CN103865923B - Preparation and application of influenza virus-carried HAdV (human adenovirus) chimeric vaccine - Google Patents
Preparation and application of influenza virus-carried HAdV (human adenovirus) chimeric vaccine Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5254—Virus avirulent or attenuated
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
- A61K2039/5256—Virus expressing foreign proteins
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- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention discloses a preparation and application of an influenza virus-carried HAdV chimeric vaccine and a DNA (deoxyribonucleic acid) molecule represented by SEQ ID No.1. According to the HAdV chimeric vaccine disclosed by the invention, HAdV infectious disease pathogens can be covered, more crowds can be protected from being harmed by influenza viruses and HAdV, and foundations are laid for producing a 'dual-purpose vaccine' or a 'multi-purpose vaccine'.
Description
Technical field
The present invention relates to a kind of preparation and its application of influenza virus for the HAdV chimerics of carrier.
Background technology
Respiratory infectious disease is remained so far causes in the world one of main causes of death, and adenovirus
(Adenovirus, AdV) and influenza virus are then important respiratory pathogens.It is well known that adenovirus is in distributed in nature
It is quite varied, it is one of encountered pathogenic of children respiratory tract diseases.The about 5%- in infantile acute upper respiratory infection below 3 years old
10% is caused by adenovirus.It is in hospital from northern China and southern various places the clinical investigation result of sick child, it was demonstrated that 3,7 type adenopathies
Poison is the main pathogen of adenovirus pneumonia, by they caused by ALRI account for the 50% of all adenovirus infections.And gland
Virus gives people class health and lives and causes serious threat safely, increasingly by society pass because its type is more, scope of causing a disease is wide
Note.At the same time, people will not forget historical three influenza outbreaks, special to the catastrophic effect that the whole world is brought
It is not that H5, H7 subtype avian influenza virus for occurring in recent years cross over the event that species barrier infects people, gives people class and beat again
Alarm bell.Therefore the prevention and control of adenovirus hominis and influenza have become the significant problem of current life science.
At present, vaccine inoculation is the most effective measure for preventing and controlling human infectious disease.In past more than 50 years, influenza
The development experience of vaccine inactivated vaccine, split vaccine, subunit vaccine, DNA vaccination, have made important for human health
Contribution.The Gripovax that in recent years U.S. and Russia develop, is that one kind reduces virulence and can be in optimal adaptation temperature
Attenuation human influenza virus's strain of the lower growth of degree, clinical trial confirm be it is safe and effective, can effective flu-prevention, the vaccine
Extensive application conquer influenza in 21 century for the mankind and provide rosy prospect.However, for human airway adenovirus,
So far the vaccine for using also is not approved for, but for the research of vaccine is always the emphasis of international concern.Although adenopathy
Malicious Vaccine Development is rapid, and the adenovirus inactivated vaccine of development, subunit vaccine and attenuated live vaccine, are mankind's Respiratory Tract Adenovirus
Immunoprophylaxis provide candidate vaccine, but not yet large-scale application.From in terms of research because adenovirus has as many as 51 serotypes,
Differing greatly between each type, the potential immunity of induction body generation is too drastic after vaccine immunity, and injecting immune can not be produced
Raw mucosa-immune and traditional attenuated live vaccine also there are problems that it is unstable and potential immune, to the development proposition of vaccine
Challenge.
In recent years, one be that antigen immune bioinformatics technique develops rapidly is body main protective adjacent for adenovirus six
Epitope is designed and generates significant impact, is that realization improves B cell generation NAT, lowers immune too drastic, lifting
The adenovirus vaccine development of protection comprehensively provides theoretical foundation and technical support；Two is that Reverse Genetics are fast-developing, is
Develop safely, effectively, the attenuated live vaccine of multivalence provide advanced technology so that influenza virus has manifested good as delivery system
Good development prospect, while the purpose for reaching " seedling is dual-purpose " or " seedling is multiplex " for embedded virus provides theoretical foundation；Three
It is that nasal-spraying immune approach is designed with generation mucous membrane and systemic immunity response, realizes immunity for embedded virus for adenovirus hominis
Cross protection, becomes the important development direction of human adenovirus vaccine.Therefore, above technology collection hold, be integrated into development safely, have
Effect, practical human adenovirus vaccine ensure there is provided science.
First, antigen immune biology information technology is developed rapidly, and the research and development for adenovirus vaccine provide New Policy
Early in the mid-50, when being attempted to the tonsillotome with surgery excision, paraphyte and setting up cell culture system, find
There is a kind of infectious agent that the epithelial cell of culture can be made regression occur, this factor is officially named adenovirus within 1956
(Adenovirus).It can cause the disease of human airway, intestines and stomach, urinary system and eye.Human adenovirus (human
Adenovirus, HAdV) belong to mammal AdV category, it is nonencapsulated double-stranded DNA virus, it is a diameter of in icosahedral structure of virus
70～90nm.A～G7 groups are divided into according to physics, chemistry, biological property, each group includes some serotypes, totally 51 type, wherein
1-8,19,21,35 serotypes are all related to serious acute respiratory disease, and 3,7 types are two kinds of pathogenic highests.It is external
There are many document reports：3rd, 7 type adenovirus cause acute respiratory distress in septic yanks' depot it is popular more
Seriously.From in terms of virus structure, HAdV capsid proteins are made up of 252 capsomeres, wherein 20 of 240 capsomere constitutive protein capsids
Face, its constitutive protein is referred to as six adjacent bodies (Hexon, 120KD), is main envelope protein；12 capsomeres are located at 20 face bodies
Drift angle, is referred to as penton (penton, 82KD)；The also fiber (fiber, 62KD) of trimer protein.Wherein, six adjacent bodies are
The major antigen albumen of adenovirus, stimulates body to produce antibody, suppresses the change of viral conformation, so as to neutralize virion.Six is adjacent
Body contains substantial amounts of antigenic determinant, including type specificity and subgenus specific antigen epitope and neutrality epitope.Base
Bottom (pedestal) includes two regions P1 and P2 area, and tower area is made up of 4 rings (1oop), i.e. Loop1, Loop2, Loop3,
Loop4 areas.In recent years, due to the structural specificity of adenovirus hexon, Neutralization and crystallization is encoded, participates in immunity anti-
Should, become the focus of Chinese scholars research.As can be seen here, HAdV is due to its special, complicated pathogenic characteristic, to HAdV's
Prevention and control propose a difficult problem.
Vaccine is the maximally effective means of control infectious disease.In mankind's Fighting Disease and the struggle of infectious disease, vaccine is played
Important function, adenovirus vaccine is no exception.The U.S. has adopted enteric-coated adenovirus attenuated live early in the sixties
Vaccine carries out immunization experiment to the adult of depot, and discovery can reduce the heating that 90 percent adenovirus causes
Symptom, but find that the NAT that this live vaccine induction is produced is very low by Serologic detection.The seventies make
Immunity is carried out with adenovirus enteric live vaccine, it is found that IgM, IgG, IgA are raised in serum, but without discovery in nasal discharge
Corresponding IgA type antibody.Although adenovirus attenuated live vaccine works well used in U.S. army, there are still in U.S. army
The phenomenon of ARD outbursts, has research to confirm using DNA chip and round pcr, after being inoculated with existing adenovirus attenuated live vaccine, connects
Kind person remains to coinfection（Co-infection）The adenovirus of other types, it was demonstrated that existing attenuated live vaccine to other types
Adenovirus does not have Cross immunogenicity ability.Therefore, HAdV subunit vaccines are duty-bound becomes the important development mesh of this area
Mark.
In the several years in past, adenovirus is widely used in gene therapy and chimeric research as a kind of genophore
In, and adenovirus infection hinders the curative effect of this gene therapy significantly, while people will not ignore this chimeric bringing
Potential side reaction.In recent years, the development of antigen immune bioinformatics sets to the adjacent body main protection antigen epi-positions of HAdV six
Meter generates significant impact, to realize that the T of protective antigens, the research and development of B cell Dominant Epitopes vaccine provide theories technique support.
There is document report application phage display small peptide technology exploration adenovirus epi-position, with 3 type adenovirus neutralizing antibodies bacteriophage is screened
Random pentadecapeptide storehouse, after the three-wheel screening, 22 phage clones of random picking are separately added into 3 type adenovirus infections
Hela cells.As a result find that 8/22 clone's thalline small peptide has protective effect to 3 type adenovirus infection Hela cells.Separately have
Person compares the adjacent body amino acid sequence of adenovirus six by computer, with reference to the polypeptide exposure of 2 type adenovirus tomographs prompting
Situation and antigenicity forecast analysis, select 937～1230,2166～2607 and 937～2607 volumes of hexon gene
Code sequence.The antigen site of the prediction that this region includes has very high homology between Respiratory Tract Adenovirus type, in adenovirus
There may be preferable exposed property on particle and may contain between type or group-specific antigen epi-position.External also someone applies Ad2
Loop1 areas expression of the antigenic determinant in B group Coxsackie virus, using Coxsackie virus as carrier, recombinant type II gland
The L1 rings of hexonmer albumen can be such that it expresses in HeLa cell inner stablities, and expressed albumen can induce generation both
Anti- adenovirus and the neutralizing antibody of anti-Coxsackie virus, and then develop the recombinant vaccine of multivalence.
2nd, the development of influenza virus Reverse Genetics, is the HAdV chimerics virus for preparing influenza virus for carrier
Strain provides Reliable guarantee
At present, reverse Genetics Technique influenza virus field extensive application and develop rapidly, 8 plasmids of influenza virus
Virus rescue system at home and abroad has become the technology of maturation so that using influenza virus as delivery system expression alien gene
Become very attractive research direction, recombinant respiratory infects the study hotspot of disease vaccine and concentrates on influenza virus.Stream
Influenza Virus have many advantages as the carrier for developing other infectious disease pathogens vaccines：(1) body can be stimulated to produce strong
Mucous membrane and systemic immunity response；(2) influenza vaccines are due to the annual production of antigenic variation needs；(3) knot of HA and NA surface proteins
Structure and function are determined can be carried out genetic manipulation and not affect their function on them；(4) influenza virus has defined
Efficient reverse genetics operating system；(5) mouse and ferret are the respiratory mucosa immunity of restructuring influenza virus vaccine Candidate Strain
Response and the research of immunoprotection provide good animal model.With other carriers such as adenovirus, retrovirus compares, and flows
Influenza Virus will not form DNA intermediate products in replicative cycle, additionally due to it will not be integrated into the chromosome of host so that its tool
There is higher security.Strategy for the operation of influenza virus gene group has：Foreign protein is fitted together to into surface glycoprotein HA and NA,
Other genetic fragment is produced, non-structural protein NS 1 is transformed.
In view of the breakthrough that influenza virus Reverse Genetics are obtained, has lot of documents report and makees influenza virus
Being successfully prepared for delivery system can reach effective immunoprotection, multivalence chimeric vaccine candidates, be the research of vaccine
Development prospect is widened.Horimoto in 2004 etc. constructs chimera virus (A/B), and the virus is containing chimera (A/B)
HA and Type B virus full length NA fragments.The research provides a kind of new thinking to develop attenuated live vaccine from single host strain.
Maeda Y in 2005 etc. produce a kind of influenza A of restructuring by reverse Genetics Technique, and the viral code area is secondary
The HA/NA ectodomains of influenza virus, can effectively breed in chicken embryo but be attenuation in mouse body.When sick with restructuring
After malicious intranasal administration mouse, the antibody of opposing influenza virus and parainfluenza virus can be all produced.The live vaccine of this bivalent
Immune protective effect be substantially better than combined vaccine.The report small peptide such as the same year Zhu NanLi is inserted into influenza virus HA antigens
" loop " ring region, subsequent research shows that protective antigens (PA) the large fragment polypeptide of B.anthracis can be inserted into stream
The functional areas of Influenza Virus H3 hypotypes HA, the recombinant influenza of chimeric HA-PA genes is hereditary Jing after mdck cell and chicken embryo are passed on
Good stability, results of animal shows that recombinant virus can cause the protein induced antibody response of HA and PA.2003
Kawaka etc. enters GFP gene integrations after the ad-hoc location of NA genes, as a result finds the efficiency of recombinant influenza particle assembling
May be up to 91%.GFP and IL-2 is successively inserted Andrej Egorov in 2005 etc. the ORFs of NS1, as a result successfully
The strains of influenza viruses of restructuring is arrived.
The above be at present using influenza virus as delivery system expression alien gene present Research, it is and domestic and international,
It is embedding for developing the HAdV that influenza virus is carrier using influenza virus gene group as the carrier for expressing HAdV virus antigen epitopes
Close vaccine at home and abroad to have not been reported.
3rd, nasal-spraying immune influenza virus is the HAdV chimerics of carrier, is to lift safety, the base of comprehensive immunoprotection
Stone
In June, 2003, Medimmune companies of the U.S. production influenza trivalent attenuated live vaccine Flumist by FDA approval and
Come into operation, it is adaptable to which 2-17 year healthy children and 18-49 year health adult, clinical trial results show that the vaccine is
Safely effectively.By 2 months 2012, U.S. FDA approval influenza tetravalence attenuated live vaccine listing, the vaccine was connect by nasal cavity
Kind, using method is easy, has wide range of applications so that with acclimatization to cold Gripovax as delivery system expression alien gene
Develop other vaccines and open the new world, bring new opportunity.Influenza virus is logical as a kind of strong vaccine carrier
Cross and deliver different antigens and can induce T, the B cell immune response of generation system and mucous membrane into nose tissue.Additionally, attenuated live
Vaccine can induce locally and systemically immunity by intranasal immunisations, thus have defence to make to the upper respiratory tract and ALRI
With.The immune response that attenuated live vaccine is induced is similar to the response to wild-type virus.For the HAdV of respiratory mucosa infection
For vaccine, injection inoculation is not preferable immunization route, inconvenient yet.Therefore, develop nasal be vaccine immunity most
Good selection.
Nasal meatus is rich in GALT, is the target site of intranasal inoculation vaccine, i.e. nose associated lymphoid tissue（NALT）, it
It is predominantly located at the GALT ring of throat, referred to as WaldeyerShi rings.Research shows, bronchia mucosal area about 150cm2, on
Chrotoplast gap is larger and closely coupled with capillary, and blood vessel and lymph node are abundant, the unit CBF about 40ml/ of schneiderian membrance
Min, than muscle, brain, liver it is all big, actually pharynx nasalis is good immune organ, there is abundant antigen presenting cell, energy
Antagonism original is effectively offered, processes, and produces mucosa-immune and systemic immunity response, and the mucosa-immune of local, moreover it is possible to cause
The interlinking of the position such as respiratory tract, alimentary canal, urogenital tract mucosal immune response, but it is most strong with respiratory tract.
Nasal-spraying immune is the field that at present research is enlivened, and also has many enterprise development intranasal inoculation vaccines in recent years, particularly
Respiratory infectious disease vaccine, such as Gripovax not only can produce local mucosa-immune by intranasal inoculation, also
With generation system immunity, and can produce with Cross immunogenicity.Therefore, the influenza virus for developing intranasal inoculation is carrier
HAdV chimerics substitute injection and have great application prospect, this be due to nasal cavity immunity have it is simple, effectively, be very suitable for
Many people's self uses, while enable body to resist two kinds of pathogenic infections of HAdV and influenza, and to HAdV epidemic diseases
Seedling type of immune response can change, and thus reach safer purpose, and the immunoprophylaxis for HAdV and influenza provides guarantee.
The content of the invention
It is an object of the invention to provide a kind of preparation and its application of influenza virus for the HAdV chimerics of carrier.
The present invention provides the DNA molecular shown in SEQ ID No.1.
A kind of recombinant virus falls within protection scope of the present invention, and the virus is prepared as follows：
By respectively containing acclimatization to cold, the PB2 genes of attenuated influenza virus, acclimatization to cold, the PB1 genes of attenuated influenza virus,
Acclimatization to cold, the PA genes of attenuated influenza virus, acclimatization to cold, the NP genes of attenuated influenza virus, acclimatization to cold, attenuated influenza virus
M genes and acclimatization to cold, the expression plasmid of the NS genes of attenuated influenza virus, and the HA genes containing target influenza virus respectively
With the expression plasmid cotransfection host cell of the NA genes of target influenza virus, cultivate and obtain recombinant influenza；
Insert or replace with optional position in the opening code-reading frame of at least one gene in gene as described below
The structural protein gene of HAdV：
（1）Acclimatization to cold, the PB2 genes of attenuated influenza virus；
（2）Acclimatization to cold, the PB1 genes of attenuated influenza virus；
（3）Acclimatization to cold, the PA genes of attenuated influenza virus；
（4）Acclimatization to cold, the NP genes of attenuated influenza virus；
（5）Acclimatization to cold, the M genes of attenuated influenza virus；
（6）Acclimatization to cold, the NS genes of attenuated influenza virus；
（7）The HA genes of target influenza virus；
（8）The NA genes of target influenza virus；
The acclimatization to cold, the PB2 genes of attenuated influenza virus, PB1 genes, PA genes, NP genes, M genes and NS genes,
And the HA genes and NA genes of target influenza virus is located on different expression plasmids；
The structural protein gene of the HAdV is the hexon of the hexon full genome of HAdV and/or HAdV
Dominant antigen epitope gene.
In above-mentioned recombinant virus, the host cell is MDCK, Vero, 293T, COS cell or MDCK/293T, MDCK/
The cell that COS is co-cultured；
The structural protein gene of the HAdV is the dominant antigen table of hexon tower the area Loop1 and Loop2 of HAdV
The fusion of position gene.
In any of the above-described described recombinant virus, the HAdV is 3 types HAdV or 7 types HAdV；
3 type HAdV is specially HAdV-3 Strain VR-3；
7 type HAdV is specially HAdV-7 Strain GZ08, GenBank accession No.GQ478341.
In any of the above-described described recombinant virus, the advantage of hexon tower the area Loop1 and Loop2 of the HAdV resists
The nucleotide sequence of the fusion of former epitope gene is as shown in SEQ ID No.1.
In any of the above-described described recombinant virus, the structural protein gene of the HAdV is inserted in the acclimatization to cold, attenuation stream
The opening code-reading frame of the NS genes of Influenza Virus from 5 ' ends between the 375th to the 376th nucleotides；
The structural protein gene of the HAdV and the acclimatization to cold, the opening code-reading frame of the NS genes of attenuated influenza virus
5 '-UAAUG-3 ' sequences are connected with from 5 ' ends between the 375th nucleotides；
And/or,
The structural protein gene of the HAdV is the opening code-reading frame of the M genes by the acclimatization to cold, attenuated influenza virus
The 223rd nucleotides from 5 ' ends replace to the 760th nucleotides；
And/or,
The structural protein gene of the HAdV is from 5 ' by the opening code-reading frame of the NA genes of the target influenza virus
Play the 184th nucleotides and replace to the 1253rd nucleotides in end.
In any of the above-described described recombinant virus, the acclimatization to cold, attenuated influenza virus are acclimatization to cold, attenuation A type influenzas
Virus or acclimatization to cold, attenuation Type B influenza virus；
The acclimatization to cold, attenuation influenza A are specially H2N2 hypotype acclimatization to colds, attenuated influenza virus, then are specially
Acclimatization to cold, attenuated influenza virus strain A/Ann Arbor/6/60 (H2N2)；
The target influenza virus is influenza A or Type B influenza virus, and it is sub- that the influenza A is specially H1
Any one in type-H16 hypotypes, then specially H1N1 subtype influenza virus, then specially strains of influenza viruses A/
California/07/2009（H1N1）；
The target influenza virus is without any process（Such as without attenuation, without acclimatization to cold）Common open country
Raw type influenza virus.
In any of the above-described described recombinant virus, the structural protein gene of the HAdV is inserted in the acclimatization to cold, attenuation stream
The opening code-reading frame of the NS genes of Influenza Virus from 5 ' ends between the 375th to the 376th nucleotides gained sequence be SEQ
In ID No.2 shown in the nucleotides of 41-1162 positions；
The structural protein gene of the HAdV is the opening code-reading frame of the M genes by the acclimatization to cold, attenuated influenza virus
The 223rd nucleotides from 5 ' ends to the 760th nucleotides to replace gained sequence be 40-759 positions in SEQ ID No.3
Shown in nucleotides；
The structural protein gene of the HAdV is last from 5 ' by the opening code-reading frame of the NA genes of the target influenza virus
It is 35-650 positions nucleotides in SEQ ID No.4 to have held the 184th nucleotides to replace gained sequence to the 1253rd nucleotides
It is shown.
When building the expression plasmid, above-mentioned PB2 genes, PB1 genes, PA genes, NP genes, M genes, NS genes, HA
Gene or NA genes, are connected with 3 ' NCR sequences of respective gene at respective 5 ' ends, are respectively connected with respective 3 ' ends each
From 5 ' NCR sequences of gene；
The plasmid that sets out of the expression plasmid is bidirectional transcription expression vector pAD3000；
When building the expression plasmid, each gene is inserted in the BsmBI sites of bidirectional transcription expression vector pAD3000.
The chimeric that any of the above-described described virus is prepared falls within protection scope of the present invention.
Above-mentioned DNA molecular answering in the product for preparing prevention and/or treating the disease that influenza virus and/or HAdV cause
With falling within protection scope of the present invention；
And/or,
Any of the above-described described virus is preparing prevention and/or is treating the product of the disease that influenza virus and/or HAdV cause
Application in product falls within protection scope of the present invention；
And/or,
Above-mentioned chimeric is in the product for preparing prevention and/or treating the disease that influenza virus and/or HAdV cause
Using falling within protection scope of the present invention；
The influenza virus is influenza A or Type B influenza virus, the influenza A be specially H1 hypotypes-
Any one in H16 hypotypes；
The HAdV is 3 types HAdV or 7 types HAdV；
3 type HAdV is specially HAdV-3 Strain VR-3；
7 type HAdV is specially HAdV-7 Strain GZ08, GenBank accession No.GQ478341.
The HAdV chimerics that the present invention is provided can cover HAdV infectious disease pathogens, protect more crowds from influenza
Virus and HAdV infection, are to realize that " seedling is dual-purpose " or " seedling is multiplex " lays a good foundation.
Description of the drawings
Fig. 1 is the restructuring strategy schematic diagram for building influenza virus for the HAdV chimerics of carrier.
Electron microscopic morphology identifications and granular size and distribution situation of the Fig. 2 for recombinant virus rFLU/HAdV/NS1.
Fig. 3 is the antibody titer measurement result of mouse immune rFLU/HAdV/NS1.
Fig. 4 is that mouse immune rFLU/HAdV/NS1 produces IFN-γ, IL-4 cellullar immunologic responses.
Fig. 5 is that mouse immune rFLU/HAdV/NS1 produces the mucosal antibodies potency for being directed to HAdV-3.
Fig. 6 is the immune protective effect that rFLU/HAdV/NS1 immune mouses are attacked adenopathy strain.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, commercially obtain.
PAD3000 document " Hoffmann E, Mahmood K, Yang CF, Webster RG, Greenberg HB,
Kemble G.Rescue of influenza B virus from eight plasmids.Proc Natl Acad
Sci.2002;99(17):11411-6. " mistake disclosed in, the public can obtain from Academy of Military Medicine, PLA.
6-8 week old BALB/c mouse is purchased from Military Medical Science Institute's Experimental Animal Center.
The ferret of 10-12 week old is purchased from Chinese Wuxi Angora An Gelu companies.
2012-2013 influenza viruses epidemic strain A/California/07/2009 then（H1N1）By national influenza center
There is provided, the public can obtain from Academy of Military Medicine, PLA.HA, NA gene of the Strain is expanded respectively, then
The BsmBI sites of bidirectional transcription/expression vector pAD3000 are inserted respectively into, structure obtains the recombinant plasmid pD- of the Strain
HA、pD-NA。
Acclimatization to cold, attenuated influenza virus strain A/Ann Arbor/6/60 (H2N2)（Write a Chinese character in simplified form A/AA/6/60）In document " Yang
P,Duan Y,Wang C,Xing L,Gao X,Tang C,Luo D,Zhao Z,Jia W,Peng D,Liu X,Wang
X.Immunogenicity and protective efficacy of a live attenuated vaccine against
the2009pandemic A H1N1in mice and ferrets.Vaccine.2011Jan17;29(4):In 698-705. "
It is disclosed, the public can obtain from Academy of Military Medicine, PLA.6 internal genes PB2, PB1 of the Strain,
PA, NP, NS, M, are inserted respectively into the BsmBI sites of bidirectional transcription/expression vector pAD3000, and structure obtains the restructuring of the virus
Plasmid pD-PB2, pD-PB1, pD-PA, pD-NP, pD-NS, pD-M.
Influenza virus A/PR/8/1934 (PR8) Strain document " Li C, Yang P, Sun Y, Li T, Wang C,
Wang Z,Zou Z,Yan Y,Wang W,Wang C,Chen Z,Xing L,Tang C,Ju X,Guo F,Deng J,Zhao
Y,Yang P,Tang J,Wang H,Zhao Z,Yin Z,Cao B,Wang X,Jiang C.IL-17response
mediates acute lung injury induced by the2009pandemic influenza A(H1N1)
virus.Cell Res.2012Mar;22(3):Mistake disclosed in 528-38. ", the public can be from Chinese People's Liberation Army's military medicine
The academy of sciences obtains.
HAdV-3 Strain VR-3, purchased from ATCC.
HAdV-7 Strain GZ08 (GenBank accession No.GQ478341) is in document " Qiu H, Li X, Tian
X,Zhou Z,Xing K,Li H,Tang N,Liu W,Bai P,Zhou R.Serotype-specific neutralizing
antibody epitopes of human adenovirus type3(HAdV-3)and HAdV-7reside in
multiple hexon hypervariable regions.J Virol.2012Aug;86(15):7964-75. " disclosed in
Cross, the public can obtain from Academy of Military Medicine, PLA.
The hexon tower area of HAdV is made up of 4 rings (1oop), i.e. Loop1, Loop2, Loop3, Loop4 area, under
State L1, L2 in embodiment and represent Loop1 and Loop2 respectively.
Experimental example 1, influenza virus is fitted together to epidemic disease for the HAdV of the chimeric HAdV-Hexon-L1/L2 dominant antigens epi-position of carrier
The preparation of seedling rFLU-HAdV/NS1
First, Hexon-L1/L2 in the HAdV of 3 types and 7 types（HAdV-Hexon-L1/L2）Dominant antigen epitope sequences such as SEQ
Shown in ID No.1, Jing zooperies and clinical patient serum are verified, meet next step requirement of experiment.
2nd, construction recombination plasmid pHexon-L1/L2-NS1
NS1 genetic fragments with acclimatization to cold, attenuated influenza virus strain A/AA/6/60 are excellent to insert HAdV-Hexon-L1/L2
The target spot of gesture antigen epitope genes, using molecular biology method construction recombination plasmid pHexon-L1/L2-NS1, specific strategy
As shown in Figure 1A.
Specifically HAdV-Hexon-L1/L2 dominant antigen epitope genes are inserted into into acclimatization to cold, attenuated influenza virus strain A/
The NS1 gene open reading frames of AA/6/60（ORF）Front 125 amino acid encoding gene at, centre add linker（5′-
UAAUG-3′）Connection, the existing terminator effects of linker, has promoter to act on again, is finally the encoding gene of NEP albumen, will
The recombinant plasmid for building is named as pHexon-L1/L2-NS1.
Step is as follows：
（One）DNA molecular shown in synthesis SEQ ID No.2, in SEQ ID No.2 the 1st to the 14th from 5 ' ends
For restriction enzyme site, the 15th to the 41st is the NCR of NS1 genes 3 ', and the 41st to the 415th is acclimatization to cold, attenuated influenza virus
The 1st to the 375th nucleotides from 5 ' ends of strain A/AA/6/60NS gene open reading frames, the 416th to the 420th
For linker, the 421st to the 699th is HAdV-Hexon-L1/L2 dominant antigen epitope genes, the 700th to the 1162nd
For NS gene open reading frames from 5 ' ends the 376th to the 838th nucleotides, the 1163rd to the 1191st be NS bases
Because of 5 ' NCR, the 1192nd to the 1203rd is restriction enzyme site.
（Two）DNA molecular shown in BsmBI digestion SEQ ID No.2, obtains genetic fragment；BsmBI digestion pAD3000,
Obtain carrier large fragment；Genetic fragment is connected with carrier large fragment, recombinant plasmid is obtained, pHexon-L1/ is named as
L2-NS1, send sequencing result correct recombinant plasmid.
3rd, by recombinant plasmid pHexon-L1/L2-NS1 and acclimatization to cold, the inside disease of attenuated influenza virus strain A/AA/6/60
Recombinant plasmid pD-PB2, pD-PB1, pD-PA, pD-NP, pD-M that virus gene skeleton PB2, PB1, PA, NP, M build respectively, and
Influenza virus epidemic strain A/California/07/2009 then（H1N1）The gene constructed recombinant plasmid pD-HA of HA, NA,
PD-NA, each 0.2 μ g of totally 8 kinds of plasmids, mixed in equal amounts adds 10 μ L transfection reagents（Effectene, purchased from Qiagen companies）Room temperature
Effect 10min, cotransfection mdck cell, 33 DEG C, 5%CO2, 48-60h is cultivated, cell suspension is obtained, by the age in days of cell suspension inoculation 9
SPF chicken embryos, 33 DEG C of culture 72h, harvest chick embryo allantoic liquid and determine HA hemagglutinative titers, and the testing result of HA hemagglutinative titers is 1:256-
512, obtain the HAdV chimerics of chimeric HAdV-Hexon-L1/L2 dominant antigen epi-positions（rFLU-HAdV/NS1）.
4th, the chimeric strain that step 3 is obtained is identified, electron microscopic observation morphology of virus, as a result as shown in Figure 2.Figure
2 show that the vaccine strain meets influenza virus representative configuration feature, and virion size is between 80-120nm.
5th, rFLU-HAdV/NS1 is inoculated with into 9-11 age in days SPF chicken embryos（Purchased from Beijing Experimental Amimal Research Centre）Pass on,
Take second generation chick embryo allantoic liquid and extract viral RNA, through RT-PCR, amplify PB2, PB1, PA, NP, HA, NA, M and NS totally 8
Genetic fragment, send company to be sequenced genetic fragment respectively, as a result consistent with expected gene order.
6th, by rFLU-HAdV/NS1 Jing chicken embryo mass propgations, ultrafiltration concentration, sucrose gradient centrifugation after purification, run SDS-
PAGE electrophoresis, after gel-colored, decolouring, can detect that correspondingly sized NP, HA1, HA2, NEP albumen, show the main of antigen
Composition is not lost.
7th, the temperature of rFLU-HAdV/NS1 is sensitive（temperature sensitive,ts）, acclimatization to cold
（coldadapted,ca）And attenuation（attenuated,att）Phenotypic examination
RFLU-HAdV/NS1 is inoculated with into mdck cell or 9-11 day instar chicken embryos, under the conditions of 25,33,37 and 39 DEG C
Culture 3 days, collects cell conditioned medium or chick embryo allantoic liquid determines virus titer.As a result show, the vaccine strain has ts, ca phenotype.
Meanwhile, the vaccine strain shows att phenotype on the ferret of BALB/c mouse and 10-12 week old.
8th, immune effects of the rFLU-HAdV/NS1 in mouse body
（One）By rFLU-HAdV/NS1 Jing ultrafiltration concentrations, SDGC after purification, the attenuation of the vaccine is prepared
Live vaccine formulation.
From 6-8 week old BALB/c mouses, it is divided into vaccine group and control group, 20 per group.
Vaccine group：By the vaccine Nasal immunization BALB/c mouse, immunity 2 times, it is spaced 2 weeks, arranging immunizing dose is
104TCID50, 105TCID50Two dosage groups, volume is 20 μ l.
Control group：PBS replaces vaccine, while immunization wayses, immune volume and immunization time are consistent with vaccine group.By vaccine
2 weeks after initial immunity, after booster immunization, the blood sampling of Jing mouse tail veins is collected by centrifugation each group mouse for group and control group
Serum.Determined using HI methods and be directed in serum wild type influenza virus A/California/07/2009（H1N1）Antibody effect
Valency, while being detected in serum for the specific antibody production of HAdV-3 Strain VR-3, knot using microneutralization method
Fruit is as shown in figure 3, in Fig. 3, * is represented compared with control group, p<0.05；* is represented compared with control group, p<0.01.
Fig. 3 A are that wild type influenza virus A/California/07/2009 is directed in serum（H1N1）Antibody titer survey
Determine result.
Fig. 3 B are for the specific antibody production measurement result of HAdV-3 Strain VR-3 in serum.
Fig. 3 shows that experimental group is directed to wild type influenza virus A/California/07/2009（H1N1）HI in and imitate
Valency and for HAdV-3 Strain VR-3 NAT respectively with the rising more notable than potency of PBS groups, show influenza virus
For the HAdV chimerics of carrier（rFLU-HAdV/NS1）After immune animal, can detect that in serum for HAdV and influenza disease
The high-caliber specific antibody titres of poison, show that body is can induce after the vaccine immunity animal to be produced for HAdV and influenza disease
The Double immune response of poison.
（Two）Using step（One）Identical method distinguishes Nasal immunization BALB/c mouse with rFLU-HAdV/NS1, and sets
Control is put, 2 weeks after booster immunization, animal is put to death, SPL, ELISPOT methods detection vaccine strain immune animal is separated
IFN-γ and IL-4 cellullar immunologic responses being produced afterwards, as a result being distinguished as shown in Figure 4 A and 4 B shown in FIG., in Fig. 4, * * are represented and control group
Compare, p<0.01.
Fig. 4 shows that mouse body can be produced for influenza virus PR8 and HAdV-3 after rFLU-HAdV/NS1 Nasal immunizations
The IFN-γ of Strain VR-3, IL-4 cell immune responses, it is extremely more notable than difference with PBS groups（p<0.01）, show recombinant virus
Body can be induced to produce preferable cell immune response.
（Three）Using step（One）Identical method rFLU-HAdV/NS1 Nasal immunization BALB/c mouses, and arrange right
According to 2 weeks after booster immunization, for the sIgA antibody of HAdV-3 Strain VR-3 in ELISA method detection mouse lung, Nasal lavage fluid
Potency, as a result distinguishes as fig. 5 a and fig. 5b, and in Fig. 5, * is represented compared with control group, p<0.05；* is represented and control group phase
Than p<0.01.
Fig. 5 shows that sIgA antibody is significantly raised in mouse nose, lung-douching fluid after rFLU-HAdV/NS1 Nasal immunizations, with
PBS groups are extremely more notable than difference（p<0.01）, show that recombinant virus can induce body to produce preferable mucosa-immune reaction.
9th, animal protection experiment
Using the method rFLU-HAdV/NS1 Nasal immunization BALB/c mouses of step 8, and control is set, is exempted from reinforcement
2 weeks after epidemic disease, respectively with Strain HAdV-3 being clinically separated（VR-3）、HAdV-7（GZ08）Attack BALB/c mouse, infectious agent
Measure as 2 × 109VPs, the lung tissue of the lung tissue and normal mouse that take each group mouse carries out pathology detection, as a result such as Fig. 6 A
It is shown.
Fig. 6 A show that the lung pathology change of PBS group mouse is obvious, visible pulmonary edema under mirror, and a large amount of inflammatory cells, lymph are thin
Born of the same parents infiltrate, and alveolar spaces are thickened, and normal mouse pulmonary morphology is intact.RFLU-HAdV/NS1 group mouse lung tissues pathology disease
Change is obviously improved compared with PBS groups, wherein 105TCID50High dose group is better than 104TCID50Low dose group.
The intrapulmonary viral copy number of each group mouse is detected using qPCR methods, as a result respectively such as Fig. 6 B and 6C institutes
Show, in Fig. 6, * is represented compared with control group, p<0.05.
Fig. 6 B and 6C show, rFLU-HAdV/NS1 group mouse lung inner virus HAdV-3（VR-3）、HAdV-7（GZ08）Answer
Number processed is substantially reduced compared with PBS groups.
As a result show, recombinant vaccine rFLU-HAdV/NS1 immunized mices obtain anti-for the immune protective of HAdV
Body.
Experimental example 2, influenza virus is fitted together to epidemic disease for the HAdV of the chimeric HAdV-Hexon-L1/L2 dominant antigens epi-position of carrier
The preparation of seedling rFLU-HAdV/M
First, in the HAdV of 3 types and 7 types HAdV-Hexon-L1/L2 dominant antigens epitope sequences as shown in SEQ ID No.1,
According to the method validation of step one in embodiment 1, meet next step requirement of experiment.
2nd, construction recombination plasmid pHexon-L1/L2-M
With acclimatization to cold, attenuated influenza virus strain A/AA/6/60 M genetic fragments as insert HAdV-Hexon-L1/L2 advantages
The target spot of antigen epitope genes, using molecular biology method construction recombination plasmid pHexon-L1/L2-M, specific strategy is as schemed
Shown in 1B.
Specifically HAdV-Hexon-L1/L2 dominant antigen epitope genes are inserted into into acclimatization to cold, attenuated influenza virus strain A/
The M gene open reading frames of AA/6/60（ORF）Front 222 nucleotides and rear 222 nucleotides between, by the weight for building
Group plasmid is named as pHexon-L1/L2-M.
Step is as follows：
（One）DNA molecular shown in synthesis SEQ ID No.3, in SEQ ID No.3 the 1st to the 15th from 5 ' ends
For restriction enzyme site, the 16th to the 40th is the NCR of M genes 3 ', and the 40th to the 261st is acclimatization to cold, attenuated influenza virus strain
A/AA/6/60M gene open reading frames from 5 ' ends from the 1st to the 222nd nucleotides, the 262nd to the 537th is
HAdV-Hexon-L1/L2 dominant antigen epitope genes, the 538th to the 759th is acclimatization to cold, attenuated influenza virus strain A/AA/
6/60M gene open reading frames from 5 ' ends from the 761st to the 982nd nucleotides, the 760th to the 782nd be M bases
Because of 5 ' NCR, the 783rd to the 794th is restriction enzyme site.
（Two）DNA molecular shown in BsmBI digestion SEQ ID No.3, obtains genetic fragment；BsmBI digestion pAD3000,
Obtain carrier large fragment；Genetic fragment is connected with carrier large fragment, recombinant plasmid is obtained, pHexon-L1/ is named as
L2-M, send sequencing result correct recombinant plasmid.
3rd, by recombinant plasmid pHexon-L1/L2-M, with acclimatization to cold, the inside disease of attenuated influenza virus strain A/AA/6/60
Recombinant plasmid pD-PB2, pD-PB1, pD-PA, pD-NP, pD-NS that virus gene skeleton PB2, PB1, PA, NP and NS build respectively,
And influenza virus epidemic strain A/California/07/2009 then（H1N1）The gene constructed recombinant plasmid pD-HA of HA, NA,
PD-NA, according to the method for step 3 in embodiment 1 by 8 kinds of plasmid co-transfection mdck cells, 37 DEG C, 5%CO2, 48-60h is cultivated,
Cell suspension is obtained, by the age in days SPF chicken embryos of cell suspension inoculation 9,35 DEG C of culture 72h, chick embryo allantoic liquid is harvested and is obtained chimeric HAdV-
The HAdV chimeric strains of Hexon-L1/L2 dominant antigen epi-positions（rFLU-HAdV/M）.
4th, as a result electron microscopic observation morphology of virus shows that the vaccine strain meets is identified to the vaccine strain that step 3 is obtained
Influenza virus representative configuration feature.
5th, rFLU-HAdV/M is inoculated with into 9-11 age in days SPF chicken embryos（Purchased from Beijing Experimental Amimal Research Centre）Pass on, take
Second generation chick embryo allantoic liquid extracts viral RNA, through RT-PCR, amplifies PB2, PB1, PA, NP, HA, NA, M and NS totally 8 bases
Because of fragment, company is sent to be sequenced genetic fragment respectively, it is as a result consistent with expected gene order.
6th, by rFLU-HAdV/M Jing chicken embryo mass propgations, ultrafiltration concentration, sucrose gradient centrifugation after purification, run SDS-PAGE
Electrophoresis, the main component of antigen is present.
7th, the temperature of rFLU-HAdV/M is sensitive（temperature sensitive,ts）, acclimatization to cold（cold
adapted,ca）And attenuation（attenuated,att）Phenotypic examination
RFLU-HAdV/M is inoculated with into mdck cell or 9-11 day instar chicken embryos, is trained under the conditions of 25,33,37 and 39 DEG C
Support 3 days, collect cell conditioned medium or chick embryo allantoic liquid determines virus titer.As a result show, the vaccine strain has ts, ca phenotype.Together
When, the vaccine strain shows att phenotype on BALB/c mouse and ferret animal.
8th, immune effects of the rFLU-HAdV/M in mouse body
By rFLU-HAdV/M Jing ultrafiltration concentrations, SDGC after purification, the attenuated live vaccine of the vaccine is prepared
Formulation.
From 6-8 week old BALB/c mouses, it is divided into vaccine group and control group, 20 per group.
The immunity of rFLU-HAdV/M is carried out to mouse according to the step of embodiment 1 eight method, while arranging control group.
By vaccine group and control group 2 weeks after initial immunity, after booster immunization, the blood sampling of Jing mouse tail veins, centrifugation
Collect the serum of each group mouse.
Determined using HI methods and be directed in serum wild type influenza virus A/California/07/2009（H1N1）It is anti-
Body potency, while detecting that the specific antibody in serum for HAdV-3 Strain VR-3 produces feelings using microneutralization method
Condition, as a result as shown in table 2.
The immune effect detection of table 2rFLU-HAdV/M
Table 2 shows, the HAdV chimerics with influenza virus as carrier（rFLU-HAdV/M）After immune animal, in serum
The antibody titer for HAdV and influenza virus is can detect that, shows that body generation is can induce after the vaccine immunity animal to be directed to
HAdV and the Double immune response of influenza virus.
Embodiment 3, influenza virus is fitted together to epidemic disease for the HAdV of the chimeric HAdV-Hexon-L1/L2 dominant antigens epi-position of carrier
The preparation of seedling rFLU-HAdV/NA
First, in the HAdV of 3 types and 7 types HAdV-Hexon-L1/L2 dominant antigens epitope sequences as shown in SEQ ID No.1,
According to the step of embodiment 1 one method validation, meet next step requirement of experiment.
2nd, construction recombination plasmid pHexon-L1/L2-NA
With A/California/07/2009（H1N1）NA genetic fragments resist for insertion HAdV-Hexon-L1/L2 advantages
The target spot of former epitope gene, using molecular biology method construction recombination plasmid pHexon-L1/L2-NA, specific strategy such as Fig. 1 C
It is shown.
Specifically HAdV-Hexon-L1/L2 dominant antigen epitope genes are inserted into into popular strains of influenza viruses H1N1 then
The NA gene open reading frames of hypotype A/California/07/2009（ORF）Front 183 nucleotides and rear 157 nucleotides
Between, the recombinant plasmid for building is named as into pHexon-L1/L2-NA.
Step is as follows：
（One）Synthesis SEQ ID No.4 shown in DNA molecular, in SEQ ID No.4 from 5 ' end the 1st to the 15th be
Restriction enzyme site, the 16th to the 35th is the NCR of NA genes 3 ', and the 35th to the 217th is influenza virus epidemic strain A/ then
California/07/2009（H1N1）NA gene open reading frames the 1st to the 183rd nucleotides from 5 ' ends,
218 to the 493rd is HAdV-Hexon-L1/L2 dominant antigen epitope genes, and the 494th to the 650th is NA gene opens
The 1254th to the 1410th nucleotides from 5 ' ends of reading frame, the 651st to the 681st is NA gene 5 ' NCR, the
682 to the 693rd is restriction enzyme site.
（Two）DNA molecular shown in BsaI digestion SEQ ID No.4, obtains genetic fragment；BsmBI digestion pAD3000, obtain
To carrier large fragment；Genetic fragment is connected with carrier large fragment, recombinant plasmid is obtained, pHexon-L1/L2- is named as
NA, send sequencing result correct recombinant plasmid.
3rd, by recombinant plasmid pHexon-L1/L2-NA, with acclimatization to cold, the inside disease of attenuated influenza virus strain A/AA/6/60
Recombinant plasmid pD-PB2, pD-PB1, pD-PA, pD-NP, pD-M that virus gene skeleton PB2, PB1, PA, NP, M and NS build respectively
And pD-NS, and influenza virus epidemic strain A/California/07/2009 then（H1N1）The gene constructed recombinant plasmids of HA
PD-HA, according to the method for step 3 in embodiment 1 by 8 kinds of plasmid co-transfection mdck cells, 37 DEG C, 5%CO2, 48-60h is cultivated,
Cell suspension is obtained, by the age in days SPF chicken embryos of cell suspension inoculation 9,33 DEG C of culture 72h, chick embryo allantoic liquid is harvested and is obtained chimeric HAdV-
The HAdV chimeric strains of Hexon-L1/L2 dominant antigen epi-positions（rFLU-HAdV/NA）.
4th, as a result electron microscopic observation morphology of virus shows that the vaccine strain meets is identified to the vaccine strain that step 3 is obtained
Influenza virus representative configuration feature.
5th, rFLU-HAdV/NA is inoculated with into 9-11 age in days SPF chicken embryos（Purchased from Beijing Experimental Amimal Research Centre）Pass on, take
Second generation chick embryo allantoic liquid extracts viral RNA, through RT-PCR, amplifies PB2, PB1, PA, NP, HA, NA, M and NS totally 8 bases
Because of fragment, company is sent to be sequenced genetic fragment respectively, it is as a result consistent with expected gene order.
6th, by rFLU-HAdV/NA Jing chicken embryo mass propgations, ultrafiltration concentration, sucrose gradient centrifugation after purification, run SDS-
PAGE electrophoresis, the main component of antigen is present.
7th, the temperature of rFLU-HAdV/NA is sensitive（temperature sensitive,ts）, acclimatization to cold（cold
adapted,ca）And attenuation（attenuated,att）Phenotypic examination
RFLU-HAdV/NA is inoculated with into mdck cell or 9-11 day instar chicken embryos, is trained under the conditions of 25,33,37 and 39 DEG C
Support 3 days, collect cell conditioned medium or chick embryo allantoic liquid determines virus titer.As a result show, the vaccine strain has ts, ca phenotype.Together
When, the vaccine strain shows att phenotype on BALB/c mouse and ferret animal.
8th, immune effects of the rFLU-HAdV/NA in mouse body
By rFLU-HAdV/NA Jing ultrafiltration concentrations, SDGC after purification, the attenuated live epidemic disease of the vaccine is prepared
Seedling formulation.
From 6-8 week old BALB/c mouses, it is divided into vaccine group and control group, 20 per group.
Method according to step 8 in embodiment 1 carries out the immunity of rFLU-HAdV/NA to mouse, while arranging control group.
By vaccine group and control group 2 weeks after initial immunity, after booster immunization, the blood sampling of Jing mouse tail veins, centrifugation
Collect the serum of each group mouse.
Determined using HI methods and be directed in serum wild type influenza virus A/California/07/2009（H1N1）It is anti-
Body potency, while detecting that the specific antibody in serum for HAdV-3 Strain VR-3 produces feelings using microneutralization method
Condition, as a result as shown in table 3.
The immune effect detection of table 3rFLU-HAdV/NA
Table 3 shows that influenza virus is the HAdV chimerics of carrier（rFLU-HAdV/NA）After immune animal, can in serum
The antibody titer for HAdV and influenza virus is detected, shows that body is can induce after the vaccine immunity animal to be produced for HAdV
With the Double immune response of influenza virus.
Claims (18)
- DNA molecular shown in 1.SEQ ID No.1.
- 2. a kind of recombinant virus, the virus is prepared as follows：By respectively containing acclimatization to cold, the PB2 genes of attenuated influenza virus, acclimatization to cold, the PB1 genes of attenuated influenza virus, cold suitable Should, the PA genes of attenuated influenza virus, acclimatization to cold, the NP genes of attenuated influenza virus, acclimatization to cold, the M bases of attenuated influenza virus Cause and acclimatization to cold, the expression plasmid of the NS genes of attenuated influenza virus, and respectively the HA genes containing target influenza virus and The expression plasmid cotransfection host cell of the NA genes of target influenza virus, culture obtains recombinant influenza；Wherein, the acclimatization to cold, the opening code-reading frame of the NS genes of attenuated influenza virus the 375th to from 5 ' ends The structural protein gene of HAdV, the structural protein gene of the HAdV and the acclimatization to cold, attenuation are inserted between 376 nucleotides The opening code-reading frame of the NS genes of influenza virus from 5 ' ends is connected with 5 '-UAAUG-3 ' sequences between the 375th nucleotides, Gained sequence is shown in 41-1162 positions nucleotides in SEQ ID No.2；Or, by the acclimatization to cold, the opening code-reading frame of the M genes of attenuated influenza virus the 223rd to the 760th from 5 ' ends Position nucleotides replaces with the structural protein gene of HAdV, and gained sequence is shown in 40-759 positions nucleotides in SEQ ID No.3；Or, by the opening code-reading frame of the NA genes of the target influenza virus from 5 ' ends the 184th to the 1253rd core Thuja acid replaces with the structural protein gene of HAdV, and gained sequence is shown in 35-650 positions nucleotides in SEQ ID No.4；The structural protein gene of the HAdV is the dominant antigen epi-position base of hexon tower the area Loop1 and Loop2 of HAdV The fusion of cause；The nucleotides of the fusion of the dominant antigen epitope gene of hexon tower the area Loop1 and Loop2 of the HAdV Sequence is as shown in SEQ ID No.1.
- 3. virus according to claim 2, it is characterised in that：The host cell is MDCK, Vero, 293T, COS cell Or the cell that MDCK/293T, MDCK/COS are co-cultured.
- 4. virus according to claim 2, it is characterised in that：The HAdV is 3 types HAdV or 7 types HAdV.
- 5. virus according to claim 4, it is characterised in that：3 type HAdV is HAdV-3 Strain VR-3；7 type HAdV be HAdV-7 Strain GZ08, GenBank accession No.GQ478341.
- 6. according to the arbitrary described virus of claim 2-5, it is characterised in that：The acclimatization to cold, attenuated influenza virus are cold suitable Influenza A or acclimatization to cold, attenuation Type B influenza virus, should be attenuated；The target influenza virus is influenza A or Type B influenza virus.
- 7. virus according to claim 6, it is characterised in that：The acclimatization to cold, attenuation influenza A are H2N2 hypotypes Acclimatization to cold, attenuated influenza virus；The influenza A is any one in H1 hypotype-H16 hypotypes.
- 8. virus according to claim 7, it is characterised in that：The H2N2 hypotypes acclimatization to cold, attenuated influenza virus are cold Adaptation, attenuated influenza virus strain A/Ann Arbor/6/60 (H2N2)；The influenza A is strains of influenza viruses A/ California/07/2009(H1N1)。
- 9. the chimeric for being prepared by the arbitrary described virus of claim 2-8.
- 10. the DNA molecular described in claim 1 is preparing prevention and/or is treating the disease that influenza virus and/or HAdV cause Application in product.
- 11. applications according to claim 10, it is characterised in that：The influenza virus is influenza A or Type B influenza Virus.
- 12. applications according to claim 11, it is characterised in that：The influenza A is in H1 hypotype-H16 hypotypes Any one；The HAdV is 3 types HAdV or 7 types HAdV.
- The arbitrary described virus of 13. claims 2-8 is preparing prevention and/or is treating the disease that influenza virus and/or HAdV cause Application in the product of disease.
- 14. applications according to claim 13, it is characterised in that：The influenza virus is influenza A or Type B influenza Virus.
- 15. applications according to claim 14, it is characterised in that：The influenza A is in H1 hypotype-H16 hypotypes Any one；The HAdV is 3 types HAdV or 7 types HAdV.
- Chimeric described in 16. claims 9 is preparing prevention and/or is treating the disease that influenza virus and/or HAdV cause Product in application.
- 17. applications according to claim 16, it is characterised in that：The influenza virus is influenza A or Type B influenza Virus.
- 18. applications according to claim 17, it is characterised in that：The influenza A is in H1 hypotype-H16 hypotypes Any one；The HAdV is 3 types HAdV or 7 types HAdV.
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