CN104232594A - Recombinant homologous avian H1N1 influenza virus inactivated vaccine strain (JS40/PR8) as well as preparation method and application of inactivated vaccine strain - Google Patents
Recombinant homologous avian H1N1 influenza virus inactivated vaccine strain (JS40/PR8) as well as preparation method and application of inactivated vaccine strain Download PDFInfo
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Abstract
The invention discloses a recombinant homologous avian H1N1 influenza virus inactivated vaccine strain (JS40/PR8) as well as a preparation method and an application of the inactivated vaccine strain. The recombinant avian H1N1 influenza virus inactivated vaccine strain takes A/swine/Jiangsu/40/2011 (H1N1) as genetic donors of surface hemagglutinin (HA) and neuraminidase (NA), and influenza virus A/PR/8/34(H1N1) as donors of six internal genes of a polymerase PB2 subunit, a polymerase PB1 subunit, a polymerase PA subunit, nucleoprotein (NP), a matrix protein (M) and non-structural protein (NS); the vaccine strain is obtained by natural recombination; and the preservation number of the vaccine strain is CCTCC NO:V201419. The invention also discloses a method for building the recombinant homologous avian H1N1 influenza virus inactivated vaccine strain, and an application of the homologous recombinant avian H1N1 influenza virus inactivated vaccine strain for preventing homologous avian H1N1 influenza of animals or people.
Description
Technical field
The present invention relates to recombination engineered vaccine, particularly, the present invention relates to a kind of for preventing the recombination engineered vaccine of animals and humans fowl type H1N1 influenza, more specifically, vaccine involved in the present invention is a kind of for preventing the recombination classes fowl type H1N1 inactivated influenza virus vaccine of animals and humans fowl type H1N1 influenza.
Background technology
Influenza virus is the togavirus that the extensive infected poultry of a kind of energy and mammiferous segmented strand RNA are formed.In the whole world, influenza often occurs sporadicly to break out assorted random pandemic form of holding concurrently, in the past few decades, the main Selection utilization of various countries remain totivirus deactivation vaccine, it mainly stimulates body to produce humoral immunization to resist the hemagglutinin surface glycoprotein of identical hypotype.
Class fowl type H1N1 is extensively popular in China swinery, and epidemic situation still expands (Yan Chen, Infect Genet Evol.2013) in continuation.In 2011, Sihong County, Jiangsu Province of China occurred that people infects class fowl type H1N1 swine influenza virus event, and this Ye Shi China first time report class fowl type H1N1 hypotype porcine influenza infects people's event (Huanliang Yang, Emerg infect 2012).Influenza A depends on host range change that virus causes because of adaptive variation, the restrictive factor of host self and the change of environmental factors across kind of propagation.In order to the development of corresponding epidemic situation, invention has been the development of nature recombinant vaccine strain.
Desirable vaccine strain should possess the antigen matching good with epidemic strain, to conditions such as chicken embryo had no pathogenicity and chicken embryo high titre growth property.But, be difficult to possess the strain of above-mentioned condition as vaccine strain from nature separation.At home, because the street strain's chicken embryo adaptability now be separated is poor, existing domestic class fowl type H1N1 influenza vaccines are caused to there is the shortcomings such as cost is high, the vaccine immunity phase is short.
Influenza virus A/PR/8/34 (H1N1) (being called for short PR8) is laboratory chicken embryo adapted strain, and namely this virus can grow to very high titre in chicken embryo, and this chicken embryo adaptability is determined by 6 internal gene of virus.In addition, when two influenza virus co-infection cell or chicken embryo, can exchange by producer, thus produce new gene recombined virus.Therefore, pass through genetic recombinant methods, the carrying out of HA, NA gene of 6 internal gene of influenza virus A/PR/8/34 (H1N1) and epidemic strain can be recombinated and obtain a new recombinant virus, this recombinant virus has the antigenicity consistent with HA and NA genetic donor strain, also has the chicken embryo high titre growth characteristics of influenza virus A/PR/8/34 (H1N1) simultaneously.
Artificial nature's recombinant technology is a kind of simple and effective structure 2:6 recombinant viral vaccine strain method, the genome of influenza virus is made up of the strand RNA fragment of 8 segmentations, when 2 viral co-infection chicken embryos or cell, its genomic fragment can exchange, thus produce some new recombinant viruses (reassortant), by the process containing corresponding antibodies positive serum, then effectively can screen the strain of 2:6 recombinant viral vaccine.(Hualan?Chen,Vaccine2003)。
Summary of the invention
Technical problem to be solved by this invention provides a kind of recombination classes fowl type H1N1 inactivated influenza virus vaccine strain and preparation method thereof.
In order to reach above object, the technology used in the present invention means are:
Inventor infects the separation of people's event locality at Jiangsu class fowl type H1N1 and obtains a strain swine influenza virus isolated strain, sequence alignment analysis finds this strain and the strain very high homology infecting people, the virus strain called after A/swine/Jiangsu/40/2011 (H1N1) this separation obtained, is called for short SW/JS40 (H1N1).
The present invention utilizes artificial recombination method, with H1N1 hypotype swine influenza virus A/swine/Jiangsu/40/2011 (H1N1) for surface antigen haemagglutinin (HA) and neuraminidase (NA) genetic donor, with influenza virus A/PR/8/34 (H1N1) for polysaccharase PB2 subunit, polysaccharase PB1 subunit, polysaccharase PA subunit, nucleoprotein (NP), the donor of stromatin (M) and Nonstructural Protein (NS) 6 internal gene, the class fowl type H1N1 inactivated influenza virus vaccine strain of 1 strain restructuring is obtained by naturally recombinating, called after influenza A A/swine/Jiangsu/40/PR8 (H1N1) (referred to as JS40/PR8), be deposited in and be positioned at China typical culture collection center, address is in the Wuhan University of Wuhan, China, its preserving number is CCTCCNO:V201419, the preservation time is on June 19th, 2014.
The present invention selects 6 internal gene that can grow to laboratory chicken embryo adapted strain A/PR/8/34 (H1N1) of very high titre in chicken embryo of influenza virus as backbone genes, and select HA and the NA gene being separated class fowl type strain A/swine/Jiangsu/40/2011 (H1N1) obtained for 2011 as surface gene, utilize nature recombination method, prepared recombinant virus JS40/PR8, this recombinant strain can as the vaccine strain of class fowl type H1N1 influenza.
Experiment confirms, JS40/PR8 is consistent with China class fowl type H1N1 influenza virus epidemic strain SW/JS40 (H1N1) antigenicity, will produce good specific protective to class fowl type H1N1 influenza virus epidemic strain SW/JS40 (H1N1).6 internal gene of this virus containing influenza virus chicken embryo high titre adapted strain A/PR/8/34 (H1N1), therefore have good chicken embryo adaptability, viral growth titre, compared with the poison of open country, can improve more than 8 times; Produce vaccine as vaccine strain, can greatly reduce production of vaccine cost, improve vaccine quality.
Therefore, further, the class fowl type H1N1 inactivated influenza virus vaccine strain that the invention allows for described restructuring preparation prevention class fowl type H1N1 influenza virus cause in the medicine of disease application.
Wherein, preferably, described medicine is for preventing the class fowl type H1N1 influenza infection of animal or human.
Further, the invention allows for a kind of method building the class fowl type H1N1 inactivated influenza virus vaccine strain of described restructuring, the method comprises:
(1) vaccine strain A/PR/8/34 (H1N1) co-inoculation chick embryo allantoic cavity is adapted to class fowl type H1N1 strains of influenza viruses A/swine/Jiangsu/40/2011 (H1N1) and chicken embryo or cell carries out nature restructuring;
(2) adapt to the antibody screening of vaccine strain A/PR/8/34 (H1N1) with anti-described chicken embryo, removing surface gene derives from the strain that described chicken embryo adapts to vaccine strain A/PR/8/34 (H1N1);
(3) by screen the strain that obtains carry out on chicken embryo 3 generation limited clone purification, Trizol is utilized to fission by force agent method, extract viral RNA, carry out reverse transcription, then RT-PCR amplification is carried out with the influenza virus A/PR/8/34 (H1N1) of design and each 8 pairs of specificity diagnostic primerses of the domestic isolate A/swine/Jiangsu/40/2011 of class fowl type H1N1 influenza virus (H1N1) virus, then, nucleic acid gel electrophoresis is carried out to amplified production, identify that the recombinant virus obtained is that HA and NA comes from the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) of class fowl type H1N1 influenza virus, 6 internal gene PB2, PB1, PA, NP, M and NS is from the recombinant virus of influenza virus A/PR/8/34 (H1N1), obtain.
In method of the present invention, preferably, described primer comprises:
Can only amplify 8 specific fragments of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) gene and can not amplify 8 pairs of specificity diagnostic primerses of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) of any fragment of influenza virus A/PR/8/34 (H1N1), primer sequence is as follows:
And 8 specific fragments of influenza virus A/PR/8/34 (H1N1) can only be amplified and 8 pairs of specificity diagnostic primerses of the influenza virus A/PR/8/34 (H1N1) of any fragment of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) can not be amplified, primer sequence is as follows:
To sum up, experiment proves, the present invention prepared a strain excellent can be used for prevent class fowl type H1N1 influenza virus cause the virus strain of disease, of the present invention is the further development research of class fowl type H1N1 influenza vaccines, has established solid basis.
Accompanying drawing explanation
Figure 1A be with JS40 and PR8 strain cDNA for template, utilize the amplification that the Auele Specific Primer of JS40 strain PB2, PB1, PA, HA, NP, NA, M, NS gene carries out;
Wherein the first to the 8th swimming lane is that the Auele Specific Primer of PB2, PB1, PA, HA, NP, NA, M, NS gene using JS40 strain is to the amplification of JS40 strain cDNA; M is DNA Marker 2000; 9th to the 16 swimming lane is the amplification that the Auele Specific Primer of PB2, PB1, PA, HA, NP, NA, M, NS gene using JS40 strain is template to PR8 strain cDNA;
Figure 1B be with JS40 and PR8 strain cDNA for template, utilize the amplification that the Auele Specific Primer of PR8 strain PB2, PB1, PA, HA, NP, NA, M, NS gene carries out;
Wherein 1 ~ 8 swimming lane is the amplification that the Auele Specific Primer of PB2, PB1, PA, HA, NP, NA, M, NS gene using PR8 strain is template to JS40 strain cDNA; M is DNA Marker 2000; 9 ~ 16 swimming lanes are the amplification that the Auele Specific Primer of PB2, PB1, PA, HA, NP, NA, M, NS gene using PR8 strain is template to PR8 strain cDNA;
Fig. 2 is the RT-PCR amplification of PB2, PB1, PA, HA, NA, NP, M and NS gene of JS40/PR8 recombinant virus;
1 ~ 8 swimming lane: recombinant virus JS40/PR8 utilizes the PCR primer of 8 pairs of specificity diagnostic primers amplifications of JS40; M:DNA molecular mass standard; 9 ~ 16 swimming lanes: recombinant virus JS40/PR8 utilizes the PCR primer of 8 pairs of specificity diagnostic primers amplifications of PR8;
The growth curve that Fig. 3 measures optimum culturing temperature 35 DEG C for restructuring JS40/PR8 virus and wild-type class fowl type H1N1 influenza virus A/swine/Jiangsu/40/2011 (H1N1) (being called for short JS40);
Fig. 4 is the body weight change situation of each group mouse after attacking poison.
Sequence illustrates:
SEQ ID No.1:JS40/PR8 HA gene;
SEQ ID No.2:JS40/PR8 NA gene;
SEQ ID No.3:JS40/PR8 PB2 gene;
SEQ ID No.4:JS40/PR8 PB1 gene;
SEQ ID No.5:JS40/PR8 PA gene;
SEQ ID No.6:JS40/PR8 NP gene;
SEQ ID No.7:JS40/PR8 M gene;
SEQ ID No.8:JS40/PR8 NS gene.
Embodiment
Further describe the present invention below in conjunction with specific embodiments and the drawings, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Experiment material
1. virus strain
A/swine/Jiangsu/40/2011 (H1N1) (being called for short JS40) (is documented in document: the foundation of the reverse genetics system of a strain class fowl type H1N1 hypotype swine influenza virus, Yang Huanliang etc., " Chinese Preventive Veterinary Medicine report ", 02 phase in 2013) preserved by this laboratory, A/PR/8/34 (H1N1) (being called for short PR8) is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's animal influenza center.
2.SPF chicken embryo and cell
The SPF chicken embryo of 9-11 age in days is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's SPF hatching room, and mdck cell (Madin-Darby canine kidney(cell line) (MDCK)) is purchased from Wuhan Virology Institute,Chinan academy of Sciences.
3. main agents
DATP, dGTP, dCTP, dTTP and rTaq archaeal dna polymerase is purchased from the precious biotechnology company limited in Dalian; RNA extracts reagent TRIzol, and mouse source ThermoScript II (MLV) test kit, DTT, RNaseOUT and archaeal dna polymerase are purchased from Invitrogen company; Glue reclaims test kit and mini-scale plasmid extracts test kit purchased from Shanghai Hua Shun biotechnology company limited; Sequencing reaction test kit (CEQTM DTCS Quick Start Kit) is purchased from BECKMAN company; Anti-A/PR/8/34 (H1N1) antiserum(antisera) is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's animal influenza center.
4. primer
Com-parison and analysis, design and synthesis can amplify 8 specific fragments of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) gene and can not amplify 16 diagnostic primerses of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) of any fragment of influenza virus A/PR/8/34 (H1N1), and synthetic primer sequence is in table 1.
The diagnostic primers sequence of 8 specific fragments of table 1 class fowl type H1N1 influenza virus A/swine/Jiangsu/40/2011 (H1N1)
In addition, design and synthesis can only amplify 8 specific fragments of influenza virus A/PR/8/34 (H1N1) and can not amplify influenza virus A/PR/8/34 (H1N1) 16 diagnostic primerses of any fragment of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1), and synthetic primer sequence is in table 2.
The diagnostic primers sequence of 8 specific fragments of table 2 influenza virus A/PR/8/34 (H1N1)
Embodiment 1: the foundation of screening, qualification recombinant virus method
Trizol is utilized to fission by force agent method, extract the RNA of influenza virus A/PR/8/34 (H1N1) and class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1), carry out reverse transcription, then do PCR reaction with the influenza virus A/PR/8/34 (H1N1) of above-mentioned design and the fragments specific diagnostic primers of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1).8 specific fragments of influenza virus A/PR/8/34 (H1N1) strain can only be amplified according to the fragments specific diagnostic primers of the gene design of influenza virus A/PR/8/34 (H1N1) and any fragment of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) strain can not be amplified, 8 specific fragments of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) strain can only be amplified according to the fragments specific diagnostic primers of the gene design of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) and any fragment of influenza virus A/PR/8/34 (H1N1) strain can not be amplified.This will prove the foundation of screening, qualification recombinant virus method.
As shown in Figure 1A, with the cDNA of the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) of class fowl type H1N1 influenza virus and influenza virus A/PR/8/34 (H1N1) strain for template, utilize 8 pairs of fragment-specific primer amplifications of the influenza virus A/swine/Jiangsu/40/2011 (H1N1) of synthesis, the cDNA of A/swine/Jiangsu/40/2011 (H1N1) is only had to amplify 8 specific fragments, and any fragment that A/PR/8/34 (H1N1) strain cDNA does not amplify.
As shown in Figure 1B, with the cDNA of the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) of class fowl type H1N1 influenza virus and influenza virus A/PR/8/34 (H1N1) strain for template, utilize influenza virus A/PR/8/34 (H1N1) 8 pairs of fragment-specific primer amplifications of synthesis, only have influenza virus A/PR/8/34 (H1N1) strain cDNA to amplify 8 specific fragments, and influenza virus domestic isolate A/swine/Jiangsu/40/2011 (H1N1) strain cDNA does not amplify any fragment.
Prove thus to have established screening method, this system can identify that 8 genes of prepared recombinant virus derive from influenza virus A/PR/8/34 (H1N1) or the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) of class fowl type H1N1 influenza virus respectively.
Embodiment 2: the preparation of recombination classes fowl type H1N1 influenza virus JS40/PR8 and qualification
1. coinfection-two viral genome are recombinated:
Domestic isolate A/the swine/Jiangsu/40/2011 (H1N1) of class fowl type H1N1 influenza virus of 100 μ l and influenza virus A/PR/8/34 (H1N1) (the being called for short PR8) mixing of 50 μ l add in 850 μ l PBS, and the SPF chick embryo allantoic cavity of co-inoculation 10 age in days carries out nature restructuring.Cultivate 24 hours for 35 DEG C, collect allantoic fluid.
2. antibody screening-removing surface gene derives from the strain of influenza virus A/PR/8/34 (H1N1):
Get allantoic fluid that 50 μ l gather in the crops under above-mentioned specific artificial condition and anti-A/PR/8/34 (H1N1) the antiserum(antisera) mixing that 600 μ l multiple proportions (4 times) are diluted, room temperature effect 30 minutes, more each extent of dilution inoculates three pieces of chicken embryos.Then, the chicken embryo of inoculation is cultivated 48 hours in 35 DEG C of incubators, measure the HA-HI test (see laboratory animal hemagglutination-inhibition test national standard (GB) (GB/T 14926.54-2001)) of chick embryo allantoic liquid of each extent of dilution inoculation, to collect and the chicken embryo inoculated after retaining the process of low dilution A/PR/8/34 (H1N1) antiserum(antisera) has the allantoic fluid of HA-HI test.Then, same procedure does secondary antiserum(antisera) process, collects equally and the chicken embryo inoculated after retaining the process of low dilution A/PR/8/34 (H1N1) antiserum(antisera) has the allantoic fluid of HA-HI test.
3. dilute the strain that purifying-removing internal gene derives from H1N1 strains of influenza viruses:
Process according to the method described above the virus obtained carry out on chicken embryo 3 generation limited clone purification, Trizol is utilized to fission by force agent method, extract viral RNA, carry out reverse transcription, then carry out RT-PCR amplification with the influenza virus A/PR/8/34 (H1N1) of design and each 8 pairs of specificity diagnostic primerses (table 1 and 2) of the domestic isolate A/swine/Jiangsu/40/2011 of class fowl type H1N1 influenza virus (H1N1) virus.Then, nucleic acid gel electrophoresis is carried out to amplified production, identify that the recombinant virus obtained is that HA and NA comes from the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) of class fowl type H1N1 influenza virus, 6 internal gene PB2, PB1, PA, NP, M and NS is from the recombinant virus (see Fig. 2) of influenza virus A/PR/8/34 (H1N1), by the recombinant virus called after A/swine/Jiangsu/40/PR8 (H1N1) obtained, be called for short JS40/PR8, be preserved in the China typical culture collection center of the Wuhan University being positioned at Wuhan, China, its preserving number is CCTCC NO:V201419.
4. the Sequence Identification of recombinant virus
Extract the RNA of recombinant virus JS40/PR8, RT-PCR amplifies 8 fragments of the full-length genome of recombinant virus JS40/PR8 respectively, carries out nucleic acid gel electrophoresis to PCR primer, measures the particular sequence of each fragment after glue reclaims.Utilize DNAStar software (DNAStar Lasergene V7.1), gene comparision is carried out to the sequence of the sequence measured and the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) of wild strain class fowl type H1N1 influenza virus and influenza virus A/PR/8/34 (H1N1), find that the genome of recombinant virus JS40/PR8 is all without any the change of Nucleotide, show that HA with NA of recombinant virus comes from that class fowl type H1N1 influenza virus is domestic is separated A/swine/Jiangsu/40/2011 (H1N1); 6 internal gene PB2, PB1, PA, NP, M and NS are from influenza virus A/PR/8/34 (H1N1).
5. the mensuration of recombinant virus JS40/PR8 growth curve
Parental virus class fowl type H1N1 influenza virus epidemic strain A/swine/Jiangsu/40/2011 (H1N1) (being called for short JS40) and recombinant virus JS40/PR8 are inoculated 5 groups of SPF chicken embryos, 24,48,72 and 96 hours are cultivated respectively under 35 DEG C of conditions, the chicken embryo getting above-mentioned cultivation different time carries out blood clotting mensuration, detects the growth curve of parent's strain A/swine/Jiangsu/40/2011 (H1N1) and recombinant virus JS40/PR8.
Recombinant virus is under 35 DEG C of culture condition as can be seen from the test results, and the hemagglutinative titer of 72 little recombinant viruses is constantly far above the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) (as shown in Figure 3) of wild strain class fowl type H1N1 influenza virus.
The strain of preparation influenza vaccines, not only require that it has good immunogenicity, and require that there are good growth characteristics aborning, as can be seen from the growth curve that restructuring JS40/PR8 virus measures under 35 DEG C of culture condition, recombinant virus growth titre is significantly improved compared with the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) of parent's class fowl type H1N1 influenza virus, and hemagglutinative titer exceeds 8 times (as shown in Figure 3).This illustrates that the present invention has prepared the recombinant virus of a height growth titre, brings huge economic interests by for later batch production.
6. the antigenicity analysis of recombinant virus JS40/PR8
Carry out intersecting HI according to the antigen of the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) of parent's strain class fowl type H1N1 influenza virus and recombinant virus and serum and test the antigenicity analysis data of (see laboratory animal hemagglutination-inhibition test national standard (GB) (GB/T14926.54-2001)), antigenic difference is less than 2 times as can be seen from the test results, after restructuring is described, virus still remains the antigenicity (see table 3) of parent's strain.
The antigenicity analysis of table 3: parent's strain A/swine/Jiangsu/40/2011 (H1N1) and recombinant virus JS40/PR8
7. the Virulence detection of recombinant virus JS40/PR8
BALB/c mouse is and host adaptive mammalian animal model pathogenic as influenza virus often.For understanding the infection ability of recombinant strain and pathogenic, the present invention adopts mouse to be animal model, by recombinant strain SW/JS/40/PR8 and parent's strain SW/JS/40/2011 respectively with 10
6tCID
50dosage via intranasal application Mice Inoculated, observes mouse survival, body inner virus and copies and the situation of body weight loss.
The SPF level female BAl BIc/c mouse (purchased from Beijing company of dimension tonneau China) in six week age, is divided into three groups at random, often organizes 8, weigh in.All mouse are respectively through CO
2after light anesthesia, first group contains 10 by intranasal inoculation 50ul
6tCID
50the virus liquid of recombinant strain SW/JS/40/PR8 (JS40/PR8), second group contains 10 by intranasal inoculation 50ul
6tCID
50the virus liquid of parent's strain SW/JS/40/2011 (JS40/11), the 3rd group of inoculation 50ul PBS is as a control group.Weigh single the body weight of each group of mouse after infecting virus every day, calculate mean body weight and observe morbidity and survival condition, namely weight loss 30% implements euthanasia.All mouse are all raised in the mouse cage having independent ventilation systems.
Often group 3d after attacking poison, randomly draws 3 survival mice, through CO
2cut open after anesthesia and kill, gather brain, concha, spleen, kidney, lungs, carry out the titration of viral level respectively; The pathological material of disease that the virus isolation and Identification of tissue is collected is contained in sterilizing EP pipe, and-70 DEG C of preservations are taken out with the grinding of sterilizing mill, made the DMEM tissue suspension of 10%, get supernatant fluid and carry out titration after centrifugal on mdck cell, according to mensuration TCID
50method carries out viral level in mensuration tissue.Mouse weighs in every day, to 14d.
Result shows that all group mouse all occur without death condition.All virus is can't detect in brain, spleen, kidney and concha.In lungs, recombinant strain SW/JS/40/PR8 viral level is lower than SW/JS/40/2011 group (table 4), because virus infection causes weight limit loss SW/JS/40/PR8 to be 5%, lower than 10% (Fig. 4) of SW/JS/40/2011 group, show that recombinant strain virulence is lower than parent's strain SW/JS/40/2011.
Table 4 liang group infecting mouse internal organs virus purification and lethal cases result
8. the immunogenicity experiments of recombinant virus JS40/PR8
Be 10 by content
8eID
50the recombinant virus JS40/PR8 of/mL with after 0.1% formalin-inactivated, to match the MONTANIDE that BIC Corp provides
tMvaccine prepared by ISA206 adjuvant, with the SPF level female BAl BIc/c mouse in 10 6 week ages for animal pattern, 0.1ml intramuscular injection immunity, after immunity, 14d detects HI antibody titers in serum, in serum, HI antibody titers is between 80-320, shows that recombinant virus JS40/PR8 has good immunogenicity.
Reference:
1.Yang?H,Qiao?C,Tang?X,Chen?Y,Xin?X,Chen?H.Human?infection?from?avian-like?influenza?A(H1N1)viruses?in?pigs,China.Emerg?Infect?Dis.2012?Jul；18(7):1144-6.
2.Chen?Y,Zhang?J,Qiao?C,Yang?H,Zhang?Y,Xin?X,Chen?H.Co-circulation?of?pandemic?2009?H1N1,classical?swine?H1N1?and?avian-like?swine?H1N1?influenza?viruses?in?pigs?in?China.Infect?Genet?Evol.2013,13:331-3382012?Nov?10.
3.Hualan?Chen，Kanta?Subbarao，David?Swayne，Qi?Chen，Xiuhua?Lu，Jacqueline?Katz，Nancy?Cox，Yumiko?Matsuoka.Generation?and?evaluation?of?an?high-growth?reassortant?H9N2?influenza?A?virus?as?apandemic?vaccine?candidate.Vaccine?。
Claims (5)
1. the class fowl type H1N1 inactivated influenza virus vaccine strain of a restructuring, it is characterized in that with H1N1 hypotype swine influenza virus A/swine/Jiangsu/40/2011 (H1N1) for surface antigen haemagglutinin (HA) and neuraminidase (NA) genetic donor, with influenza virus A/PR/8/34 (H1N1) for polysaccharase PB2 subunit, polysaccharase PB1 subunit, polysaccharase PA subunit, nucleoprotein (NP), the donor of stromatin (M) and Nonstructural Protein (NS) 6 internal gene, obtain by naturally recombinating, class fowl type H1N1 inactivated influenza virus vaccine strain called after influenza A A/swine/Jiangsu/40/PR8 (H1N1) of the restructuring obtained, be deposited in China typical culture collection center, address is in Wuhan University, its preserving number is CCTCC NO:V201419.
2. the class fowl type H1N1 inactivated influenza virus vaccine strain of restructuring according to claim 1 preparation prevention class fowl type H1N1 influenza virus cause in the medicine of disease application.
3. application according to claim 2, wherein said medicine is for preventing the class fowl type H1N1 influenza infection of animal or human.
4. build a method for the class fowl type H1N1 inactivated influenza virus vaccine strain of restructuring according to claim 1, the method comprises:
(1) vaccine strain A/PR/8/34 (H1N1) co-inoculation chick embryo allantoic cavity is adapted to class fowl type H1N1 strains of influenza viruses A/swine/Jiangsu/40/2011 (H1N1) and chicken embryo or cell carries out nature restructuring;
(2) adapt to the antibody screening of vaccine strain A/PR/8/34 (H1N1) with anti-described chicken embryo, removing surface gene derives from the strain that described chicken embryo adapts to vaccine strain A/PR/8/34 (H1N1);
(3) by screen the strain that obtains carry out on chicken embryo 3 generation limited clone purification, Trizol is utilized to fission by force agent method, extract viral RNA, carry out reverse transcription, then RT-PCR amplification is carried out with the influenza virus A/PR/8/34 (H1N1) of design and each 8 pairs of specificity diagnostic primerses of the domestic isolate A/swine/Jiangsu/40/2011 of class fowl type H1N1 influenza virus (H1N1) virus, then, nucleic acid gel electrophoresis is carried out to amplified production, identify that the recombinant virus obtained is that HA and NA comes from the domestic isolate A/swine/Jiangsu/40/2011 (H1N1) of class fowl type H1N1 influenza virus, 6 internal gene PB2, PB1, PA, NP, M and NS is from the recombinant virus of influenza virus A/PR/8/34 (H1N1), obtain.
5. method as claimed in claim 4, it is characterized in that, described primer comprises:
Can only amplify 8 specific fragments of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) gene and can not amplify 8 pairs of specificity diagnostic primerses of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) of any fragment of influenza virus A/PR/8/34 (H1N1), primer sequence is as follows:
And 8 specific fragments of influenza virus A/PR/8/34 (H1N1) can only be amplified and 8 pairs of specificity diagnostic primerses of the influenza virus A/PR/8/34 (H1N1) of any fragment of class fowl type H1N1 influenza isolate A/swine/Jiangsu/40/2011 (H1N1) can not be amplified, primer sequence is as follows:
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105754961A (en) * | 2016-03-30 | 2016-07-13 | 中国农业科学院哈尔滨兽医研究所 | Recombinant replication-deficient adenovirus expressing avian-like H1N1 swine influenza virus HA protein and constructing method and application thereof |
| CN105821009A (en) * | 2016-04-05 | 2016-08-03 | 中国人民解放军第三〇二医院 | Construction and application of liver cancer targeted oncolytic influenza viruses |
| CN110305854A (en) * | 2019-07-18 | 2019-10-08 | 山东中医药大学 | A kind of recombination H3N2 subtype influenza virus, construction method and application carrying luciferase |
| CN110760486A (en) * | 2019-11-29 | 2020-02-07 | 山东省农业科学院家禽研究所 | H13N8 recombinant influenza virus and application thereof |
| CN111032861A (en) * | 2017-08-28 | 2020-04-17 | 一般财团法人阪大微生物病研究会 | Staged preparation method of reassortant influenza virus |
| CN112877300A (en) * | 2021-02-10 | 2021-06-01 | 中国农业大学 | Preparation of G4 avian European subtype H1N1 inactivated vaccine for swine influenza virus |
| CN112961836A (en) * | 2021-02-25 | 2021-06-15 | 新疆农业大学 | E new BEV virulent strain and separation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1546658A (en) * | 2003-12-02 | 2004-11-17 | 中国农业科学院哈尔滨兽医研究所 | Artificially recombinant H9N2 hypotype influenza virus and its uses |
| CN102234637A (en) * | 2010-04-23 | 2011-11-09 | 中国农业科学院哈尔滨兽医研究所 | Preparation for reconstruction influenza A H1N1 virus inactivated vaccine strain (SC/PR8), and use thereof |
| WO2014019990A1 (en) * | 2012-08-03 | 2014-02-06 | Sanofi Pasteur | Production of infectious influenza viruses |
| CN103602638A (en) * | 2013-11-20 | 2014-02-26 | 中国农业科学院哈尔滨兽医研究所 | Recombinant equine influenza virus strain, preparation method thereof and vaccine prepared from recombinant equine influenza virus strain |
-
2014
- 2014-09-11 CN CN201410460909.5A patent/CN104232594B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1546658A (en) * | 2003-12-02 | 2004-11-17 | 中国农业科学院哈尔滨兽医研究所 | Artificially recombinant H9N2 hypotype influenza virus and its uses |
| CN102234637A (en) * | 2010-04-23 | 2011-11-09 | 中国农业科学院哈尔滨兽医研究所 | Preparation for reconstruction influenza A H1N1 virus inactivated vaccine strain (SC/PR8), and use thereof |
| WO2014019990A1 (en) * | 2012-08-03 | 2014-02-06 | Sanofi Pasteur | Production of infectious influenza viruses |
| CN103602638A (en) * | 2013-11-20 | 2014-02-26 | 中国农业科学院哈尔滨兽医研究所 | Recombinant equine influenza virus strain, preparation method thereof and vaccine prepared from recombinant equine influenza virus strain |
Non-Patent Citations (4)
| Title |
|---|
| P PALESE，MB RITCHEY，JL SCHULMAN，ED KILBOURNE: "Genetic composition of a high-yielding influenza A virus recombinant: a vaccine strain against "swine" influenza.", 《SCIENCE》 * |
| 孟沙沙，等: "一株类禽型H1N1 亚型猪流感病毒的反向遗传系统的建立", 《中国预防兽医学报》 * |
| 施建忠，等: "重组高繁殖力疫苗株H5N2(H5/PR8)病毒的制备和鉴定", 《中国预防兽医学报》 * |
| 杨涛,等: "重组细胞高产型H3N2 猪流感病毒株的拯救", 《病毒学报》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105754961A (en) * | 2016-03-30 | 2016-07-13 | 中国农业科学院哈尔滨兽医研究所 | Recombinant replication-deficient adenovirus expressing avian-like H1N1 swine influenza virus HA protein and constructing method and application thereof |
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| CN111032861B (en) * | 2017-08-28 | 2023-09-12 | 一般财团法人阪大微生物病研究会 | Staged preparation method of reassortant influenza virus |
| CN110305854A (en) * | 2019-07-18 | 2019-10-08 | 山东中医药大学 | A kind of recombination H3N2 subtype influenza virus, construction method and application carrying luciferase |
| CN110305854B (en) * | 2019-07-18 | 2023-09-15 | 山东中医药大学 | Recombinant H3N2 subtype influenza virus carrying luciferase, construction method and application |
| CN110760486A (en) * | 2019-11-29 | 2020-02-07 | 山东省农业科学院家禽研究所 | H13N8 recombinant influenza virus and application thereof |
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| CN112961836B (en) * | 2021-02-25 | 2023-07-04 | 新疆农业大学 | E-strain BEV novel virulent strain, and separation method and application thereof |
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