CN100571775C - Avian influenza virus marking vaccine and its production and application - Google Patents

Avian influenza virus marking vaccine and its production and application Download PDF

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CN100571775C
CN100571775C CNB2006101144087A CN200610114408A CN100571775C CN 100571775 C CN100571775 C CN 100571775C CN B2006101144087 A CNB2006101144087 A CN B2006101144087A CN 200610114408 A CN200610114408 A CN 200610114408A CN 100571775 C CN100571775 C CN 100571775C
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virus
gene
vaccine
influenza virus
avian influenza
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CN1970081A (en
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陈化兰
田国彬
姜永萍
步志高
于康震
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Harbin Veterinary Research Institute of CAAS
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Abstract

The present invention relates to the H5N1 subtype avian influenza virus marker vaccine that a kind of cervical region at the NA gene has inserted the advantage B cell epitope 5B19 of murine hepatitis virus S2 glycoprotein, more specifically, it is H5N1 subtype avian influenza virus marker vaccine strain H5N1/PR8-5B19.The invention also discloses the method and of vaccine and the application in bird flu epidemiological surveillance of this marker vaccine of the described H5N1 subtype avian influenza virus marker vaccine of preparation at the preparation birds flu-preventing.

Description

Avian influenza virus marking vaccine and its production and application
Technical field
The present invention relates to recombinant viral vaccine field, particularly, the present invention relates to the H5N1 subtype avian influenza virus marker vaccine that a kind of cervical region at the NA gene has inserted the advantage B cell epitope 5B19 of murine hepatitis virus S2 glycoprotein, more specifically, it is H5N1 subtype avian influenza virus marker vaccine strain H5N1/PR8-5B19.The invention also discloses the method and of vaccine and the application in bird flu epidemiological surveillance of this marker vaccine of the described H5N1 subtype avian influenza virus marker vaccine of preparation at the preparation birds flu-preventing.
Background technology
1.A the reverse genetic technology of type influenza virus
In recent years, the influenza virus Progress on Molecular Biology is very fast, shows that mainly molecular epidemiology, influenza virus are duplicated and fields such as regulation and control, influenza vectors transformation and application, influenza virus treatment and prevention.Wherein influenza virus is duplicated and regulatory mechanism is to disclose influenza virus infectious and pathogenic mechanism, and research is the basis of treatment and preventive measure effectively, and the progress in this field has benefited from the foundation and the application of influenza virus reverse genetic manipulation technology.
The research group of Conzelmann utilized clone's cDNA to rescue first and has obtained minus-stranded rna virus in 1994--rabies virus (Schnell, et al, 1994).They have cloned the cDNA with the complementary positive chain RNA of viral genome (cRNA) sequence, are placed under the control of the promoter of t7 rna polymerase and hepatitis ribozyme sequence, thereby have made up recombiant plasmid.After with the recombinant vaccinia virus infection cell of expressing t7 rna polymerase, with the recombiant plasmid transfecting eukaryotic cells that makes up, reuse protein expressing plasmid cotransfection to be providing the transcription complex of virus, and these all place under the control of promoter of t7 rna polymerase.Utilize this method, research worker is rescued other member (Baron, et al, 1997 of having obtained Rhabdoviridae and orthomyxoviridae family in succession; Clarke, et al, 2000; Cassen, et al, 2000), and very fast the promotion rescued the research that obtains to gene element sections RNA viruses.
Bridgen and Elliott utilized the method for Conzelmann research group to rescue and have obtained Bunyamwera virus (Bridgen, et al, 1996) in 1996, its genome is made up of three strand RNA fragments.Obtain for genome non-segmented negative virus and rescuing of segmented virus like this, all obtained success.Influenza virus then is an exception, except being produced polymerase protein and nucleoprotein by clone's cDNA, also will form eight viral RNA fragments by it, so it rescue to obtain and exist special complexity.
To the encode cDNA of eight genetic fragments of A/WSN/33 (H1N1) virus of the research group of Kawaoka in 1999 places between the terminator sequence of human RNA polymerase I promoter and Mus rna plymerase i, and then rescue and obtained influenza virus, finally overcome from clone's cDNA and rescued the technology barrier (Neumann that obtains influenza virus, et al, 1999).With constructed plasmid transfection 293T cell, intracellular rna plymerase i is transcribed eight RNA fragments that produced virus.In initial experiment, transfection simultaneously the expression plasmid of 9 kinds of structural protein.Although will be with these 17 kinds of plasmids (eight kinds are used to produce viral RNA, and nine kinds are used for expressing protein) transfered cell simultaneously, the viral yield that obtains of rescuing very high, in the supernatant of every microlitre cultured cell, can reach 1 * 10 7Individual infectious virus particle.This research group only utilizes 12 plasmids by improving systematic parameter in the nearest research, comprises eight rna transcription plasmids and four protein expressing plasmids (PB1, PB2, PA and NP) transfectional cell, has obtained to surpass 1 * 10 8Individual virion.This mainly is because the transfection efficiency height of 293T cell makes a large amount of cells all obtain whole 12 plasmids duplicating with initial virus.In addition, rna plymerase i extensively exists in auxocyte, has guaranteed duplicating of constructed plasmid.Except having very high efficient, the rna plymerase i system is very simple, only needs dna clone and rotaring dyeing technology, and this can both realize at many virusology laboratorys.Also report a kind of method that obtains influenza virus of simply rescuing at Fodor in 1999 etc., utilized the sequence of hepatitis to substitute the terminator of rna plymerase i, guaranteed the verity (Fodor, et al, 1999) of the vRNA 3 ' end that produced.
Hoffmamn etc. modified the rna plymerase i system in 2000, and with its called after rna plymerase i/II system (Hoffmann, et al, 2000).In this system, coding viral gene cDNA fragment is reversed between the promoter and terminator sequence that is cloned in rna plymerase i, this complete transcript unit places under the control of the promoter (CMV promoter) of rna plymerase ii and polyadenylic acid sequence with forward orientation again, thereby has formed a bidirectional transcription system.Transcribing of rna plymerase i produced negative strand viruses vRNA in this system, and transcribing of rna plymerase ii then formed positive strand virus mRNA, so both produced vRNA by identical template, also produced mRNA, thereby do not make up protein expressing plasmid at needs.This systematic research has great importance, and only utilizes 8 plasmids just can rescue and obtains out influenza virus, has significantly reduced applied plasmid quantity, and this has improved virus for the low cell of transfection efficiency and has rescued the efficient that obtains.In rna plymerase i/II system, the synthetic of the expression of virus protein and vRNA finished by identical cDNA template, obtain the influenza virus that lacks or contain lethal mutation on one or several genetic fragment so this system can not rescue, 12 pUC pUCs just are more suitable in this case.These the two kinds researchs that are influenza virus based on 8 plasmids and the 12 plasmid reverse genetic operating systems of plasmid provide favourable instrument in a word.
2. influenza virus reverse genetic The Application of Technology
Attractive, the most potential application of influenza virus reverse genetic operating system utilizes it to carry out production of vaccine exactly.The current inactivated vaccine that adopts can effectively alleviate the syndrome that influenza is brought, but can not prevention infection.Cause weak vaccine in a kind of cold adaptation of the U.S. at present and entered the clinical pilot scale stage; compare with the inactivated vaccine of routine, it can provide better protection to the child, still the remarkable (Maassab of the difference on effect of two kinds of vaccines for the adult; et al, 1999).This in addition vaccine only has a few amino acid whose replacement, though its Phenotype is stable in clinical experiment, still can not avoids taking place virulence and returns strong danger in case apply on a large scale.
Utilize the rna plymerase i system to rescue and obtain influenza virus, can on six proteic genes in inside of coding virus, import and a plurality ofly cause weak sudden change, thereby produce the vaccine strain of influenza virus.The recombinant virus (Ozaki, et al, 2004 that utilize reverse genetic operating system to produce to have current trend strain HA and NA; Li et al, 1999).People such as Subbarrao utilize the reverse genetic operating system of 12 plasmids, rescue and obtained molecular modification and cause weak H5N1/PR8 recombinant virus, wherein contain the HA of Hong Kong H5N1 subtype avian influenza virus in 1997 A/HK/491/97 and 6 internal gene (Subbarao of NA gene and high titre Embryo Gallus domesticus adapted strain A/Puerto Rico/8/34 (PR8), et al, 2003).The HA gene of this recombinant virus has been removed the basic amino acid of HA cracking site through molecular modification; thereby lost highly pathogenic to chicken and Embryo Gallus domesticus and mice; and having kept the characteristics of high titre Embryo Gallus domesticus growth property, the inactivated vaccine of preparation can provide protection completely to mice.Chen etc. utilize the reverse genetic operating system of 12 plasmids; rescue and obtained the HA with China's Mainland H9N2 subtype avian influenza virus and the recombinant virus of NA gene and six internal gene of A/Puerto Rico/8/34 (PR8); prepared vaccine has good immunogenicity; can produce high-caliber HI antibody after the immunity; even the virus of A/Hong Kong/1073/99 with different subgroups carries out counteracting toxic substances; immunoprotection (Chen, et al, 2003) completely still can be provided.
Influenza virus also is a kind of very potential carrier, utilizes it exogenous gene can be imported mammiferous cell.In fact, in depending on the reverse genetic operating system of helper virus, influenza virus can be held exogenous gene.For several relatively shorter polypeptide, can be inserted on the segmental antigen site of influenza virus HA as proteic v3 ring of HIV-1 virus gp120 and the conservative fragment of gp41 albumen born of the same parents internal area camber, thereby induce for the segmental immunoreation of external source (Li, et al, 1993; Muster, et al, 1994,1995).In order to express the foreign protein of total length, the gene of genes of interest and influenza virus can be carried out physical connection, external source gene transcription and translation are (Garcia-Sastre et al, 1994 of carrying out as the part of influenza virus gene in this method; Percy et al, 1994).Another kind method be with exogenous gene as one independently fragment express.Enami etc. rescued NS 1 albumen that has obtained encoding wild type in 1991, but did not contain the proteic recombinant virus of NS2 (Enami, et al, 1991).In this reverse genetic operating system, the helper virus of being adopted contains temperature-sensitive mutation on NS1.So when virus is cultivated under unfavorable temperature, just can effectively duplicate with regard to nine genetic fragments of needs, this shows that influenza virus can contain the genome more than eight genetic fragments when packing.In order to guarantee that genes of interest not having can to continue existence in the genome influenza virus under the condition of selection pressure, can suddenly change to the promoter of influenza virus gene, thereby cause the excess of genes of interest to duplicate or transcribe.
Exogenous gene is imported recipient cell safely and effectively, can (virus like particles VLPs) realizes, does not comprise virulent whole eight kinds of RNA in VLPs by virus-like particle.In this reverse genetic operating system, encoding influenza virus protein gene and have the virus-like particle that has effectively formed influenza virus with the expression of the reporter gene of Influenza Virus RNA similar.Gomez-Puertas etc. utilized this system at 1999 and Neumann etc. in 2000, all structural protein and the polymerase protein and the NP albumen of virus have been expressed, simultaneously exogenous gene has been imported recipient cell, in cell, can give expression to polymerase protein and NP albumen, conversely again can initial genes of interest duplicating and transcribing, thereby make its effectively expressing (Gomez-Puertas, et al, 1999; Neumann, et al, 2000).Because do not contain the vRNA of the virus structural protein of encoding in VLPs, so can not produce the progeny virus particle that makes new advances after gene imports, this has just guaranteed the safety of this system.In addition, owing to there is the influenza virus of 15 HA hypotypes and 9 NA hypotypes just can alleviate the danger that produces the immunity opposing for the influenza vectors oneself protein, thereby can use VLPs to import exogenous gene repeatedly for utilizing.
Utilize the rna plymerase i system purpose sudden change can be imported the genome of influenza virus, this has just opened up new approach for the not preceding influenza virus biological study of secular stagnation, feature such as viral regulating and controlling sequence, structure-function relationship, the close preferendum of pathogenic and host cell etc., especially viral pathogenicity Molecular Study.
3. bird flu virus and marker vaccine
Bird flu (Avian Influenza, AI) be by bird flu virus (Avian Influenza Virus, AIV) birds that causes infects and/or disease syndrome, AIV belongs on taxonomy: viral boundary (Vira)----orthomyxoviridae family (Orthomyxoviridae)----Influenza Virus (Influenza Virus A and B)----bird flu virus (AvianInfluenzaVirus).Bird flu virus belongs to the A type influenza virus of orthomyxoviridae family's Influenza Virus, and genome is made up of 8 sub-thread strand RNA fragments.Its surface texture albumen hemagglutinin (HA) is different with neuraminidase (NA) antigenicity, is divided into different subtype.Hemagglutinin (HA) is the main immunogen protein of bird flu virus, it can induce body to produce antibody-mediated specific humoral immune response, thereby the antibody of anti-HA can combining or the viral infection that neutralizes of the fusion process of virus envelope and endocytosis body film by viral interference and sialic acid receptor.The aminoacid sequence of the pathogenicity of AIV and its surface texture albumen HA cracking site is closely related.The HA cracking site of low pathogenicity AIV has only a basic amino acids arginine (R), this structures shape can only in respiratory system, breed behind these viral infection animals, because have only airway epithelial cell to contain the protease of the special similar pancreatin of a kind of arginine, the HA0 that cracking site can be contained single basic amino acids arginine is cracked into activated HA1 and HA2, starts the absorption and the replicative cycle of virus.Highly pathogenicity H5 and H7 hypotype AIV HA cracking site contain successive a plurality of alkaline amino acid residue-RKKR-, the protease identification and the cracking that can extensively be existed in the various kinds of cell in the body, therefore have tissue tropism widely, just can cause the general diffusion and cause rapid death in case infect.But compare with the subtype influenza viruses such as H1, H2, H3 and H9 of infected person, highly pathogenic H5 and H7 hypotype AIV are more huge to the mankind's potential hazard, in case because promptly may general diffusion and rapid causing death after the infected person.
Marker vaccine (marker vaccine) or be referred to as DIVA vaccine (differentiating infected fromvaccinated animals), be exactly after giving animal immune with a kind of vaccine, by the serology detection method that matches with it, immune animal and natural infected animal can be made a distinction (Babiuk, et al, 1999).The bird flu virus inactivated whole virus vaccines is the vaccine that is most widely used in the world at present; mainly be because its immunogenicity is strong; antigen component is complete; the immunoprotection time is long; but inactivated whole virus vaccines also has some shortcomings of self, especially influences the monitoring and the Epidemiological study of bird flu, so the urgent immunity can only be used for bird flu and break out the time reduces economic loss; and do not advocate immunity still there being the place that epidemic situation takes place, therefore limited its application to a great extent.Just because the unsurmountable shortcoming of conventional inactivated avian influenza vaccine makes exploitation deactivation marker vaccine necessitate.
During 1999-2001 Italy breaks out the H7N1 subtype avian influenza, carry out the inactivated vaccine immunity and come birds flu-preventing, but used vaccine is not homologous H7N1 subtype virus, but prepare inactivated vaccine with allogenic Pakistani H7N3 separated strain (A/CK/Pakistan/95/H7N3), its objective is it as a kind of natural marker vaccine, DIVA vaccine (Capua more accurately says so, et al, 2002).Because the H7 hypotype HA in vaccine is as main immunoprotection antigen; immunoprotection to the H7N1 subtype avian influenza virus can be provided; and in vaccine, contain the N3 hypotype NA gene that is different from the N1 hypotype; then produce antibody after the immunity at N3NA; when the H7N1 subtype viral infection takes place, then produce N1NA antibody, can and infect poultry with immune poultry by means of diagnostic kit and make a distinction.Utilize this marker vaccine Italy effectively eliminated by the H7N1 subtype avian influenza virus cause popular.But the marker vaccine of this strategy can only use under the single situation of viral prevalence background, if a regional inner virus popularity complexity, various subtype avian influenza virus coexistences, just be difficult to select the NA hypotype of suitable conduct nature labelling, therefore better strategy is that the influenza virus gene fragment is carried out molecular modification, add ectogenic label, the poultry of vaccine immunity and natural infection bird flu is distinguished by the detection of label.
Summary of the invention
As mentioned above, the influenza virus vaccine development that is established as of influenza virus reverse genetic operating system provides strong instrument, utilize this technology in this research, manually rescue and obtained molecular modification and cause weak H5N1 subtype avian influenza virus marker vaccine strain H5N1/PR8-5B19, the first strain H5N1 subtype avian influenza virus A/Goose/Guangdong/1/96 that its HA and NA gene source arrive in isolated in China, internal gene derives from high titre Embryo Gallus domesticus adapted strain A/PuertoRico/8/34 (PR8).A little less than the HA gene of H5N1/PR8-5B19 causes through molecular modification, have the HA cracking site feature of Lowly Pathogenic Avian Influenza Virus, the cervical region of NA gene has inserted the advantage B cell epitope 5B19 of Mouse hepatitis virus S2 glycoprotein as the external source label.This vaccine strain not only has good Embryo Gallus domesticus growth characteristics and to the low pathogenicity of chicken and Embryo Gallus domesticus; prepared inactivated vaccine can provide immunoprotection completely to the attack of H5N1 subtype avian influenza virus; and utilize serological method can distinguish vaccine immune chicken and natural infection chicken behind the vaccine immunity, be used for the anti-system of H5N1 subtype avian influenza virus.
Therefore, the purpose of this invention is to provide the following:
1. inserted the H5N1 subtype avian influenza virus marker vaccine of the advantage B cell epitope 5B19 of murine hepatitis virus S2 glycoprotein at the cervical region of NA gene, the aminoacid sequence of the advantage B cell epitope 5B19 of wherein said murine hepatitis virus S2 glycoprotein is shown in SEQ ID NO.1.
2. according to above-mentioned 1 H5N1 subtype avian influenza virus marker vaccine, its method preparation by may further comprise the steps:
(1) with the protein expressing plasmid of transcribing plasmid, pol gene, the HA genetic transcription plasmid of the negative strand viruses vRNA of H5N1 subtype avian influenza virus with insert NA gene transcription plasmid and the transfection reagent immixture of the advantage B cell epitope 5B19 of murine hepatitis virus S2 glycoprotein;
(2) host cell of the described virus replication permission of the mixture cotransfection of plasmid after will acting on and transfection reagent is cultivated the host cell after the transfection;
(3) cell after the results transfection, the inoculated into chick embryo allantoic cavity is rescued and is obtained recombinant virus.
3. according to above-mentioned 2 H5N1 subtype avian influenza virus marker vaccine, wherein said HA gene is the saltant HA gene that manually lacks the continuous a plurality of basic amino acids in cracking site place.
4. according to above-mentioned 1 H5N1 subtype avian influenza virus marker vaccine, it is H5N1 subtype avian influenza virus marker vaccine strain H5N1/PR8-5B19.
5. according to above-mentioned 4 H5N1 subtype avian influenza virus marker vaccine, its method preparation by may further comprise the steps:
(1) vRNA is transcribed plasmid pPOLI-PB2, pPOLI-PB1, pPOLI-PA, pPOLI-NP, pPOLI-M and pPOLI-NS, the protein expressing plasmid pcDNA-PB2 of four pol genes, pcDNA-PB1, pcDNA-PA and pcDNA-NP and molecular modification cause weak HA gene bidirectional transcription plasmid pBD-MutHA and insert the NA gene bidirectional transcription plasmid pBD-NA-5B19 and the transfection reagent immixture of foreign epitope;
(2) host cell of the described virus replication permission of the mixture cotransfection of plasmid after will acting on and transfection reagent is cultivated the host cell after the transfection;
(3) cell after the results transfection, the inoculated into chick embryo allantoic cavity is rescued and is obtained recombinant virus,
Wherein preferred described host cell is the 293T cell.
6. treat and/or prevent application in the medicine of the disease that bird flu virus causes according to the H5N1 subtype avian influenza virus marker vaccine of any one among the above-mentioned 1-5 in preparation.
7. according to any one the application of H5N1 subtype avian influenza virus marker vaccine in the bird flu epidemiological surveillance among the above-mentioned 1-5.
8. according to above-mentioned 7 application, wherein said bird flu epidemiological surveillance comprises the step of utilizing the ELISA method to detect the antibody of intravital HI antibody of target organism and 5B19 epi-position, and wherein said target organism has been applied described H5N 1 subtype avian influenza virus marker vaccine.
9. inactivated avian influenza vaccine, it comprises according to any one H5N1 subtype avian influenza virus marker vaccine among the above-mentioned 1-5.
10. test kit that is used to distinguish avian influenza vaccine immunity poultry and bird flu natural infection poultry, it comprises according to any one H5N1 subtype avian influenza virus marker vaccine among the above-mentioned 1-5.
Breaking out to world's aviculture of H5N1 subtype avian influenza causes crushing blow, and has important public hygienics meaning.Vaccine immunity is the important technical of the highly pathogenic H5N1 subtype avian influenza of anti-system, can with other the immune measure of natural infection phase region to the control of bird flu with eradicate extremely important.This research and utilization reverse genetic manipulation technology is manually rescued and has been obtained H5N1 hypotype recombinant influenza vaccine strain H5N1/PR8-5B19, its 6 internal gene derive from high titre Embryo Gallus domesticus adapted strain PR8, and HA and NA gene source are in the first strain H5N1 subtype avian influenza virus GS/GD/1/96 of isolated in China.Remove a plurality of continuous basic amino acid of HA gene cracking site by molecular modification, made its HA gene expression characteristics that has possessed low pathogenicity AIV, between NA gene cervical region 49-67 position, inserted the advantage B cell epitope 5B19 of murine hepatitis virus S2 glycoprotein.
Do not cause death in 96 hours behind the H5N1/PR8-5B19 vaccine strain virus inoculation Embryo Gallus domesticus, it is not pathogenic to the SPF chicken that nasal cavity and vein infect the back, has the biological safety of height.Vaccine strain virus has the Embryo Gallus domesticus growth characteristics of height, and 72 hours virus replications peak behind inoculated into chick embryo, and allantoic fluid blood clotting valency is 10.5log2.Virus continuous passage 14 times in Embryo Gallus domesticus, pathogenicity is not returned by force, the 5B19 epi-position stable existence that inserts in the NA gene.
The H5N1/PR8-5B19 vaccine strain has good immunogenicity, and behind the single immunization, the 2nd week can detect HI antibody, and the HI antibody horizontal peaks during the 4th week, does not also occur during to 12 weeks significantly descending; Booster immunization once can obviously improve antibody horizontal behind the single immunization, and the HI antibody titer can reach 11.5log2 when the peak.Attack to highly pathogenic H5N1 hypotype AIV behind the vaccine immunity provides protection fully, does not fall ill behind the immune chicken counteracting toxic substances, and is not dead, not toxin expelling.
16 amino acid short peptides with the 5B19 epi-position of synthetic have been set up the indirect ELISA method that detects anti-5B19 epitope antibodies as envelope antigen.The result shows behind the H5N1/PR8-5B19 inactivated vaccine single immunization in the 3rd week 10 chickens has 2 chickens to produce the ELISA antibody, 7 chicken ELISA antibody test positives are arranged in the 6th week and 12 when all, and behind the booster immunization 10 all chickens all produce can detected anti-5B19 antibody.
This research utilize first based on the reverse genetic manipulation technology of plasmid artificial constructed the strain of H5N1 subtype avian influenza virus marker vaccine.Can induce high-caliber HI antibody behind the deactivation marker vaccine single immunization, provide immunoprotection completely immune chicken.All immune chicken serums all can detect the antibody that is directed to the 5B19 epi-position behind the booster immunization.Therefore, the application of H5N1/PR8-5B19 deactivation marker vaccine can be distinguished marker vaccine immunity chicken and natural infection chicken, is used for the anti-system of H5N1 high pathogenic avian influenza.
Description of drawings
Fig. 1 .pBD carrier sketch;
Fig. 2. molecular modification causes the structure of weak HA gene bidirectional transcription plasmid pBD-MutHA;
The structure of Fig. 3 .pUC-MutHA plasmid and PCR identify;
Fig. 4 .pBD-MutHA plasmid PCR and enzyme action are identified;
Fig. 5 .pBD-MutHA carrier sketch;
Fig. 6. reorganization NA gene bidirectional transcription plasmid pBD-NA-5B19 makes up;
Fig. 7 .pBD-NA-5B19 plasmid construction and PCR and enzyme action are identified;
Fig. 8 .pBD-NA-5B19 carrier sketch;
Fig. 9. the enzyme assay of marker vaccine strain neuraminic acid;
The Embryo Gallus domesticus growth characteristics of Figure 10 .H5N1/R8-5B19 Strain;
The hereditary stability of Figure 11 .H5N1/PR8-5B19 vaccine strain NA-5B19 gene;
Figure 12 .H5N1/PR8-5B19 inactivated vaccine single immunization (A) and booster immunization (B) back HI detect;
The nucleotide sequence of Figure 13 .pBD carrier (SEQ ID NO.2);
Figure 14. plasmid pPOLI-PB2 nucleotide sequence;
Figure 15. plasmid pPOLI-PB1 nucleotide sequence;
Figure 16. plasmid pPOLI-PA nucleotide sequence;
Figure 17. plasmid pPOLI-NP nucleotide sequence;
Figure 18. plasmid pPOLI-NS nucleotide sequence;
Figure 19. plasmid pPOLI-M nucleotide sequence;
Figure 20. plasmid pcDNA-PB2 nucleotide sequence;
Figure 21. plasmid pcDNA-PB1 nucleotide sequence;
Figure 22. plasmid pcDNA-PA nucleotide sequence;
Figure 23. plasmid pcDNA-NP nucleotide sequence;
Figure 24. plasmid pBD-NA nucleotide sequence (SEQ ID NO.3);
Figure 25 .H5N1/PR8-5B19 viruses molecule is modified and is caused weak HA gene M utHA (SEQ ID NO.4);
Figure 26 .H5N1/PR8-5B19 virus analysis is modified NA gene NA-5B19.
The specific embodiment
Hereinafter describe reference example in detail the present invention, described embodiment only is intended as illustrative explanation the present invention, rather than intention limits the scope of the invention.Scope of the present invention is specifically limited by accompanying Claim.
Embodiment 1 molecule causes the production of weak H5N1 subtype avian influenza virus marker vaccine strain H5N1/PR8-5B19
1 materials and methods
1.1 Strain
A/Goose/Guangdong/1/96 (H5N1) is (available from the Harbin veterinary institute) (GS/GD/1/96)
1.2SPF chicken and SPF Embryo Gallus domesticus
9-11 age in days SPF Embryo Gallus domesticus and 4-8 SPF chicken in age in week are available from Harbin veterinary institute Experimental Animal Center.All SPF chicken tests are all carried out in the negative pressure isolator.
1.3 plasmid vector and transfection plasmid
PBD carrier (collection of illustrative plates is seen Fig. 1) (Zejun Li, Hualan Chen, Peirong Jiao, Guohua Deng, GuobinTian, Yanbing Li, Erich Hoffnann, Robert G.Webster, Yumiko Matsuoka, and KangzhenYu.Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian MouseModel.J.Virol.79:12058-12064.) make up by ageing doctor Lan, with the unidirectional plasmid vector pPolIsapIRib (Pleschka that transcribes, S., R.Jaskunas, O.G.Engelhardt, T.Zurcher, P.Palese, and A.Garcia-Sastre.1996.A plasmid-based reverse genetics system for influenza.A virus.J.Virol.70:4188-4192.) the XbaI enzyme cutting fragment that comprises polymerase I promoter-SapI cloning site-Mus source ribozyme sequence in, oppositely insert the XbaI site of pCI (Promega) plasmid, form the bidirectional transcription expression plasmid carrier that rna plymerase ii promoter (CMV) → viral RNA transcription stop signals sequence Rib → influenza virus gene group cDNA (5 ' → 3 ') → human RNA polymerase I promoter → mRNA tanscription termination PolyA signal sequence (SV40) constitutes, utilize this carrier can transcribe out influenza virus minus strand vRNA and normal chain mRNA simultaneously.
Utilized in this research through 12 plasmid reverse genetic operating systems after modifying, wherein the vRNA of six internal gene transcribes plasmid pPOLI-PB2, and pPOLI-PB 1, pPOLI-PA, pPOLI-NP, the protein expressing plasmid pcDNA-PB2 of pPOLI-M and pPOLI-NS and four pol genes, pcDNA-PB1, pcDNA-PA and pcDNA-NP (sequence is seen below accompanying drawing respectively) all come from people source strains of influenza viruses A/Puerto Rico/8/34 (A/RR/8/34), are that Kanta doctor Subbarao by Center for Disease Control (CDC) of the United States Federal is so kind as to give (Subbarao, K., Chen, H., Swayne, D., Mingay, L., Fodor, E., Brownlee, G., Xu, X., Lu, X., Katz, J., Cox, N., Matsuoka, Y.Evaluation of a genetically modified reassortant H5N1 influenza A virusvaccine candidate generated by plasmid-based genetics.Virology 2003,305 (1): 192-200).The NA gene bidirectional transcription plasmid pBD-NA-5B19 that molecular modification causes weak HA gene bidirectional transcription plasmid pBD-MutHA and insertion foreign epitope makes up in this research, derive from China first strain H5N1 subtype avian influenza virus strain A/Goose/Guangdong/1/96 (H5N1) (GS/GD/1/96), wherein pBD-NA-5B 19 is at pBD-NA (sequence is seen accompanying drawing) (Zejun Li, Yongping Jiang, Zhigao Bu, Guobin Tian, Fenfju Zhao, AiqingWang, Kangzhen Yu and Hualan Chen.The NS1 gene contributes to the virulence and hostrange of H5N1 avian influenza viruses in chickens.Accepted by J.Virol.) makes up on (doctor Li Zejun provides) basis.
1.4 main agents and instrument
The big fragment of DNA Polymerase I Kleonw, T4DNA polymerase, T4DNA ligase, restriction endonuclease SapI,, XhoI, NotI, available from NEB company, NsiI is available from MBI company (Lithuania), EcoRI, XbaI, HindIII, BamHI, dATP, dGTP, dCTP and dTTP, r-Taq archaeal dna polymerase purchase the precious biological engineering company limited in Dalian.RNA extracts reagent Trizol LS, Mus source reverse transcription (MLV) test kit, and DTT, RNaseOUT and Pfx high-fidelity DNA polymerase, transfection reagent box Lipofectamine 2000, serum-free medium Opti-MEM is available from Invitrogen company.Glue reclaims test kit and reclaims test kit available from Shanghai China Shun biological engineering company limited with a small amount of plasmid.Sequencing reaction test kit (CEQTM DTCS Quick Start Kit) is available from BECKMAN company.Transfection plasmid extraction kit (Perfectprep Plasmid Mini) is available from Eppendorf company (U.S.).
The anti-chicken two of the rabbit of used 45% gelatin and HRP labelling resists available from Sigma company in the ELISA experiment; the 5B19 polypeptide is given birth to worker's biological engineering company limited synthesizing and purifying by Shanghai; OPD (o-Phenylenediamine, o-phenylenediamine) is available from Shanghai China Shun biological engineering company limited.96 hole enzyme reaction plates are available from Orange company.Microplate reader Model 680Microplate Reader is available from BIO-RAD company.
1.5 viral RNA extraction, reverse transcription and PCR
GS/GD/1/96 kind poison 10 5Collect the dead germ allantoic fluid in 24-36 hour behind the dilution inoculation 9-10 age in days SPF Embryo Gallus domesticus, carry out RNA and extract and reverse transcription, the cDNA that produces virus (gets 250 μ l virus allantoic fluid, add 750 μ l TrizolLS lysates, mixing, room temperature effect 10 minutes is adding 200 μ l chloroforms, mixing, room temperature was placed 5 minutes, and centrifugal 15 minutes of 12000g, 4 ℃ shift supernatant to new centrifuge tube, the isopropanol precipitating that adds 500 μ l, room temperature was placed 10 minutes.12000g abandons supernatant after centrifugal 10 minutes, behind the precipitation vacuum drying, is resuspended in the DEPC treating water.After RNA extracted, reverse transcription added each composition (the reverse transcription primer sequence is 5 ' AGC AAA AGC AGG 3 ') by the explanation of Mus source reverse transcription (MLV) test kit, and 37 ℃ of reaction 2-3h are directly used in PCR.)。PCR carries out under the effect of Pfx high-fidelity DNA polymerase, add each composition by the test kit description, 94 ℃ of degeneration 5min, enter following circulation, 94 ℃ of 1min of degeneration, annealing does not coexist between 52-65 ℃ according to primer, time is 1min, extend 68 ℃ of 2-3min, totally 35 circulations, 72 ℃ are extended 10min.
1.6 sequencing
In the PCR pipe, respectively PCR is identified positive plasmid and an amount of ddH 2O mixes, 96 ℃ are heated pre-degeneration 3 minutes, naturally cold adds sequencing primer (5pmol/ μ l) and DTCS Quick Start Master Mix 2 μ l after room temperature, the PCR that checks order behind mixing reaction, the PCR response procedures is: 96 ℃ of 20sec, 50 ℃ of 20sec, 60 ℃ of 4min, 35 circulations remain on 4 ℃ at last.Reaction terminating liquid (the NaOAC 2ul of 3M PH5.2 that adds 2.5ul after the PCR reaction is finished, the EDTA2ul of 100mM PH8.0, the Glycogen 1ul of 20mg/ml, instant joining) cessation reaction, add 30ul 95% ice ethanol precipitation DNA then, mixing, 4 ℃, the centrifugal immediately 20min of 12000rpm.After carefully outwelling supernatant, with the residual salt of 100-200ul 70% ice twice eluting of ethanol, 4 ℃, the centrifugal 5min of 12000rpm.After vacuum is drained or room temperature dries DNA,, carefully add in the CEQ sample plate hole, add a dropstone wax oil at last in the sample plate hole with the abundant dissolving DNA precipitation of 30ul Methanamide.(Bechman America) upward checks order to clone's recombiant plasmid, utilizes DNASTAR software (DNASTAR.Inc.) that the sequence that obtains is analyzed afterwards at the BechmanCEQ8000 sequenator to adopt dideoxy chain termination immediately.
1.7 primer
Used primer comprises used primer in pBD-MutHA and the pBD-NA-5B19 bidirectional transcription plasmid construction process in the experiment, and is synthetic by the rich inferior biological engineering company limited in Shanghai.Specifically see Table 1.
The used primer of table 1 transfection plasmid construction
1.8HA and the structure of NA transfection plasmid pBD-MutHA and pBD-NA-5B19
1.8.1 the structure of bidirectional transcription plasmid pBD-MutHA
CDNA with GS/GD/1/96 virus is a template, carry out the PCR reaction respectively with two couples of primer HA1-F1/HA1-R1 and HA2-F1/HA2-R1, amplify HA1 and HA2 fragment, purpose is to remove a plurality of continuous basic amino acid of HA cracking site, makes it obtain the HA gene expression characteristics of Lowly Pathogenic Avian Influenza Virus.Get 5ulPCR product electrophoretic examinations amplification on 1% agarose gel, reclaim the test kit recovery with a small amount of glue respectively for two genetic fragment HA1 and HA2 then.
HA1 and HA2 fragment are carried out double digestion for the HA1 fragment with BamHI and XhoI after reclaiming, HA2 fragment usefulness XhoI and Hind III enzyme action, and pUC18 (available from precious biotechnology engineering (Dalian) company limited) carried out double digestion with BamH I and Hind III.Then will be through the HA1 of enzyme action, HA2 two fragments and pUC18 carrier are with 3: 3: 1 mixed, 16 ℃, with T4DNA ligase effect 16h, transform JM109 competence escherichia coli then according to a conventional method, choose bacterium, the upgrading grain carries out electrophoresis, carries out PCR with primer HAF1/HAR1 and identifies.Be accredited as male plasmid with being dissolved in the sterilization deionized water of 50 μ L behind the plasmid extraction kit extraction plasmid in a small amount, be used for sequencing reaction.
Identify positive plasmid pUC-MutHA positive and that order-checking is correct for PCR, carry out PCR with primer MutHAF1/MutHAR1, outside 5 ' and 3 ' end sequence of this primer and HA gene fitted like a glove, forward primer 5 ' end increased by two base CC, and downstream primer 5 ' end increases TT.The PCR product is handled 30min for 12 ℃ with the T4DNA polymerase in the presence of dCTP and dTTP, 75 ℃ of 20min make enzyme deactivation, and glue reclaims the test kit recovery and is used for coupled reaction.
The pBD carrier uses the SapI enzyme at 37 ℃ of following enzyme action effect 3h, reclaim test kit with glue and reclaim, use the big fragment of DNA Polymerase I Kleonw in 25 ℃ of effect 25min then under the condition of dGTP and dATP existence, 75 ℃ of 20min make enzyme deactivation, glue reclaims test kit and reclaims, and is standby.
After PCR product and pBD vehicle treated are good, according to 1: 3-1: 6 mixed, 16 ℃, with T4DNA ligase effect 16h, transform the JM109 competent cell, choose bacterium, the upgrading grain carries out electrophoresis, carries out PCR with primer HAF1/HAR1 and identifies.Be accredited as male plasmid with being dissolved in the sterilization deionized water of 50 μ L behind the plasmid extraction kit extraction plasmid in a small amount, be used for sequencing reaction.
1.8.2 containing the bidirectional transcription plasmid pBD-NA-5B19 of murine hepatitis virus (MHV) 5B19 epi-position makes up
There is not disappearance in the NA gene of GS/GD/1/96 at cervical region, in order to make up the reorganization NA gene that has the external source label, we are with murine hepatitis virus (Murine Hepatitis virus, MHV) the advantage B cell epitope 5B19 of S2 glycoprotein inserts NA gene cervical region, to replace the 49-67 amino acids of wild type NA gene cervical region.The aminoacid sequence of 5B19 epi-position is " SPLLGCIGSTCAEDGN ", and nucleotides sequence is classified 5 '-AGT CCC CTG CTG GGA TGCATC GGC TCC ACC TGT GCC GAG GAC GGC AAC-3 ' as.
PBD-NA is a template with NA gene bidirectional transcription plasmid, utilizes two couples of primer NA-5B19-F1/NA-5B19-R1 and NA-5B19-F2/NA-5B19-R2 to carry out PCR, and amplification contains the nucleotide fragments Fragment1 and the Fragment2 of part 5B19 epi-position respectively.The PCR product total length of F1/R1 should be 306bp, and 5 ' end has unique EcoRI site, and 3 ' end has unique NsiI site and contains the upstream portion of 5B19 epi-position; The PCR product of F2/R2 should be 1553bp, and 5 ' end has unique NsiI site and contains the downstream part of 5B19 epi-position, and 3 ' end has unique NotI site.Get 5ul PCR product electrophoretic examinations amplification on 1% agarose gel.
The PCR product of two fragment Fragment 1 and Fragment 2 is reclaimed the recovery of (in a small amount) test kit description operating procedure according to glue.Fragment1 EcoRI and NsiI, Fragment 2 usefulness NsiI and NotI enzyme action, and the pBD-NA plasmid carried out double digestion with EcoRI and NotI, then will be through the Fragment1 of enzyme action, Fragment2 two fragments and pBD-NA carrier were with 3: 3: 1 mixed, 16 ℃, with T4DNA ligase effect 16h, transform the JM109 competent cell then according to a conventional method, choose bacterium, the upgrading grain carries out electrophoresis, carry out PCR with primer Marker174U24/Marker1200L25 and NAF1/NAR1 and identify, and carry out enzyme action and identify.Be accredited as male plasmid and be used to carry out sequencing reaction.
1.9 molecule causes weak rescuing of H5N1 subtype avian influenza virus marker vaccine strain H5N1/PR8-5B19 and obtains
1.9.1H5N1/PR8-5B19 rescue and obtain
293T cell (human embryonic kidney cell, CRL-11268 is from ATCC) is incubated in the Tissue Culture Flask with the DMEM culture fluid that contains 10% hyclone earlier, passes in six orifice plates after 5 generations of continuous passage are above, places 37 ℃, 5%CO 2Cultivate in the incubator.Treat that the 293T cell grows to 80% when being paved with, and carries out transfection by Lipofectamine 2000 test kit operation instructions.
For the artificial strain of H5N1 subtype avian influenza virus marker vaccine, the 12 plasmid reverse genetic operating systems that utilize process to modify made.Above-mentioned 6 kinds of unidirectional bidirectional transcription plasmid pBD-MutHA and pBD-NA-5B19 that make up in plasmid and 4 kinds of albumen expression plasmids and this research that transcribe that derive from reassortant virus, each plasmid is got 1 μ g and is added abundant mix homogeneously in the 50 μ l OPTi-MEM culture medium, get 12 μ l Lipofectamine, 2000 transfection reagents simultaneously and add in the 250 μ l OPTi-MEM culture medium, left standstill 5 minutes in room temperature behind the mixing.The OPTi-MEM culture medium that will contain transfection reagent and plasmid was then mixed, in room temperature effect 30 minutes.
After treating that transfection reagent and plasmid effect are finished, the cell culture fluid in six orifice plates is sopped up, and wash twice with the OPTi-MEM culture medium; every then hole adds the OPTi-MEM culture fluid of 1ml; and in the hole with transfection reagent and plasmid mixture adding 293T cell six orifice plates, in 37 ℃, 5%CO 2Cultivate in the incubator.Transfection after 24 hours is changed the transfection reagent in the culture plate, washes once with OPTi-MEM then, to remove residual transfection reagent, adds 2ml OPTi-MEM at last and (contains 0.5 μ g/ml TPCK-Trypsin, Sigma), continue to cultivate 48-72 hour.Transfection after finishing is got off the cell purge, the back inoculation 9-11 age in days SPF Embryo Gallus domesticus that is uniformly dispersed, each chick embryo allantoic cavity inoculation 0.2ml transfectional cell suspension.Inoculate back 48 hours and collect chick embryo allantoic liquid, the chicken red blood cell with 0.5% carries out hemagglutination test (HA), collects allantoic fluid for the Embryo Gallus domesticus that the blood clotting valency is arranged, and carries out virus and identifies.
Transfection rescue obtain H5N1/PR8-5B19 virus in, utilize this system to rescue and obtained H5N1/PR8 virus, it is identical with H5N1/PR8-5B19 that transfection and virus are rescued the process of obtaining, but used NA gene bidirectional transcription plasmid is pBD-NA, does not contain the 5B19 epi-position in the NA gene.
1.9.2H5N1/PR8-5B19 the evaluation of viral HA and NA gene
Utilize reverse genetic operating system manually to make after the H5N1/PR8-5B19 virus, need identify, rescued the HA and the NA gene that obtain virus to determine and whether comprise the molecular modification that adds to some extent rescuing the virus that obtains.H5N1/PR8-5B19 and H5N1/PR8 virus inoculation SPF Embryo Gallus domesticus, collect chick embryo allantoic liquid behind the 48h, extract the RNA of virus, utilize the Auele Specific Primer Uni-12 (sequence is 5 ' AGC AAA AGC AGG 3 ') of influenza virus to carry out the cDNA that reverse transcription produces virus then.Utilize the Auele Specific Primer HAF1/HAR1 and the NAF1/NAR1 of HA and NA gene to carry out pcr amplification, the PCR product is identified the back recovery through agarose gel (1%) electrophoresis, carries out the order-checking of PCR product with CEQTM DTCS-QUICK Star Kit then.
2 results
2.1pBD-MutHA plasmid construction
The GS/GD/1/96 virus of wild type is high pathogenic avian influenza virus, cause weak vaccine strain in order to make up molecular modification, the method of employing PCR lacks a plurality of successive basic amino acid of HA gene cracking site, make it obtain the HA gene expression characteristics of Lowly Pathogenic Avian Influenza Virus, concrete construction strategy as shown in Figure 2.
2.1.1pUC-MutHA the structure of plasmid
CDNA with GS/GD/1/96 virus is a template, utilizes two couples of primer HA1-F1/HA1-R1 and HA2-F1/HA2-R1 to carry out the PCR reaction respectively, amplifies HA1 and HA2 fragment.Get 5ul PCR product and check amplification on 1% agarose gel electrophoresis, the size of fragment HA1 and HA2 respectively is: 1078bp and 752bp (see figure 3), electrophoresis result conforms to expection.
After HA1 and HA2 fragment reclaimed, with BamHI and XhoI double digestion, the HA2 fragment was with XhoI and Hind III enzyme action for the HA1 fragment, and pUC18 is carried out double digestion with BamH I and Hind III.Three's enzyme action connects conversion after reclaiming, and extracts plasmid, carries out PCR with primer HAF1/HAR1 and identifies (Fig. 3).The wild type HA full length gene of GS/GD/1/96 is 1778bp, and through should be 1767bp after the sudden change, the PCR electrophoresis result is consistent with expection.Be accredited as male plasmid and after plasmid recovery test kit extracts plasmid in a small amount, be dissolved in the sterilization deionized water of 50 μ L, be used for sequencing reaction.Sequencing result shows, the HA gene is 1767bp in the pUC-MutHA plasmid, the HA gene that inserts in this plasmid is different with wild gene, and the aminoacid sequence of its HA cracking site is sported-RETR-by-RERRRKKR-, and the latter is typical H5 hypotype low pathogenicity bird flu virus HA characterization of molecules.And, to be not intended to sport CGA by AGA near arginine (R) codon of cracking site, and so just avoid in the process that virus goes down to posterity, inserting new basic amino acid at the HA cracking site again virulence is returned by force owing to the nucleotide sequence reproduce effect (Polymerase stuttering) of polymerase protein.
2.12pBD-MutHA plasmid construction
With plasmid pUC-MutHA is template, carries out PCR with primer MutHAF1/MutHAR1, the MutHA gene of amplification total length.The PCR product reclaims the test kit recovery with glue and is used for coupled reaction after using the T4DNA polymerase to handle in the presence of dCTP and the dTTP.The pBD carrier is used the SapI enzyme to handle the back at 37 ℃ of following enzyme action and is reclaimed the test kit recovery with glue, handles with DNA Polymerase I Kleonw under the situation of dGTP and dATP existence then, and glue reclaims the test kit recovery.After PCR product and pBD vehicle treated are good, connect conversion, extract plasmid, carry out electrophoresis, carry out PCR with primer HAF1/HAR1 and identify that the result is positive.Carry out enzyme action simultaneously and identify, and the enzyme action product is carried out agarose gel (1%) electrophoresis.The result, with the XhoI enzyme action obtained 1.2Kb and 4.9kb two bands, obtained 1.5kb with XbaI enzyme cutting, 0.6kb and the band of 4.0kb, obtained the fragment of linearizing 6.1kb during with the HindIII enzyme action, the gained result all with expect the analysis result consistent (Fig. 4) of the pBD-MutHA that obtains.Be accredited as male plasmid with being dissolved in the sterilization deionized water of 50 μ L behind the plasmid extraction kit extraction plasmid in a small amount, be used for sequencing reaction.Sequencing result shows that the HA gene is 1767bp (sequence is seen accompanying drawing) in the pBD-MutHA plasmid, and is identical with pUC-MutHA, does not undergo mutation, and the aminoacid sequence of HA cracking site is-RETR-that pBD-MutHA plasmid sketch is seen Fig. 5.
2.2pBD-NA-5B19 plasmid construction
In order to make up the strain of H5N1 subtype avian influenza virus marker vaccine, with 16 the aminoacid insertion NA gene cervical regions of PCR method with MHV S2 glycoprotein 5B19 epi-position, as the external source label, concrete construction strategy as shown in Figure 6 with this.With pBD-NA is template, utilizes two couples of primer NA-5B19-F1/NA-5B19-R1 and NA-5B19-F2/NA-5B19-R2 to carry out PCR, and amplification contains the nucleotide fragments Fragment1 and the Fragment2 of part 5B19 epi-position respectively.The PCR product total length of F1/R1 should be 306bp, and the PCR product of F2/R2 should be 1553bp.Get 5ul PCR product and carrying out electrophoresis detection, the size of fragment Fragment1 and Fragment2 respectively is as a result: 0.3kb and 1.5kb left and right sides (see figure 7) conform to expection.After two fragments reclaim, Fragment1 EcoRI and NsiI, Fragment2 NsiI and NotI enzyme action, and the pBD-NA plasmid carried out double digestion with EcoRI and NotI, the three is connected conversion, extract plasmid, carry out PCR evaluation and enzyme action evaluation with primer NAF1/NAR1 and Marker174U24/Marker1200L25.The result, obtained during with the BamHI enzyme action 0.8Kb and 5.0kb two bands, obtained the band of 1.8kb and 4.0kb, obtained the fragment of linearizing 5.8kb during with the NsiI enzyme action with XbaI enzyme cutting, the gained result all with the consistent (see figure 7) of analysis result of the pBD-NA-5B19 that obtains of expection, pBD-NA-5B19 plasmid sketch is seen Fig. 8 (the NA-5B19 sequence is seen accompanying drawing).
2.3H5N1/PR8-5B19 rescue and obtain and virus is identified
Change liquid after 24 hours behind the 12 kinds of common transfection 293T of plasmid cells, 200 μ l OPTI-MEM (containing 0.5 μ g/ml TPCK-Trypsin) are added in every hole, continue to cultivate 48-72 hour.With transfectional cell suspension inoculation SPF Embryo Gallus domesticus, measure the hemagglutination activity of allantoic fluid after 48 hours then.For the allantoic fluid that the blood clotting valency is arranged with 10 4After the dilution, inoculation SPF Embryo Gallus domesticus was collected allantoic fluid after 48 hours, extracted viral RNA, carried out RT-PCR, and HA and NA gene are carried out sequencing, rescued to obtain in viral HA and the NA gene whether comprise the molecular modification that is added with evaluation.The sequencing results shows that the HA full length gene is 1767bp, consists of-RETR-at the aminoacid of HA cracking site, is the HA gene expression characteristics of low pathogenicity bird flu virus; The NA full length gene is 1446bp, has substituted 20 aminoacid sequences of wild type NA gene at NA gene cervical region with 16 amino acid whose 5B19 epi-positions, and therefore the NA gene that produces has lacked 4 aminoacid than wild type gene, and is all viral consistent with the vaccine strain of expecting.
The biological characteristics of embodiment 2H5N1/PR8-5B19 vaccine strain
1. material and method
1.1H5N1/PR8-5B19 vaccine strain is pathogenic to chicken
In intravenous inoculation pathogenic index (IVPI) determination test, choose 2 groups 10 6 the week age SPF chicken, the H5N1/PR8-5B19 of 1: 10 times of dilution of every chicken intravenous inoculation 0.2ml or H5N1/PR8 virus allantoic fluid are inoculated and are observed morbidity and death condition in back 10 days, calculate IVPI.
In the nasal cavity infection experiment, choose 2 groups 10 6 the week age SPF chicken, every chicken inoculates 0.1ml10 with nasal 6EID 50H5N1/PR8-5B19 or H5N1/PR8 virus allantoic fluid.Infect back 1,3,5,7,9 days collection throat swabs carry out virus with the cloaca swab to be separated, and observes incidence.Infected back 21 days, and gathered the determination of serum blood clotting and suppress (HI) antibody.
1.2 marker vaccine strain NA neuraminic acid enzyme assay
The NA of influenza virus has the neuraminic acid enzymatic activity, acts on the myosin substrate, can discharge sialic acid, and arsenite can stop this enzymatic activity.Thiobarbituricacid combines with certain proportion with free sialic acid and produces pink.In order to verify that inserting foreign epitope has or not influence to the neuraminic acid enzymatic activity of H5N1/PR8-5B19 virus later on, utilizes the neuraminic acid enzyme test to detect.Concrete operating procedure is as follows:
1. with PBS H5N1/PR8-5B19 is become 10 with the H5N1/PR8 viral dilution 7.0EID 50The viral dosage of/0.1ml on this basis, doubly carries out doubling dilution with 0.5log10 (1: 3.16), is respectively stock solution, 10 -0.5, 10 -1, 10 -1.5, 10 -2, 10 -2.5, 10 -3, 10 -3.5Dilution.
2. virus sample: each dilution virus of 50 μ l+50 μ l PBS+100 μ l myosins; Negative control: 100 μ lPBS+100 μ l myosins.Sample add good after, shake test tube, seal the mouth of pipe, 37 ℃ water-bath 16-18 hour.
3. after the water-bath, room temperature cooling 2 minutes adds 0.1ml periodates solution in every pipe.The concussion test tube, room temperature was placed 20 minutes.
4. every pipe adds 1.0ml arsenite solution, and it is brown that liquid is, and thermal agitation is to brown disappearance, and liquid in pipe becomes white.
5. every pipe adds 2.5ml thiobarbituricacid solution, and thermal agitation placed on the test tube rack boiling water bath 15 minutes fast.Thiobarbituricacid combines the pulverize redness with free sialic acid with certain proportion.
6. ice bath cooling test tube leaves water-bath after the cooling, adds warrenoff solution 5ml, builds the mouth of pipe, tighten, centrifugal 10 minutes of 200rpm, the butanols of carefully drawing the upper strata in cuvette, the OD value at spectrophotometric determination 549nm place.Before surveying virus sample, do blank with the myosin negative control group.
7. judge the NA activity by drawing the NA activity curve, abscissa is the viral dilution degree, and vertical coordinate is the OD value at 549nm place.
1.3H5N1/PR8-5B19 the Embryo Gallus domesticus growth characteristics of vaccine strain
In order to measure the Embryo Gallus domesticus growth characteristics of H5N1/PR8-5B19 vaccine strain, select H5N1/PR8-5B19 and H5N1/PR8 virus, with the dose inoculation 9-11 day instar chicken embryo of each Embryo Gallus domesticus 103EID50.Respectively 18,24, respectively got 10 Embryo Gallus domesticus in 30,36,42,48,54,60,72 and 96 hours after the egg inoculation.With every group of 10 chick embryo allantoic liquids with equal mixed after, measure allantoic fluid blood clotting valency, draw viral growth curves then, observe the growth characteristics of virus.
1.4H5N1/PR8-5B19 the hereditary stability of HA and NA in the vaccine strain
A little less than the HA gene of H5N1/PR8-5B19 vaccine strain causes through molecular modification, the NA gene has inserted foreign epitope 5B19 through modifying, in order to verify, the H5N1/PR8-5B19 vaccine strain is carried out going down to posterity for 14 times on the SPF Embryo Gallus domesticus through the HA after modifying and the hereditary stability of NA gene.For can 5B19 epi-position that verify insertion stable existence in the NA gene, whether can lose because virus goes down to posterity, choose the 1st respectively, 5, the viral allantoic fluid in 10,12,14 generations, utilize Trisol LS reagent to extract viral RNA, carry out the cDNA of reverse transcription generation virus with the Auele Specific Primer Uni-12 of influenza virus.Utilize the Auele Specific Primer of NA-5B19 gene to carry out pcr amplification then, primer sequence is: Marker 174U24/Marker 1200L25, carry out electrophoresis detection then.
2. result
2.1H5N1/PR8-5B19 vaccine strain is pathogenic to chicken
After with sterilization PBS H5N1/PR8-5B19 and H5N1/PR8 virus allantoic fluid being done 1: 10 times of dilution, measure respectively for 6 the week age SPF chicken intravenous inoculation pathogenic index (IVPI), this two strain is as a result manually rescued the virus that obtains chicken is the low pathogenicity strain, any disease symptom does not appear in infection in back 10 days, also do not have death, IVPI is 0.Gather to infect back 21 days serum, detect HI antibody with hemagglutination inhibition test, the result shows all 10 chicken HI antibody horizontals between 7-9log2, shows that virus duplicates in the chicken body.
H5N1/PR8-5B19 and H5N1/PR8 virus allantoic fluid are respectively with 10 6EID 50Viral dosage nasal cavity infect 6 age in week the SPF chicken, test chicken is no abnormal in back 14 days of infection, does not fall ill, and is not dead, takes to infect back 1,3,5,7,9 days throat swab carries out virus with the cloaca swab to be separated, the result is all negative.Infected back 21 days, and gathered the determination of serum blood clotting and suppress (HI) antibody, the result in 10 chickens of every kind of viral infection, have only 2 chickens detect very low-level HI antibody (＜3log2).The analysis showed that two strain virus can not duplicate in the respiratory tract chicken after nasal cavity infects, the low-level HI antibody that indivedual chickens are produced is produced as the antigenic stimulus body by virus inoculation itself.
2.2 marker vaccine strain H5N1/PR8-5B19 neuraminidase (NA) determination of activity
In order to determine after NA gene cervical region inserts ectogenic 5B19 epi-position, whether can reduce the neuraminic acid enzymatic activity of H5N1/PR8-5B19 vaccine strain NA, H5N1/PR8 is carried out gradient dilution detection neuraminic acid enzymatic activity with the H5N1/PR8-5B19 viral dilution after containing identical viral infection dosage, as shown in Figure 9.
H5N1/PR8 is identical with the genetic background of H5N1/PR8-5B19 two viruses, just inserted the 5B19 epi-position of ectogenic MHV at the NA of H5N1/PR8-5B19 gene cervical region, as can be seen from Figure 9, the neuraminic acid enzymatic activity of H5N1/PR8-5B19 virus is starkly lower than H5N1/PR8.For example the viral dilution degree is when 1.5log10, and the OD value of the 549nm of H5N1/PR8 still is 1.31, and H5N1/PR8-5B19 then has been reduced to 0.097.
2.4.3H5N1/PR8-5B19 the Embryo Gallus domesticus growth characteristics of vaccine strain
The neuraminic acid enzymatic activity of H5N1/PR8-5B19 virus for determining whether therefore to influence the growth characteristics of virus, is carried out the Embryo Gallus domesticus proliferation test of virus because of having inserted the 5B19 epi-position at NA gene cervical region and having reduced.Choose H5N1/PR8-5B19 and H5N1/PR8 virus, respectively with allantoic cavity approach inoculation 9-11 day instar chicken embryo, each egg inoculation 10 3EID 50Viral dosage.Respectively 18,24, respectively got 10 Embryo Gallus domesticus in 30,36,42,48,54,60,72 and 96 hours after the egg inoculation, with every group of 10 chick embryo allantoic liquids with equal mixed after, measure allantoic fluid blood clotting valency, draw viral growth curves, the results are shown in Figure 10.
H5N1/PR8-5B19 and H5N1/PR8 virus all do not cause death in 96 hours behind inoculated into chick embryo, show that two strain virus are the low pathogenicity strain to Embryo Gallus domesticus.As shown in figure 10, H5N1/PR8 18 hours allantoic fluids behind inoculated into chick embryo do not detect hemagglutination activity, blood clotting then appears in twenty-four-hour urine capsule liquid after inoculation, the HA valency is 2log2, and after this along with virus duplicating in Embryo Gallus domesticus, the blood clotting valency of allantoic fluid improves rapidly, 72 hours growth titres are for the highest behind inoculated into chick embryo, reach 11log2, decline is arranged slightly at 96 hours allantoic fluid blood clotting valencys, but still at 10.5log2.H5N1/PR8-5B19 18 hours allantoic fluids behind inoculated into chick embryo do not have hemagglutination activity yet, at twenty-four-hour urine capsule liquid blood clotting valency is 1log2, the blood clotting valency of allantoic fluid improves rapidly subsequently, also reaches summit of growth in 72 hours behind inoculated into chick embryo, and allantoic fluid blood clotting valency is 10.5log2.In general, the growth titre of H5N1/PR8 virus in Embryo Gallus domesticus will be higher than H5N1/PR8-5B19, but do not have significant difference between the two.Inserted ectogenic 5B19 epi-position in the H5N1/PR8-5B19NA gene though the result shows, caused the neuraminic acid enzymatic activity of virus to reduce, the marker vaccine strain virus still has good Embryo Gallus domesticus growth characteristics.
2.4.4H5N1/PR8-5B19 the hereditary stability of vaccine strain
The feature that has had the HA gene cracking site of Lowly Pathogenic Avian Influenza Virus a little less than the HA gene of H5N1/PR8-5B19 vaccine strain causes through molecular modification, the NA gene has inserted foreign epitope 5B19 through modifying, for verify the molecular modification that in HA and NA gene, adds can be along with going down to posterity of virus stable existence, the H5N1/PR8-5B19 vaccine strain is carried out continuous 14 times go down to posterity on the SPF Embryo Gallus domesticus.The H5N1/PR8-5B19 Strain does not cause death all the time to Embryo Gallus domesticus in the process of going down to posterity, and shows that the low pathogenicity feature of HA gene is maintained, and virulence do not occur and returns strong phenomenon, shows that this vaccine strain has the biological safety of height.
For can 5B19 epi-position that verify insertion stable existence in the NA gene, whether can lose because virus goes down to posterity, choose the 1st respectively, 5,10,12, the viral allantoic fluid in 14 generations, extract viral RNA, carry out RT-PCR, carry out pcr amplification with the Auele Specific Primer Marker 174U24/Marker 1200L25 of NA-5B19 gene.Electrophoresis result as shown in figure 11, the result has obtained being about the band of 1kb, with the expection big or small 1051bp consistent.Negative control is selected the viral allantoic fluid of H5N1/PR8, and PCR does not amplify the fragment of purpose size.The result shows, the 5B19 epi-position of being inserted can be in H5N1/PR8-5B19 virus stable existence, can be used as the production that stable foreign epitope label is used for marker vaccine.
The 3 one kinds of preliminary foundation that can distinguish the ELISA detection method of H5N1/PR8-5B19 marker vaccine immune serum and non-marked vaccine immunity serum or natural infection chicken serum of embodiment
1. materials and methods
1.1ELISA operation sequence
In order to utilize the chicken of serological method separator vaccine immune chicken and non-marked vaccine immune chicken or natural infection H5N1 subtype avian influenza virus, set up a kind of polypeptidase connection immunoadsorption experimental technique of indirect method, concrete steps are as follows:
1. antigen coated: 16 aminoacid (" SPLLGCIGSTCAEDGN ") of synthetic murine hepatitis virus 5B19 epi-position are used antigen as bag.After polypeptide is synthetic, be dissolved in the distilled water, polypeptide be diluted to finite concentration with the carbonate buffer solution of pH 9.2.Add 100 μ l polypeptide coating buffers in every hole of ELISA Plate, bag was by 16-24 hour in 4 ℃ of refrigerators.
2. sealing: after antigen coated, the coating buffer in the ELISA Plate is discarded, clean three times with the PBST washing liquid.Every then hole adds 5% gelatin 100 μ l sealing sample well, and sealing is 2 hours in 37 ℃ of wet boxes.
3. add an antiserum: after the ELISA Plate sealing, discard confining liquid, with PBST washing liquid washing three times.After one antiserum done certain multiple dilution with PBST, every hole added 100 μ l, and effect is 90 minutes in 37 ℃ of wet boxes.
4. adding ELIAS secondary antibody: an antiserum effect after finishing discards it, with PBST washing liquid washing three times.With PBST (Phosphate Buffered Saline/Tween) the anti-chicken antibody of the rabbit of horseradish peroxidase-labeled is done 1: 5000 times of dilution then, every hole adds 100 μ l, and effect is 90 minutes in 37 ℃ of wet boxes.
5. add the substrate colour developing: after two anti-effects finish it is discarded, with PBST washing liquid washing three times.Every then hole adds the OPD substrate that 100 μ l prepare.Colour developing is 10 to 15 minutes in 37 ℃ of wet boxes.
6. cessation reaction: after colour developing finished, every hole added the H of 50 μ l 2M 2SO 4The stop buffer cessation reaction.
7. measure optical density OD value: utilize microplate reader to measure the OD value of 490nm after chromogenic reaction stops.
1.2 antigen coated concentration and serum dilution test
Be cushioned liquid with bag the 5B19 polypeptide of synthetic is diluted to 2 μ g/100 μ l, 1 μ g/100 μ l, 0.5 μ g/100 μ l, four variable concentrations of 0.25 μ g/100 μ l, bag be by enzyme reaction plate, every hole 100 μ l, and each titre two hole, 4 ℃ are spent the night.Of PBST the anti-chicken serum of crossing of H5N1/PR8-5B19 marker vaccine immune chicken serum and natural infection H5N1 subtype avian influenza virus was made respectively 1: 50,1: 100 and 1: 200, square formation was formed in 1: 400 times of dilution.After antigen coated the finishing, in 37 ℃ of sealings 2 hours, after the washing, add different dilution antiserums with confining liquid, 1.5 hours after scouring of 37 ℃ of effects, every then hole adds the ELIAS secondary antibody of 100 μ l dilution in 1: 5000 and carries out according to above-mentioned ELISA program.Set not increase serum matched group simultaneously, with same program determination.
1.3 criterion test
After antigen and serum dilution are determined, 50 parts of anti-chicken serums excessively of H5N1 bird flu virus experimental infection and H5N1 hypotype non-marked vaccine immunity serum are carried out ELISA mensuration, the result is carried out statistical analysis according to determined method.
2. result
2.5.1 determining of antigen coated concentration and serum dilution
The suitableeest bag of table 2 antigen is determined by concentration and serum optimal dilution
Figure C20061011440800241
The 5B19 polypeptide antigen wraps by Sptting plate with 2-0.25 μ g/100 μ l, serum is doubling dilution from 1: 50 to 1: 400,1: 5000 times of dilution of ELIAS secondary antibody, from square formation result of the test (table 2), along with the increase of serum diluting multiple and the reduction of antigen coated concentration, the OD value of serum decreases, when serum dilution at 1: 100, the polypeptide bag by concentration when the 1 μ g/100 μ l, positive serum OD value approaches 1.0, negative serum OD value＜0.2, and the ratio (P/N) of positive and negative serum value is for the highest, so the dilution factor of polypeptide and positive serum is a best operating condition at this moment.In view of the above, determine that the bag of polypeptide antigen is 1 μ g/100 μ l by concentration, the optimum dilution degree of serum is 1: 100.
2.5.2 determining of criterion
Choose the anti-chicken serum of crossing behind 50 parts of H5N1 subtype avian influenza virus non-marked vaccine immunity serum and the H5N1 subtype avian influenza virus experimental infection, carrying out ELISA according to the method for being set up detects, the result shows that the OD value of 50 parts of serum is between 0.123 to 0.267, sample mean and standard deviation are respectively X=0.188, SD=0.036, obtain confidence interval upper limit X+3SD=0.298 ≈ 0.30, with 0.30 the upper limit as non-marked vaccine immune chicken or natural infection chicken serum OD value, according to Principle of Statistics, when blood serum sample OD value＞X+3SD, can on 99.9% level, be judged to be the positive.Therefore we draw indirect ELISA criterion, and promptly the OD value of testing sample is positive greater than 0.30, be less than or equal to 0.30 negative.
The test of embodiment 4H5N1/PR8-5B19 vaccine immunity
1. materials and methods
1.1 the preparation of inactivated vaccine
After the dilution of H5N1/PR8-5B19 virus allantoic fluid, with allantoic cavity approach inoculation 9-11 day instar chicken embryo, each egg inoculation 10 3EID 50Viral dosage, inoculation back is continued hatching and is collected chick embryo allantoic liquid after 48 hours, measures the blood clotting valency between 9-11 log2.Through removing tissue and cell debris behind the 1000rpm/min low-speed centrifugal, get supernatant and add formalin in 4 ℃ of deactivations 72 hours with 0.25% ratio.In order to verify inactivating efficacy, get the allantoic fluid inoculated into chick embryo after the deactivation, each egg inoculation 0.1ml is in 37 ℃ of allantoic fluid blood clottings that detect inoculated into chick embryo after cultivating 48 hours, it is all negative that the result shows after the deactivation that the inoculated into chick embryo allantoic fluid HA of institute detects, and illustrates that deactivation is complete.After the deactivation according to 93: 7: 150 (allantoic fluid: tween: mixed mineral oil), utilize vortex mixer to stir repeatedly reaching adequately emulsified purpose, till not scattering on the water surface, so far vaccine promptly prepares and finishes when oozing with syringe.
1.2 HI antibody and 5B19 epi-position ELISA detection of antibodies behind the vaccine immunity
In order to determine the immunogenicity of H5N1/PR8-5B19 deactivation marker vaccine, and the chicken that can distinguish marker vaccine immunity chicken and non-marked vaccine immune chicken and natural infection H5N1 subtype avian influenza virus by polypeptide ELISA detection method, carry out the vaccine immunity test of SPF chicken.With 4 the week age SPF chicken be divided into 2 groups at random, 10 every group.Use the immunity of H5N1/PR8-5B19 deactivation marker vaccine for first group, every chicken 0.4ml divides 2 intramuscular injection through shank.Second group of usefulness, the immunity of H5N1/PR8-5B19 deactivation marker vaccine, every chicken 0.4ml is after immune two weeks, with same approach and dosage booster immunization once.Gather immune serum after the immunity weekly, 12 weeks after immunity, detect the HI antibody horizontal, detect immunity 3 weeks of back with the ELISA method simultaneously, 6 weeks and be directed to the ELISA antibody of 5B19 epi-position 12 weeks.
1.3H5N1/PR8-5B19 inactivated vaccine is to the immunoprotection test of SPF chicken
In order to estimate the immune protective effect of H5N1/PR8-5B19 deactivation marker vaccine, choose 10 4 SPF chicken muscle injection in age in week 0.4ml inactivated vaccines, set up 10 non-immune SPF chicken contrasts simultaneously.Immunity three weeks of back, gather serum, measure HI antibody, then with 10 6EID 50The H5N1 of dosage is strong, and malicious GS/GD/1/96 via intranasal application approach is attacked; observe morbidity and death condition behind the counteracting toxic substances; to determine the immune protective effect of vaccine; and 3 days larynx swab and cloaca swab behind the collection counteracting toxic substances; then gather the swab on the dead same day for chicken dead in 3 days, in 9-11 day instar chicken embryo, carry out titration of virus after the cotton swab collection.
2. result
HI antibody and 5B19 epi-position ELISA detection of antibodies after the marker vaccine immunity
2.1HI antibody test
As shown in figure 12, H5N1/PR8-5B19 deactivation marker vaccine with the 0.4ml single immunization after, a Zhou Junwei detects HI antibody behind 10 SPF chicken immunes, all chickens of 2 week all can detect HI antibody in the immunity back, HI antibody horizontal 4 weeks after immunity peak, for about 8log2, after detecting immunity 12 when week the HI antibody horizontal do not occur significantly descending, still more than 7.5log2.First week did not detect HI antibody yet after second group of test chicken immunity, immunity back during two weeks antibody horizontal reach about 5log2, then with same dosage booster immunization, continue to detect 12 weeks after the immunity, 5 weeks of antibody horizontal after the immunity first time peak, can reach 11.5log2, then at the peak last very long, the HI antibody horizontal of all chickens is still about 10log2 when detecting for 12 weeks.Above-mentioned experimental result shows that H5N1/PR8-5B19 deactivation marker vaccine has good immunogenicity, can induce to produce high-caliber HI antibody.
2.2 be directed to the ELISA antibody test of 5B19 epi-position
ELISA antibody test behind table 3 single immunization and the booster immunization
The antibody that is directed to the 5B19 epi-position with the polypeptide ELISA method set up after to single immunization and booster immunization in the immune chicken serum in 3,6 and 12 weeks detects, and the result is as shown in table 3.3 all 10 experimental chickens have only two chickens to produce ELISA antibody behind the single immunization, 6 weeks of immunity back and then had 7 chickens to produce ELISA antibody in 12 weeks, still have 3 chickens not detect antibody to the end.In the test group of immunity back 2 all booster immunizations first time, 3 weeks after immunity (with the immunity calculating first time) have 4 chickens to produce the ELISA antibody, and 6 weeks after immunity and 12 all 10 all chickens have all produced the antibody that is directed to the 5B19 epi-position.In addition with HI antibody relatively, be directed to that the ELISA antibody of 5B19 epi-position occurs more a little later, after immunity, had only indivedual chickens to produce antibody in 3 weeks.
2.3 marker vaccine immunoprotection test
With H5N1/PR8-5B19 deactivation marker vaccine immunity SPF chicken in age in 10 4 weeks, every chicken muscle injection 0.4ml inactivated vaccine is set up 10 non-immune SPF chicken contrasts simultaneously.Three weeks after the immunity, gather serum, measure HI antibody, HI antibody can reach more than the 8log2 after three weeks of H5N1/PR8-5B 19 deactivation marker vaccines immunity as a result, and matched group is the HI negative antibody.Then with 10 6EID 50The H5N1 of dosage is strong, and malicious GS/GD/1/96 via intranasal application approach is attacked, any clinical symptoms does not appear behind the marker vaccine immunity chicken counteracting toxic substances, there is not death yet, three days larynx swabs and cloaca swab all are not separated to virus behind the counteracting toxic substances, and 10 chickens of matched group are all dead in 6 days behind counteracting toxic substances, and larynx swab and cloaca swab all are separated to the virus of very high titre.Experimental result explanation marker vaccine provides protection (table 4) fully to immune chicken.
Table 4H5N1/PR8-5B19 inactivated vaccine is to the immunoprotection of SPF chicken
Figure C20061011440800281
The H5N1 subtype highly pathogenic avian influenza virus is popular in extensive range, has caused serious harm to aviculture, especially starts from H5N1 subtype avian influenza that Korea S sweeps across whole south east asia the end of the year 2003 and is very popular and has brought catastrophic strike to aviculture especially.Wild birds and duck, aquatic birds such as goose are the natural reservoir (of bird flu viruses) of influenza virus, influenza virus stasigenesis in these host, and further propagate and give poultry (Chin, et al, 2002; Webster, et al, 2002).This breadboard H5N1 of studies show that subtype avian influenza virus can exist in healthy duck body and any clinical symptoms (Chen, et al, 2004) not occur, and this is feasible, and fundamentally to eliminate bird flu virus seemingly impossible.Therefore, except strengthening epidemiological surveillance and bio-safety measure, vaccine immunity is active measures and the key link that anti-system bird flu takes place.
At present, in worldwide, the oil-emulsion inactivated vaccinating agent of totivirus is the avian influenza vaccine (Subbarao, et al, 2003) that is most widely used.The inactivated vaccine safety is good, and antigen component is complete, and immunogenicity is strong, can provide good immune protection to vaccinated flock.The use of oil seepage has effectively been controlled the diffusion of nineteen ninety-five Mexico HPAI epidemic situation and has further been spread (Garcia, et al, 1998).In China, H9N2 and H5N1 hypotype inactivated vaccine have also obtained using widely, for positive role has been played in the control of bird flu epidemic situation.Ideal inactivated vaccine kind strain should possess the condition of following 4 aspects: the first, good antigen specific aim, and promptly vaccine strain virus should have and the corresponding to antigenicity of popular virus; The second, height biological safety, vaccine strain virus should not have pathogenic to chicken and other animal and people; Three, height Embryo Gallus domesticus growth adaptability, vaccine strain should effectively duplicate on Embryo Gallus domesticus, and prepared vaccine antigen content height can be guaranteed immune effect.Four, the application of vaccine can not disturbed the investigation of epidemic situation and the epidemiological surveillance of bird flu.
Isolated in China to the H5N1 subtype avian influenza virus be highly pathogenic strain, and the part strain has obtained mammiferous highly pathogenic (Chen, et al, 2004), behind the inoculated into chick embryo, deadly Embryo Gallus domesticus that can be very fast, virus is difficult to duplicate the titre that reaches higher in Embryo Gallus domesticus, therefore it both can't be guaranteed the biological safety of vaccine as vaccine strain, can not guarantee the effective antigen amount and the immune effect of vaccine strain.Though do not have such shortcoming, lack again and the consistent antigenicity of China's epidemic isolates, so also be not suitable for as producing the anti-desirable strain of making China's avian influenza vaccine from the standard vaccine strain of external introduction from the attenuated vaccine strain of external introduction.
Therefore, in order to make up the Embryo Gallus domesticus growth adaptability that both has height, biological safety and good antigen specific aim, can not disturb the H5N1 subtype avian influenza virus inactivated vaccine strain of epidemiological surveillance again, manually rescue in this research and obtained H5N1 hypotype recombinant influenza strain H5N1/PR8-5B19, its six internal gene derive from high titre Embryo Gallus domesticus adapted strain A/Puerto Rico/8/34 (PR8) (H1N1), and HA and NA gene source are in the first strain H5N1 subtype avian influenza virus GS/GD/1/96 of isolated in China.Removed a plurality of successive basic amino acid of HA gene cracking site by molecular modification, made it possess the HA gene expression characteristics of Lowly Pathogenic Avian Influenza Virus.Inserted the advantage B cell epitope 5B19 of murine hepatitis virus S2 glycoprotein at the cervical region of NA gene.H5N1/PR8-5B19 serves as a mark that vaccine and a kind of polypeptidase connection immunoadsorption experimental technique combines can separator vaccine immune chicken and natural infection chicken.Its theoretical foundation is:
1.A/PR/8/34 be the most frequently used internal gene donor strain, so as to improving the growth characteristics of recombinant vaccine strain
Human influenza virus's vaccine strain of using in the world is the trivalent inactivated influenza virus vaccine at present, comprises H1N1, H3N2 hypotype A type influenza virus, and Type B influenza virus (Subbarao, et al, 2003).Wherein included H1N1 and H3N2 influenza virus all are that 6+2 resets influenza virus, just have two the surperficial gene HA and the NA of epidemic isolates, other six internal gene PB2, PB1, PA, NP, M and NS are provided by high titre Embryo Gallus domesticus adapted strain A/PR/8/34.Constructed like this recombinant influenza had both had the main immunogenic gene HA of epidemic isolates and NA and epidemic isolates and had had corresponding to antigen matching, and the immunoprotection to epidemic isolates can be provided to greatest extent.On the other hand, A/PR/8/34 is the Embryo Gallus domesticus growth adaptation strain after repeatedly going down to posterity, and does not cause death after the Embryo Gallus domesticus growth, possesses the high characteristics of good vaccine strain growth titre.The high Embryo Gallus domesticus growth characteristics of A/PR/8/34 mainly are by M gene-determined (Kilbourne, et al, 1969).
2.GS/GD/1/96 virus has the existing good antigen concordance of H5N1 subtype avian influenza virus with China
To China's different times since 1996; different regions; the isolating a large amount of H5 subtype highly pathogenic avian influenza virus of different hosts have carried out the antigenicity analysis of system; confirm that obviously variation does not take place institute's toxic strain on the HA gene that plays main immanoprotection action; with China's isolating the earliest goose source GS/GD/1/96 Strain antigenicity basically identical (Tian Guobin etc., 2004).
3. influenza virus HA gene is that molecular modification causes weak main target gene
The H5N1 subtype avian influenza virus is highly pathogenicity to chicken, and Embryo Gallus domesticus is high lethal (Shortridge, et al, 1998; Suarez, et al, 1998; Subbarao, et al, 1998).The viral allantoic fluid of cultivating in the 9-11 day instar chicken embryo is the main mode that influenza virus vaccine is produced.Although can prolong brooding time and improve its growth performance on Embryo Gallus domesticus by improving the incubation temperature of Embryo Gallus domesticus, the growth titre of H5N1 subtype avian influenza virus on Embryo Gallus domesticus is difficult to be improved, and is unfavorable for production of vaccine (Takada, et al, 1999).It is that bird flu virus has highly pathogenic important determiner and essential condition (Steinhauer, et al, 1999 that HA gene cracking site has a plurality of successive basic amino acids; Taubenberger, 1998), the HA cracking site of low pathogenicity AIV has only basic amino acid one arginine (R), and the HA cracking site of highly pathogenicity AIV contains continuous a plurality of basic amino acid.A plurality of basic amino acids of HA cracking site have improved the tissue tropism of high pathogenic avian influenza virus, can duplicate at a plurality of vitals of body, therefore cause the general and the fatal disease (Senne et al, 1996) of infected chicken.Therefore a Critical policies of vaccine design is exactly a plurality of basic amino acids of removing HA gene cracking site by the method for sudden change, make it have the HA gene expression characteristics of low pathogenicity bird flu virus, the influenza virus vaccine strain that is produced then becomes the low pathogenicity strain that Embryo Gallus domesticus is not had lethal.According to this kind strategy, American scholar has made up 1997 Hong Kong H5N1 hypotype human influenza virus HA gene molecules modifications in succession and has caused weak cold adaptation recombinant virus and the high titre adaptation of Embryo Gallus domesticus recombinant virus (Subbarao, et al, 2003; Li, et al, 1999).In this research, the RERRRKKR-of GS/GD/1/96HA gene cracking site is sported-RETR-, 4 basic amino acids have been lacked, and a lysine (K) sported threonine (T), to be not intended to sport CGA near the arginine codon AGA of cracking site, to avoid inserting a plurality of basic amino acids at the cracking site place again owing to the nucleotide sequence reproduce effect of polymerase.This situation sees Mexican highly pathogenic H5N2 subtype avian influenza virus of nineteen ninety-five, has only low pathogenicity virus in the groove initial, but along with the popular HPAI that then occurred, by analysis, be owing to the RNA sequence at the HA of cracking site gene has formed hairpin structure, be inserted into the hairpin structure of RNA after polymerase protein duplicates nucleotide sequence AAAGAA and increased by two extra basic amino acid lysines (K) and arginine (R), and make it to sport (the Garcia that has high pathogenic avian influenza virus, et al, 1996).
The NA gene of influenza virus be carry out ideal basis that molecular modification makes up the marker vaccine strain because of
In recent years, marker vaccine is widely used in the elimination plan of viral infectious in veterinary applications, more and more obtains paying attention to.But the strategy commonly used of marker vaccine design is exactly to have immunogenic antigen gene by modifying nonessential in the deletion vaccine strain virus.The basis of the detection method that matches with it is exactly to detect in the animal serum of marker vaccine immunity less than being directed to the antigenic antibody of disappearance, then contain the antigenic antibody of disappearance in the animal serum of natural infection, therefore just can make a distinction the animal of the animal of marker vaccine immunity and natural infection.This strategy has obtained application in the design of a lot of viral vaccines, such as pseudorabies virus, and rinderpest virus, (van Oirschot, et al, 1996 such as typical swine fever virus; Walsh, et al, 2000; Van Gennip, et al, 2002; Widkojoatmodjo, et al, 2000).Be exactly the antibody response that this antigen can produce quick and longer duration in infection animal wherein, in the animal serum of marker vaccine immunity, then can not detect at this antigenic antibody response as the antigenic major criterion of the disappearance of selected marker.
Above-mentioned strategy is seemingly inapplicable for the design of influenza virus marker vaccine.Because the HA gene is main immunogenic gene in influenza virus, be the major antigen composition of inducing protective immunity, if the vaccine strain that its disappearance is produced will lose corresponding immanoprotection action thereupon.An alternative method for the design of influenza virus marker vaccine is exactly to use the epi-position of irrelevant virus to substitute the part of a certain gene of influenza virus.The NA gene of influenza virus has such advantage, can replace the nonessential district of a part of NA gene with foreign epitope.
5. the reverse genetic operating system of influenza virus is the effective ways that make up good influenza virus vaccine strain
Utilization is the influenza virus gene group to be modified to make up have the best approach and the effective way of required feature vaccine strain based on the reverse genetic operating system of plasmid.Utilize this technology, can be a due feature set of good vaccine strain institute.The influenza virus vaccine strain that obtains of rescuing has six internal gene of reassortant virus and HA and the NA gene of H5N1 virus strain GS/GD/1/96.This vaccine strain had both had high titre Embryo Gallus domesticus growth property, has low pathogenicity again to chicken, to the not lethal of Embryo Gallus domesticus, and inserted the characteristic that the foreign epitope label can be used as marker vaccine at the NA gene, therefore also just possessed a good vaccine strain the various conditions that should possess.
Embryo Gallus domesticus does not cause death in 96 hours behind the H5N1/PR8-5B19 vaccine strain virus inoculation, though inserted the neuraminic acid enzymatic activity that ectogenic epi-position has obviously reduced NA at NA gene cervical region, but this not influence virus duplicating in Embryo Gallus domesticus, the blood clotting valency of 72 hours allantoic fluids can reach 10.5lo2 after infecting Embryo Gallus domesticus, it is not pathogenic to chicken that nasal cavity infection and vein infect the back, virus virulence do not occur and returns by force in Embryo Gallus domesticus goes down to posterity process, the molecular modification stable existence that shows the HA gene, and the MHV virus 5 B19 epi-position of inserting NA gene cervical region in virus goes down to posterity process does not lack, stablized as ectogenic label and to be gone down to posterity, guaranteed that therefore it causes the various conditions of weak marker vaccine strain as molecular modification.
After the immunity of H5N1/PR8-5B19 inactivated vaccine, second week can detect HI antibody, and in 4 weeks behind the single immunization, the HI antibody horizontal peaks, and significantly decline does not also appear in 12 weeks after immunity; Booster immunization once can obviously improve the HI antibody horizontal that immunity is produced behind the single immunization, the HI antibody titer can reach 11.5log2 when the peak, and can be at the peak period last very long, these show that the H5N1/PR8-5B19 inactivated vaccine has good immunogenicity.Attack to highly pathogenic H5N1 subtype avian influenza virus behind the vaccine immunity provides protection fully, does not fall ill behind the immune chicken counteracting toxic substances, and is not dead, and all is not separated to virus in throat swab and cloaca swab, guaranteed that it is as the vaccine effectiveness of application.
In order to distinguish immune animal and infection animal, what is more important detects the ELISA antibody that is directed to marker vaccine strain 5B19 epi-position.The 5B19 epi-position is the advantage B cell epitope in the murine hepatitis virus S2 glycoprotein, and this epi-position has very strong immunogenicity (Koolen, et al, 1990; Luytjes, et al, 1987,1989; Talbot, et al, 1984), and be used for the research (Mebatsion, et al, 2002) of newcastle disease virus marker vaccine.
16 aminoacid of synthetic MHV 5B19 epi-position as antigen coated elisa plate, carry out ELISA experiment with indirect method with it, detect the ELISA antibody that is directed to this epi-position in the immune chicken serum.In the negative serum sample being detected the criterion experiment of determining the ELISA detection method, the ELISA of 50 parts of negative chicken serums detects the OD value between 0.123-0.267, and negative value is high slightly.Possible reason is, the aminoacid at two ends has non-specific in 16 aminoacid of 5B19 epitope, therefore can with some the composition generation non-specific binding in the negative serum, further improving one's methods is 10 aminoacid " LLGCIGSTCA " that play the role of a nucleus in only synthetic this epi-position, bag is carried out ELISA by Sptting plate after perhaps utilizing prokaryotic expression system to express this epi-position, further optimizes the ELISA detection method with this.However, utilize the ELISA method of being set up, effectively separator vaccine immune chicken and natural infection chicken or non-marked vaccine immune chicken.The result shows, there is the part chicken to produce ELISA antibody in the 3rd week behind the marker vaccine single immunization, in the 6th week and 12 all 10 chickens, 7 chicken ELISA antibody test positives are arranged, ELISA antibody does not appear in other 3 chickens, and with vaccine immunity after the HI antibody that produced relatively, ELISA antibody occur more a little later.Tracing it to its cause may be in the structure of vaccine strain, and the 5B19 epi-position has been inserted NA gene cervical region, and this position is under the covering of the huge head of NA molecule, therefore makes the epi-position of inserting be not easy to be discerned by immune system.But, when behind single immunization with same approach and dosage booster immunization once, 4 chickens produce antibody during 3 weeks after immunity, when in 6 weeks and 12 weeks detections, 10 all chickens all detect antibody.Therefore should determine rational immune programme for children for the application of marker vaccine, and further determine immune duration, optimum immuning dose etc.In addition; by this favourable instrument that influenza virus gene group is modified of reverse genetic operating system; can be by further exploring; HA and NA gene for influenza virus carry out molecular modification; optimize foreign epitope and insert the site; make up the immanoprotection action with HA and NA of duplicating of appreciable impact influenza virus neither, also can produce very strong influenza virus marker vaccine strain at the foreign epitope antibody response.
Conclusion
1. utilize reverse genetic operating system artificial constructed a H5N1 subtype avian influenza virus marker vaccine strain H5N1/PR8-5B19 strain low pathogenicity, that the height Embryo Gallus domesticus adapts to.Virulence was not returned by force after virus repeatedly went down to posterity, but and its molecular marker 5B19 sequence genetic stability.
2. the small peptide that contains the 5B19 sequence with synthetic is an antigen, has set up the ELISA detection method of anti-5B19 epitope antibodies.
Produce high-caliber HI antibody 3.H5N1/PR8-5B19 can induce behind the once immune SPF chicken of inactivated vaccine, provide protection fully to the attack of H5N1 hypotype highly pathogenic avian influenza virus, promptly immune chicken does not fall ill, and is not dead, not toxin expelling.In just exempting from back 2 all booster immunizations once, all chickens all can produce can detected anti-5B19 antibody with routine immunization dosage.Therefore can H5N1/PR8-5B19 be the marker vaccine that the vaccine strain development can be differentiated mutually with natural infection, be used for the anti-system of H5N1 subtype avian influenza.
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17.Neumann，G.，Hughes，M.T.，Kawaoka，Y.Influenza?A?virus?NS2?protein?mediates?vRNPnuclear?export?through?NES-indePendent?interactionwith?hCRM1.EMBO?J.2000，19(24)：6751-6758.
18.Neumann，G.，Watanabe，T.，Kawaoka，Y.Plasmid-d?riven?formation?of?influenza?virus-likeparticles.J.Virol.2000，74(1)：547-551.
19.Neumann，G.，Watanabe，T.，Ito，H.，Watanabe，S.，Goto，H.，Gao，P.，Hughes，M.，Perez，D.，Donis，R.，Hoffmann，E.，Hobom，G.，Kawaka，Y.Generation?of?influenza?A?viruses?entirelyfrom?cloned?cDNAs.Proc.Natl.Acad.Sci.USA?1999，96(16)：9345-9350.
20.Ozaki，H.，Govorkova，E.A.，Li，C.，Xiong，X.，Webster，R.G.，Webby，R.J.Generation?ofhigh-yielding?influenza?A?viruses?in?African?green?monkey?kidney?cells?by?reverse?genetics.J.Virol.2004，78(4)：1851-1857.
21.Perey，N.，Barclay，W.S.，Garcia-Sastre，A.，Palese，P.Expression?of?a?foreign?protein?byinfluenza?A?virus.J.Virol.1994，68(7)：4486-4492.
22.Schnell，M.J.，Mebatsion，T.，Conzelmann，K.K.Infectious?rabies?viruses?from?cloned?cDNA.EMBO?J.1994，13(18)：4195-4203.
23.Subbarao，K.，Chen，H.，Swayne，D.，Mingay，L.，Fodor，E.，Brownlee，G.，Xu，X.，Lu，X.，Katz，J.，Cox，N.，Matsuoka，Y.Evaluation?of?a?genetically?modified?reassortant?H5N1?influenza?Avirus?vaccine?candidate?generated?by?plasmid-based?genetics.Virology?2003，305(1)：192-200.
Sequence table
＜110〉Harbin Veterinary Medicine Inst., China Academy of Agriculture
＜120〉avian influenza virus marking vaccine and its production and application
<130>IB064088
<160>4
<170>PatentIn?version?3.1
<210>1
<211>16
<212>PRT
＜213〉mice
<400>1
Ser?Pro?Leu?Leu?Gly?Cys?Ile?Gly?Ser?Thr?Cys?Ala?Glu?Asp?Gly?Asn
1 5 10 15
<210>2
<211>4550
<212>DNA
＜213〉plasmid sequence
<400>2
ttggccattg?catacgttgt?atctatatca?taatatgtac?atttatattg?gctcatgtcc 60
aatatgaccg?ccatgttggc?attgattatt?gactagttat?taatagtaat?caattacggg 120
gtcattagtt?catagcccat?atatggagtt?ccgcgttaca?taacttacgg?taaatggccc 180
gcctggctga?ccgcccaacg?acccccgccc?attgacgtca?ataatgacgt?atgttcccat 240
agtaacgcca?atagggactt?tccattgacg?tcaatgggtg?gagtatttac?ggtaaactgc 300
ccacttggca?gtacatcaag?tgtatcatat?gccaagtccg?ccccctattg?acgtcaatga 360
cggtaaatgg?cccgcctggc?attatgccca?gtacatgacc?ttacgggact?ttcctacttg 420
gcagtacatc?tacgtattag?tcatcgctat?taccatggtg?atgcggtttt?ggcagtacac 480
caatgggcgt?ggatagcggt?ttgactcacg?gggatttcca?agtctccacc?ccattgacgt 540
caatgggagt?ttgttttggc?accaaaatca?acgggacttt?ccaaaatgtc?gtaataaccc 600
cgccccgttg?acgcaaatgg?gcggtaggcg?tgtacggtgg?gaggtctata?taagcagagc 660
tcgtttagtg?aaccgtcaga?tcactagaag?ctttattgcg?gtagtttatc?acagttaaat 720
tgctaacgca?gtcagtgctt?ctgacacaac?agtctcgaac?ttaagctgca?gaagttggtc 780
gtgaggcact?gggcaggtaa?gtatcaaggt?tacaagacag?gtttaaggag?accaatagaa 840
actgggcttg?tcgagacaga?gaagactctt?gcgtttctga?taggcaccta?ttggtcttac 900
tgacatccac?tttgcctttc?tctccacagg?tgtccactcc?cagttcaatt?acagctctta 960
aggctagagt?acttaatacg?actcactata?ggctagcctc?gagaattcac?gcgtggtacc?1020
tctagagtcc?cattcgccat?taccgagggg?acggtcccct?cggaatgttg?cccagccggc?1080
gccagcgagg?aggctgggac?catgccggcc?agaagagcca?gatctggagc?tgctggtgca?1140
gcaataaagg?gagtcgggac?gatggtaatg?gaactaattc?ggatgataaa?gcgaggcatt?1200
aatgaccgga?acttctggag?aggcgagaat?ggacgaagaa?caaggattgc?atatgagaga 1260
atgtgcaaca?tcctcaaagg?gaaatttcaa?acagcagcac?aaaaagcaat?gatggatcag 1320
gtgcgagaaa?gcagaaatcc?tgggaatgct?gaaattgaag?atctggctct?tccaataacc 1380
cggcggccca?aaatgccgac?tcggagcgaa?agatatacct?cccccggggc?cgggaggtcg 1440
cgtcaccgac?cacgccgccg?gcccaggcga?cgcgcgacac?ggacacctgt?ccccaaaaac 1500
gccaccatcg?cagccacaca?cggagcgccc?ggggccctct?ggtcaacccc?aggacacacg 1560
cgggagcagc?gccgggccgg?ggacgccctc?ccggccgccc?gtgccacacg?caggggccgg 1620
cccgtctaga?gtcgacccgg?gcggccgctt?cgagcagaca?tgataagata?cattgatgag 1680
tttggacaaa?ccacaactag?aatgcagtga?aaaaaatgct?ttatttgtga?aatttgtgat 1740
gctattgctt?tatttgtaac?cattataagc?tgcaataaac?aagttaacaa?caacaattgc 1800
attcatttta?tgtttcaggt?tcagggggag?atgtgggagg?ttttttaaag?caagtaaaac 1860
ctctacaaat?gtggtaaaat?cgataaggat?ccgggctggc?gtaatagcga?agaggcccgc 1920
accgatcgcc?cttcccaaca?gttgcgcagc?ctgaatggcg?aatggacgcg?ccctgtagcg 1980
gcgcattaag?cgcggcgggt?gtggtggtta?cgcgcagcgt?gaccgctaca?cttgccagcg 2040
ccctagcgcc?cgctcctttc?gctttcttcc?cttcctttct?cgccacgttc?gccggctttc 2100
cccgtcaagc?tctaaatcgg?gggctccctt?tagggttccg?atttagtgct?ttacggcacc 2160
tcgaccccaa?aaaacttgat?tagggtgatg?gttcacgtag?tgggccatcg?ccctgataga 2220
cggtttttcg?ccctttgacg?ttggagtcca?cgttctttaa?tagtggactc?ttgttccaaa 2280
ctggaacaac?actcaaccct?atctcggtct?attcttttga?tttataaggg?attttgccga 2340
tttcggccta?ttggttaaaa?aatgagctga?tttaacaaaa?atttaacgcg?aattttaaca 2400
aaatattaac?gcttacaatt?tcctgatgcg?gtattttctc?cttacgcatc?tgtgcggtat 2460
ttcacaccgc?atatggtgca?ctctcagtac?aatctgctct?gatgccgcat?agttaagcca 2520
gccccgacac?ccgccaacac?ccgctgacgc?gccctgacgg?gcttgtctgc?tcccggcatc 2580
cgcttacaga?caagctgtga?ccgtctccgg?gagctgcatg?tgtcagaggt?tttcaccgtc 2640
atcaccgaaa?cgcgcgagac?gaaagggcct?cgtgatacgc?ctatttttat?aggttaatgt 2700
catgataata?atggtttctt?agacgtcagg?tggcactttt?cggggaaatg?tgcgcggaac 2760
ccctatttgt?ttatttttct?aaatacattc?aaatatgtat?ccgctcatga?gacaataacc 2820
ctgataaatg?cttcaataat?attgaaaaag?gaagagtatg?agtattcaac?atttccgtgt 2880
cgcccttatt?cccttttttg?cggcattttg?ccttcctgtt?tttgctcacc?cagaaacgct 2940
ggtgaaagta?aaagatgctg?aagatcagtt?gggtgcacga?gtgggttaca?tcgaactgga 3000
tctcaacagc?ggtaagatcc?ttgagagttt?tcgccccgaa?gaacgttttc?caatgatgag 3060
cacttttaaa?gttctgctat?gtggcgcggt?attatcccgt?attgacgccg?ggcaagagca 3120
actcggtcgc?cgcatacact?attctcagaa?tgacttggtt?gagtactcac?cagtcacaga 3180
aaagcatctt?acggatggca?tgacagtaag?agaattatgc?agtgctgcca?taaccatgag 3240
tgataacact?gcggccaact?tacttctgac?aacgatcgga?ggaccgaagg?agctaaccgc 3300
ttttttgcac?aacatggggg?atcatgtaac?tcgccttgat?cgttgggaac?cggagctgaa 3360
tgaagccata?ccaaacgacg?agcgtgacac?cacgatgcct?gtagcaatgg?caacaacgtt 3420
gcgcaaacta?ttaactggcg?aactacttac?tctagcttcc?cggcaacaat?taatagactg 3480
gatggaggcg?gataaagttg?caggaccact?tctgcgctcg?gcccttccgg?ctggctggtt 3540
tattgctgat?aaatctggag?ccggtgagcg?tgggtctcgc?ggtatcattg?cagcactggg 3600
gccagatggt?aagccctccc?gtatcgtagt?tatctacacg?acggggagtc?aggcaactat 3660
ggatgaacga?aatagacaga?tcgctgagat?aggtgcctca?ctgattaagc?attggtaact 3720
gtcagaccaa?gtttactcat?atatacttta?gattgattta?aaacttcatt?tttaatttaa 3780
aaggatctag?gtgaagatcc?tttttgataa?tctcatgacc?aaaatccctt?aacgtgagtt 3840
ttcgttccac?tgagcgtcag?accccgtaga?aaagatcaaa?ggatcttctt?gagatccttt 3900
ttttctgcgc?gtaatctgct?gcttgcaaac?aaaaaaacca?ccgctaccag?cggtggtttg 3960
tttgccggat?caagagctac?caactctttt?tccgaaggta?actggcttca?gcagagcgca 4020
gataccaaat?actgttcttc?tagtgtagcc?gtagttaggc?caccacttca?agaactctgt 4080
agcaccgcct?acatacctcg?ctctgctaat?cctgttacca?gtggctgctg?ccagtggcga 4140
taagtcgtgt?cttaccgggt?tggactcaag?acgatagtta?ccggataagg?cgcagcggtc 4200
gggctgaacg?gggggttcgt?gcacacagcc?cagcttggag?cgaacgacct?acaccgaact 4260
gagataccta?cagcgtgagc?tatgagaaag?cgccacgctt?cccgaaggga?gaaaggcgga 4320
caggtatccg?gtaagcggca?gggtcggaac?aggagagcgc?acgagggagc?ttccaggggg 4380
aaacgcctgg?tatctttata?gtcctgtcgg?gtttcgccac?ctctgacttg?agcgtcgatt 4440
tttgtgatgc?tcgtcagggg?ggcggagcct?atggaaaaac?gccagcaacg?cggccttttt 4500
acggttcctg?gccttttgct?ggccttttgc?tcacatggct?cgacagatct 4550
<210>3
<211>5805
<212>DNA
＜213〉plasmid sequence
<400>3
tcaatattgg?ccattagcca?tattattcat?tggttatata?gcataaatca?atattggcta 60
ttggccattg?catacgttgt?atctatatca?taatatgtac?atttatattg?gctcatgtcc 120
aatatgaccg?ccatgttggc?attgattatt?gactagttat?taatagtaat?caattacggg 180
gtcattagtt?catagcccat?atatggagtt?ccgcgttaca?taacttacgg?taaatggccc 240
gcctggctga?ccgcccaacg?acccccgccc?attgacgtca?ataatgacgt?atgttcccat 300
agtaacgcca?atagggactt?tccattgacg?tcaatgggtg?gagtatttac?ggtaaactgc 360
ccacttggca?gtacatcaag?tgtatcatat?gccaagtccg?ccccctattg?acgtcaatga 420
cggtaaatgg?cccgcctggc?attatgccca?gtacatgacc?ttacgggact?ttcctacttg 480
gcagtacatc?tacgtattag?tcatcgctat?taccatggtg?atgcggtttt?ggcagtacac 540
caatgggcgt?ggatagcggt?ttgactcacg?gggatttcca?agtctccacc?ccattgacgt 600
caatgggagt?ttgttttggc?accaaaatca?acgggacttt?ccaaaatgtc?gtaataaccc 660
cgccccgttg?acgcaaatgg?gcggtaggcg?tgtacggtgg?gaggtctata?taagcagagc 720
tcgtttagtg?aaccgtcaga?tcactagaag?ctttattgcg?gtagtttatc?acagttaaat 780
tgctaacgca?gtcagtgctt?ctgacacaac?agtctcgaac?ttaagctgca?gaagttggtc 840
gtgaggcact?gggcaggtaa?gtatcaaggt?tacaagacag?gtttaaggag?accaatagaa 900
actgggcttg?tcgagacaga?gaagactctt?gcgtttctga?taggcaccta?ttggtcttac 960
tgacatccac?tttgcctttc?tctccacagg?tgtccactcc?cagttcaatt?acagctctta 1020
aggctagagt?acttaatacg?actcactata?ggctagcctc?gagaattcac?gcgtggtacc 1080
tctagagtcc?cattcgccat?taccgagggg?acggtcccct?cggaatgttg?cccagccggc 1140
gccagcgagg?aggctgggac?catgccggcc?agcaaaagca?ggagattaaa?atgaatccaa 1200
atcagaagat?aataaccatt?ggatcaatct?gtatggtagt?tgggataatt?agcttgatgt 1260
tacaaattgg?gaacataatc?tcaatatggg?tcagtcattc?aattcagaca?gggaatcaac 1320
accaagctga?accatgcaat?caaagcatta?ttacttatga?aaacaacacc?tgggtaaatc 1380
aaacatatgt?caacatcagc?aataccaatt?ttcttactga?aaaagctgtg?gcttcagtaa 1440
cattagcggg?caattcatct?ctttgcccca?ttagcggatg?ggctgtacac?agtaaggaca 1500
acggtataag?aatcggttcc?aagggggatg?tgtttgttat?aagagagccg?ttcatctcat 1560
gctcccactt?ggaatgcaga?actttctttt?tgactcaggg?agccttgctg?aatgacaagc 1620
actccaatgg?gaccgtcaaa?gacagaagcc?ctcacagaac?attgatgagt?tgtcctgtgg 1680
gtgaggctcc?ctccccatat?aactcaaggt?ttgagtctgt?tgcttggtcg?gcaagtgctt 1740
gccatgatgg?caccagttgg?ttgacaattg?gaatttctgg?cccagacaat?ggggctgtgg 1800
ctgtattgaa?atacaacggc?ataataacag?acactatcaa?gagttggagg?aacaacatac 1860
tgagaactca?agagtctgaa?tgtgcatgtg?taaatggctc?ttgctttact?gtaatgactg 1920
acggaccaag?taatgggcag?gcctcatata?agatcttcaa?aatggaaaaa?gggaaagtag 1980
ttaaatcagt?cgaattgaat?gcccctaatt?atcactatga?ggagtgctcc?tgttatcctg 2040
atgctggcga?aatcacatgt?gtgtgcaggg?ataattggca?tggctcaaat?cggccatggg 2100
tatctttcaa?tcaaaatttg?gagtatcaaa?taggatatat?atgcagtgga?gttttcggag 2160
acaatccacg?ccccaatgat?ggaacaggca?gttgtggtcc?ggtgtcccct?aacggggcat 2220
atggagtaaa?agggttttca?tttaaatacg?gcaatggtgt?ttggatcggg?agaaccaaaa 2280
gcactaattc?caggagcggc?tttgaaatga?tttgggatcc?aaatgggtgg?actggaacgg 2340
acagtagctt?ctcggtgaaa?caagatatcg?tagcaataac?tgattggtca?ggatatagcg 2400
ggagttttgt?ccagcatcca?gaactgacag?gattagattg?cataagacct?tgtttctggg 2460
ttgagctaat?cagagggcgg?cccaaagaga?gcacaatttg?gactagtggg?agcagcatat 2520
ctttttgtgg?tgtaaatagt?gacactgtgg?gttggtcttg?gccagacgat?gccgagttgc 2580
cattcaccat?tgacaagtag?tttgttcaaa?aaactccttg?tttctactaa?taacccggcg 2640
gcccaaaatg?ccgactcgga?gcgaaagata?tacctccccc?ggggccggga?ggtcgcgtca 2700
ccgaccacgc?cgccggccca?ggcgacgcgc?gacacggaca?cctgtcccca?aaaacgccac 2760
catcgcagcc?acacacggag?cgcccggggc?cctctggtca?accccaggac?acacgcggga 2820
gcagcgccgg?gccggggacg?ccctcccggc?cgcccgtgcc?acacgcaggg?gccggcccgt 2880
ctagagtcga?cccgggcggc?cgcttcgagc?agacatgata?agatacattg?atgagtttgg 2940
acaaaccaca?actagaatgc?agtgaaaaaa?atgctttatt?tgtgaaattt?gtgatgctat 3000
tgctttattt?gtaaccatta?taagctgcaa?taaacaagtt?aacaacaaca?attgcattca 3060
ttttatgttt?caggttcagg?gggagatgtg?ggaggttttt?taaagcaagt?aaaacctcta 3120
caaatgtggt?aaaatcgata?aggatccggg?ctggcgtaat?agcgaagagg?cccgcaccga 3180
tcgcccttcc?caacagttgc?gcagcctgaa?tggcgaatgg?acgcgccctg?tagcggcgca 3240
ttaagcgcgg?cgggtgtggt?ggttacgcgc?agcgtgaccg?ctacacttgc?cagcgcccta 3300
gcgcccgctc?ctttcgcttt?cttcccttcc?tttctcgcca?cgttcgccgg?ctttccccgt 3360
caagctctaa?atcgggggct?ccctttaggg?ttccgattta?gtgctttacg?gcacctcgac 3420
cccaaaaaac?ttgattaggg?tgatggttca?cgtagtgggc?catcgccctg?atagacggtt 3480
tttcgccctt?tgacgttgga?gtccacgttc?tttaatagtg?gactcttgtt?ccaaactgga 3540
acaacactca?accctatctc?ggtctattct?tttgatttat?aagggatttt?gccgatttcg 3600
gcctattggt?taaaaaatga?gctgatttaa?caaaaattta?acgcgaattt?taacaaaata 3660
ttaacgctta?caatttcctg?atgcggtatt?ttctccttac?gcatctgtgc?ggtatttcac 3720
accgcatatg?gtgcactctc?agtacaatct?gctctgatgc?cgcatagtta?agccagcccc 3780
gacacccgcc?aacacccgct?gacgcgccct?gacgggcttg?tctgctcccg?gcatccgctt 3840
acagacaagc?tgtgaccgtc?tccgggagct?gcatgtgtca?gaggttttca?ccgtcatcac 3900
cgaaacgcgc?gagacgaaag?ggcctcgtga?tacgcctatt?tttataggtt?aatgtcatga 3960
taataatggt?ttcttagacg?tcaggtggca?cttttcgggg?aaatgtgcgc?ggaaccccta 4020
tttgtttatt?tttctaaata?cattcaaata?tgtatccgct?catgagacaa?taaccctgat 4080
aaatgcttca?ataatattga?aaaaggaaga?gtatgagtat?tcaacatttc?cgtgtcgccc 4140
ttattccctt?ttttgcggca?ttttgccttc?ctgtttttgc?tcacccagaa?acgctggtga 4200
aagtaaaaga?tgctgaagat?cagttgggtg?cacgagtggg?ttacatcgaa?ctggatctca 4260
acagcggtaa?gatccttgag?agttttcgcc?ccgaagaacg?ttttccaatg?atgagcactt 4320
ttaaagttct?gctatgtggc?gcggtattat?cccgtattga?cgccgggcaa?gagcaactcg 4380
gtcgccgcat?acactattct?cagaatgact?tggttgagta?ctcaccagtc?acagaaaagc 4440
atcttacgga?tggcatgaca?gtaagagaat?tatgcagtgc?tgccataacc?atgagtgata 4500
acactgcggc?caacttactt?ctgacaacga?tcggaggacc?gaaggagcta?accgcttttt 4560
tgcacaacat?gggggatcat?gtaactcgcc?ttgatcgttg?ggaaccggag?ctgaatgaag 4620
ccataccaaa?cgacgagcgt?gacaccacga?tgcctgtagc?aatggcaaca?acgttgcgca 4680
aactattaac?tggcgaacta?cttactctag?cttcccggca?acaattaata?gactggatgg 4740
aggcggataa?agttgcagga?ccacttctgc?gctcggccct?tccggctggc?tggtttattg 4800
ctgataaatc?tggagccggt?gagcgtgggt?ctcgcggtat?cattgcagca?ctggggccag 4860
atggtaagcc?ctcccgtatc?gtagttatct?acacgacggg?gagtcaggca?actatggatg 4920
aacgaaatag?acagatcgct?gagataggtg?cctcactgat?taagcattgg?taactgtcag 4980
accaagttta?ctcatatata?ctttagattg?atttaaaact?tcatttttaa?tttaaaagga 5040
tctaggtgaa?gatccttttt?gataatctca?tgaccaaaat?cccttaacgt?gagttttcgt 5100
tccactgagc?gtcagacccc?gtagaaaaga?tcaaaggatc?ttcttgagat?cctttttttc 5160
tgcgcgtaat?ctgctgcttg?caaacaaaaa?aaccaccgct?accagcggtg?gtttgtttgc 5220
cggatcaaga?gctaccaact?ctttttccga?aggtaactgg?cttcagcaga?gcgcagatac 5280
caaatactgt?tcttctagtg?tagccgtagt?taggccacca?cttcaagaac?tctgtagcac 5340
cgcctacata?cctcgctctg?ctaatcctgt?taccagtggc?tgctgccagt?ggcgataagt 5400
cgtgtcttac?cgggttggac?tcaagacgat?agttaccgga?taaggcgcag?cggtcgggct 5460
gaacgggggg?ttcgtgcaca?cagcccagct?tggagcgaac?gacctacacc?gaactgagat 5520
acctacagcg?tgagctatga?gaaagcgcca?cgcttcccga?agggagaaag?gcggacaggt 5580
atccggtaag?cggcagggtc?ggaacaggag?agcgcacgag?ggagcttcca?gggggaaacg 5640
cctggtatct?ttatagtcct?gtcgggtttc?gccacctctg?acttgagcgt?cgatttttgt 5700
gatgctcgtc?aggggggcgg?agcctatgga?aaaacgccag?caacgcggcc?tttttacggt 5760
tcctggcctt?ttgctggcct?tttgctcaca?tggctcgaca?gatct 5805
<210>4
<211>1767
<212>DNA
＜213〉artificial sequence
<400>4
agcaaaagca?ggggtctgat?ctgttaaaat?ggagaaaata?gtgcttcttc?ttgcaatagt 60
cagtcttgtc?aaaagtgatc?agatttgcat?tggttaccat?gcaaacaact?cgacagagca 120
ggttgacaca?ataatggaaa?agaacgttac?tgttacacat?gcccaagaca?tactggaaaa 180
gacacacaat?gggaagctct?gcgatctaaa?tggagtgaag?cctctcattt?tgagagattg 240
tagtgtagct?ggatggctcc?tcggaaaccc?tatgtgtgac?gaattcatca?atgtgccgga 300
atggtcttac?atagtggaga?aggccagtcc?agccaatgac?ctctgttacc?caggggattt 360
caacgactat?gaagaactga?aacacctatt?gagcagaaca?aaccattttg?agaaaattca 420
gatcatcccc?aaaagttctt?ggtccaatca?tgatgcctca?tcaggggtga?gctcagcatg 480
tccataccat?gggaggtcct?cctttttcag?aaatgtggta?tggcttatca?aaaagaacag 540
tgcataccca?acaataaaga?ggagctacaa?taataccaac?caagaagatc?ttttagtact 600
gtgggggatt?caccatccta?atgatgcggc?agagcagaca?aagctctatc?aaaacccaac 660
cacttacatt?tccgttggaa?catcaacact?gaaccagaga?ttggttccag?aaatagctac 720
tagacccaaa?gtaaacgggc?aaagtggaag?aatggagttc?ttctggacaa?ttttaaagcc 780
gaatgatgcc?atcaatttcg?agagtaatgg?aaatttcatt?gctccagaat?atgcatacaa 840
aattgtcaag?aaaggggact?cagcaattat?gaaaagtgaa?ttggaatatg?gtaactgcaa 900
caccaagtgt?caaactccaa?tgggggcgat?aaactctagt?atgccattcc?acaacataca 960
ccccctcacc?atcggggaat?gccccaaata?tgtgaaatca?aacagattag?tccttgcgac?1020
tggactcaga?aatacccctc?aaagagagac?tcgaggacta?tttggagcta?tagcaggttt?1080
tatagaggga?ggatggcagg?gaatggtaga?tggttggtat?gggtaccgcc?atagcaatga?1140
gcaggggagt?ggatacgctg?cagacaaaga?atccacccaa?aaggcaatag?atggagtcac?1200
caataaggtc?aactcgatca?ttgacaaaat?gaacactcag?tttgaggccg?ttggaaggga?1260
atttaataac?ttggaaagga?ggatagagaa?tttaaacaag?cagatggaag?acggactcct?1320
agatgtctgg?acttataatg?ctgaacttct?ggttctcatg?gaaaatgaga?gaactctaga?1380
ctttcatgac?tcaaatgtca?agaaccttta?tgacaaggtc?cgactacagc?ttagggataa?1440
tgcaaaggag?ctgggtaatg?gttgtttcga?gttctatcac?aaatgtgata?atgaatgtat?1500
ggaaagtgta?aaaaacggaa?cgtatgacta?cccgcagtat?tcagaagaag?caagactaaa?1560
cagagaggaa?ataagtggag?taaaattgga?atcaatggga?acttaccaaa?tactgtcaat?1620
ttattcaaca?gtggcgagtt?ccctagcact?ggcaatcatg?gtagctggtc?tatctttatg?1680
gatgtgctcc?aatggatcgt?tacaatgcag?aatttgcatt?taaatttgtg?agttcagctt?1740
gtagttaaaa?acacccttgt?ttctact 1767

Claims (10)

1. inserted the H5N1 subtype avian influenza virus marker vaccine H5N1/PR8-5B19 of the advantage B cell epitope 5B19 of murine hepatitis virus S2 glycoprotein at the cervical region of NA gene, the aminoacid sequence of the advantage B cell epitope 5B19 of wherein said murine hepatitis virus S2 glycoprotein is shown in SEQ ID NO.1, and it is the cervical region 49-67 position of NA gene that the cervical region of wherein said NA gene inserts the site.
2. according to the H5N1 subtype avian influenza virus marker vaccine of claim 1, it prepares by the method that may further comprise the steps:
(1) with the protein expressing plasmid of transcribing plasmid, pol gene, the HA genetic transcription plasmid of the negative strand viruses vRNA of H5N1 subtype avian influenza virus with insert NA gene transcription plasmid and the transfection reagent immixture of the advantage B cell epitope 5B19 of murine hepatitis virus S2 glycoprotein;
(2) host cell of the described virus replication permission of the mixture cotransfection of plasmid after will acting on and transfection reagent is cultivated the host cell after the transfection;
(3) cell after the results transfection, the inoculated into chick embryo allantoic cavity is rescued and is obtained recombinant virus.
3. according to the H5N1 subtype avian influenza virus marker vaccine of claim 2, wherein said HA gene is the saltant HA gene that manually lacks the continuous a plurality of basic amino acids in cracking site place.
4. according to the H5N1 subtype avian influenza virus marker vaccine of claim 1, it prepares by the method that may further comprise the steps:
(1) vRNA is transcribed plasmid pPOLI-PB2, pPOLI-PB1, pPOLI-PA, pPOLI-NP, pPOLI-M and pPOLI-NS, the protein expressing plasmid pcDNA-PB2 of four pol genes, pcDNA-PB1, pcDNA-PA and pcDNA-NP and molecular modification cause weak HA gene bidirectional transcription plasmid pBD-MutHA and insert the NA gene bidirectional transcription plasmid pBD-NA-5B19 and the transfection reagent immixture of foreign epitope;
(2) host cell of the described virus replication permission of the mixture cotransfection of plasmid after will acting on and transfection reagent is cultivated the host cell after the transfection;
(3) cell after the results transfection, the inoculated into chick embryo allantoic cavity is rescued and is obtained recombinant virus.
5. according to the H5N1 subtype avian influenza virus marker vaccine of claim 4, wherein said host cell is the 293T cell.
6. treat and/or prevent application in the medicine of the disease that bird flu virus causes according to the H5N1 subtype avian influenza virus marker vaccine of any one among the claim 1-5 in preparation.
7. be used for the application of the reagent of bird flu epidemiological surveillance in preparation according to the H5N1 subtype avian influenza virus marker vaccine of any one among the claim 1-5.
8. according to the application of claim 7, the wherein said reagent that is used for the bird flu epidemiological surveillance utilizes the ELISA method to detect the antibody of intravital HI antibody of target organism and 5B19 epi-position, and wherein said target organism has been applied described H5N1 subtype avian influenza virus marker vaccine.
9. inactivated avian influenza vaccine, it comprises according to any one H5N1 subtype avian influenza virus marker vaccine among the claim 1-5.
10. test kit that is used to distinguish avian influenza vaccine immunity poultry and bird flu natural infection poultry, it comprises according to any one H5N1 subtype avian influenza virus marker vaccine among the claim 1-5.
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