mutation of M gene, the vaccine candidate strain has good gene stability, and attenuation characteristic and has strong immunogenicity, can induce a specific serum antibody of influenza virus and a mucous membrane antibody, and can induce different subgroups for generating T cell and quantity of CD8＜+＞T cells for secreting the IFN-gamma. Under current production capability and technology, immunization population can be effectively enlarged, and the influenza virus attenuated live vaccine has important social meaning and usage value during a flu period.
Description
Technical field
The present invention relates to field of biological product, more particularly, is related to a kind of attenuated influenza virus live vaccine and its preparation
Method.
Background technology
Influenza virus prevalence can substantially be divided into pandemics, localized epidemics and distribute three kinds.Since in April, 2009,
A kind of novel influenza（H1N1）Begin to extend in Mexico and cause flu outbreak in world wide rapidly
[Mexico flu deaths raise fears of global epidemic.MSNBC.2009-04-24.].By the end of
On May 26th, 2011, cause 1,353,141 people infection altogether in worldwide, death toll is 15,934 people.
The maximally effective means of flu-prevention viral prevalence are vaccination.
Compared with traditional inactivated vaccine, attenuated influenza virus live vaccine has more advantages, and it is original that it remains virus
It is amount of activated, after inoculation crowd can by virus upper respiratory tract replicate simulation natural infection, body can be induced to produce
More fully immunne response, obtains experimenter and is more extensively effectively protected.Simultaneously attenuated live vaccine can exist in body
Long period, therefore cause the time of effective immunne response more strong and persistently long, rareer is some attenuated live vaccines
Body can be induced to produce stronger cross-immune reaction, have certain intersection to the popular strain virus that surface antigen occurs drift
Protective effect.Additionally, its using method is easy easily.
Now, the attenuated influenza virus live vaccine of listing mainly includes cold adaptation attenuated live vaccine and the E Luo for developing in the U.S.
This attenuated live vaccine developed.Vaccine replication capacity in 37 DEG C of the normal body temperature of people is greatly limited, to people not
With or with relatively low pathogenic ability, but the strains of influenza viruses of effective duplication can be carried out at 25 DEG C and 33 DEG C, have
Cold adaptation and temperature sensitive characteristic [Ambrose CS, Luke C, Coelingh K.Current status of live
attenuated influenza vaccine in the United States for seasonal and pandemic
influenza.Influenza Other Respi Viruses.2008,2(6):193-202.].But due to patent and know
Know the restriction and protection of property right, the attenuated live vaccine of the type does not also have in China's approval listing.
The stromatin of influenza virus, plays an important role in its replicative cycle, and matrix prote m1 is influenza disease
The most albumen of content in malicious granule, take part in the composition of viral shell skeleton, and in addition it can also be combined with the RNA of virus, with RNP
Composite bulk phase interaction, play an important role [Sha B, L uo M.Structure of a in the duplication and infection of virus
bifunctional membrane-RNA binding protein,influenza virus matrix protein M
1.Nat Struct Bio l.1997,4(3):239-244.].M2 albumen has the work of pH value in ion channel and adjusting film
With the effect can affect the conformation of HA albumen, so as to affect sprouting and infecting [Takeuch i K, L amb R for virus
A.Influenza virus M 2 protein ion channel activity stabilizes the native form
of fowl plague virus hemagglutinin during intracellular transport.J
Virol.1994,68(2):911-919.].For the above-mentioned reasons, the present invention have studied and prepare influenza virus based on M gene mutation
The methods and techniques of attenuated live vaccine.
The content of the invention
First technical problem to be solved by this invention is to provide a kind of attenuated influenza virus live vaccine.
Second technical problem to be solved by this invention is to provide a kind of preparation side of attenuated influenza virus live vaccine
Method.
The M genes that 3rd technical problem to be solved by this invention is to provide mutation are preparing flu-prevention vaccine system
Application in agent.
In order to solve above-mentioned first technical problem, the invention provides a kind of attenuated influenza virus live vaccine, its preservation
Numbering is：CCTCC NO:V201302.
In order to solve above-mentioned second technical problem, the invention provides the preparation method of attenuated influenza virus live vaccine,
Characterized in that, comprising the following steps：
（1）The RNA of virus is extracted from the chick embryo allantoic liquid of inoculation influenza virus, is prepared using the technology of RT-PCR
CDNA, expands the genetic fragment of influenza virus, is building up to respectively on expression plasmid；
（2）M site-directed point mutations, are building up on plasmid and sequence verification, the such as SEQ ID of the M gene orders after mutation
Shown in NO.1；
（3）The M gene plasmids of mutation and other unmutated gene plasmids transfect 293T cells jointly；
（4）Cells and supernatant inoculated into chick embryo, checking stability, attenuation, and safety and efficiency evaluation.
Used as a preferred version, the influenza virus are influenza A.
In order to solve above-mentioned 3rd technical problem, the invention provides the M genes of mutation are preparing flu-prevention vaccine
Application in preparation, it is characterised in that the M gene orders of the mutation are as shown in SEQ ID NO.1.
Used as a preferred version, the immunization wayses of the bacterin preparation are via intranasal application immunity.
Used as another preferred version, the influenza virus are influenza A.
The claimed attenuated influenza virus live vaccine of the present invention is named as influenza A viruss Influenza Virus A
M-PR8（Call M-PR8 in the following text）, it is deposited in China typical culture collection center（Preservation address：Wuhan City, Hubei Province Wuchang District Luo Ka
The Wuhan University of hill path 16 China typical culture collection center postcode 430072）, preservation date is on March 5th, 2013,
Deposit number is CCTCC NO:V201302.
Beneficial effects of the present invention：The invention provides a kind of attenuated influenza virus live vaccine, the mutation based on M genes and
Prepare, the vaccine candidate strain has preferable gene stability, attenuation, with stronger immunogenicity, induces machine
The strong immunne response of body, can not only induce influenza virus Specific serum antibodies and mucoantibody, it is also possible to which induction produces T
The different subgroups and the CD8 of secretion of gamma-IFN of cell+T cell quantity, under existing production capacity and technology, can be effective
Expansion immunity crowd, there is important social meaning and use value in flu outbreak.
Description of the drawings
Fig. 1：The Technology Roadmap that the present invention is implemented.
Fig. 2：Level of growth of the contrast M-PR8 and PR8 influenza virus on mdck cell, is inoculated with respectively on mdck cell
The PR8 and M-PR8 of 0.001MOI, harvests respectively 24h after inoculation, the sample of 48h, 72h, 96h, using median tissue infective dose
Method detect the virus titer of each sample.* represent to compare with PR8 groups have significant difference（P ＜ 0.05）.
Fig. 3：Lung virus titer, per group of 3 6-8 week old mices, collunarium is inoculated with 20 μ l 105TCID50 dosage MPR8 or
PR8 virus liquids, after 3 days mice is put to death, and using the PBS (containing 0.1%BSA) of 2ml trachea-alveolar is rinsed, and is infected using median tissue
The method of dosage detects the virus titer of each sample, and * represents to compare with PR8 groups have significant difference（P ＜ 0.05）.
Fig. 4:The M-PR8 vaccine immunities of various dose detect the secretion level of IFN-γ in its splenocyte after three weeks.Per group
3 6-8 week old mices, difference collunarium is inoculated with 20 μ l 10TCID50, and 100 TCID50,1000TCID50 dosage M-PR8 is viral
Liquid, matched group collunarium is inoculated with the PBS of 20 μ l, and mice is put to death after 21 days, and separating Morr. cell detects spleen using ELISPOT assay
The secretion level of IFN-γ in cell, * represents to compare with matched group have significant difference（P ＜ 0.05）.
Fig. 5:The frequency of CD3+CD4+T cells, A, negative control in the splenocyte of the mice after analysis vaccine immunity；B,
10TCID50 immune group；C, 100TCID50 immune group；D, 1000TCID50 immune group.
Fig. 6：The frequency of CD3+CD8+T cells, A, negative control in the splenocyte of the mice after analysis vaccine immunity；B,
10TCID50 immune group；C, 100TCID50 immune group；D, 1000TCID50 immune group.
Fig. 7:The M-PR8 immune mouses of various dose are in 10 × LD50Body weight becomes after the homologous viral PR8 lethal challenges of dosage
Change.
Fig. 8:The M-PR8 immune mouses of various dose are in 10 × LD50Survival after the homologous viral PR8 lethal challenges of dosage
Rate.
Fig. 9:The M-PR8 immune mouses of various dose are in 10 × LD50Body weight after the heterologus virus H9N2 lethal challenges of dosage
Change.
Figure 10:The M-PR8 immune mouses of various dose are in 10 × LD50Depositing after the heterologus virus H9N2 lethal challenges of dosage
Motility rate.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.Experimental technique used in following embodiments for example without
Specified otherwise, is conventional method.Material used, reagent etc. in following embodiments, if no special instructions, can be from business way
Footpath obtains.It should be understood that these embodiments are only illustrative of the invention and is not intended to limit the scope of the invention.In the following example
The experimental technique of unreceipted actual conditions, generally according to normal condition, such as Sambrook et al., molecular cloning：Laboratory handss
Volume (New York:Cold Spring Harbor Laboratory Press, 1989) described in condition, or according to manufacture
Condition proposed by manufacturer.
The preparation of the attenuated influenza virus live vaccine of embodiment 1.
The RNA of virus is extracted from the chick embryo allantoic liquid of inoculation A/Puerto Rico/8/34 (H1N1) virus, using RT-
The technology of PCR prepares cDNA, expands eight genetic fragments of PB2, PB1, PA, HA, NP, NA, M, NS of influenza virus.Using overlap
The method of PCR introduces respectively mutation on M genes, and the M gene orders after mutation are as shown in SEQ ID NO.1.In transfection reagent
Lower eight plasmids of liposome Lipofectamine2000 auxiliary transfect 293T cells jointly.After 72 hours, sample, inoculation 10 are harvested
Day instar chicken embryo, after 48-72 hours, harvests chick embryo allantoic liquid, blood coagulation tests and gene extracting, sequence verification virus sequence it is correct
Property.The strain is named as M-PR8.
M-PR8 strain M gene orders（Sequence after mutation）
agcgaaagca tgtagatatt gaaagatgag tcttctaacc gaggtcgaaa cgtacgtact
ctctatcatc ccgtcagggc ccctgaaagc cgagatcgca cagagacttg aagatgtctt tgcagggaag
aacactgatc ttgaggttct catggaatgg ctaaagacaa gaccaatcct gtcacctctg actaagggga
ttttaggatt tgtgttcacg ctcaccgtgc ccagtgagcg aggactgcag cgtagacgct ttgtccaaaa
tgcccttaat gggaacgggg atccaaataa catggacaaa gcagttaaac tgtataggaa gctcaagagg
gagataacat tccatggggc caaagaaatc tcactcagtt attctgctgg tgcacttgcc agttgtatgg
gcctcatata caacaggatg ggggctgtga ccactgaagt ggcatttggc ctggtatgtg caacctgtga
acagattgct gactcccagc atcggtctca taggcaaatg gtgacaacaa ccaatccact aatcagacat
gagaacagaa tggttttagc cagcactaca gctaaggcta tggagcaaat ggctggatcg agtgagcaag
cagcagaggc catggaggtt gctagtcagg ctagacaaat ggtgcaagcg atgagaacca ttgggactca
tcctagctcc agtgctggtc tgaaaaatga tcttcttgaa aatttgcagg cctatcagaa acgaatgggg
gtgcagatgc aacggttcaa gtgatcctct cgctattgcc gcaaatatca ttgggatctt gcacttgaca
ttgtggattc ttgatcgtct ttttttcaaa tgcatttacc gtcgctttaa atacggactg aaaggagggc
cttctacgga aggagtgcca aagtctatga gggaagaata tcgaaaggaa cagcagagtg ctgtggatgc
tgacgatggt cattttgtca gcatagagct ggagtaaaaa actacattgt ttctact。
Described M gene mutation sequences, are applicable not only to described A type Gripovaxs, other type influenzas
Attenuated live vaccine also within use range, such as Type B Gripovax.
The research of embodiment 2.M-PR8 biological characteristicses
1) stable gene Journal of Sex Research：M-PR8 Strain is inoculated with mdck cell, it is continuous to pass for 10 generations, collect the 1st, 5,10
For cells and supernatant, extracted total RNA, reverse transcription is building up on pMD18-T Vector into cDNA, PCR amplification genes, carries out
Sequencing, software compares gene, and can identification mutant strain stably pass on.
2）Growth characteristics research on Strain cellular level：According to 0.001MOI virus inoculations, virus infection is determined respectively
The TCID of 24,48,72,96 hours afterwards50, for contrasting the growing state of M-PR8 viruses and PR8 viruses.
3）The research of attenuation：With pentobarbital sodium intraperitoneal anesthesia mice, every mice per nasal 20 μ L 10 of instillation5
TCID50Virus liquid, after 3 days, the disconnected neck of anesthesia is put to death, and aseptic dissection takes lung, Bronchio- alveolar wash with viral dilution liquid and is filled
Washing liquid 3 times, centrifugation, subpackage detects mouse lung virus titer with cell 50 3nfective dose.
4）The research of attenuated influenza virus live vaccine Candidate Strain M-PR8 viral immunogenics：
Immune programme for children and step：The female BAl BIc of 6-8 week old/c mices are divided into 4 groups：1. M-PR8 Strain
（1000TCID50）, 2. M-PR8 Strain（100TCID50）3. M-PR8 Strain（10TCID50）4. PBS blank control groups.
21 days after primary immune response, blood and nasal wash are taken, for antibody titer detection, and while separating spleen cell, for ELISPOT
The T cell and flow cytomery T cell subgroup of detection secretion of gamma-IFN.
A antibody tests：
（1）96 hole elisa Plates are coated with the PR8 inactivated virus vaccines of 10 μ g/ml, 4 DEG C, overnight incubation.
（2）After washing three times with PBST, 100 μ l PBST solution are added（Containing 8% defatted milk powder）, 37 DEG C are placed 1 hour.
（3）, using ELISA Plate is added after confining liquid dilution, 37 DEG C are reacted 1 hour for serum or nasal wash.
（4）PBST is washed three times, and the anti-work night of sheep anti mouse two of 100 μ l labelling HRP is added per hole, and 37 DEG C are placed 1 hour.
（5）PBST is washed three times, and 100 μ l TMB liquid, room temperature reaction 15-30 minutes are added per hole.
（6）The H of 50 μ l 2M is added per hole2SO4Terminating reaction, the OD values gone out with microplate reader detection 450nm, record data,
2SD is added with the meansigma methodss of matched group, as the criterion of positive findingses.
B Enzyme linked immunospots（ELISPOT）：
In order to detect the cellullar immunologic response level of mice, using ELISPOT mouse spleen lymphocyte secretion is have detected
Cytokine IFN-γ situation, be simply described below：
（1）The activation of pre-coated plate：200 μ l DMEM are added per hole, 10min is stored at room temperature, liquid is deducted.
（2）Add cell suspension：100 μ l concentration are added to be 1 × 10 per hole3Cells/ μ l spleen mononuclear cells.
（3）Add stimulus object：3 multiple holes only add 100 μ l DMEM, as background negative control, in positive control hole 10 are added
The PMA and Ionomycin of μ l working concentrations, adds 10 μ l DMEM, experimental group to add the μ g/ml's of final concentration 10 in negative control hole
PR8 totiviruss inactivated vaccines.
（4）Culture：37 DEG C, 5%CO224hr is cultivated in cell culture incubator.
（5）Cell lysis：Topple over the culture medium and cell in pulling, add the deionized water of 200 μ l pre-coolings, 4 DEG C of placements
10min, hypotonic lysis cell.
（6）Board-washing：Liquid in hole is got rid of, with 200 μ l/ holes 1 × Washing buffer 5 times are washed.
（7）Detection antibody：The one of the biotin labeling that 100 μ l of addition have diluted per hole resists, and 37 DEG C are incubated 1 hour.
（8）Board-washing：Liquid in hole is got rid of, with 200 μ l/ holes 1 × Washing buffer 6 times are washed.
（9）Enzyme-linked Avidin incubation：The enzyme table Avidin working solution for adding 100 μ l to dilute per hole, 37 DEG C of incubations 1 are little
When.
（10）Board-washing：Liquid in hole is got rid of, with 200 μ l/ holes 1 × Washing buffer 5 times are washed.
（11）Colour developing：The AEC nitrite ions of 100 μ matching while using, room temperature lucifuge colour developing 30min or so are added per hole.
（12）Terminate：Liquid in pouring aperture, opens base, is washed with deionized 5 times positive and negatives, and plate is positioned over into cool place
Place's room temperature is dried.
（13）ELISPOT spot counts, statistical data analysis.
ELISPOT spot counts be by up to section be Bioisystech Co., Ltd's reading.
The T cell subgroup of C flow cytomery CD4+, CD8+：
（1）Splenocyte specimen：The Falcon pipes for taking lid are numbered respectively, the negative control of numbering 1, numbering 2CD3, numbering
3CD3+CD4, numbering 4CD3+CD8, numbering 5CD3+CD4+CD8+, this six manage for for adjust compensation and voltage, remaining
Experimental group carry out labelling respectively, for detecting, 500 μ l 10 are often separately added in pipe6Individual corresponding cell.
（2）Falcon pipes are taken out, is washed twice with 2%FCS-PBS, 1000rpm, be centrifuged 5 minutes.Slowly suction out supernatant.
（3）1 μ g CD3APC, 1 μ g CD4 FITC or 1 μ gCD8PE fluorescent antibodys are separately added into according to pipe subscript note, are tested
Group adds three kinds of fluorescent antibodys, mixes, and room temperature lucifuge is incubated 15 minutes.
（4）Often pipe adds 2ml to contain 3%PBS-PBS, 1000rpm, is centrifuged 5 minutes, washes twice, leaves 200 μ l supernatants, uses rifle
Piping and druming uniform cell.
（5）Upper machine testing：Under CellQuest softwares, 100,000 cells are obtained.
（6）Data statistic analysis
The attenuated influenza virus live vaccine M-PR8 Candidate Strains of embodiment 3. can be provided for homologous virus protection
Immune programme for children and step：The female BAl BIc of 6-8 week old/c mices are divided into points 4 groups：1. M-PR8 Strain
（1000TCID50）, 2. M-PR8 Strain（100TCID50）3. M-PR8 Strain（10TCID50）4. PBS blank control groups.
Once, the PR8 counteracting toxic substances of 10LD50 after 21 days 3 days after virus attack, take broncho-alveolar lavage liquid and determine virus titer for immunity,
Remaining observation body weight change and survival rate.
The measure of virus titer：Trachea-lung washing liquid is made into 10 times of dilutions of series, with each dilution factor infection 96 holes are incubated at
Mdck cell in plate（Density is about 80%～90%）, each dilution factor 4 cell holes of parallel inoculation, and infection cell is placed in
CO2In incubator.After 37 DEG C of culture 72hr, hemagglutination test detects the titre of virus at a temperature of each, uses Reed-Muench methods
Calculate TCID50.Each experimental group virus titer is come table with the meansigma methodss ± SD of the virus titer of per group of all 5 mice samples
Show.
The attenuated influenza virus live vaccine Candidate Strain M-PR8 of embodiment 4. can be provided for the protection of different subtype virus
Immune programme for children and step：The female BAl BIc of 6-8 week old/c mices are divided into 3 groups：1. M-PR8 Strain
（1000TCID50）, 2. M-PR8 Strain（100TCID50）3. M-PR8 Strain（10TCID50）4. PBS blank control groups.
Once, the H9N2 counteracting toxic substances of 10LD50 after 21 days 3 days after virus attack, take broncho-alveolar lavage liquid and determine virus drop for immunity
Degree, remaining observation body weight change and survival rate.
Experimental result：
1）Strain hereditary stability is determined：M-PR8 Strain inoculation mdck cell carries out the generation of continuous passage 10, takes respectively
1st, 5,10 generation gene sequencing, compares gene order, as a result shows that gene order is not undergone mutation, it was demonstrated that strain have compared with
Good hereditary stability.
2）M-PR8 Strain growth characteristics researchs：By 0.001MOI virus inoculations, determine respectively 24,48 after virus infection,
The virus titer of 72,96 hours, for contrasting the growing state of M-PR8 Strain and PR8 Strain.As shown in Fig. 2 after inoculation
24 hours, the growth of 2 strain virus did not had obvious difference.When 72 hours, M-PR8 Strain is compared with PR8 Strain, and it is sick
Malicious titre reduces about more than 300 times, it was demonstrated that the replication capacity of virus has a larger decrease, and preliminary proof M-PR8 is viral
Strain has the characteristic for weakening virulence.
3) research of attenuation：Every mice per nasal 20 μ l 10 of instillation5M-PR8 the or PR8 virus liquids of TCID50,3
Tian Houqu pulmonarys bronchial perfusate, determines virus titer.As a result the average virus titer of instillation M-PR8 Strain groups is shown
log103.5TCID50/ml, the average titer of PR8 infection group and viral infection groups is log106.4TCID50/ml, the two has significance
Difference, illustrates that duplications of the M-PR8 in respiratory tract receives effectively suppression, it is shown that the strain has attenuation or weak poison
Characteristic（Fig. 3）
4）M-PR8 Strain immunogenicities
a：ELISA antibody：
In order to evaluate shell M-PR8 attenuated live vaccines can inducing mouse produce specific antibody response ability, mouse immune
Afterwards 21 days methods using eyeball venous blood sampling collect serum, and by ELISA method PR8 specific IgG antibodies in serum are determined.
As a result show（Table 1）, 1000TCID50M-PR8 groups have highest serum antibody and nasal wash antibody, and the increasing of antibody titer
Plus there is dose-dependence.
The M-PR8 vaccines of the intranasal immunizations various dose of table 1 induce the antibody response level that body is produced in mice
Note：The antibody response that per group of 3 mices induce in M-PR8 vaccine immune mouses.After immunity three weeks, sample is collected
This, using ELISA detection antibody titres.
a:As a result represented with the meansigma methodss ± SD of 3 mices.
b：Expression has significant difference, p ＜ 0.05 compared with matched group.
c：Expression has significant difference, p ＜ 0.05 compared with 10TCID50 immune group.
d:Expression has significant difference, p ＜ 0.05 compared with 100TCID50 immune group.
e:Representative is not detected by.
b：Enzyme linked immunospot：
As a result show（Fig. 4）, the splenocytes numbers of each immune group secretion of gamma-IFN increase with the increase of immunizing dose,
The splenocytes numbers of the secretion of gamma-IFN of 1000TCID50 vaccine immunities group induction have the difference of significance with matched group（P ＜
0.05）.
The T cell subgroup of C flow cytomery CD4+, CD8+：
As a result show（Fig. 5,6）, all immune group dawn Mus T cell subgroup (CD4+T, CD8+T cell) suffer from increase
Long, the level for this demonstrating cellular immunization has dose dependent.Wherein CD3+CD4+T cell subsets of matched group, CD3+CD8
Ratio shared by+T cell is respectively 3.29%, the CD3+CD4+T cells of 1.42%, 1000TCID50 vaccine immunity groups, CD3+CD8
Ratio shared by+T cell cell is respectively 10.62%, 4.53%.The CD3+CD4+T cells of 100TCID50 vaccine immunity groups, CD3
Ratio shared by+CD8+T cell is respectively 6.60%, 3.43%.The CD3+CD4+T cells of 10TCID50 vaccine immunity groups,
Ratio shared by CD3+CD8+T cell is respectively 3.68%, 2.98%.
4）：Attenuated influenza virus live vaccine Candidate Strain M-PR8 can provide the protection a virus titers for homologous viral PR8：
As a result show（Table 2）, using during PR8 virus attacks, the average Pneumovirinae titre of PBS groups is about 107TCID50/ ml and
104.6TCID50/ml.Immune 100TCID50Mice Mean pulmonary virus titer and nasal wash virus titer respectively may be about
105.4TCID50/ ml and 103.7TCID50/ ml, with the difference that PBS control group has significance.Immune 1000TCID50Mice is average
Lung virus titer and nasal wash virus titer respectively may be about 104.2TCID50/ ml and 102.5TCID50/ ml, with other groups
There is the difference of significance.
After the M-PR8 of the immune various dose of table 2. is viral, the effect of the homologous PR8 virus attacks of mice opposing fatal dose
Note：Per group of 3 6-8 week old mices, difference collunarium inoculation 20 μ l 10TCID50,100 TCID50,1000TCID50
Dosage M-PR8 virus liquids, matched group collunarium is inoculated with the PBS of 20 μ l, and the PR8 virus attacks of the 21st day 10LD50 are put to death on the 24th day
Mice, harvests trachea-alveola washing liquid and nasal wash, and using median tissue infective dose method virus titer is determined.
a：As a result represented with the meansigma methodss ± SD of 3 mices.
b：Expression has significant difference, p ＜ 0.05 compared with matched group
c:Expression has significant difference, p ＜ 0.05 compared with 10TCID50 immune group.
d：Expression has significant difference, p ＜ 0.05 compared with 100TCID50 immune group.
B body weight change and survival rate：
Matched group is all dead at the 7th day, 1000TCID50 immune group survival rate 100%, and 100TCID50 immune group is deposited
Motility rate 30%.By the body weight change for observing mice, 1000TCID50 immune group is found after counteracting toxic substances, the body weight change of mice is very
It is little, original body weight was returned at 2 weeks or so, and 100TCID50 immune group weight recoveries are relatively slow.
5）：Attenuated influenza virus live vaccine Candidate Strain M-PR8 can provide the protection for different subtype virus H9N2
A virus titers：
As shown in table 3, as a result show that, with the increase of immunizing dose, the virus titer of mouse lung is decreased, it is and right
Compare according to group, the lung virus titer and nasal wash virus titer of 1000TCID50 groups have the difference of significance（P<
0.05）.
After the M-PR8 of the immune various dose of table 3. is viral, the different hypotype H9N2 virus attack of mice opposing fatal dose
Effect
Note：Per group of 3 6-8 week old mices, respectively collunarium inoculation volume is 10TCID50, the 100TCID50 of 20 μ l,
The M-PR8 virus liquids of 1000TCID50 dosage, matched group collunarium is inoculated with the PBS of 20 μ l, and the H9N2 viruses of the 21st day 10LD50 are attacked
Hit, put to death mice within the 24th day, harvest trachea-alveola washing liquid and nasal wash, using median tissue infective dose method disease is determined
Malicious titre.
a：As a result represented with the meansigma methodss ± SD of 3 mices.
b：Expression has significant difference, p ＜ 0.05 compared with matched group.
c:Expression has significant difference, p ＜ 0.05 compared with 10TCID50 immune group.
d：Expression has significant difference, p ＜ 0.05 compared with 100TCID50 immune group.
B body weight change and survival rate：
As a result as Fig. 9 and Figure 10 shows, after matched group and all of immune group counteracting toxic substances a certain degree of clinic is all occurred in that
Symptom, but, 1000TCID50 immune group weight loss lesser extents, and reply it is very fast.In terms of survival rate,
1000TCID50 immune group is survived completely, protective rate 100%, 100TCID50 immune group survival rate 30%, and 10TCID50 immune group is deposited
Motility rate 10%, PBS groups are completely dead.
Claims (4)
1. a kind of attenuated influenza virus live vaccine, its deposit number is：CCTCC NO:V201302.
2. the preparation method of attenuated influenza virus live vaccine described in claim 1, it is characterised in that comprise the following steps：
(1) RNA of virus is extracted from the chick embryo allantoic liquid of inoculation influenza A, using the technology of RT-PCR cDNA is prepared,
The genetic fragment of amplification influenza virus, is building up to respectively on expression plasmid；
(2) M site-directed point mutations, are building up on plasmid and sequence verification, the such as SEQ ID NO.1 institutes of the M gene orders after mutation
Show；
(3) the M gene plasmids and other unmutated gene plasmids of mutation transfects 293T cells jointly；
(4) cells and supernatant inoculated into chick embryo, checking stability, attenuation, and safety and efficiency evaluation.
3. application of the M genes of mutation in prevention A type influenza vaccine formulations are prepared, it is characterised in that the M genes of the mutation
Sequence is as shown in SEQ ID NO.1.
4. application of the M genes being mutated according to claim 3 in prevention A type influenza vaccine formulations are prepared, its feature exists
In the immunization wayses of the bacterin preparation are via intranasal application immunity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310090753.1A CN104056276B (en) | 2013-03-20 | 2013-03-20 | Influenza virus attenuated live vaccine and preparation method thereof |
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