CN103865923A - 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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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 influenza virus is preparation and the application thereof of the HAdV chimeric of carrier.
Background technology
Respiratory infectious disease remains so far and causes in the world one of main causes of death, and adenovirus (Adenovirus, AdV) and influenza virus are important respiratory pathogens.As everyone knows, adenovirus is very extensive in distributed in nature, is one of encountered pathogenic of children respiratory tract diseases.Below 3 years old, in infantile acute upper respiratory infection, about 5%-10% is caused by adenovirus.Be in hospital from northern China and southern various places sick child's clinical investigation result, confirms that 3,7 type adenovirus are main pathogen of adenovirus pneumonia, and the lower respiratory infection being caused by them accounts for 50% of all adenovirus infections.And adenovirus is because its type is many, the scope of causing a disease is wide, cause serious threat to human health and life security, be more and more subject to social concerns.Meanwhile, people can not forget historical three influenza outbreaks, the catastrophic effect bringing to the whole world, and the H5 particularly occurring in recent years, H7 subtype avian influenza virus are crossed over species barrier infection people's event, have again beaten alarm bell to the mankind.Therefore the prevention and control of adenovirus hominis and influenza have become the significant problem of current life science.
At present, vaccine inoculation is prevention and the effective measure of controlling human infectious disease.In in the past more than 50 year, the development experience of influenza vaccines inactivated vaccine, split vaccine, subunit vaccine, DNA vaccination, for human health has been made significant contribution.The Gripovax of the U.S. and Russia's development in recent years, it is a kind of attenuation human influenza virus strain that has reduced virulence and can grow at optimal adaptation temperature, it is safe and effective that clinical experiment confirms, effectively flu-prevention, the widespread use of this vaccine provides rosy prospect for the mankind conquer influenza in 21 century.But, for human airway adenovirus, the vaccine of use that also do not go through so far, but be the emphasis of international concern for the research of vaccine always.Although adenovirus vaccine development is rapidly, adenovirus inactivated vaccine, subunit vaccine and the attenuated live vaccine of development, for the immunoprophylaxis of mankind's Respiratory Tract Adenovirus provides candidate vaccine, not yet large-scale application.See because adenovirus has more than 51 serotypes from research, differing greatly between each type, after vaccine immunity, bringing out body, to produce potential immunity too drastic, and injecting immune can not produce mucosa-immune and traditional attenuated live vaccine and also have the problems such as unstable and potential immunity is too drastic, has proposed challenge to the development of vaccine.
In recent years, the one, antigen immune bioinformatics technique develops rapidly as for the adjacent body main protection antigen of adenovirus six epi-position, design has produced great effect, for realization improves B cell generation NAT, attenuating immunity adenovirus vaccine development too drastic, lifting all-round protection provides theoretical basis and technical support; The 2nd, Reverse Genetics fast development, attenuated live vaccine safe for developing, effective, multivalence provides modern technique, make influenza virus manifest good development prospect as delivery system, the object that simultaneously reaches " seedling is dual-purpose " or " seedling is multiplex " for embedded virus provides theoretical foundation; The 3rd, to be embedded virus design and have the mucous membrane of generation and immune cross protection is replied, realized to systemic immunity for adenovirus hominis nasal-spraying immune approach, becomes human adenovirus vaccine's important development direction.Therefore, above technology collection holds, is integrated into safe, effective, the practical human adenovirus vaccine of development science guarantee is provided.
One, developing rapidly of antigen immune biology information technology, for the research and development of adenovirus vaccine provide New Policy
As far back as the mid-50, when people attempt to set up cell culture system with the tonsilla of excision, vegetation, find that there is a kind of infectious and can make the epithelial cell of cultivating occur regression, within 1956, this factor is adenovirus (Adenovirus) by definite designation.It can cause the disease of human airway, gi tract, urinary system and eye.Human adenovirus (human adenovirus, HAdV) belongs to Mammals AdV and belongs to, and for nonencapsulated double-stranded DNA virus, is icosahedral structure of virus, and diameter is 70～90nm.Be divided into A～G7 group according to physics, chemistry, biological property, each group comprises some serotype, totally 51 types, wherein 1-8,19,21,35 serotypes all with serious acute respiratory disease-related, and 3,7 types are pathogenic the highest two kinds.Also there are many bibliographical informations abroad: 3,7 type adenovirus cause that acute respiratory distress is popular comparatively serious in septic yanks' depot.From virus structure, HAdV capsid protein is made up of 252 capsomeres, wherein 20 faces of 240 capsomere constitutive protein capsids, and its constitutive protein is called as six adjacent bodies (Hexon, 120KD), is main envelope protein; 12 capsomeres are positioned at the drift angle of 20 bodies, are called as penton (penton, 82KD); Also has the fiber (fiber, 62KD) of trimer protein.Wherein, six adjacent bodies are the major antigen albumen of adenovirus, stimulate body produce antibody, suppress the change of viral conformation, thus in and virosome.Six adjacent bodies contain a large amount of antigenic determinants, comprise type specificity and subgenus specific antigens epi-position and neutrality epitope.Substrate (pedestal) comprises P1He P2 district, two regions, and tower district is made up of 4 rings (1oop), i.e. Loop1, Loop2, Loop3, Loop4 district.In recent years, due to the structure specificity of adenovirus hexon, in coding and epitope, participate in immune response, become the focus of Chinese scholars research.As can be seen here, HAdV, due to its special, complicated etiology characteristic, has proposed a difficult problem to the prevention and control of HAdV.
Vaccine is to control the most effective means of transmissible disease.In the struggle of mankind's Fighting Disease and transmissible disease, vaccine has been brought into play vital role, and adenovirus vaccine is no exception.The U.S. has adopted enteric-coated adenovirus attenuated live vaccine to carry out immunization experiment to the grownup of depot as far back as the sixties, discovery can reduce the heating paresthesia that 90 percent adenovirus causes, but is detected and found that the NAT of this living vaccine induction generation is very low by serology.The seventies uses adenovirus enteric living vaccine to carry out immunity, finds that IgM, IgG in serum, IgA raise, but in nasal discharge, does not find corresponding IgA type antibody.Although adenovirus attenuated live vaccine result of use in U.S. army is good; but in U.S. army, still exist the phenomenon of ARD outburst; there is research to adopt DNA chip and round pcr to confirm; inoculate after existing adenovirus attenuated live vaccine; inoculator still can other type of coinfection (Co-infection) adenovirus, prove that the adenovirus to other type of existing attenuated live vaccine does not have cross immunity protective capability.Therefore, HAdV subunit vaccine is duty-bound becomes the important development target of this area.
In several years, adenovirus is widely used in gene therapy and chimeric research as a kind of genophore in the past, and adenovirus infection has hindered the curative effect of this gene therapy greatly, and people can not ignore the potential side reaction that this chimeric brings simultaneously.In recent years, to the adjacent body main protection antigen of HAdV six epi-position, design has produced great effect in the development of antigen immune information biology, for the T, the research and development of B cell Dominant Epitopes vaccine that realize protective antigen provide theories technique support.There is bibliographical information application phage display small peptide technology exploration adenovirus epi-position, with 3 type adenovirus neutralizing antibody screening phage random pentadecapeptide storehouses, after three-wheel screening, random picking 22 phage clones, add respectively the Hela cell of 3 type adenovirus infections.Found that 8/22 clone's thalline small peptide has provide protection to 3 type adenovirus infection Hela cells.Separately there is scholar to compare the adjacent body aminoacid sequence of adenovirus six by computer, in conjunction with polypeptide exposure and the antigenicity forecast analysis of 2 type adenovirus tomograph promptings, select 937～1230,2166～2607 and 937～2607 encoding sequences of hexon gene.The antigen site of the prediction that this region includes has very high homology between Respiratory Tract Adenovirus type, may have good exposure and may contain between type or group-specific antigen epi-position on adenovirus particles.The expression of the external also antigenic determinant in Loop1 district that someone applies Ad2 in B group Coxsackie virus, using Coxsackie virus as carrier, the L1 ring of recombinant type II adenovirus hexon can make its HeLa cell inner stablity express, expressed albumen can be induced the neutralizing antibody of the not only anti-adenovirus of generation but also anti-Coxsackie virus, and then the recombinant vaccine of development multivalence.
Two, the development of influenza virus Reverse Genetics, the HAdV chimeric virus strain that is carrier for preparation influenza virus provides Reliable guarantee
At present, reverse Genetics Technique is in the widespread use in influenza virus field and develop rapidly, the 8 plasmid virus rescue systems of influenza virus have become proven technique at home and abroad, making becomes very attractive research direction using influenza virus as delivery system expression alien gene, the study hotspot of recombinant respiratory transmissible disease vaccine concentrates on it influenza virus.Influenza virus has many advantages as the carrier of other infectious disease pathogens vaccine of exploitation: (1) can stimulate body to produce strong mucous membrane and systemic immunity is replied; (2) influenza vaccines are because antigenic variation needs annual production; (3) structure and function of HA and NA surface protein has determined to carry out genetic manipulation and not affect their function them; (4) influenza virus has formed efficient reverse genetics operating system; (5) mouse and ferret provide good animal model for the research of restructuring influenza virus vaccine Candidate Strain respiratory mucosa immunne response and immunoprotection.As adenovirus, retrovirus is compared with other carriers, and influenza virus can not form DNA intermediate product at replicative cycle, can not be integrated in addition host's karyomit(e) due to it, makes it have higher security.Strategy for influenza virus gene group operation has: foreign protein chimeric enter surface glycoprotein HA and NA, produce other gene fragment, transformation non-structural protein NS 1.
The breakthrough obtaining in view of influenza virus Reverse Genetics; existing lot of documents report has successfully been prepared and can have been reached chimeric Candidate Strain effective immunoprotection, multivalence influenza virus as delivery system, for development prospect has been widened in the research of vaccine.Horimoto in 2004 etc. have built mosaic virus (A/B), the HA that this virus contains mosaic (A/B) and Type B virus total length NA fragment.This research is for providing a kind of new thinking from single host strain exploitation attenuated live vaccine.Maeda Y in 2005 etc. produce a kind of A type influenza virus of restructuring by reverse Genetics Technique, and the HA/NA ectodomain that this viral coding region is parainfluenza virus can effectively be bred but in Mice Body, is attenuation in chicken embryo.When with after recombinant virus intranasal administration mouse, all can produce the antibody of opposing influenza virus and parainfluenza virus.The immune protective effect of the living vaccine of this bivalent is obviously better than combined vaccine.The same year, the little peptide of report such as Zhu NanLi can insert " loop " ring region of influenza virus HA antigen; studies show that subsequently; protective antigen (PA) the large fragment polypeptide of B.anthracis can be inserted into the functional zone of influenza virus H3 hypotype HA; the recombinant influenza of chimeric HA-PA gene is after mdck cell and chicken embryo go down to posterity; genetic stability is good; results of animal shows, recombinant virus can cause HA and the protein induced antibody response of PA.Kawaka in 2003 etc. enter GFP gene integration after the specific position of NA gene, found that the efficiency of recombinant influenza particle assembling can be up to 91%.Andrej Egorov in 2005 etc. are successively by the open reading frame of GFP and IL-2 insertion NS1, and result has successfully obtained the strains of influenza viruses of restructuring.
The above is the current present Research using influenza virus as delivery system expression alien gene, and both at home and abroad, using influenza virus gene group as the carrier of expressing HAdV virus antigen epitope, be that the HAdV chimeric of carrier at home and abroad have not been reported for developing influenza virus.
Three, nasal-spraying immune influenza virus is the HAdV chimeric of carrier, is the foundation stone that promotes safety, comprehensive immunoprotection
In June, 2003, the influenza trivalent attenuated live vaccine Flumist that Medimmune company of the U.S. produces is ratified by FDA and comes into operation, be applicable to healthy children of 2-17 year and 18-49 year health adult, clinical experiment result shows that this vaccine is safety and effectively.To in February, 2012, the listing of U.S. FDA approval influenza tetravalence attenuated live vaccine, this vaccine passes through intranasal inoculation, using method is easy, have wide range of applications, make to develop other vaccines taking acclimatization to cold Gripovax as delivery system expression alien gene and opened up the new world, brought new opportunity.Influenza virus enters nose tissue and can induce the T of generation system and mucous membrane, B cellullar immunologic response by sending different antigen as a kind of strong vaccine carrier.In addition, attenuated live vaccine, by immunity in nose, can be induced part and general immunity, thereby the upper respiratory tract and lower respiratory infection are had to defense reaction.The immunne response that attenuated live vaccine induces is similar to replying wild-type virus.The HAdV vaccine infecting for respiratory mucosa, injection inoculation is not desirable immunization route, inconvenient yet.Therefore, development nasal is the optimal selection of vaccine immunity.
Nasal meatus is rich in lymphoid tissue, is the target site of intranasal inoculation vaccine, i.e. the relevant lymphoid tissue (NALT) of nose, and it is mainly positioned at the lymphoid tissue ring of throat, is called WaldeyerShi ring.Research shows, the about 150cm2 of bronchia mucosal area, epithelial cell gap is large and be closely connected with capillary vessel, blood vessel and lymphoglandula are abundant, the about 40ml/min of unit volume of blood flow of nasal mucosa, all large than muscle, brain, liver, in fact pharynx nasalis is good immune organ, there is abundant antigen presenting cell, energy antagonism is former effectively offers, processes, and produce mucosa-immune and systemic immunity and reply, and local mucosa-immune, can also cause the interlinking of the position mucosal immune responses such as respiratory tract, digestive tube, urogenital tract, but the strongest with respiratory tract.
Nasal-spraying immune is the active field of studying at present; in recent years also there are many enterprise development intranasal inoculation vaccines; particularly respiratory infectious disease vaccine; as Gripovax passes through intranasal inoculation; not only can produce local mucosa-immune; can also produce systemic immunity, and generation has cross immunity protection.Therefore, the HAdV chimeric that the influenza virus of exploitation intranasal inoculation is carrier substitutes injection and has great application prospect, this is because nasal cavity immunity has simply, oneself effective, that be very suitable for numerous crowds uses, make body can resist HAdV and two kinds of pathogenic infections of influenza simultaneously, and can change HAdV vaccine immune response type, reach thus safer object, for the immunoprophylaxis of HAdV and influenza is given security.
Summary of the invention
The object of this invention is to provide a kind of influenza virus is preparation and the application thereof of the HAdV chimeric of carrier.
The invention provides the DNA molecular shown in SEQ ID No.1.
A kind of recombinant virus also belongs to protection scope of the present invention, and this virus is prepared as follows:
The expression plasmid of NS gene of the M gene of NP gene, acclimatization to cold, attenuated influenza virus of PA gene, acclimatization to cold, attenuated influenza virus of PB1 gene, acclimatization to cold, attenuated influenza virus of PB2 gene, acclimatization to cold, attenuated influenza virus of acclimatization to cold, attenuated influenza virus and acclimatization to cold, attenuated influenza virus will be contained respectively, and contain respectively the expression plasmid cotransfection host cell of the HA gene of target influenza virus and the NA gene of target influenza virus, cultivate and obtain recombinant influenza;
The structural protein gene of HAdV is inserted or replaced with in optional position in the opening code-reading frame of at least one gene in gene as described below:
(1) the PB2 gene of acclimatization to cold, attenuated influenza virus;
(2) the PB1 gene of acclimatization to cold, attenuated influenza virus;
(3) the PA gene of acclimatization to cold, attenuated influenza virus;
(4) the NP gene of acclimatization to cold, attenuated influenza virus;
(5) the M gene of acclimatization to cold, attenuated influenza virus;
(6) the NS gene of acclimatization to cold, attenuated influenza virus;
(7) the HA gene of target influenza virus;
(8) the NA gene of target influenza virus;
PB2 gene, PB1 gene, PA gene, NP gene, M gene and the NS gene of described acclimatization to cold, attenuated influenza virus, and the HA gene of target influenza virus and NA gene are positioned on different expression plasmids;
The structural protein gene of described HAdV is the dominant antigen epitope gene of the hexon of the full gene of the hexon of HAdV and/or HAdV.
In above-mentioned recombinant virus, described host cell is MDCK, Vero, 293T, COS cell or MDCK/293T, MDCK/COS cultured cells altogether;
The structural protein gene of described HAdV is the fusion gene of the hexon tower district Loop1 of HAdV and the dominant antigen epitope gene of Loop2.
In above-mentioned arbitrary described recombinant virus, described HAdV is 3 type HAdV or 7 type HAdV;
Described 3 type HAdV are specially HAdV-3 virus strain VR-3;
Described 7 type HAdV are specially HAdV-7 virus strain GZ08, GenBank accession No.GQ478341.
In above-mentioned arbitrary described recombinant virus, the nucleotide sequence of the fusion gene of the hexon tower district Loop1 of described HAdV and the dominant antigen epitope gene of Loop2 is as shown in SEQ ID No.1.
In above-mentioned arbitrary described recombinant virus, the structural protein gene of described HAdV be inserted in the NS gene of described acclimatization to cold, attenuated influenza virus opening code-reading frame from 5 ' end between the 375th to the 376th Nucleotide;
The opening code-reading frame of the NS gene of the structural protein gene of described HAdV and described acclimatization to cold, attenuated influenza virus from 5 ' end, between the 375th Nucleotide, be connected with 5 '-UAAUG-3 ' sequence;
And/or,
The structural protein gene of described HAdV is that 760 Nucleotide of the 223rd Nucleotide to the from 5 ' end of the opening code-reading frame of the M gene of described acclimatization to cold, attenuated influenza virus are replaced;
And/or,
The structural protein gene of described HAdV is that 1253 Nucleotide of the 184th Nucleotide to the from 5 ' end of the opening code-reading frame of the NA gene of described target influenza virus are replaced.
In above-mentioned arbitrary described recombinant virus, described acclimatization to cold, attenuated influenza virus are acclimatization to cold, attenuation A type influenza virus or acclimatization to cold, attenuation Type B influenza virus;
Described acclimatization to cold, attenuation A type influenza virus are specially the acclimatization to cold of H2N2 hypotype, attenuated influenza virus, then are specially acclimatization to cold, attenuated influenza virus strain A/Ann Arbor/6/60 (H2N2);
Described target influenza virus is A type influenza virus or Type B influenza virus, described A type influenza virus is specially any one in H1 hypotype-H16 hypotype, be specially again H1N1 subtype influenza virus, then be specially strains of influenza viruses A/California/07/2009(H1N1);
Described target influenza virus is the common wild-type influenza virus that does not pass through (as do not passed through attenuation, not passing through acclimatization to cold) of any processing.
In above-mentioned arbitrary described recombinant virus, the structural protein gene of described HAdV be inserted in the NS gene of described acclimatization to cold, attenuated influenza virus opening code-reading frame from 5 ' end between the 375th to the 376th Nucleotide institute's calling sequence be in SEQ ID No.2 shown in the Nucleotide of 41-1162 position;
The structural protein gene of described HAdV is that 760 Nucleotide of the 223rd Nucleotide to the from 5 ' end of the opening code-reading frame of the M gene of described acclimatization to cold, attenuated influenza virus are replaced to institute's calling sequences is in SEQ ID No.3 shown in the Nucleotide of 40-759 position;
It is in SEQ ID No.4 shown in the Nucleotide of 35-650 position that 1253 Nucleotide of the 184th Nucleotide to the from 5 ' end of the opening code-reading frame of the NA gene of described target influenza virus are replaced institute's calling sequences by the structural protein gene of described HAdV.
While building described expression plasmid, above-mentioned PB2 gene, PB1 gene, PA gene, NP gene, M gene, NS gene, HA gene or NA gene, all be connected with 3 ' NCR sequence of gene separately at 5 ' end separately, be all connected with 5 ' NCR sequence of gene separately at 3 ' end separately;
The plasmid that sets out of described expression plasmid is bidirectional transcription expression vector pAD3000;
While building described expression plasmid, all each gene is inserted in to the BsmBI site of bidirectional transcription expression vector pAD3000.
The chimeric that above-mentioned arbitrary described virus prepares also belongs to protection scope of the present invention.
The application that above-mentioned DNA molecular prevents and/or treats in the product of the disease that influenza virus and/or HAdV cause in preparation also belongs to protection scope of the present invention;
And/or,
The application that above-mentioned arbitrary described virus prevents and/or treats in the product of the disease that influenza virus and/or HAdV cause in preparation also belongs to protection scope of the present invention;
And/or,
The application that above-mentioned chimeric prevents and/or treats in the product of the disease that influenza virus and/or HAdV cause in preparation also belongs to protection scope of the present invention;
Described influenza virus is A type influenza virus or Type B influenza virus, and described A type influenza virus is specially any one in H1 hypotype-H16 hypotype;
Described HAdV is 3 type HAdV or 7 type HAdV;
Described 3 type HAdV are specially HAdV-3 virus strain VR-3;
Described 7 type HAdV are specially HAdV-7 virus strain GZ08, GenBank accession No.GQ478341.
HAdV chimeric provided by the invention can cover HAdV infectious disease pathogens, protects more crowds to avoid influenza virus and HAdV infection, lays a good foundation for realizing " seedling is dual-purpose " or " seedling is multiplex ".
Brief description of the drawings
Fig. 1 is the restructuring strategy schematic diagram that builds the HAdV chimeric that influenza virus is carrier.
Fig. 2 is electron microscopic morphology qualification and granular size and the distribution situation of 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 response.
Fig. 5 is that mouse immune rFLU/HAdV/NS1 generation is tired for the mucosal antibodies of HAdV-3.
Fig. 6 is the immune protective effect that rFLU/HAdV/NS1 immune mouse is attacked adenopathy strain.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
PAD3000 is at 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. " in disclosed, the public can obtain from Academy of Military Medicine, PLA.
6-8 BALB/c mouse in age in week is purchased from Military Medical Science Institute's Experimental Animal Center.
The ferret in age in 10-12 week is purchased from Chinese Wuxi Angora An Gelu company.
2012-2013 influenza virus is epidemic strain A/California/07/2009(H1N1 then) provided by national influenza center, the public can obtain from Academy of Military Medicine, PLA.The HA of this virus strain that increases respectively, NA gene, then be inserted into respectively the BsmBI site of bidirectional transcription/expression vector pAD3000, build the recombinant plasmid pD-HA, the pD-NA that obtain this virus strain.
Acclimatization to cold, attenuated influenza virus strain A/Ann Arbor/6/60 (H2N2) (writing a Chinese character in simplified form A/AA/6/60) are at 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): 698-705. " in, disclosed, the public can obtain from Academy of Military Medicine, PLA.6 internal gene PB2, PB1 of this virus strain, PA, NP, NS, M, be inserted into respectively the BsmBI site of bidirectional transcription/expression vector pAD3000, builds and obtain this viral recombinant plasmid pD-PB2, pD-PB1, pD-PA, pD-NP, pD-NS, pD-M.
Influenza virus A/PR/8/1934 (PR8) virus strain is at 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): 528-38. " in, disclosed, the public can obtain from Academy of Military Medicine, PLA.
HAdV-3 virus strain VR-3, purchased from ATCC.
HAdV-7 virus strain GZ08 (GenBank accession No.GQ478341) is at 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. " in, disclosed, the public can obtain from Academy of Military Medicine, PLA.
The hexon tower district of HAdV is made up of 4 rings (1oop), i.e. Loop1, Loop2, Loop3, Loop4 district, and in following embodiment, L1, L2 represent respectively Loop1 and Loop2.
The preparation of the HAdV chimeric rFLU-HAdV/NS1 of the chimeric HAdV-Hexon-L1/L2 dominant antigen epi-position that experimental example 1, influenza virus are carrier
One, Hexon-L1/L2(HAdV-Hexon-L1/L2 in the HAdV of 3 types and 7 types) dominant antigen epitope sequences as shown in SEQ ID No.1, through the checking of experimentation on animals and clinical patient serum, meet next step requirement of experiment.
Two, construction recombination plasmid pHexon-L1/L2-NS1
Taking the NS1 gene fragment of acclimatization to cold, attenuated influenza virus strain A/AA/6/60 as inserting the target spot of HAdV-Hexon-L1/L2 dominant antigen epitope gene, utilize molecular biology method construction recombination plasmid pHexon-L1/L2-NS1, specific strategy is as shown in Figure 1A.
Specifically HAdV-Hexon-L1/L2 dominant antigen epitope gene is inserted into the front 125 amino acid whose encoding gene places of the NS1 gene open reading frame (ORF) of acclimatization to cold, attenuated influenza virus strain A/AA/6/60, centre adds linker(5 '-UAAUG-3 ') connect, the existing terminator effect of linker, there is again promotor effect, finally the encoding gene of NEP albumen, by the recombinant plasmid called after pHexon-L1/L2-NS1 building.
Step is as follows:
(1) DNA molecular shown in synthetic SEQ ID No.2, in SEQ ID No.2 from 5 ' end the 1st to the 14th be restriction enzyme site, the 15th to the 41st is NS1 gene 3 ' NCR, the 41st to the 415th is acclimatization to cold, the 1st to the 375th Nucleotide from 5 ' end of attenuated influenza virus strain A/AA/6/60NS gene opening code-reading frame, the 416th to the 420th is linker, the 421st to the 699th is HAdV-Hexon-L1/L2 dominant antigen epitope gene, the 700th to the 1162nd is the 376th to the 838th Nucleotide from 5 ' end of NS gene opening code-reading frame, the 1163rd to the 1191st is NS gene 5 ' NCR, the 1192nd to the 1203rd is restriction enzyme site.
(2) BsmBI enzyme is cut the DNA molecular shown in SEQ ID No.2, obtains gene fragment; BsmBI enzyme is cut pAD3000, obtains carrier large fragment; Gene fragment is connected with carrier large fragment, obtains recombinant plasmid, by its called after pHexon-L1/L2-NS1, send sequencing result correct recombinant plasmid.
Three, by recombinant plasmid pHexon-L1/L2-NS1 and acclimatization to cold, the inside virogene skeleton PB2 of attenuated influenza virus strain A/AA/6/60, PB1, PA, NP, the recombinant plasmid pD-PB2 that M builds respectively, pD-PB1, pD-PA, pD-NP, pD-M, and influenza virus epidemic strain A/California/07/2009(H1N1 then) HA, the recombinant plasmid pD-HA that NA is gene constructed, pD-NA, totally 8 kinds of each 0.2 μ g of plasmid, balanced mix adds 10 μ L transfection reagent (Effectene, purchased from Qiagen company) room temperature effect 10min, cotransfection mdck cell, 33 DEG C, 5%CO
2cultivate 48-60h, obtain cell suspension, by cell suspension inoculation 9 age in days SPF chicken embryos, cultivate 72h for 33 DEG C, results chick embryo allantoic liquid is measured HA hemagglutinative titer, and the detected result of HA hemagglutinative titer is 1:256-512, obtains the HAdV chimeric (rFLU-HAdV/NS1) of chimeric HAdV-Hexon-L1/L2 dominant antigen epi-position.
Four, chimeric strain step 3 being obtained is identified, electron microscopic observation morphology of virus, and result is as shown in Figure 2.Fig. 2 shows that this vaccine strain meets influenza virus representative configuration feature, and virion size is between 80-120nm.
Five, rFLU-HAdV/NS1 being inoculated to 9-11 age in days SPF chicken embryo (purchased from Beijing Experimental Amimal Research Centre) goes down to posterity, get s-generation chick embryo allantoic liquid and extract viral RNA, through RT-PCR, amplify PB2, PB1, PA, NP, HA, NA, M and NS totally 8 gene fragments, respectively gene fragment is sent to company's order-checking, result is consistent with expection gene order.
Six, rFLU-HAdV/NS1 is cultivated in a large number through chicken embryo, after ultrafiltration and concentration, sucrose gradient centrifugation purifying, run SDS-PAGE electrophoresis, after gel-colored, decolouring, NP, HA1, HA2, the NEP albumen that corresponding size can be detected, show that the main component of antigen is not lost.
Seven, the temperature sensitive of rFLU-HAdV/NS1 (temperature sensitive, ts), acclimatization to cold (coldadapted, ca) and attenuation (attenuated, att) Phenotypic examination
RFLU-HAdV/NS1 is inoculated to mdck cell or 9-11 day instar chicken embryo, and respectively at cultivating 3 days under 25,33,37 and 39 DEG C of conditions, collecting cell supernatant or chick embryo allantoic liquid are measured virus titer.Result shows, this vaccine strain has ts, ca phenotype.Meanwhile, this vaccine strain shows attenuation phenotype on BALB/c mouse and the 10-12 ferret in age in week.
Eight, the immune effect of rFLU-HAdV/NS1 in Mice Body
(1) by rFLU-HAdV/NS1 after ultrafiltration and concentration, sucrose density gradient centrifugation purifying, prepare the attenuated live vaccine formulation of this vaccine.
Select 6-8 BALB/c mouse in age in week, be divided into vaccine group and control group, 20 every group.
Vaccine group: by this vaccine collunarium immunity BALB/c mouse, immunity 2 times, 2 weeks, interval, it is 10 that immunizing dose is set
4tCID
50, 10
5tCID
50two dosage groups, volume is 20 μ l.
Control group: PBS replaces vaccine, and immunization ways, immune volume and immunity time are consistent with vaccine group simultaneously.By vaccine group and control group respectively at initial immunity after, after booster immunization 2 weeks, through mouse tail vein blood sampling, the serum of each group of mouse of centrifugal collection.Adopt HI method to measure in serum for wild-type influenza virus A/California/07/2009(H1N1) antibody titer, adopt microneutralization method to detect the specific antibody production for HAdV-3 virus strain VR-3 in serum simultaneously, result as shown in Figure 3, in Fig. 3, * represent compared with control group p<0.05; * represents compared with control group, p<0.01.
Fig. 3 A is for wild-type influenza virus A/California/07/2009(H1N1 in serum) antibody titer measurement result.
Fig. 3 B is the specific antibody production measurement result for HAdV-3 virus strain VR-3 in serum.
Fig. 3 shows, experimental group is for wild-type influenza virus A/California/07/2009(H1N1) HI neutralization tire and for the NAT of HAdV-3 virus strain VR-3 respectively with PBS group than the remarkable rising of tiring, after showing that influenza virus is HAdV chimeric (rFLU-HAdV/NS1) immune animal of carrier, the high-caliber specific antibody titres for HAdV and influenza virus in serum, can be detected, show can induce body to produce the dual immunne response for HAdV and influenza virus after this vaccine immunity animal.
(2) adopt the method rFLU-HAdV/NS1 collunarium immunity BALB/c mouse respectively that step () is identical, and contrast is set, after booster immunization 2 weeks, put to death animal, separate splenic lymphocyte, ELISPOT method produces IFN-γ and IL-4 cellullar immunologic response after detecting vaccine strain immune animal, result respectively as shown in Figure 4 A and 4 B shown in FIG., in Fig. 4, * * represents compared with control group, p<0.01.
Fig. 4 shows, after the immunity of rFLU-HAdV/NS1 collunarium, mouse body can produce IFN-γ, the IL-4 cell immune response for influenza virus PR8 and HAdV-3 virus strain VR-3, with significantly (p<0.01) of PBS group ratio heteropole, show that recombinant virus can induce body to produce good cell immune response.
(3) adopt the method rFLU-HAdV/NS1 collunarium immunity BALB/c mouse that step () is identical, and contrast is set, after booster immunization 2 weeks, ELISA method detects the sIgA antibody titer for HAdV-3 virus strain VR-3 in mouse lung, Nasal lavage fluid, result is respectively as shown in Fig. 5 A and Fig. 5 B, in Fig. 5, * represents compared with control group, p<0.05; * represents compared with control group, p<0.01.
Fig. 5 shows, after the immunity of rFLU-HAdV/NS1 collunarium, in mouse nose, lung-douching fluid, sIgA antibody obviously raises, and with significantly (p<0.01) of PBS group ratio heteropole, shows that recombinant virus can induce body to produce good mucosa-immune reaction.
Nine, protection of animal experiment
Adopt the method rFLU-HAdV/NS1 collunarium immunity BALB/c mouse of step 8, and contrast be set, after booster immunization 2 weeks, use respectively the virus strain HAdV-3(VR-3 of clinical separation), HAdV-7(GZ08) attack BALB/c mouse, infective dose is 2 × 10
9vPs, gets the lung tissue of each group of mouse and the lung tissue of normal mouse is carried out pathology detection, and result as shown in Figure 6A.
Fig. 6 A shows, the lung pathology of PBS group mouse changes obviously, visible pulmonary edema under mirror, and a large amount of inflammatory cells, lymphocytic infiltration, alveolar interstitial thickens, and normal mouse pulmonary morphology is intact.RFLU-HAdV/NS1 group mouse lung histopathology pathology is obviously improved compared with PBS group, and wherein 10
5tCID
50high dose group is better than 10
4tCID
50low dose group.
Adopt qPCR method to detect the lung inner virus copy number of each group of mouse, result is respectively as shown in Fig. 6 B and 6C, and in Fig. 6, * represents 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) the number that copies all obviously reduce compared with PBS group.
Result shows, after recombiant vaccine rFLU-HAdV/NS1 immunity, mouse has obtained the immunoprotective antibody for HAdV.
The preparation of the HAdV chimeric rFLU-HAdV/M of the chimeric HAdV-Hexon-L1/L2 dominant antigen epi-position that experimental example 2, influenza virus are carrier
One, in the HAdV of 3 types and 7 types, HAdV-Hexon-L1/L2 dominant antigen epitope sequences, as shown in SEQ ID No.1, according to the method validation of step 1 in embodiment 1, meets next step requirement of experiment.
Two, construction recombination plasmid pHexon-L1/L2-M
Taking the M gene fragment of acclimatization to cold, attenuated influenza virus strain A/AA/6/60 as inserting the target spot of HAdV-Hexon-L1/L2 dominant antigen epitope gene, utilize molecular biology method construction recombination plasmid pHexon-L1/L2-M, specific strategy is as shown in Figure 1B.
Specifically HAdV-Hexon-L1/L2 dominant antigen epitope gene is inserted between front 222 Nucleotide and rear 222 Nucleotide of M gene open reading frame (ORF) of acclimatization to cold, attenuated influenza virus strain A/AA/6/60, by the recombinant plasmid called after pHexon-L1/L2-M building.
Step is as follows:
(1) DNA molecular shown in synthetic SEQ ID No.3, in SEQ ID No.3 from 5 ' end the 1st to the 15th be restriction enzyme site, the 16th to the 40th is M gene 3 ' NCR, the 40th to the 261st is acclimatization to cold, attenuated influenza virus strain A/AA/6/60M gene opening code-reading frame from 5 ' end from the 1st to the 222nd Nucleotide, the 262nd to the 537th is HAdV-Hexon-L1/L2 dominant antigen epitope gene, the 538th to the 759th is acclimatization to cold, attenuated influenza virus strain A/AA/6/60M gene opening code-reading frame from 5 ' end from the 761st to the 982nd Nucleotide, the 760th to the 782nd is M gene 5 ' NCR, the 783rd to the 794th is restriction enzyme site.
(2) BsmBI enzyme is cut the DNA molecular shown in SEQ ID No.3, obtains gene fragment; BsmBI enzyme is cut pAD3000, obtains carrier large fragment; Gene fragment is connected with carrier large fragment, obtains recombinant plasmid, by its called after pHexon-L1/L2-M, send sequencing result correct recombinant plasmid.
Three, by recombinant plasmid pHexon-L1/L2-M, recombinant plasmid pD-PB2, the pD-PB1, pD-PA, pD-NP, the pD-NS that build respectively with inside virogene skeleton PB2, PB1, PA, NP and the NS of acclimatization to cold, attenuated influenza virus strain A/AA/6/60, and influenza virus epidemic strain A/California/07/2009(H1N1 then) gene constructed recombinant plasmid pD-HA, the pD-NA of HA, NA, according to the method for step 3 in embodiment 1 by 8 kinds of plasmid co-transfection mdck cells, 37 DEG C, 5%CO
2, cultivate 48-60h, obtain cell suspension, by cell suspension inoculation 9 age in days SPF chicken embryos, cultivate 72h for 35 DEG C, results chick embryo allantoic liquid obtains the HAdV chimeric strain (rFLU-HAdV/M) of chimeric HAdV-Hexon-L1/L2 dominant antigen epi-position.
Four, vaccine strain step 3 being obtained is identified, electron microscopic observation morphology of virus, and result shows that this vaccine strain meets influenza virus representative configuration feature.
Five, rFLU-HAdV/M being inoculated to 9-11 age in days SPF chicken embryo (purchased from Beijing Experimental Amimal Research Centre) goes down to posterity, get s-generation chick embryo allantoic liquid and extract viral RNA, through RT-PCR, amplify PB2, PB1, PA, NP, HA, NA, M and NS totally 8 gene fragments, respectively gene fragment is sent to company's order-checking, result is consistent with expection gene order.
Six, rFLU-HAdV/M is cultivated in a large number through chicken embryo, after ultrafiltration and concentration, sucrose gradient centrifugation purifying, run SDS-PAGE electrophoresis, the main component of antigen exists.
Seven, the temperature sensitive of rFLU-HAdV/M (temperature sensitive, ts), acclimatization to cold (cold adapted, ca) and attenuation (attenuated, att) Phenotypic examination
RFLU-HAdV/M is inoculated to mdck cell or 9-11 day instar chicken embryo, and respectively at cultivating 3 days under 25,33,37 and 39 DEG C of conditions, collecting cell supernatant or chick embryo allantoic liquid are measured virus titer.Result shows, this vaccine strain has ts, ca phenotype.Meanwhile, this vaccine strain shows attenuation phenotype on BALB/c mouse and ferret animal.
Eight, the immune effect of rFLU-HAdV/M in Mice Body
RFLU-HAdV/M, after ultrafiltration and concentration, sucrose density gradient centrifugation purifying, is prepared to the attenuated live vaccine formulation of this vaccine.
Select 6-8 BALB/c mouse in age in week, be divided into vaccine group and control group, 20 every group.
According to the method for the step 8 of embodiment 1, mouse is carried out the immunity of rFLU-HAdV/M, control group is set simultaneously.
By vaccine group and control group respectively at initial immunity after, after booster immunization 2 weeks, through mouse tail vein blood sampling, the serum of each group of mouse of centrifugal collection.
Adopt HI method to measure in serum for wild-type influenza virus A/California/07/2009(H1N1) antibody titer, adopt microneutralization method to detect the specific antibody production for HAdV-3 virus strain VR-3 in serum, result is as shown in table 2 simultaneously.
The immune effect of table 2rFLU-HAdV/M detects
Table 2 shows, after HAdV chimeric (rFLU-HAdV/M) immune animal taking influenza virus as carrier, in serum, the antibody titer for HAdV and influenza virus can be detected, show can induce body to produce the dual immunne response for HAdV and influenza virus after this vaccine immunity animal.
The preparation of the HAdV chimeric rFLU-HAdV/NA of the chimeric HAdV-Hexon-L1/L2 dominant antigen epi-position that embodiment 3, influenza virus are carrier
One, in the HAdV of 3 types and 7 types, HAdV-Hexon-L1/L2 dominant antigen epitope sequences, as shown in SEQ ID No.1, according to the method validation of the step 1 of embodiment 1, meets next step requirement of experiment.
Two, construction recombination plasmid pHexon-L1/L2-NA
Taking A/California/07/2009(H1N1) NA gene fragment as inserting the target spot of HAdV-Hexon-L1/L2 dominant antigen epitope gene, utilize molecular biology method construction recombination plasmid pHexon-L1/L2-NA, specific strategy as shown in Figure 1 C.
Specifically HAdV-Hexon-L1/L2 dominant antigen epitope gene is inserted between front 183 Nucleotide and rear 157 Nucleotide of the NA gene open reading frame (ORF) of popular strains of influenza viruses H1N1 hypotype A/California/07/2009 then, by the recombinant plasmid called after pHexon-L1/L2-NA building.
Step is as follows:
(1) DNA molecular shown in synthetic SEQ ID No.4, in SEQ ID No.4 from 5 ' end the 1st to the 15th be restriction enzyme site, the 16th to the 35th is NA gene 3 ' NCR, the 35th to the 217th for influenza virus epidemic strain A/California/07/2009(H1N1 then) the 1st to the 183rd Nucleotide from 5 ' end of NA gene opening code-reading frame, the 218th to the 493rd is HAdV-Hexon-L1/L2 dominant antigen epitope gene, the 494th to the 650th is the 1254th to the 1410th Nucleotide from 5 ' end of NA gene opening code-reading frame, the 651st to the 681st is NA gene 5 ' NCR, the 682nd to the 693rd is restriction enzyme site.
(2) BsaI enzyme is cut the DNA molecular shown in SEQ ID No.4, obtains gene fragment; BsmBI enzyme is cut pAD3000, obtains carrier large fragment; Gene fragment is connected with carrier large fragment, obtains recombinant plasmid, by its called after pHexon-L1/L2-NA, send sequencing result correct recombinant plasmid.
Three, by recombinant plasmid pHexon-L1/L2-NA, recombinant plasmid pD-PB2, the pD-PB1, pD-PA, pD-NP, pD-M and the pD-NS that build respectively with inside virogene skeleton PB2, PB1, PA, NP, M and the NS of acclimatization to cold, attenuated influenza virus strain A/AA/6/60, and influenza virus epidemic strain A/California/07/2009(H1N1 then) the gene constructed recombinant plasmid pD-HA of HA, according to the method for step 3 in embodiment 1 by 8 kinds of plasmid co-transfection mdck cells, 37 DEG C, 5%CO
2, cultivate 48-60h, obtain cell suspension, by cell suspension inoculation 9 age in days SPF chicken embryos, cultivate 72h for 33 DEG C, results chick embryo allantoic liquid obtains the HAdV chimeric strain (rFLU-HAdV/NA) of chimeric HAdV-Hexon-L1/L2 dominant antigen epi-position.
Four, vaccine strain step 3 being obtained is identified, electron microscopic observation morphology of virus, and result shows that this vaccine strain meets influenza virus representative configuration feature.
Five, rFLU-HAdV/NA being inoculated to 9-11 age in days SPF chicken embryo (purchased from Beijing Experimental Amimal Research Centre) goes down to posterity, get s-generation chick embryo allantoic liquid and extract viral RNA, through RT-PCR, amplify PB2, PB1, PA, NP, HA, NA, M and NS totally 8 gene fragments, respectively gene fragment is sent to company's order-checking, result is consistent with expection gene order.
Six, rFLU-HAdV/NA is cultivated in a large number through chicken embryo, after ultrafiltration and concentration, sucrose gradient centrifugation purifying, run SDS-PAGE electrophoresis, the main component of antigen exists.
Seven, the temperature sensitive of rFLU-HAdV/NA (temperature sensitive, ts), acclimatization to cold (cold adapted, ca) and attenuation (attenuated, att) Phenotypic examination
RFLU-HAdV/NA is inoculated to mdck cell or 9-11 day instar chicken embryo, and respectively at cultivating 3 days under 25,33,37 and 39 DEG C of conditions, collecting cell supernatant or chick embryo allantoic liquid are measured virus titer.Result shows, this vaccine strain has ts, ca phenotype.Meanwhile, this vaccine strain shows attenuation phenotype on BALB/c mouse and ferret animal.
Eight, the immune effect of rFLU-HAdV/NA in Mice Body
RFLU-HAdV/NA, after ultrafiltration and concentration, sucrose density gradient centrifugation purifying, is prepared to the attenuated live vaccine formulation of this vaccine.
Select 6-8 BALB/c mouse in age in week, be divided into vaccine group and control group, 20 every group.
According to the method for step 8 in embodiment 1, mouse is carried out the immunity of rFLU-HAdV/NA, control group is set simultaneously.
By vaccine group and control group respectively at initial immunity after, after booster immunization 2 weeks, through mouse tail vein blood sampling, the serum of each group of mouse of centrifugal collection.
Adopt HI method to measure in serum for wild-type influenza virus A/California/07/2009(H1N1) antibody titer, adopt microneutralization method to detect the specific antibody production for HAdV-3 virus strain VR-3 in serum, result is as shown in table 3 simultaneously.
The immune effect of table 3rFLU-HAdV/NA detects
Table 3 shows, after influenza virus is HAdV chimeric (rFLU-HAdV/NA) immune animal of carrier, in serum, the antibody titer for HAdV and influenza virus can be detected, show can induce body to produce the dual immunne response for HAdV and influenza virus after this vaccine immunity animal.
Claims (10)
- DNA molecular shown in 1.SEQ ID No.1.
- 2. a recombinant virus, this virus is prepared as follows:The expression plasmid of NS gene of the M gene of NP gene, acclimatization to cold, attenuated influenza virus of PA gene, acclimatization to cold, attenuated influenza virus of PB1 gene, acclimatization to cold, attenuated influenza virus of PB2 gene, acclimatization to cold, attenuated influenza virus of acclimatization to cold, attenuated influenza virus and acclimatization to cold, attenuated influenza virus will be contained respectively, and contain respectively the expression plasmid cotransfection host cell of the HA gene of target influenza virus and the NA gene of target influenza virus, cultivate and obtain recombinant influenza;The structural protein gene of HAdV is inserted or replaced with in optional position in the opening code-reading frame of at least one gene in gene as described below:(1) the PB2 gene of acclimatization to cold, attenuated influenza virus;(2) the PB1 gene of acclimatization to cold, attenuated influenza virus;(3) the PA gene of acclimatization to cold, attenuated influenza virus;(4) the NP gene of acclimatization to cold, attenuated influenza virus;(5) the M gene of acclimatization to cold, attenuated influenza virus;(6) the NS gene of acclimatization to cold, attenuated influenza virus;(7) the HA gene of target influenza virus;(8) the NA gene of target influenza virus;The structural protein gene of described HAdV is the dominant antigen epitope gene of the hexon of the full gene of the hexon of HAdV and/or HAdV.
- 3. virus according to claim 2, is characterized in that: described host cell is MDCK, Vero, 293T, COS cell or MDCK/293T, MDCK/COS cultured cells altogether;The structural protein gene of described HAdV is the fusion gene of the hexon tower district Loop1 of HAdV and the dominant antigen epitope gene of Loop2.
- 4. according to the virus described in claim 2 or 3, it is characterized in that: described HAdV is 3 type HAdV or 7 type HAdV;Described 3 type HAdV are specially HAdV-3 virus strain VR-3;Described 7 type HAdV are specially HAdV-7 virus strain GZ08, GenBank accession No.GQ478341.
- 5. according to the arbitrary described virus of claim 2-4, it is characterized in that: the nucleotide sequence of the fusion gene of the hexon tower district Loop1 of described HAdV and the dominant antigen epitope gene of Loop2 is as shown in SEQ ID No.1.
- 6. according to the arbitrary described virus of claim 2-5, it is characterized in that: the structural protein gene of described HAdV be inserted in the NS gene of described acclimatization to cold, attenuated influenza virus opening code-reading frame from 5 ' end between the 375th to the 376th Nucleotide;The opening code-reading frame of the NS gene of the structural protein gene of described HAdV and described acclimatization to cold, attenuated influenza virus from 5 ' end, between the 375th Nucleotide, be connected with 5 '-UAAUG-3 ' sequence;And/or,The structural protein gene of described HAdV is that 760 Nucleotide of the 223rd Nucleotide to the from 5 ' end of the opening code-reading frame of the M gene of described acclimatization to cold, attenuated influenza virus are replaced;And/or,The structural protein gene of described HAdV is that 1253 Nucleotide of the 184th Nucleotide to the from 5 ' end of the opening code-reading frame of the NA gene of described target influenza virus are replaced.
- 7. according to the arbitrary described virus of claim 2-6, it is characterized in that: described acclimatization to cold, attenuated influenza virus are acclimatization to cold, attenuation A type influenza virus or acclimatization to cold, attenuation Type B influenza virus;Described acclimatization to cold, attenuation A type influenza virus are specially the acclimatization to cold of H2N2 hypotype, attenuated influenza virus, then are specially acclimatization to cold, attenuated influenza virus strain A/Ann Arbor/6/60 (H2N2);Described target influenza virus is A type influenza virus or Type B influenza virus, described A type influenza virus is specially any one in H1 hypotype-H16 hypotype, be specially again H1N1 subtype influenza virus, then be specially strains of influenza viruses A/California/07/2009(H1N1).
- 8. according to the arbitrary described virus of claim 2-7, it is characterized in that: the structural protein gene of described HAdV be inserted in the NS gene of described acclimatization to cold, attenuated influenza virus opening code-reading frame from 5 ' end between the 375th to the 376th Nucleotide institute's calling sequence be in SEQ ID No.2 shown in the Nucleotide of 41-1162 position;The structural protein gene of described HAdV is that 760 Nucleotide of the 223rd Nucleotide to the from 5 ' end of the opening code-reading frame of the M gene of described acclimatization to cold, attenuated influenza virus are replaced to institute's calling sequences is in SEQ ID No.3 shown in the Nucleotide of 40-759 position;It is in SEQ ID No.4 shown in the Nucleotide of 35-650 position that 1253 Nucleotide of the 184th Nucleotide to the from 5 ' end of the opening code-reading frame of the NA gene of described target influenza virus are replaced institute's calling sequences by the structural protein gene of described HAdV.
- 9. the chimeric being prepared by the arbitrary described virus of claim 2-8.
- 10. DNA molecular claimed in claim 1 prevents and/or treats the application in the product of the disease that influenza virus and/or HAdV cause in preparation;And/or,The arbitrary described virus of claim 2-8 prevents and/or treats the application in the product of the disease that influenza virus and/or HAdV cause in preparation;And/or,Chimeric claimed in claim 9 prevents and/or treats the application in the product of the disease that influenza virus and/or HAdV cause in preparation;Described influenza virus is A type influenza virus or Type B influenza virus, and described A type influenza virus is specially any one in H1 hypotype-H16 hypotype;Described HAdV is 3 type HAdV or 7 type HAdV.
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