CN104804099A - Recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine - Google Patents
Recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine Download PDFInfo
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Abstract
The invention relates to preparation and application of a recombinant H9N2 subtype avian influenza (Avian influenza H9N2) enhanced multi-epitope vaccine. The vaccine takes neutralizing epitope, Th epitope, CTL epitope and B cell epitope of the major structural protein hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and matrix protein 2 (M2) of H9N2 subtype avian influenza virus as the frame structure, after being in flexible linker connection and series connection with cell factor interleukin 18 (chIL-18), the frame structure is then cloned into a pRSETB carrier to be transformed into escherichia coli, and then processes like fermentation, purification and emulsification are carried out, so that an avian influenza enhanced multi-epitope vaccine with ideal immunogenicity is achieved. Animal experiments indicate that the recombinant H9N2 subtype avian influenza enhanced multi-epitope vaccine is not only good in safety, but also can activate effective humoral immune and cellular immune responses.
Description
Technical field
The invention belongs to biotechnology genetically engineered field, relate generally to the reinforced polyepitope vaccines preparation and application of a kind of restructuring H9N2 subtype avian influenza.Particularly, utilize gene recombination technology, by major structural protein: the neutralizing epitope of hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and stromatin 2 (M2), Th epi-position, CTL epi-position and B cell epi-position are connected with cytokine fowl IL-18, and be cloned into carrier, transform Host Strains, by fermentation, the preparation of purifying, emulsifying process, obtain H9N2 subtype avian influenza polyepitope vaccines and this vaccine application in the avian infectious disease H9N2 subtype avian influenza of prevention of recombinating.
Background technology
Influenza virus can be divided into A, B and C tri-type.Wherein the distribution range of Type B and C type influenza virus is less, endanger lighter, and the harm of influenza A is the most serious, except the infection mankind, other Mammals and birds (Webster, 1992) can also be infected, and worldwide flu outbreak can be caused, great strike (Al-Mazrou A etal, 1991 are caused to the development of human health and livestock industry; ).Hemagglutinin (hemagglutinin, and neuraminidase (neuraminidase HA), NA) be the main surface glycoprotein of influenza virus 2 kinds and surface antigen, have hypospecificity, according to the difference of HA and NA, influenza A can be divided into again 17 kinds of HA (H1-H17) and 10 kinds of NA (N1-N10) hypotype (Zhu, 2013), wherein H1N1, H2N2 and H3N2 popular in the mankind (Earn, 2002; Fouchier, 2004).LPAIV is wide-scale distribution worldwide, and breaks through species barrier and be transmitted to people and pig (Alice, 2011; Ge, 2009; Park, 2011).1966, H9N2 hypotype AIV separates first from U.S. turkey group, since nineteen ninety-seven, and H9N2 hypotype AIV spread and epidemic in the bird in Asia, North America, the Middle East, Europe And Africa area, break through species barrier once in a while and be transmitted to people (Alice, 2011; Ge, 2009; Park, 2011).Henceforth H9N2 hypotype AIV is always in the foster fowl area wide-scale distribution that China is main.Though H9N2 hypotype AIV belongs to LPAIV, it can cause the decline of respiratory symptom, laying rate, immunosuppression and excite other virus or bacteria mixed infection, causes huge financial loss (Chen, 2012 to China's aviculture; Wu, 2010).
H9 subtype influenza virus is distributed widely in all over the world, particularly in Eurasia.According to the relation of its gene evolution, it is the same with other hypotype, can be divided into North America series and Eurasia series.Have been found that subbreed row (Guan Y, 1999) having three Eurasian series at least at present, respectively with Qa/HK/Gl/97, DK/HK/Y280/97, DK/HK/Y439/97 for representative.
The genome of influenza virus is made up of 8 gene fragments, the albumen of coding 12 kinds or more altogether, wherein 8 kinds is structural protein, comprise polysaccharase B2 (polymerase B2, PB2), polysaccharase B1 (polymerase B1, PB1), polysaccharase A (polymerase A, PA), HA, NP, NA, M1 and M2, Nonstructural Protein (nonstructural protein, NS) NS1 and NS2 is comprised, the PA-X of PA genes encoding is a kind of fusion rotein (Shi of influenza virus, 2012), PB1-F2 (the chen W relevant to the apoptosis that influenza virus particles mediates of PB1 genes encoding, 2001).
The red corpuscle of HA energy aggegation many animals, it is a kind of surface protein of influenza virus, it can be incorporated on the sialic acid receptor of host cell surface, help the antigenicity also changing virus in cell entry host cell, and then the supervision of escape host immune system, main determining factor (Guo, 2000 of virus variation, virulence and host specificity; Kimble, 2011).HA gene is the antigen producing neutralizing antibody; induction body immune system produces provide protection; it is the main determining factor of virus variation, virulence and host specificity; but also body can be stimulated to produce cytotoxic T lymphocyte (CTL) reaction, be that AIV induces body to produce the target antigen of protectiveness humoral immune reaction.The change of HA Argine Monohydrochloride and virus antigenicity make a variation closely related, and may change host specificity (Krause, 2010 of virus; Carrat, 2007).
NA albumen is another important surface antigen of influenza virus particles, has immunogenicity, can induce the generation of corresponding antibodies.The corresponding antibodies that NA brings out does not belong to neutralizing antibody, but can suppress virus diffusion (Hay, 1998) in vivo.The sialic acid of NA albumen energy cracking receptor surface glycoprotein end, makes progeny virus ion discharge from cell, is conducive to the propagation (Castrucci, 1993) of virus.
NP is the structural protein of being encoded by genomic fragment 5, is the main component forming nucleocapsid.It is generally acknowledged, NP is conservative structural protein, and in virus evolution process, aberration rate is very low, has type specificity.According to its antigenic difference, influenza virus can be divided into A type, Type B, C type.NP also has effect in the host specificity determining virus, is a fragment the most conservative in influenza virus gene group, has type specificity.NP is a kind of multifunctional protein, can form the nucleocapsid of virus, jointly form RNP complex body, prevent vRNA from decomposing, also have important effect (Bullido et al., 2000) in virus transcription and reproduction process with polysaccharase and RNA.NP is nucleoprotein, is rich in essence, sweet, Serine, is a kind of basic protein, causes host cell immunne response (Lamb, 1982; Portela, 2002), being the main shaft of volution nucleocapsid, is also the identification target position of CTL (cellulotoxic lymphocyte).
M is hydrophobic proteins, be rich in arginine, comprise M1 and M2 two kinds of protein (Treanor, 1990), the wherein extracellular region M2e sequence high conservative of M2, although M2 antibody does not have Neutralization effect, mouse experiment shows, use M2e monoclonal antibody passive immunization obviously can reduce the virus titer (Jegerlehner, 2004) of mouse nose and lung.Research shows (0kuda, 2001 that the cell killing (ADCC) that M2e antibody mediates mainly through antibody-dependant cell plays a role; Neirynck, 1999; Fan, 2004).
Two surface glycoproteins of influenza virus easily morph; the protection antibody that host produces is also for these two kinds of albumen; humoral immunization can impel antigenic variation (Corti again; 2011); especially some region is as the antigenic variation of HAI, causes the appearance of new epidemic isolates or some life-span short sub-evolutionary branching.And the ctl response that cellular immunization limits as MHC I is mainly for relatively more conservative inside albumen (Forrest, 2008), and have cross reactivity between different subtype and strain, the epi-position of CTL also can morph, the selection of the evolution of influenza virus then mainly antibody.Influenza infection early stage, the cellular immunization such as CTL and cytokine aspect plays a role in limiting virus diffusion etc.Phase after infection, the generation of neutralizing antibody recovers to play an important role to the removing of virus and host.Although HA and NA is the antigenic albumen of influenza virus most, only has and select conservative composition, antigen could be reduced and change the impact brought, thus resist the attack of most of strain, play provide protection widely (Ekiert, 2009; Ernst, 2006; ).
Current, the prevention of human influenza and bird flu depends on inactivated vaccine and the long-term use experience of attenuated live vaccine proves that they are safely and effectively, and has played huge effect.Vaccine immunity enhances the resistivity of body, decreases discharge and the propagation of virus.The vaccine that present stage is used for anti-bird flu processed is mainly the deactivation vaccine relying on chicken embryo propagation to produce, although there is lot of advantages in traditional deactivation vaccine, but still have many weak points, as disturbed epidemiological surveillance, rely on chicken embryo propagation cost high, go down to posterity in chicken embryo and easily morph, burn chicken embryo residual body pollution environment etc., and deactivation vaccine and living virus vaccine all cannot successfully manage present influenza virus makes a variation rapid, many subtype influenzas deposit popular, that kind boundary is day by day fuzzy trend.Thus the development strengthening new generation vaccine is further needed.
Interleukin-18 (Interleukin-18, IL-18) be the novel cell immune-regulating factor (Okamura of a kind of nineteen ninety-five reported first, 1995), Schneider in 2000 etc. (Schneider, 2000) obtain ChIL-18 cDNA first.Interleukin 18 has inducing T cell and NK cell produces IFN-γ (Kohno, 1997), promote T cell propagation, strengthen the various biological function (Micallef such as the cytotoxic activity of Thl cell and NK cell, 1996), in enhancing is immune, antitumor etc., there are great potential using value (Kanda, 2000; Marshall, 2006), at immune response, a kind of important cytokine especially in cellullar immunologic response, can be used as immunological adjuvant (Degen, 2005 of vaccine; Puehler, 2003).
Summary of the invention
The present invention is using the neutralizing epitope of H9N2 subtype avian influenza virus major structural protein hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and stromatin 2 (M2), Th epi-position, CTL epi-position and B cell epi-position as vaccine skeleton construction, with avian cytokines interleukin 18 (chIL-18) gene tandem, after expression in escherichia coli, through the technique such as protein purification, emulsification, obtain the reinforced polyepitope vaccines of restructuring H9N2 subtype avian influenza with Desirable immunogenic.Effective humoral immunization and cell immune response can be induced after this vaccine immunity target animals.
An object of the present invention there are provided a kind of newly can be used for prevent the reinforced polyepitope vaccines polypeptide of the restructuring of H9N2 subtype avian influenza and vaccine composition thereof; Two of object of the present invention there are provided structure and the preparation method of the reinforced polyepitope vaccines of described bird flu; Three of object of the present invention there are provided the engineering strain can expressing the reinforced polyepitope vaccines of described bird flu; Four of object of the present invention there are provided the preparation method of described polyepitope vaccines; Five of object of the present invention there are provided the purposes of the reinforced polyepitope vaccines of described bird flu in prevention H9N2 subtype avian influenza.
In first aspect, the invention provides a kind of reinforced polyepitope vaccines polypeptide of restructuring H9N2 subtype avian influenza for preventing and composition thereof.It contains the neutralizing epitope of major structural protein hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and stromatin 2 (M2), Th epi-position, CTL epi-position with, B cell epi-position and avian cytokines interleukin 18 (chIL-18).The reinforced polyepitope vaccines albumen of described bird flu or polypeptide or pharmacy acceptable salt and the carrier required for antigen expressed neoepitope Western.Carrier also can comprise the sequence of each epitope of coding separately, and series connection can be undertaken by genetic engineering method.Described vaccine also comprises nonimmune active substance, be the connection portion of each polypeptide, not there is the immunogenicity of epitope, not there is any adjuvanticity yet, mainly contain purification tag, joint peptide, chemically modified part, N end signal peptide and C and hold polyadenylic acid etc.Described pharmacy acceptable salt refers to nontoxicity, stimulation and transformation reactions, is applicable to the salt of human or animal tissues.Inactive substance and pharmacy acceptable salt are well known to those skilled in the art.Restructuring H9N2 subtype avian influenza reinforced polyepitope vaccines polypeptid acid sequence is as follows:
SCEEIAVCAVRLRENLCLYFEDDELECDAFCKDKTIKRFFRNVNSQLLVVRPDLNVAAFEDVTDQEVKSGSGMYFDIHCYKTTAPSAGMPVASSVQVEDKSYYMCCEKEHGKMVVRFREGEVPKDIPGESNIIFFKKTFTSCSSKAFKFEYSLEQGMFLAFEEEDSLRKLILKKLPREDEVDETTKFVTSHNERHNLGDIPGCKVAEYKNWSKPGGLNNKHSNGTTHDRIPGSGTPRDDGSSSSSNCIDPNNECAAYLTQKNNAYPTQDAQYTNNQEGSGNGTYNRRKYQEESKLERQGDIPGCKQVRESRNPGGSGRSNENPAHKGSGNTEGRTSDMGGPSCKRGPSTEGVPESMREEYRQEQ
In second aspect, the invention provides a kind of nucleic acid molecule, the bird flu reinforced polyepitope vaccines polypeptide of its coding described in first aspect present invention.Nucleotide of the present invention can be rna form, DNA form, many epitopes tandem sequence and fowl interleukin 18 sequence is synthesized by synthetic mode, then connect rear clone through genetically engineered operation and enter carrier, be transformed into intestinal bacteria, after screening, fermentation, purifying, obtain bird flu polyepitope vaccines polypeptide.Conventional molecular biology manipulations can be carried out in the present invention to this nucleic acid, as: PCR, digestion with restriction enzyme, connection etc., nucleic acid design 5 ' end and 3 ' end all add restriction enzyme site.Nucleotide sequence in preferred the present invention is as follows:
agc tgt gaa gag atc gct gtg tgt gca gta cgg ctt aga gaa aac ctc tgc ctc tat ttt gaa gat gatgag ctg gaa tgc gat gcc ttt tgt aag gat aaa act atc aaa cga ttc ttt cga aac gtc aat agc cag ttg cttgtg gtt cgt cca gat tta aac gtg gca gct ttt gaa gat gta aca gat cag gag gtg aaa tct ggc agt ggaatg tac ttc gac att cac tgt tac aaa acc acc gcg cct tca gca ggg atg cct gtt gca tcc agc gtc caggta gaa gat aag agt tac tac atg tgt tgt gag aaa gag cat ggg aaa atg gtt gtt cga ttt agg gaa ggagaa gtt ccc aaa gac att cct ggt gaa agt aac atc ata ttt ttc aaa aag aca ttt aca tct tgc agc tcc aaggct ttt aag ttc gag tac tca ctt gaa caa gga atg ttc ttg gcc ttt gag gaa gaa gac tcc tta aga aaa ctaatt tta aag aaa ctg ccg aga gaa gat gaa gtt gat gaa acc aca aaa ttc gta aca agt cat aat gaa aggcac aac cta ggt gat atc cca ggt tgc aag gtg gca gaa tac aag aat tgg tca aaa cca ggt ggt ctg aataac aag cac tca aat ggc act aca cat gat aga att cct ggt tct ggt aca cca aga gat gat ggt agc tccagc agc agc aac tgc ata gac cct aat aac gaa tgt gca gca tac ttg acc caa aag aac aac gct tac cctact cag gac gcc caa tac aca aat aat caa gaa ggt tct ggt aac ggg acc tac aac aga agg aag tat caagag gag tca aaa tta gaa aga cag ggt gat atc cca ggt tgc aag caa gtg cgg gaa agc aga aat cctggt ggt tct ggt aga tca aat gag aat cca gca cat aag ggt tct ggt aac act gaa ggc agg aca tcc gacatg ggt ggt cca tct tgt aaa aga ggg cct tct acg gaa gga gta cct gag tct atg agg gaa gag tat cggcag gaa cag
In the third aspect, the invention provides a kind of carrier, it is except containing the reinforced polyepitope vaccines nucleic acid molecule of coding H9N2 subtype avian influenza described in second aspect present invention, also containing being connected with this nucleotide sequence is exercisable, at the expression controlling elements needed for procaryotic cell expression (transcribe and translate).The most basic expression controlling elements comprises promotor, transcription terminator, enhanser, selected marker etc., and these controlling elements are known in the art.In the present invention, preferred e. coli bl21 (DE3, Plys) is as expression vector.
In fourth aspect, the invention provides a kind of host cell, it contains the carrier described in third aspect present invention.Host cell through to transform or transfection contains the gene order of proteins encoded of the present invention, after then there is good Inheritance and expression stability after testing, can be used for fermentation expression produce needed for the reinforced polyepitope vaccines polypeptide of H9N2 subtype avian influenza.
In the 5th, the invention provides the preparation method of the reinforced polyepitope vaccines of a kind of H9N2 subtype avian influenza, it comprises the following steps: engineering bacterium fermentation expresses H9N2 subtype avian influenza vaccine polypeptide, through thick purifying and polishing purification technique and follow-up emulsifying process, the polypeptide required for acquisition.The method wherein related to includes, but are not limited to bacterial cell disruption, inclusion body washing, centrifugal, sex change, affinity chromatography, hydrophobic chromatography, anion-exchange chromatography, reverse-phase chromatography, renaturation, emulsification etc.The preparation method related in the present invention is well known to those skilled in the art.
In the 6th, the invention provides a kind of for preventing the reinforced polyepitope vaccines of the recombinant fowl influenza of H9N2 subtype avian influenza, it comprises polypeptide described in first aspect present invention and pharmaceutically acceptable carrier.Described polyepitope vaccines can prevent the outburst of H9N2 subtype avian influenza.Pharmaceutically acceptable carrier of the present invention is immunostimulant or immunological adjuvant, and preferred immunological adjuvant is import white-oil adjuvant.
In the 7th, the invention provides the application of the restructuring H9N2 subtype avian influenza polyepitope vaccines described in the 6th aspect.Vaccine can necessarily effective dose intramuscular injection; intracutaneous or inoculated with subcutaneous injections animal; enough effective humoral immunization and cell immune response (see embodiment five, six, seven, eight, nine, ten) can be produced; neutralizing antibody is stimulated to produce; inducing peripheral blood and spleen organize CD4+ and CD8+T lymphopoiesis; there is provided antiviral activity simultaneously, significantly reduce toxin expelling, watch for animals and avoid the attack of H9N2 subtype avian influenza virus epidemic isolates.In addition, in embodiments of the invention, by carrying out laboratory safety test to vaccine, show that restructuring H9N2 subtype avian influenza polyepitope vaccines of the present invention is safe (see embodiment four).
In addition, it is pointed out that on the basis of the contextual disclosure of the application, the aspect that other have substantive distinguishing features of the present invention is apparent concerning the ordinary skill people of this area.In addition, the present invention which also uses open source literature, and their entire contents is all included in and carried out reference herein.
Accompanying drawing explanation
Following accompanying drawing for illustration of specific embodiment of the invention scheme, and is not used in the scope of the invention limiting and defined by claims.Fig. 1 recombinates H9N2 subtype avian influenza reinforced polyepitope vaccines expression plasmid pRSETB-chIL18-AIV (H9N2) design of graphics; Fig. 2 pRSETB-chIL18-AIV (H9N2) vector plasmid cleavage map, wherein swimming lane 1 is DNAmarker, molecular weight is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp from top to bottom, swimming lane 2 is digested plasmid, swimming lane 3 is non-digested plasmid, and swimming lane 4 is empty plasmid; Fig. 3 SDS-PAGE detects figure, and wherein swimming lane 1 is not for induce control sample, and swimming lane 2 is albumen Marker, is followed successively by 97KD, 66KD, 43KD, 31KD, 20KD, 14KD from top to bottom, and swimming lane 3,4 is induced samples, and arrow indication is the target protein of expressing; Fig. 4 Westernblot detects figure, and wherein swimming lane 1 is pre-dyed marker, is followed successively by 200KD, 140KD, 100KD, 80KD, 60KD, 50KD, 40KD, 30KD, 20KD from top to bottom, albumen for the purpose of swimming lane 2, and swimming lane 3 is negative control.Fig. 5 is that fermented sample SDSPAGE schemes: wherein swimming lane 1 is albumen Marker, and be followed successively by 97KD, 66KD, 43KD, 31KD, 20KD, 14KD from top to bottom, swimming lane 2 is non-induced samples, and swimming lane 3 is positive control, swimming lane 4 fermentation inducement samples; Fig. 6 is fermented sample Westernblot detection figure, wherein swimming lane 1 is pre-dyed marker, is followed successively by 200KD, 140KD, 100KD, 80KD, 60KD, 50KD, 40KD, 30KD, 20KD from top to bottom, and swimming lane 2 is positive control, swimming lane 3 is fermentation purification of samples, and swimming lane 4 is negative control.Fig. 7 is that ELISA method detects M2 protein I gG antibody titre results in serum; Fig. 8 is each experimental group lymphocyte stimulation indices detected result;
Embodiment
It is only exemplary description that concrete test method described in embodiment describes, and for elaborating the present invention, but does not form limitation of the scope of the invention, to be manyly changed to known by those skilled in the art according to of the present invention.
The mentality of designing of embodiment one H9N2 subtype avian influenza polyepitope vaccines albumen
The present invention is according to current domestic H9N2 subtype avian influenza Major Epidemic strain structural protein hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and stromatin 2 (M2) aminoacid sequence, utilize the strain of relevant biological information software DNASTAR, BIMAS and SYFPEITHI pop to carry out neutralizing epitope, Th epi-position, CTL epi-position and B cell epitope analysis, introduce avian cytokines interleukin 18 (chIL-18) as adjuvant molecules simultaneously.By after designed epi-position and the series connection of interleukin 18 molecular polypeptide in intestinal bacteria coexpression, by fermentation, purifying, the technique such as emulsification, obtain the reinforced polyepitope vaccines of H9N2 subtype avian influenza with Desirable immunogenic.The vaccine utilizing the present invention to prepare can effectively prevent H9N2 subtype avian influenza.
Comprehensive analysis domestic H9N2 subtype avian influenza virus epidemic strain genome sequence, antigenic structure, Advance of Epidemiological Research, be optimized design to recombinant fowl influenza polyepitope vaccines.The wetting ability that the present invention utilizes bioinformatics software to carry out its structural protein, antigenicity, plasticity-, the secondary structure of surface accessibility and Garnier-Robson is analyzed, predict possible B cell antigen epi-position, on the basis of CTL epi-position and t cell epitope, according to the similarity of epitope position and aminoacid sequence, analyze each epidemic isolates and have epitope, and with reference to the sequence information in GenBank, the epitope of prediction is compared, the conservative property of further analysis epitope in different virus strain, thus determine the Thelper antigen epitope polypeptide relevant to NA albumen 3 sections, with cell antigen epitope polypeptide 2 sections during HA albumen is relevant, the CTL antigen epitope polypeptide 3 sections that NP albumen is relevant, the B cell antigen epitope polypeptide 1 section that M2 albumen is relevant, the skeleton structure of vaccine will be formed after all epi-position series connection, add molecule adjuvant at skeleton nitrogen end simultaneously.The overall structure of this vaccine is:
Molecular Adjuvant(chIL 18)-NA Thelper Epitopel-NA Thelper Epitope2-NAThelper Epitope3-HA SN B Cell Epitope 1-HA SN B Cell Epitope2-NP CTL Epitope 1-NP CTL Epitope 2-NP CTL Epitope 3-M2B Cell Epitope
The structure of embodiment two coli expression carrier and expression strain
The polypeptide-coding nucleotide designed is served the synthesis of extra large handsome biotech company, nucleotide fragments two ends devise BamH I (5 ' end) and HindIII (3 ' end) restriction enzyme site respectively, being cloned on pMD18T carrier after this segment condense respectively, sequencing confirms to insert gene fragment consistent with implementation sequence (see sequence table).By recombinant plasmid called after pMD18T-chIL18-AIV (H9N2) respectively.With corresponding restriction enzyme, plasmid is carried out enzyme and cut process, coli expression carrier selects the pRSETB plasmid of Invitrogen company, also identical restriction enzyme ferment treatment is used, enzyme tangent condition: 10 μ l reaction systems, add 2 μ l plasmids in system, restriction enzyme is 5 activity units (New England Biolabs), adds 10 × damping fluid 1 μ l, deionized water polishing, 37 DEG C of enzymes cut 1.5 hours.Enzyme adds 1 μ l 200mM EDTA termination reaction after cutting end.In 1% agarose gel electrophoresis, electrophoresis 30 minutes.Under ultraviolet lamp, 2.86kb pRSETB plasmid and 1100bp chIL18-AIV (H9N2) fragment are cut, reclaim test kit specification sheets according to Qiagen company gel and carry out glue recovery.According to carrier: ratio independent and expression vector mixing by multi-epitope nucleotide fragments of fragment 1:2 ~ 3, reaction system 15 μ l, connected by T4DNA ligase enzyme, 16 DEG C of connections are spent the night, obtain recombinant plasmid called after pRSETB-chIL18-AIV (H9N2) respectively, (see Fig. 1), transform competent E. coli BL21 (DE3) pLysS.
Transform: pRSETB-chIL18-AIV (H9N2) is put thawed on ice, add 2 μ l ligation liquid, again mix, ice-water bath 30 minutes, 42 DEG C 30 seconds, then ice bath is put back to rapidly 1.5 minutes, add 1mL LB nutrient solution, 37 DEG C, quiescent culture 1 hour, 4000g low-temperature centrifugation 10 abandons supernatant second, with the resuspended thalline of 200 μ lLB substratum; Bacterium liquid is spread evenly across on the LB agar plate containing 100 μ g/mL penbritins, is inverted in 37 DEG C of thermostat containers and cultivates 12 ~ 16 hours, until Clone formation.
Qualification: the mono-clonal on picking flat board is in LB substratum, 37 DEG C, 200rpm shakes cultivation 12 hours, extract plasmid, restriction endonuclease BamH I and HindIII is used to carry out double digestion respectively, can cut out the clone of corresponding avian influenza vaccine gene size fragment, 1100bp, tentatively can be defined as positive colony (see Fig. 2); Positive colony carries out determined dna sequence and verifies its exactness (see sequence table) further.
Abduction delivering.By positive colony incubated overnight, morning next day, by 1: 100 switching, cultivates after 3 hours, adds 0.2mM IPTG, continues cultivation 4 hours, prepares sample; Conventional SDS-PAGE testing goal protein expression situation---at 45KD (see Fig. 3), see specific band for correct clone; Get correct clone, amplification culture, SDS-PAGE confirms to express correctly, and it expresses accuracy (see Fig. 4) to use conventional Western-blot to confirm further; After above-mentioned structure and qualification program, the positive colony selected can be carried out the foundation of original species word bank as engineering bacteria, bacterial classification name pRSETB-chIL18-AIV (H9N2)/BL21 (DE3, Plys).
The fermentation of embodiment three engineering bacteria, purifying and emulsification
Production bacterial classification is got in fermentation, is inoculated in (containing 100 μ g/mL penbritins) in 2mL LB liquid nutrient medium, 37 DEG C, 200rpm shaking culture 12 hours activated spawn.Access shaking flask with the inoculum size of 1: 100 again, 37 DEG C of shaking culture, to OD600=3, can be inoculated into fermentor tank in 10% ratio.Fermentation substratum is semisynthetic medium, prepares with distilled water.Correct dissolved oxygen and pH value electrode, open tank body and stir, revolution is 300rpm, the online sterilizing of tank body, when culture-liquid temp is down to 37.0 DEG C in tank, demarcates pH and dissolved oxygen (OD) zero point.Leavening temperature is 37.0 ± 0.1 DEG C, dissolved oxygen controls about 40%, pH controls 7.0, flow feeding 500mL during cultivation thalline OD600=1.0 ~ 1.2 after inoculation, within after feed supplement 1 hour, add IPTG (final concentration is 0.2mM) abduction delivering, continuous induction secondary fermentation in 5 hours terminates, and sampling is SDS-PAGE and is examined and determine expression (see Fig. 5).
The thalline that purifying will be collected, ultrasonic with carrying out after occlusion body washing lotion I (1%Triton X-100,20mMTris-cl PH8.0) suspendible, 2000W ultrasonic degradation 1 hour.4 DEG C, 12000rpm collected by centrifugation occlusion body, and wash occlusion body with occlusion body washing lotion II (1%DOC, 4M urea, 20mMTris-cl PH8.0) suspendible twice ultrasonic, secondary low-temperature centrifugation collects occlusion body.Occlusion body precipitation uses 8M urea, and 0.3% β-ME, 20mM Tris-cl (pH=8.00) mix, and stirring at room temperature 4 hours, 8000rpm low-temperature centrifugation 30min, discards precipitation.Metaprotein 1: 100 dilutes, and renaturation solution Tris (PH8.0) buffer system, adds 0.3M arginine, and 4 DEG C are stirred renaturation 24 hours.The 20mM phosphoric acid buffer of renaturation solution pH=8.0,0.5M sodium-chlor, 20mM imidazoles, affinity column in balance, with the 20mM phosphoric acid buffer of pH=8.0,0.5M sodium-chlor, 0.5M imidazoles wash-out; Must to recombinate H9N2 subtype avian influenza polyepitope vaccines work in-process stoste.
The PBS of the work in-process sterilizing of purifying is diluted to 100 μ g/mL by emulsification.Follow the example of the Montanide ISA 50V2 adjuvant of Guo Sai BIC Corp, through 121 DEG C, sterilizing 15 minutes, for subsequent use.By oil phase: aqueous phase=50: the proportions of 50, first oil phase is added in emulsion tank, start stirrer and slowly stir with the speed of 80 ~ 100r/min, slowly add aqueous phase, stir 2min again after adding, then with 5500r/min high-speed circulating emulsification 9min, make the single-phase vaccine of water-in-oil.
Embodiment four is recombinated H9N2 subtype avian influenza polyepitope vaccines safety testing
1 experimental animal 30 age in days SPF chicken 20 plumage.
2 vaccines are provided by research and development centre of company, and lot number is 140321,140323,140326.
Test adopts the test design of single-factor completely random, at random 20 30 age in days experimental animals is divided into 4 groups at random, 3 lot number groups and control group, often organizes 5, without weight differences between group.Immune group, in every chicken leg portion intramuscular injection vaccine 0.3mL/ head, is observed to 10 days, and control group uses physiological saline emulsion 0.3mL.After immunity, observe that adopting of each laboratory animal is raised, drunk water, spirit whether normally, whether have toxicity symptom, whether produce anaphylaxis, whether dead or occur other abnormal conditions.Before immunity and after off-test, all animals is weighed.Weighing results carries out statistical analysis.
3 results
3.1 clinical observation
All do not observe any anaphylaxis or toxicity symptom after 3 immune group animal immunes, the spirit of SPF chicken, adopt raise, drink water, activity, all are normal for ight soil etc., there is not obvious local inflammation equivalent damage clinical side reaction, and occur without death.
3.2 body weight change
The results are shown in Table 1, compared with the control, three immune group the weight of animals increase and control group difference remarkable (P > 0.05), show that the body weight of the immunity restructuring reinforced polyepitope vaccines of H9N2 subtype avian influenza to animal has no adverse effects.
Table 1 recombinates the reinforced polyepitope vaccines of H9N2 subtype avian influenza to the impact of SPF chicken body weight change
| Lot number | Body weight (g) before immunity | Immunity latter 10 days body weight (g) | Day weight gain (g) | P |
| 140321 | 246.34±23.37 | 435.84±41.33 | 18.95±3.52 | ＞0.05 |
| 140323 | 252.91±25.45 | 422.63±44.92 | 16.97±2.66 | ＞0.05 |
| 140326 | 248.06±21.62 | 431.49±41.05 | 18.43±1.84 | ＞0.05 |
| Control group | 262.54±24.88 | 433.78±38.86 | 17.12±2.57 | - |
The grouping of embodiment five animal experiment and immunity
1 vaccine is with to attack poison viral
Recombiant vaccine is provided by Hongqiao Ming Qin research and development centre, and lot number is that 140321,140323,140326, H9N2 inactivated vaccines (Re-2 strain) are so kind as to give by pharmaceutical development center, Yongshun, Guangdong, and lot number is 2014011.
2 experimental animals
One age in days SPF chicken 50, raises two weeks in advance, conforms.
3 groupings are divided into 5 groups with immunity, often organize 10.
SPF chicken in two week age is divided into 5 groups, polyepitope vaccines group, inactivated vaccine group and PBS control group.Immune group is intramuscular injection polyepitope vaccines and inactivated vaccine (Re-2 strain) respectively, and only, head exempts from latter 14 days same dose booster immunizations to 0.15mL/, and PBS control group immunity PBS emulsion, 0.15mL/ only.Respectively at immunity blood sampling in latter 7 days, 14 days, 21 days, 28 days and for M2 protein I gG antibody test, HI antibody titers detects and lymphopoiesis stimulation index measures, and within latter 28 days, carries out challenge test in immunity, detects for toxin expelling.
Embodiment six M2 protein I gG antibody test
Gather the serum of 1,2,3,4 week after first immunisation, by end dilution ELISA detection specificity IgG antibody.Microwell plate (Nunc Maxisorp, Nalge Nunc International, Denmark) the vaccine protein bag quilt of purifying, 2 ~ 8 DEG C are spent the night; Antiserum(antisera), in 37 DEG C of closed 1h, is done 1: 10,1: 10 by 5% skimmed milk
2, 1: 10
3, 1: 10
4, 1: 10
5, 1: 10
6, 1: 10
7, 1: 10
8doubly dilution, uses the serum of immune PBS as negative control simultaneously, adds microwell plate, 100 μ L/ holes, 1h is hatched, then by the ELIAS secondary antibody (1: 10000, Sigma, St louis) of the anti-chicken IgG-HRP of rabbit in 37 DEG C, add microwell plate, 100 μ L/ holes, hatch 1h in 37 DEG C.The colour developing of TAB substrate lucifuge 10min, 2MH
2s0
4termination reaction, detects absorbance under 450nm wavelength.Using the dilution inverse of highest serum as antibody titers, its average absorbance value (>=0.2) higher than the average absorbance value+2SD of preimmune serum, as cutoff value.
Can as seen from Figure 2, the 1st week after head exempts from, immune group animal just there occurs antibody male rotary, and average titer is respectively 1: 10
2with 1: 10
3, in 2,3,4 subsequently, a week antibody horizontal raises successively, and to the 4th week, four final mean antibody levels of immunity reached 1: 10
5above, wherein recombiant vaccine 140323 groups reaches: 1: 10
6above, can't detect antibody in the serum of contrary PBS control group mice.Above result shows that restructuring H9N2 bird flu polyepitope vaccines that the present invention designs can the specificity M2 protein I gG antibody of induced high levels.
Embodiment seven blood clotting suppresses the detection of (HI) antibody
The preparation of 11% red cell suspension
Gather the Trisodium Citrate salt solution anti-freezing of 2% of experimental group chicken blood, add the brine red corpuscle of 5 times of volumes, the centrifugal 10min of 1000rpm, after washing three times continuously, add the resuspended red corpuscle of physiological saline of cell pack 100 times of volumes, shake up rearmounted 4 DEG C for subsequent use.
2 Microhemagglutinations test (HA)
Viral hemoagglutination titration: get 96 hole V-arrangement micro-reaction plates, adds 25 μ L PBS with micropipet in 1 ~ 12 every hole, hole, drips 8 rows altogether, and 25 μ L PBS are added in the 1st row hole of rear four rows again.Draw 25 μ L standard avian influenza antigen (Harbin dimension section) to join in the 1st row hole, blow and beat 3 ~ 5 fully mixings.Be added to the 2nd row hole from the 1st row hole antigen liquid drawn after 25 μ L mixings, drawing 25 μ L after mixing joins in the 3rd row hole, carry out serial doubling dilution successively to the 11st row hole, finally respectively draw 25 μ L from the 11st row hole and abandon it, if the 12nd row hole is red corpuscle contrast.Right-to-left adds the chicken erythrocyte suspension of 25 μ L 1% successively to each hole.Sptting plate is placed in 1min that micro oscillator vibrates, room temperature (20 ~ 25 DEG C) leaves standstill observations after 30min, occurs that the most high dilution of the virus of 100% red cell agglutination is the viral agglutination valency of this sample.
3 microdose cytopathogenic effect assay (HI)
1 to the 11 hole of often arranging in V-shaped 96 hole blood clotting suppressing plate adds 25 μ L PBS solution, and the 12nd hole adds 50 μ L PBS solution as negative control; Add the tested serum of 25 μ L in the 1st hole, fully shift out 25 μ L after mixing and add to the 2nd hole, the like, doubling dilution is to the 10th hole, and the 10th hole discards 25 μ L, if the 11st hole is virus control, the 12nd hole is red corpuscle contrast.Respectively add 25 μ L 4 unit antigens in 1st ~ 11 holes, tapping Sptting plate, makes reactant mix, left at room temperature 30min.Right-to-left adds the chicken erythrocyte suspension of 25 μ L 1% successively to each hole.Sptting plate is placed in 1min that micro oscillator vibrates, room temperature (20 ~ 25 DEG C) leaves standstill observations after 40min, gets final product result of determination when red corpuscle control wells becomes obvious button shape to sink at the bottom of hole.
4 results
The HI antibody horizontal of all immune group raised gradually along with the time, to latter 28 days of immunity, the HI antibody titers of recombinant multi-epitope vaccine group is all more than 10, and inactivated vaccine group is 9.9, does not form significant difference (P > 0.05) between immune group.All immune group and PBS control group form pole significant difference (P < 0.01) (see table 2).
After table 2 SPF chicken immune, the HI antibody horizontal of different time sections detects
Note: be designated as difference extremely significantly (P < 0.01) with on the different letter of column data.
Embodiment eight lymphopoiesis stimulation index measures
Latter 28 days of immunity, asepticly takes each group of SPF chicken periphery anticoagulation 2mL, and mixes gently with isopyknic Hank ' s liquid, and the anticoagulation diluted slowly is added the centrifuge tube containing 4mL lymphocyte separation medium.Under normal temperature, the centrifugal 15min of 2000rpm.Carefully shift the white cellular layer be positioned in the middle of centrifuge tube with pipettor, and add the H9N2 (Re-2 strain) through uv irradiating deactivation, 4 DEG C, 2000rpm, centrifugal 10min, in triplicate, after cell counting, adjust number to 1 × 10 of every tube cell
6cells/mL.Getting 100 μ L has adjusted in cell diluent to 96 orifice plate of concentration, sets concanavalin A (ConA) positive control, negative control and blank group simultaneously, and often organize three repetitions, every hole final volume is 200 μ L.Be placed in 5%CO
2incubator, after 37 DEG C of cultivation 44h, every hole adds 10 μ L 5.0mg/mL MTT solution, and after continuing to cultivate 4h, under normal temperature, the centrifugal 5min of 2000rpm, discards 100 μ L supernatants, add 100mL DMSO lysate, 37 DEG C of lucifuges vibration 15min.Measure the absorbancy at 570nm place, by formulae discovery lymphopoiesis stimulation index (stimulation index, SI), SI=(test class value-negative control value)/negative control value.
Result
Similar with hemagglutination inhibition reaction, observe the lymphocyte proliferation activity of highest level in epitope polypeptide vaccine group and inactivated vaccine group, see Fig. 8.The T lymphopoiesis of control group is starkly lower than other immune group (P < 0.05), the stimulation index mean level (ML) of recombination epitope vaccine group a little more than inactivated vaccine group, but does not present significant difference (P > 0.05).
After embodiment nine attacks poison, toxin expelling rate detects
1RNA gathers throat and cloacal swabs in the 3rd day, 5 days, 7 days after being extracted from and attacking poison respectively, the toxin expelling situation of each group of test chicken is detected: brush,throat and cloacal swabs are put into the 0.01moL/L pH7.0 ~ 7.4PBS filling 1mL sterilizing respectively and (includes penicillin 2000IU/mL by RT-PCR method, Streptomycin sulphate 2mg/mL) in, add a cover, number.Each sample virus total RNA is extracted according to Trizol test kit specification sheets.
The synthetic system RNA 6 μ L of 2cDNA adds random primer (10 μm of ol/L) 2 μ L, dNTP (10mmol/Leach) 2 μ L, RNase inhibitor (RNasin) 0.5 μ L (40U/ μ L), AMV ThermoScript II 0.5 μ L (5U/ μ L) and 5 × RT damping fluid 4ml, finally mends to 20 μ L with DEPC water.42 DEG C of reverse transcriptions 1 hour, then 94 DEG C of 10min.
3PCR increases
According to the gene order of the H9N2 strain that Genebank has delivered, with the HAl gene-specific primer a pair of Primer 5.0 software design 2 couples of H9N2, upstream primer Primer1:5 '-AGCAAAAGCAGGGGAA-3 ', downstream primer Primer2:5 '-TTGTGGAACGGCAATGTGGTG-3 '.
4PCR reaction system includes each 1 μ L of P1, P2 primer, dNTP2 μ L (10mmol/L), Taq DNA polymerase 1 μ L (5U/ μ L), cDNA3 μ L, 10 × PCR damping fluid 5 μ L, finally mends to 50 μ L with distilled water.Slightly do centrifugal after, carry out pcr amplification.PCR optimum reaction condition: 95 DEG C of denaturation 5min, 95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s, circulation 30cycles; Last 72 DEG C extend 10min.
5 differentiate: carry out conventional agarose gel electrophoresis, observations under 80 ~ 100v, 30min gel imaging system, and at 938bp place, visible obviously amplified fragments is positive findings, i.e. toxin expelling.
6 results
After attacking poison as can be seen from Table 3, the 3rd day toxin expelling rate is the highest, and larynx tracheae toxin expelling rate is higher than cloaca toxin expelling rate; Recombiant vaccine immune group toxin expelling rate is minimum, and occurred 2 toxin expellings except 140323 groups at 3 days, toxin expelling situation does not all appear in other recombiant vaccine lot number groups, and inactivated vaccine group apparently higher than recombiant vaccine group, does not find toxin expelling after 7 days the 3rd day toxin expelling rate.
After poison attacked by table 3, the virus of each group cotton swab detects result
Note: toxin expelling rate=toxin expelling size of animal/this test group of animals total amount
The detection of embodiment ten immune mouse t lymphocyte subset class quantity
1 laboratory animal grouping and sampling 90 mouse are divided into 5 groups at random, often organize 18, and only, immune group subcutaneous injection PBS emulsion 200 μ L/ only for immune group every subcutaneous injection vaccine 200 μ L/.After head exempts from, two weeks booster immunizations once, and immunization method and dosage are exempted from consistent with head.25th day, 32 days, 39 days, 46 days each time periods after head exempts from, slaughter 3 mouse, pluck eyeball and get blood for each group.Anticoagulation is separated lymphocyte and does t lymphocyte subset alanysis, and aseptic its spleen of getting does t lymphocyte subset alanysis simultaneously.
The separation blood sampling 0.5ml of 2 peripheral blood lymphocytes, add Hank ' s liquid 0.5ml, mixing, is added on 1ml lymphocytes separating solution face gently.Centrifugal 15 minutes of 2000r/min, lymphocyte in the middle of careful collection, with Hank ' s liquid centrifuge washing cell precipitation 2 times, then with RPMI1640 substratum (containing Sodium.alpha.-ketopropionate 1mmol/L, beta-mercaptoethanol 2 × 10
-6mol/L, penicillin 100U/ml, Streptomycin sulphate 100 μ g/ml, calf serum 10%), cell dilution is become 1 × 10
7individual/ml, makes single cell suspension.
The preparation of 3 spleen list lymphocyte suspensions is aseptic takes mouse spleen, adds 3ml Hank ' s, (200 order) shreds little copper mesh, extruding, and cell suspension on copper mesh is collected sterile centrifugation tube, and centrifugal 5 minutes of 1500r/min, abandons supernatant liquor.Precipitate 2 times with Hank ' s liquid centrifuge washing pipe floor cells, cell dilution is become 2 × 10 by (2) in the same way
7individual/ml, makes single cell suspension.
The detection of 4T lymphocyte call subtype quantity
Anticoagulation 0.1ml is got in the pre-treatment of 4.1 peripheral blood lymphocytes, adds 8ml erythrocyte cracked liquid, room temperature effect 10 minutes, and centrifugal 10 minutes of 1500r/min, abandons supernatant liquor, add 5ml PBS, suspendible, and centrifugal 10 minutes of 1500r/min repeats 2 times.
The pre-treatment extracting spleen cell suspension 0.1ml of 4.2 splenic lymphocyte, adds 5ml PBS, centrifugal 10 minutes of 1500r/min, repeats 2 times with 0.5ml PBS suspension liquid.
The lymphocytic fluorescent mark FITC of 4.3T marks rat anti-mouse monoclonal antibody (0.1mg/ml), dilutes 10 times (0.01mg/ml).Often pipe obtained cell suspension 0.5ml, dilutes monoclonal antibody 10 μ l (0.1 μ g) respectively, and 4 DEG C act on 1 hour, and PBS damping fluid washes 1 time, is suspended and test sample by pipe floor cells 1ml PBS.
4.4FACS detects and data processing FACS detects 3000 cells, and the data obtained carries out statistical procedures, calculates its mean value.
5 results
5.1CD4+T the change of lymphocyte call subtype quantity
5.1.1 the change recombinant multi-epitope vaccine immunity group peripheral blood CD4+T lymphocyte number overall trend of peripheral blood CD4+T lymphocyte quantity is for increase gradually with immunization time, start slightly to decline after arriving peak by 32 days, and the peripheral blood CD4+T lymphocyte of inactivated vaccine combination PBS control group does not present the growth trend changed with the change of immunization time.Polyepitope vaccines group peripheral blood CD4+T lymphocyte number is significantly higher than deactivation vaccine group cell count (P < 0.05) and PBS cellular control unit number (P < 0.05), and inactivated vaccine group peripheral blood CD4+T lymphocyte number and PBS control group are without significant difference (P > 0.05) (be shown in table 4).
Table 4 vaccine immune mouse peripheral blood CD4+T cellular change
| Grouping | 25 days | 32 days | 39 days | 46 days |
| Restructuring 140321 | 31.9±2.04 | 42.5±1.81 | 33.6±2.93 | 35.8±3.34 |
| Restructuring 140323 | 33.1±3.72 | 42.8±3.15 | 35.2±2.62 | 36.5±1.92 |
| Restructuring 140326 | 32.6±1.69 | 43.4±3.27 | 34.9±2.54 | 35.2±2.88 |
| Deactivation 2014011 | 22.8±1.74 | 20.5±1.55 | 20.1±1.95 | 21.7±2.02 |
| PBS control group | 15.9±1.16 | 16.3±2.36 | 15.5±0.97 | 14.4±1.61 |
5.1.2 the change recombiant vaccine spleen CD4+T lymphocyte quantity overall trend of spleen CD4+T lymphocyte quantity is for increase gradually with immunization time, start after arriving peak by 39 days to decline, to 46 days, without significant difference (P > 0.05) between immune group, but inactivated vaccine group and PBS control group do not change with the change of immunization time this consistent with the detected result of peripheral blood lymphocyte quantity (see table 5).After immunity during 25 ~ 39 days, recombination epitope vaccine group spleen CD4+T lymphocyte number is significantly higher than deactivation vaccine group cell count (P < 0.05) and PBS control group (P < 0.01), and inactivated vaccine group is overall does not present significant difference (P > 0.05) higher than PBS control group, this is consistent with peripheral blood CD4+T lymphocyte number detected result.
Table 5 vaccine immune mouse spleen CD4+T cellular change
| Grouping | 25 days | 32 days | 39 days | 46 days |
| Restructuring 140321 | 22.3±2.52 | 38.4±1.75 | 31.5±2.18 | 18.7±1.49 |
| Restructuring 140323 | 23.1±1.83 | 37.9±3.35 | 30.6±2.86 | 17.3±1.91 |
| Restructuring 140326 | 21.5±2.08 | 38.1±2.17 | 30.3±3.84 | 18.2±3.42 |
| Deactivation 2014011 | 13.8±1.15 | 12.4±2.36 | 13.1±2.09 | 12.9±1.65 |
| PBS control group | 10.1±1.09 | 9.8±1.23 | 10.5±1.81 | 9.2±0.84 |
5.2CD8+T the change of lymphocyte call subtype quantity
5.2.1 the change recombinant multi-epitope vaccine immunity group peripheral blood CD8+T lymphocyte number of peripheral blood CD8+T lymphocyte quantity increases along with immunization time and increases, do not present downtrending, to 46 days, quantity and do not present significant difference (P > 0.05) for 39 days, and inactivated vaccine group and PBS control group CD8+T lymphocyte quantity do not present obvious increase and decrease.Latter 25 days of immunity, recombinant multi-epitope vaccine group CD8+T lymphocyte quantity and inactivated vaccine group without significant difference (P > 0.05), but apparently higher than control group (P < 0.05); Latter 32 ~ 46 days of immunity, recombinant multi-epitope vaccine group CD8+T lymphocyte quantity is apparently higher than inactivated vaccine group and PBS control group, and difference extremely significantly (P < 0.01) (see table 6).
The lymphocytic change of table 6 vaccine immune mouse peripheral blood CD8+T
| Grouping | 25 days | 32 days | 39 days | 46 days |
| Restructuring 140321 | 11.09±1.62 | 17.85±2.44 | 23.64±2.18 | 24.36±3.02 |
| Restructuring 140323 | 12.11±2.85 | 18.93±1.73 | 24.72±2.09 | 25.07±2.16 |
| Restructuring 140326 | 11.76±1.17 | 17.52±2.38 | 24.33±1.95 | 24.15±2.63 |
| Deactivation 2014011 | 10.45±1.81 | 10.91±1.65 | 11.08±2.73 | 10.83±1.48 |
| PBS control group | 7.99±0.83 | 8.25±1.16 | 7.72±1.22 | 7.69±0.95 |
5.2.2 the change recombinant multi-epitope vaccine immunity group spleen CD8+T lymphocyte number of spleen CD8+T lymphocyte quantity is higher than inactivated vaccine group and PBS control group, and present pole significant difference (P < 0.01), although inactivated vaccine group is higher than PBS control group, but do not present significant difference (P > 0.05), and the CD8+T lymphocyte quantity of inactivated vaccine group and PBS control group does not change along with time variations, this is consistent with peripheral blood CD8+T lymphocyte number detected result.To present ascendant trend different always from peripheral blood, and spleen organizes CD8+T lymphocyte quantity to peak at 32 days, after this falls after rise rapidly, to 46 days, a little more than inactivated vaccine group, but without significant difference (P > 0.05).(see table 7).
Table 7 vaccine immune mouse spleen organizes the lymphocytic change of CD8+T
| Grouping | 25 days | 32 days | 39 days | 46 days |
| Restructuring 140321 | 15.82±1.33 | 23.15±2.64 | 15.29±1.92 | 7.08±1.48 |
| Restructuring 140323 | 13.38±2.54 | 22.47±3.08 | 14.82±2.29 | 6.55±2.03 |
| Restructuring 140326 | 15.35±1.62 | 22.81±2.83 | 14.93±1.25 | 6.74±0.81 |
| Deactivation 2014011 | 6.94±1.43 | 6.58±0.77 | 6.79±0.96 | 6.46±1.85 |
| PBS control group | 4.43±0.97 | 4.62±1.45 | 5.01±0.83 | 4.59±0.67 |
Claims (7)
1. a H9N2 subtype avian influenza polyepitope vaccines fusion rotein, its aminoacid sequence is SEQ ID No.2.
2. a nucleic acid molecule, its coding claim 1 described fusion rotein.
3. a carrier, it contains nucleic acid molecule according to claim 2.
4. a host cell, it contains carrier according to claim 3.
5. nucleic acid molecule according to claim 2, its concrete sequence is as follows:
agc tgt gaa gag atc gct gtg tgt gca gta cgg ctt aga gaa aac ctc tgc ctc tat ttt gaa gat gat gag ctg
gaa tgc gat gcc ttt tgt aag gat aaa act atc aaa cga ttc ttt cga aac gtc aat agc cag ttg ctt gtg gtt cgt
cca gat tta aac gtg gca gct ttt gaa gat gta aca gat cag gag gtg aaa tct ggc agt gga atg tac ttc gac att
cac tgt tac aaa acc acc gcg cct tca gca ggg atg cct gtt gca tcc agc gtc cag gta gaa gat aag agt tac
tac atg tgt tgt gag aaa gag cat ggg aaa atg gtt gtt cga ttt agg gaa gga gaa gtt ccc aaa gac att cct
ggt gaa agt aac atc ata ttt ttc aaa aag aca ttt aca tct tgc agc tcc aag gct ttt aag ttc gag tac tca ctt
gaa caa gga atg ttc ttg gcc ttt gag gaa gaa gac tcc tta aga aaa cta att tta aag aaa ctg ccg aga gaa
gat gaa gtt gat gaa acc aca aaa ttc gta aca agt cat aat gaa agg cac aac cta ggt gat atc cca ggt tgc
aag gtg gca gaa tac aag aat tgg tca aaa cca ggt ggt ctg aat aac aag cac tca aat ggc act aca cat gat
aga att cct ggt tct ggt aca cca aga gat gat ggt agc tcc agc agc agc aac tgc ata gac cct aat aac gaa
tgt gca gca tac ttg acc caa aag aac aac gct tac cct act cag gac gcc caa tac aca aat aat caa gaa ggt
tct ggt aac ggg acc tac aac aga agg aag tat caa gag gag tca aaa tta gaa aga cag ggt gat atc cca ggt
tgc aag caa gtg cgg gaa agc aga aat cct ggt ggt tct ggt aga tca aat gag aat cca gca cat aag ggt tct
ggt aac act gaa ggc agg aca tcc gac atg ggt ggt cca tct tgt aaa aga ggg cct tct acg gaa gga gta cct
gag tct atg agg gaa gag tat cgg cag gaa cag
6., for preventing a vaccine for H9N2 subtype avian influenza, it comprises fusion rotein according to claim 1 and pharmaceutically acceptable carrier.
7. the application of fusion rotein according to claim 1 in the reinforced polyepitope vaccines of preparation restructuring H9N2 subtype avian influenza.
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| CN105968174A (en) * | 2016-05-13 | 2016-09-28 | 青岛蔚蓝生物制品有限公司 | Avian influenza H9N2 subtype virus HA protein linear antigen epitope polypeptide |
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| CN105968174A (en) * | 2016-05-13 | 2016-09-28 | 青岛蔚蓝生物制品有限公司 | Avian influenza H9N2 subtype virus HA protein linear antigen epitope polypeptide |
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