CN104232594B - Recombination classes fowl type H1N1 inactivated influenza virus vaccines strain (JS40/PR8) and its preparation method and application - Google Patents
Recombination classes fowl type H1N1 inactivated influenza virus vaccines strain (JS40/PR8) and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of recombination classes fowl type H1N1 inactivated influenza virus vaccines strain and its preparation method and application.A kind of recombination classes fowl type H1N1 inactivated influenza virus vaccines strain of the invention, its be with A/swine/Jiangsu/40/2011 (H1N1) as surface antigen haemagglutinin (HA) and neuraminidase (NA) genetic donor, with influenza virus A/PR/8/34 (H1N1) for polymerase PB2 subunits, polymerase PB1 subunits, polymerase PA subunits, nucleoprotein (NP), 6 donors of internal gene of stromatin (M) and non-structural protein (NS), obtained by restructuring naturally, preserving number is CCTCC NO：V201419.Application the invention also discloses the method and the strain of recombination classes fowl type H1N1 inactivated influenza virus vaccines for building the recombination classes fowl type H1N1 inactivated influenza virus vaccines strain to prevention animal or the class fowl type H1N1 influenzas of people.
Description
Technical field
The present invention relates to recombination engineered vaccine, in particular it relates to a kind of for preventing animals and humans fowl
The recombination engineered vaccine of type H1N1 influenzas, more specifically, vaccine involved in the present invention be it is a kind of for prevent animal and
The recombination classes fowl type H1N1 inactivated influenza virus vaccines of mankind's fowl type H1N1 influenzas.
Background technology
Influenza virus is the cyst membrane disease of the segmented strand RNA composition of a kind of extensive infected poultry of energy and mammal
Poison.In the whole world, influenza often sporadicly to break out and miscellaneous random pandemic form occurs, in the past few decades in, various countries
The main Selection utilization of family is still totivirus inactivated vaccine, and it mainly stimulates body to produce humoral immunity to resist the blood of identical hypotype
Solidifying element surface glycoprotein.
Class fowl type H1N1 is popular extensive in China swinery, and epidemic situation still is continuing to expand (Yan Chen, Infect
Genet Evol.2013).In 2011, Jiangsu Province of China Sihong County occurred in that people infects class fowl type H1N1 swine influenza virus things
Part, this is also China report class fowl type H1N1 hypotypes swine flu infection people's event (Huanliang Yang, Emerg for the first time
infect 2012).Influenza A host range change, host caused by kind of a propagation depends on virus because of adaptive variation
The change of itself restrictive factor and environmental factor.For the development of correspondence epidemic situation, the present invention has carried out natural recombinant vaccine
The development of strain.
Preferable vaccine strain should possess the antigen matching good with epidemic strain, to chicken embryo had no pathogenicity and chicken embryo high titre
The conditions such as growth.However, it is difficult to separate the strain for possessing above-mentioned condition as vaccine strain from nature.At home, due to existing
Street strain's chicken embryo adaptability that ground is separate is poor, causes the class fowl type H1N1 influenza vaccines of the existing country to there is high cost, vaccine
The shortcomings of duration of immunity is short.
Influenza virus A/PR/8/34 (H1N1) (abbreviation PR8) is that laboratory chicken embryo adapted strain, the i.e. virus can be in chicken embryos
Very high titre is grown to, this chicken embryo adaptability is determined by 6 internal genes of virus.In addition, when two influenza viruses are common
Infection one cell or chicken embryo when, can producer exchange, so as to produce new gene recombined virus.Therefore, by heredity weight
Prescription method, can recombinate the carrying out of HA, NA gene of 6 internal genes and epidemic strain of influenza virus A/PR/8/34 (H1N1)
And a new recombinant virus is obtained, the recombinant virus has the antigenicity consistent with HA and NA genetic donor strains, while also
Chicken embryo high titre growth characteristics with influenza virus A/PR/8/34 (H1N1).
Artificial nature's recombinant technique is a kind of simple and effective structure 2:6 recombinant viral vaccines strain method, influenza virus
Genome is made up of the strand RNA fragment of 8 segmentations, when 2 viral co-infection chicken embryos or cell, its genomic fragment meeting
Exchange, so that some new recombinant viruses (reassortant) are produced, by the place containing corresponding antibodies positive serum
Reason, then can effectively screen 2:The strain of 6 recombinant viral vaccines.(Hualan Chen,Vaccine2003).
The content of the invention
The technical problems to be solved by the invention provide a kind of strain of recombination classes fowl type H1N1 inactivated influenza virus vaccines and its
Preparation method.
In order to reach object above, the technology used in the present invention means are：
Inventor separates in the Jiangsu class fowl type H1N1 local isolated one plant of swine influenza virus of infection people event
Strain, sequence alignment analysis find the strain very high homology of the strain and infection people, and the isolated Strain is named as
A/swine/Jiangsu/40/2011 (H1N1), abbreviation SW/JS40 (H1N1).
The present invention utilizes artificial recombination method, with H1N1 hypotype swine influenza viruses A/swine/Jiangsu/40/2011
(H1N1) it is surface antigen haemagglutinin (HA) and neuraminidase (NA) genetic donor, with influenza virus A/PR/8/34 (H1N1)
It is polymerase PB2 subunits, polymerase PB1 subunits, polymerase PA subunits, nucleoprotein (NP), stromatin (M) and non-structural protein
(NS) 6 donors of internal gene, the class fowl type H1N1 inactivated influenza virus vaccines of 1 plant weight group have been obtained by restructuring naturally
Strain, is named as influenza A A/swine/Jiangsu/40/PR8 (H1N1) (referred to as JS40/PR8), is deposited in being located at
State's Type Tissue Collection, in the Wuhan University of Wuhan, China, its preserving number is CCTCCNO for address：V201419, preservation
Time is on June 19th, 2014.
The laboratory chicken embryo adapted strain A/PR/8/ that can chicken embryo in grow to very high titre of the present invention from influenza virus
6 internal genes of 34 (H1N1) select isolated class fowl type strain A/swine/ in 2011 as backbone genes
HA the and NA genes of Jiangsu/40/2011 (H1N1), using nature recombination method, are prepared for recombinant virus as surface gene
JS40/PR8, the recombinant strain is by can be as the vaccine strain of class fowl type H1N1 influenzas.
It is experimentally confirmed that JS40/PR8 and China class fowl type H1N1 influenza virus epidemic strain SW/JS40 (H1N1) antigenicity one
Cause, good specific protective will be produced to class fowl type H1N1 influenza virus epidemic strains SW/JS40 (H1N1).The virus contains stream
6 internal genes of Influenza Virus chicken embryo high titre adapted strain A/PR/8/34 (H1N1), therefore with good chicken embryo adaptability,
Viral growth titre can improve more than 8 times compared with wild poison；Vaccine is produced as vaccine strain, production of vaccine can be substantially reduced
Cost, improves vaccine quality.
Therefore, further, the invention allows for the class fowl type H1N1 inactivated influenza virus vaccines strain of described restructuring
Application in the medicine of disease caused by prevention class fowl type H1N1 influenza viruses is prepared.
Wherein, it is preferred that the medicine is used to prevent the class fowl type H1N1 influenza infections of animal or people.
Further, the invention allows for a kind of class fowl type H1N1 inactivated influenza virus epidemic diseases of the restructuring described in structure
The method of seedling strain, the method includes：
(1) vaccine is adapted to class fowl type H1N1 strains of influenza viruses A/swine/Jiangsu/40/2011 (H1N1) and chicken embryo
Strain A/PR/8/34 (H1N1) co-inoculation chick embryo allantoic cavities or cell carry out nature restructuring；
(2) antibody screening of vaccine strain A/PR/8/34 (H1N1) is adapted to the anti-chicken embryo, surface gene is removed and is derived from
The chicken embryo adapts to the strain of vaccine strain A/PR/8/34 (H1N1)；
(3) will screen the strain for obtaining carries out 3 generations limited clone purification in chicken embryo, agent method of being fissioned by force using Trizol,
Viral RNA is extracted, reverse transcription is carried out, then with influenza virus A/PR/8/34 (H1N1) and class fowl type H1N1 the influenzas disease of design
Each 8 pairs specific diagnostic primerses of malicious country's separation strains A/swine/Jiangsu/40/2011 (H1N1) virus carry out RT-PCR
Amplification, then, nucleic acid gel electrophoresis is carried out to amplified production, identifies that the recombinant virus for obtaining comes from class fowl type for HA and NA
H1N1 influenza viruses country's separation strains A/swine/Jiangsu/40/2011 (H1N1), 6 internal genes PB2, PB1, PA, NP,
Recombinant viruses of the M and NS from influenza virus A/PR/8/34 (H1N1), obtains final product.
In method of the present invention, it is preferred that described primer includes：
8 of class fowl type H1N1 influenza separation strains A/swine/Jiangsu/40/2011 (H1N1) gene can only be amplified
Specific fragment and the class fowl type H1N1 influenza separation strains of any fragment of influenza virus A/PR/8/34 (H1N1) can not be amplified
8 couple specificity diagnostic primerses of A/swine/Jiangsu/40/2011 (H1N1), primer sequence is as follows：
And 8 specific fragments of influenza virus A/PR/8/34 (H1N1) can only be amplified and class fowl can not be amplified
Influenza virus A/the PR/8/34 of any fragment of type H1N1 influenza separation strains A/swine/Jiangsu/40/2011 (H1N1)
(H1N1) 8 couple specificity diagnostic primerses, primer sequence is as follows：
To sum up, it is demonstrated experimentally that the present invention has prepared one plant excellent can be used to prevent class fowl type H1N1 influenza viruses
The Strain of caused disease, of the invention is the further developmental research of class fowl type H1N1 influenza vaccines, has established solid
Basis.
Brief description of the drawings
Figure 1A is with JS40 and PR8 strains cDNA as template, using JS40 strains PB2, PB1, PA, HA, NP, NA, M, NS
The amplification that the specific primer of gene is carried out；
Wherein first to the 8th swimming lane is to use the special of PB2, PB1, PA, HA, NP, NA, M, NS gene of JS40 strains
The amplification of property primer pair JS40 strains cDNA；M is DNA Marker 2000；9th to the 16th swimming lane is to use JS40 strains
PB2, PB1, PA, HA, NP, NA, M, NS gene specific primer to PR8 strains cDNA for template amplification；
Figure 1B is with JS40 and PR8 strains cDNA as template, using PR8 strain PB2, PB1, PA, HA, NP, NA, M, NS bases
The amplification that the specific primer of cause is carried out；
Wherein 1~8 swimming lane is the specific primer pair using PB2, PB1, PA, HA, NP, NA, M, NS gene of PR8 strains
JS40 strains cDNA is the amplification of template；M is DNA Marker 2000；9~16 swimming lanes be using the PB2 of PR8 strains, PB1,
The specific primer of PA, HA, NP, NA, M, NS gene is the amplification of template to PR8 strains cDNA；
Fig. 2 is the RT-PCR amplifications of PB2, PB1, PA, HA, NA, NP, M and NS gene of JS40/PR8 recombinant viruses；
1~8 swimming lane：The PCR primer that recombinant virus JS40/PR8 is expanded using 8 couple specificity diagnostic primerses of JS40；M：
DNA molecular quality standard；9~16 swimming lanes：The PCR that recombinant virus JS40/PR8 is expanded using 8 couple specificity diagnostic primerses of PR8
Product；
Fig. 3 is restructuring JS40/PR8 viruses and wild type class fowl type H1N1 influenza virus As/swine/Jiangsu/40/
2011 (H1N1) (abbreviation JS40) are in 35 DEG C of growth curves of measure of optimum culturing temperature；
Fig. 4 is the changes of weight situation for attacking each group mouse after poison.
Sequence explanation：
SEQ ID No.1：JS40/PR8 HA genes；
SEQ ID No.2：JS40/PR8 NA genes；
SEQ ID No.3：JS40/PR8 PB2 genes；
SEQ ID No.4：JS40/PR8 PB1 genes；
SEQ ID No.5：JS40/PR8 PA genes；
SEQ ID No.6：JS40/PR8 NP genes；
SEQ ID No.7：JS40/PR8 M genes；
SEQ ID No.8：JS40/PR8 NS genes.
Specific embodiment
The present invention is further described with reference to specific embodiments and the drawings, advantages of the present invention and feature will be with
Description and it is apparent.But these embodiments are only exemplary, do not constitute any limitation to the scope of the present invention.This area
Technical staff should be understood that without departing from the spirit and scope of the invention can to the details of technical solution of the present invention and
Form is modified or is replaced, but these modifications and replacement are each fallen within protection scope of the present invention.
Experiment material
1. Strain
A/swine/Jiangsu/40/2011 (H1N1) (abbreviation JS40) (is documented in document：One plant of class fowl type H1N1 hypotype
The foundation of the reverse genetics system of swine influenza virus, Yang Huanliang etc.,《Chinese Preventive Veterinary Medicine report》, 02 phase in 2013) and by this reality
Room preservation is tested, A/PR/8/34 (H1N1) (abbreviation PR8) is purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's animal influenza
The heart.
2.SPF chicken embryos and cell
The SPF chicken embryos of 9-11 ages in days are purchased from Harbin Veterinary Medicine Inst., China Academy of Agriculture's SPF hatchings room, MDCK
Cell (MDCK) is purchased from Wuhan Virology Institute,Chinan academy of Sciences.
3. main agents
DATP, dGTP, dCTP, dTTP and rTaq archaeal dna polymerase are purchased from Dalian treasured bioengineering Co., Ltd；RNA is extracted
Reagent TRIzol, mouse source reverse transcriptase (MLV) kit, DTT, RNaseOUT and archaeal dna polymerase are purchased from Invitrogen companies；
Glue reclaim kit is purchased from Shanghai Hua Shun bioengineering Co., Ltd with mini-scale plasmid extracts kit；Sequencing reaction kit
(CEQTM DTCS Quick Start Kit) is purchased from BECKMAN companies；Anti- A/PR/8/34 (H1N1) antiserum is purchased from Chinese agriculture
Industry academy of sciences Harbin veterinary institute animal influenza center.
4. primer
Com-parison and analysis, design synthesis can amplify class fowl type H1N1 influenza separation strains A/swine/Jiangsu/40/2011
(H1N1) 8 specific fragments of gene and the class fowl of any fragment of influenza virus A/PR/8/34 (H1N1) can not be amplified
16 diagnostic primerses of type H1N1 influenza separation strains A/swine/Jiangsu/40/2011 (H1N1), synthetic primer sequence is shown in Table
1。
8 specific fragments of the class fowl type H1N1 influenza virus As of table 1/swine/Jiangsu/40/2011 (H1N1)
Diagnostic primerses sequence
In addition, design synthesis can only amplify 8 specific fragments of influenza virus A/PR/8/34 (H1N1) and can not expand
Increase the influenza virus A of any fragment for class fowl type H1N1 influenza separation strains A/swine/Jiangsu/40/2011 (H1N1)/
16 diagnostic primerses of PR/8/34 (H1N1), synthetic primer sequence is shown in Table 2.
The diagnostic primerses sequence of 8 specific fragments of 2 influenza virus As of table/PR/8/34 (H1N1)
Embodiment 1：Screening, the foundation of identification recombinant virus method
Fissioned by force using Trizol agent method, extract influenza virus A/PR/8/34 (H1N1) and separated with class fowl type H1N1 influenzas
The RNA of strain A/swine/Jiangsu/40/2011 (H1N1), carries out reverse transcription, then with the influenza virus A/PR/ of above-mentioned design
The fragments specific of 8/34 (H1N1) and class fowl type H1N1 influenza separation strains A/swine/Jiangsu/40/2011 (H1N1) differentiates
Primer does PCR reactions.The fragments specific diagnostic primerses of the gene design according to influenza virus A/PR/8/34 (H1N1) can only expand
Increase 8 specific fragments influenza virus A/PR/8/34 (H1N1) strain and the separation of class fowl type H1N1 influenzas can not be amplified
Any fragment of strain A/swine/Jiangsu/40/2011 (H1N1) strain, according to class fowl type H1N1 influenza separation strains A/
The fragments specific diagnostic primerses of the gene design of swine/Jiangsu/40/2011 (H1N1) can only amplify class fowl type H1N1
8 specific fragments of influenza separation strains A/swine/Jiangsu/40/2011 (H1N1) strain and can not amplify influenza disease
Any fragment of malicious A/PR/8/34 (H1N1) strain.This will demonstrate that screening, the foundation of identification recombinant virus method.
As shown in Figure 1A, with the domestic separation strains A/swine/Jiangsu/40/2011 of class fowl type H1N1 influenza viruses
(H1N1) and influenza virus A/PR/8/34 (H1N1) strain cDNA be template, using synthesis influenza virus A/swine/
8 pairs of fragment-specific primers amplification of Jiangsu/40/2011 (H1N1), only A/swine/Jiangsu/40/2011
(H1N1) cDNA has amplified 8 specific fragments, and any that A/PR/8/34 (H1N1) strains cDNA is not amplified
Section.
As shown in Figure 1B, with the domestic separation strains A/swine/Jiangsu/40/2011 of class fowl type H1N1 influenza viruses
(H1N1) and influenza virus A/PR/8/34 (H1N1) strain cDNA be template, using synthesis influenza virus A/PR/8/34
(H1N1) 8 pairs of fragment-specific primer amplifications, only influenza virus A/PR/8/34 (H1N1) strains cDNA has amplified 8 spies
Specific fragment, and influenza virus country separation strains A/swine/Jiangsu/40/2011 (H1N1) strains cDNA is not amplified
Any fragment.
Thus prove to have been set up screening technique, this system can identify 8 genes of prepared recombinant virus
Influenza virus A/PR/8/34 (H1N1) or the domestic separation strains A/swine/ of class fowl type H1N1 influenza viruses is derived from respectively
Jiangsu/40/2011(H1N1)。
Embodiment 2：The preparation of recombination classes fowl type H1N1 influenza viruses JS40/PR8 and identification
1. coinfection-recombinate two viral genomes：
Class fowl type H1N1 influenza viruses country's separation strains A/swine/Jiangsu/40/2011 (H1N1) of 100 μ l and 50 μ
Influenza virus A/PR/8/34 (H1N1) (abbreviation PR8) mixing of l is added in 850 μ l PBS, the SPF chickens of the age in days of co-inoculation 10
Embryo allantoic cavity carries out nature restructuring.35 DEG C are cultivated 24 hours, collect allantoic fluid.
2. antibody screening-removing surface gene derives from the strain of influenza virus A/PR/8/34 (H1N1)：
Take (4 times) anti-A/PR/ of dilution of allantoic fluid and 600 μ l multiple proportions that 50 μ l are harvested under above-mentioned specific artificial condition
8/34 (H1N1) antiserum mixes, and room temperature is acted on 30 minutes, then each dilution factor is inoculated with three pieces of chicken embryos.Then, the chicken embryo of inoculation
Cultivated 48 hours in 35 DEG C of incubators, determine the HA-HI test of chick embryo allantoic liquid of each dilution factor inoculation (referring to experimental animal
Hemagglutination-inhibition test national standard (GB) (GB/T 14926.54-2001)), collect and retain the A/PR/8/34 of low dilution factor
(H1N1) chicken embryo being inoculated with after antiserum treatment has the allantoic fluid of HA-HI test.Then, same procedure is done at secondary antiserum
Reason, the chicken embryo being inoculated with after A/PR/8/34 (H1N1) antiserum treatment for equally collecting and retaining low dilution factor has the urine of HA-HI test
Cyst fluid.
3. dilution purifying-removing internal gene derives from the strain of H1N1 strains of influenza viruses：
The virus that treatment is obtained according to the method described above carries out 3 generations limited clone purification in chicken embryo, is split by force using Trizol
Become agent method, extract viral RNA, carry out reverse transcription, then with the influenza virus A/PR/8/34 (H1N1) and class fowl type H1N1 of design
Each 8 pairs specific diagnostic primerses (tables 1 of influenza virus country's separation strains A/swine/Jiangsu/40/2011 (H1N1) virus
With 2) carry out RT-PCR amplifications.Then, nucleic acid gel electrophoresis is carried out to amplified production, identifies that the recombinant virus for obtaining is HA and NA
Come from class fowl type H1N1 influenza viruses country's separation strains A/swine/Jiangsu/40/2011 (H1N1), 6 internal genes
The recombinant virus (see Fig. 2) of PB2, PB1, PA, NP, M and NS from influenza virus A/PR/8/34 (H1N1), the restructuring that will be obtained
Viral nomenclature is A/swine/Jiangsu/40/PR8 (H1N1), abbreviation JS40/PR8, is preserved in positioned at the Wuhan of Wuhan, China
The China typical culture collection center of university, its preserving number is CCTCC NO：V201419.
4. the Sequence Identification of recombinant virus
The RNA of recombinant virus JS40/PR8 is extracted, RT-PCR amplifies the full-length genome of recombinant virus JS40/PR8 respectively
8 fragments, nucleic acid gel electrophoresis is carried out to PCR primer, the particular sequence of each fragment is determined after glue reclaim.Utilize
DNAStar softwares (DNAStar Lasergene V7.1), to the sequence for determining and wild strain class fowl type H1N1 influenza viruses
The sequence of domestic separation strains A/swine/Jiangsu/40/2011 (H1N1) and influenza virus A/PR/8/34 (H1N1) carries out sequence
Row compare, and find the genome of recombinant virus JS40/PR8 without the change of any nucleotides, show the HA of recombinant virus with
NA comes from the domestic separation A/swine/Jiangsu/40/2011 (H1N1) of class fowl type H1N1 influenza viruses；6 internal genes
PB2, PB1, PA, NP, M and NS come from influenza virus A/PR/8/34 (H1N1).
5. the measure of recombinant virus JS40/PR8 growth curves
By parental virus class fowl type H1N1 influenza virus epidemic strains A/swine/Jiangsu/40/2011 (H1N1) (referred to as
JS40) and recombinant virus JS40/PR8 be inoculated with 5 groups of SPF chicken embryos, cultivated respectively under the conditions of 35 DEG C 24,48,72 and 96 hours, take
The chicken embryo of above-mentioned culture different time carries out blood clotting measure, detection parent's strain A/swine/Jiangsu/40/2011 (H1N1)
With the growth curve of recombinant virus JS40/PR8.
Recombinant virus is under 35 DEG C of condition of culture as can be seen from the test results, the hemagglutinative titer of recombinant virus at 72 hours
Far above wild strain class fowl type H1N1 influenza viruses country separation strains A/swine/Jiangsu/40/2011 (H1N1) (such as Fig. 3
It is shown).
Influenza vaccines strain is prepared, not only requires that it has good immunogenicity, and require have aborning well
Growth characteristics, from restructuring JS40/PR8 virus under 35 DEG C of condition of culture determine growth curve can be seen that recombinant virus
Growth titre has substantially compared with parent class fowl type H1N1 influenza viruses country's separation strains A/swine/Jiangsu/40/2011 (H1N1)
Improve, hemagglutinative titer is higher by 8 times (as shown in Figure 3).This explanation present invention has prepared the recombinant virus of a height growth titre,
Huge economic interests will be brought for later batch production.
6. antigenicity analysis of recombinant virus JS40/PR8
According to parent strain class fowl type H1N1 influenza viruses country's separation strains A/swine/Jiangsu/40/2011 (H1N1)
Intersect HI experiments (referring to experimental animal hemagglutination-inhibition test national standard (GB) (GB/ with the antigen and serum of recombinant virus
T14926.54-2001 antigenicity analysis data)), as can be seen from the test results antigenic difference be less than 2 times, illustrate recombinate
Afterwards, virus still remains the antigenicity (referring to table 3) of parent's strain.
Table 3：Parent's strain A/swine/Jiangsu/40/2011 (H1N1) divides with the antigenicity of recombinant virus JS40/PR8
Analysis
7. the Virulence detection of recombinant virus JS40/PR8
BALB/c mouse is through the mammalian animal model with host's adaptability pathogenic frequently as influenza virus.Recombinated to understand
The infection ability of strain and pathogenic, it is animal model that the present invention uses mouse, by recombinant strain SW/JS/40/PR8 and parent
Strain SW/JS/40/2011 is respectively with 106TCID50Dosage via intranasal application Mice Inoculated, observation mouse survival, internal virus replication and
The situation of body weight loss.
The SPF grades of female BAl BIc/c mouse (purchased from Beijing company of dimension tonneau China) of six week old, are randomly divided into three groups, every group 8
Only, weigh in.All mouse are respectively through CO2After light anesthesia, first group contains 10 by intranasal inoculation 50ul6TCID50's
The virus liquid of recombinant strain SW/JS/40/PR8 (JS40/PR8), second group contains 10 by intranasal inoculation 50ul6TCID50Parent
The virus liquid of this strain SW/JS/40/2011 (JS40/11), the 3rd group of inoculation 50ul PBS is as a control group.After infection virus
The single body weight of each group mouse being weighed daily, average weight being calculated and is observed morbidity and survival condition, Body weight loss 30% is real
Apply euthanasia.All of mouse is raised in the mouse cage for possess independent ventilation systems.
Every group of 3d after poison is attacked, randomly selects 3 survival mices, through CO2Cutd open after anesthesia and killed, collection brain, concha, spleen
Dirty, 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 mounted in sterilizing EP
Guan Zhong, -70 DEG C of preservations are taken out and are ground with sterilizing mill, are made 10% DMEM tissue suspensions, supernatant fluid is taken after centrifugation and is existed
Titrated on mdck cell, according to measure TCID50Method is measured viral level in tissue.Mouse weighs in daily,
To 14d.
Result shows that all group mouse occur without death condition.Disease is can't detect in brain, spleen, kidney and concha
Poison.Recombinant strain SW/JS/40/PR8 viral levels are less than SW/JS/40/2011 groups (table 4) in lungs, because virus infection is made
It is 5% into weight limit loss SW/JS/40/PR8, less than 10% (Fig. 4) of SW/JS/40/2011 groups, shows that recombinant strain is caused
Sick power is less than parent's strain SW/JS/40/2011.
4 two groups of infecting mouse internal organs virus purifications of table and lethal cases result
8. immunogenicity experiments of recombinant virus JS40/PR8
It is 10 by content8EID50After the recombinant virus JS40/PR8 of/mL is with 0.1% formalin-inactivated, carried with matching BIC Corp
The MONTANIDE of confessionTMISA206 adjuvants prepare vaccine, are animal pattern with 10 SPF of 6 week old grades of female BAl BIc/c mouse,
0.1ml intramuscular injection be immunized, be immunized after 14d detection serum in HI antibody titers, in serum HI antibody titers between 80-320,
Show that recombinant virus JS40/PR8 has good immunogenicity.
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Claims (5)
1. the class fowl type H1N1 inactivated influenza virus vaccines strain of a kind of restructuring, it is characterised in that with H1N1 hypotype swine influenza viruses A/
Swine/Jiangsu/40/2011 (H1N1) is surface antigen haemagglutinin (HA) and neuraminidase (NA) genetic donor, to flow
Influenza Virus A/PR/8/34 (H1N1) is polymerase PB2 subunits, polymerase PB1 subunits, polymerase PA subunits, nucleoprotein (NP), base
6 donors of internal gene of matter albumen (M) and non-structural protein (NS), are obtained, the class of resulting restructuring by restructuring naturally
The strain of fowl type H1N1 inactivated influenza virus vaccines is named as influenza A A/swine/Jiangsu/40/PR8 (H1N1), preservation
In China typical culture collection center, in Wuhan University, its preserving number is CCTCC NO for address：V201419.
2. the class fowl type H1N1 inactivated influenza virus vaccines strain of the restructuring described in claim 1 is preparing prevention class fowl type H1N1 streams
Application in the medicine of disease caused by Influenza Virus.
3. application according to claim 2, wherein the medicine is used to prevent the class fowl type H1N1 influenzas disease of animal or people
Poison infection.
4. it is a kind of build claim 1 described in restructuring class fowl type H1N1 inactivated influenza virus vaccines strain method, the method
Including：
(1) vaccine strain A/ is adapted to class fowl type H1N1 strains of influenza viruses A/swine/Jiangsu/40/2011 (H1N1) and chicken embryo
PR/8/34 (H1N1) co-inoculation chick embryo allantoic cavities or cell carry out nature restructuring；
(2) antibody screening of vaccine strain A/PR/8/34 (H1N1) is adapted to the anti-chicken embryo, surface gene is removed from described
Chicken embryo adapts to the strain of vaccine strain A/PR/8/34 (H1N1)；
(3) will screen the strain for obtaining carries out 3 generations limited clone purification in chicken embryo, and agent method of being fissioned by force using Trizol is extracted
Viral RNA, carries out reverse transcription, then influenza virus A/the PR/8/34 (H1N1) with design and class fowl type H1N1 influenza viruses state
Each 8 pairs specific diagnostic primerses of interior separation strains A/swine/Jiangsu/40/2011 (H1N1) virus carry out RT-PCR amplifications,
Then, nucleic acid gel electrophoresis is carried out to amplified production, identifies that the recombinant virus for obtaining comes from class fowl type H1N1 streams for HA and NA
Influenza Virus country's separation strains A/swine/Jiangsu/40/2011 (H1N1), 6 internal genes PB2, PB1, PA, NP, M and NS
Recombinant virus from influenza virus A/PR/8/34 (H1N1), obtains final product.
5. method as claimed in claim 4, it is characterised in that described primer includes：
8 that class fowl type H1N1 influenza separation strains A/swine/Jiangsu/40/2011 (H1N1) gene can only be amplified are special
Property fragment and the class fowl type H1N1 influenza separation strains A/ of any fragment of influenza virus A/PR/8/34 (H1N1) can not be amplified
8 couple specificity diagnostic primerses of swine/Jiangsu/40/2011 (H1N1), primer sequence is as follows：
And 8 specific fragments of influenza virus A/PR/8/34 (H1N1) can only be amplified and class fowl type can not be amplified
Influenza virus A/the PR/8/34 of any fragment of H1N1 influenza separation strains A/swine/Jiangsu/40/2011 (H1N1)
(H1N1) 8 couple specificity diagnostic primerses, primer sequence is as follows：
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