CN104804099B - A kind of reinforced polyepitope vaccines of recombination H9N2 subtype avian influenza - Google Patents

Abstract

The present invention relates to a kind of preparation and application of recombination H9N2 subtype avian influenza (Avian influenza (H9N2), AI) reinforced polyepitope vaccines.The vaccine is using neutralizing epitope, Th epitope, CTL epitope and the B cell epitope of H9N2 subtype avian influenza virus major structural protein hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and stromatin 2 (M2) as vaccine frame structure, by connecting again with cell factor fowl interleukin 18 (chIL-18) after flexible linker connection, Escherichia coli are converted after being cloned into pRSETB carrier, through techniques such as everfermentation, purifying, emulsifications, the reinforced polyepitope vaccines of bird flu with Desirable immunogenic are obtained.Animal experiments show that recombinating the reinforced polyepitope vaccines of H9N2 subtype avian influenza not only good security, but also effective humoral immunity and cell immune response can be excited.

Description

A kind of reinforced polyepitope vaccines of recombination H9N2 subtype avian influenza

Technical field

The invention belongs to biotechnology genetic engineering fields, and it is reinforced more to relate generally to a kind of recombination H9N2 subtype avian influenza Epiposition vaccine preparation and application.Specifically, using gene recombination technology, by major structural protein: hemagglutinin (HA), neuraminic acid Enzyme (NA), the neutralizing epitope of nucleocapsid protein (NP) and stromatin 2 (M2), Th epitope, CTL epitope and B cell epitope with Cell factor fowl IL-18 series connection, and it is cloned into carrier, host strain is converted, is prepared through everfermentation, purifying, emulsifying process, obtains weight Group H9N2 subtype avian influenza polyepitope vaccines and the vaccine are preventing the application in avian infectious disease H9N2 subtype avian influenza.

Background technique

Influenza virus can be divided into tri- type of A, B and C.Wherein the distribution of Type B and c-type influenza virus is smaller, and harm is lighter, And the harm of influenza A is the most serious, in addition to infecting the mankind, can also infect other mammals and birds (Webster, 1992), and worldwide flu outbreak can be caused, the development of human health and animal husbandry is caused great Strike (Al-Mazrou A etal, 1991；).Hemagglutinin (hemagglutinin, HA) and neuraminidase (neuraminidase, NA) is 2 kinds of main surface glycoproteins of influenza virus and surface antigen, has hypospecificity, according to The difference of HA and NA, influenza A can be divided into 17 kinds of HA (H1-H17) and 10 kinds of NA (N1-N10) hypotypes (Zhu, 2013) again, Wherein H1N1, H2N2 and H3N2 it is popular in the mankind (Earn, 2002；Fouchier, 2004).LPAIV is worldwide wide General propagation, and break through species barrier be transmitted to people and pig (Alice, 2011；Ge, 2009；Park, 2011).1966, H9N2 was sub- Type AIV is separated from U.S. turkey group for the first time, and since nineteen ninety-seven, H9N2 hypotype AIV is in Asia, North America, the Middle East, Europe With spread and epidemic in the birds of African Territories, once in a while break through species barrier be transmitted to people (Alice, 2011；Ge, 2009；Park, 2011).Henceforth H9N2 hypotype AIV is always in the main feeding fowl area wide-scale distribution in China.Though H9N2 hypotype AIV belongs to LPAIV, but it can cause respiratory symptom, laying rate decline, immunosupress and the other viruses of excitation or bacterium mixing sense Dye, to China's aviculture cause huge economic loss (Chen, 2012；Wu, 2010).

H9 subtype influenza virus is distributed widely in all over the world, especially in Eurasia.According to the pass of its gene evolution System, it can be divided into North America series and Eurasia series as other hypotypes.Have now been found that at least there are three Eurasian series Subbreed column (Guan Y, 1999), respectively using Qa/HK/Gl/97, DK/HK/Y280/97, DK/HK/Y439/97 as representative.

The genome of influenza virus is made of 8 genetic fragments, encodes 12 kinds or more of albumen altogether, wherein 8 kinds are knots Structure albumen, including polymerase B2 (polymerase B2, PB2), polymerase B1 (polymerase B1, PB1), polymerase A (polymerase A, PA), HA, NP, NA, M1 and M2, non-structural protein (nonstructural protein, NS) includes NS1 And the PA-X of NS2, PA gene coding is a kind of fusion protein (Shi, 2012) of influenza virus, the PB1-F2 of PB1 gene coding (chen W, 2001) related to the Apoptosis that influenza virus particles mediate.

HA can be aggregated the red blood cell of many animals, be a kind of surface protein of influenza virus, it can be incorporated into host cell On the sialic acid receptor on surface, the antigenicity of virus is helped in cell entry host cell and changes, and then host immune of escaping The monitoring of system, be virus variation, virulence and host specificity main determining factor (Guo, 2000；Kimble, 2011).HA Gene is the antigen for generating neutralizing antibody, and induction body immune system generates protective effect, is that virus variation, virulence and host are special Anisotropic main determining factor, but also body can be stimulated to generate cytotoxic T lymphocyte (CTL) reaction, it is AIV induction The target antigen of body generation protectiveness humoral immune reaction.The change of HA Argine Monohydrochloride and the close phase of virus antigenicity variation Close, and may change viral host specificity (Krause, 2010；Carrat, 2007).

NA albumen is another important surface antigen of influenza virus particles, has immunogenicity, be can induce corresponding The generation of antibody.The corresponding antibodies that NA induces are not belonging to neutralizing antibody, but can inhibit the diffusion (Hay, 1998) of virus in vivo. NA albumen can crack the sialic acid of receptor surface glycoprotein end, release progeny virus ion from cell, be conducive to The propagation (Castrucci, 1993) of virus.

NP is the structural proteins encoded by genomic fragment 5, is the main component for constituting nucleocapsid.It is generally believed that NP is Conservative structural proteins, aberration rate is very low during virus evolution, has type specificity.It, can according to its antigenic difference Influenza virus is divided into A type, Type B, c-type.NP also has effect in terms of the host specificity for determining virus, is influenza virus gene A segment the most conservative in group, has type specificity.NP is a kind of multifunctional protein, can form the nucleocapsid of virus, with RNP complex is collectively formed in polymerase and RNA, prevents vRNA from decomposing, and also has in virus transcription and reproduction process important Effect (Bullido et al., 2000).NP is nucleoprotein, is rich in smart, sweet, serine, is a kind of basic protein, causes host Cellullar immunologic response (Lamb, 1982；Portela, 2002), it is the main shaft and CTL (cell toxicant lymph of spiral shape nucleocapsid Cell) identification target position.

M is hydrophobic proteins, is rich in arginine, including two kinds of protein of M1 and M2 (Treanor, 1990), wherein M2 Extracellular region M2e sequence is highly conserved, although M2 antibody does not have neutralization activity, mouse experiment shows passively to exempt from using M2e monoclonal antibody Epidemic disease can be substantially reduced the virus titer (Jegerlehner, 2004) of mouse nose and lung.Research shows that M2e antibody is main By antibody-dependant cell mediate cell killing (ADCC) play a role (0kuda, 2001；Neirynck, 1999；Fan, 2004)。

Two surface glycoproteins of influenza virus easily morph, and the protection antibody that host generates is also for both Albumen, humoral immunity can promote antigenic variation (Corti, 2011), the especially antigenic variation of some regions such as HAI again, lead Cause the appearance of new popular strain or short sub- evolutionary branching of certain service life.And the ctl response master of cellular immunity such as MHC I limitation If for than more conservative inside albumen (Forrest, 2008), and having cross reactivity between different subtype and strain, CTL Epitope can also morph, influenza virus evolution then be mainly antibody selection.The early stage of influenza infection, CTL and It plays a role in terms of limiting virus in terms of the cellular immunities such as cell factor.Phase after infection, the production of neutralizing antibody It is raw that the removing of virus and host are restored to play an important role.Although HA and NA are the most antigenic albumen of influenza virus, only The ingredient for having selection conservative, just can be reduced antigen variation bring influences, to resist the attack of most of strains, plays extensively Protective effect (Ekiert, 2009；Ernst, 2006；).

Currently, the prevention of human influenza and bird flu depend on inactivated vaccine and attenuated live vaccine it is long-term use warp Going through proof, they are safely and effectively, and to have played huge effect.Vaccine immunity enhances the resistivity of body, reduces The discharge and propagation of virus.It is mainly at this stage the inactivated vaccine for relying on chicken embryo proliferation production for the vaccine of anti-bird flu processed, though So there are lot of advantages for traditional inactivated vaccine, but still there are many shortcomings, such as interfere epidemiological surveillance, are proliferated by chicken embryo It is at high cost, it passes in chicken embryo and easily morphs, burn chicken embryo residuum pollution environment etc., and inactivated vaccine can not have with living virus vaccine Effect makes a variation to present influenza virus and rapid, more subtype influenzas and deposits trend popular, that kind boundary is increasingly fuzzy.Thus need Further strengthen the development of new generation vaccine.

Interleukin-18 (Interleukin-18, IL-18) is that a kind of novel cell that nineteen ninety-five is reported for the first time is immune Regulatory factor (Okamura, 1995), Schneider in 2000 etc. (Schneider, 2000) obtain ChIL-18 cDNA for the first time. There is interleukin 18 inducing T cell and NK cell to generate IFN-γ (Kohno, 1997), promotes T cell proliferation, enhances Thl cell And the multiple biological functions such as cytotoxic activity of NK cell (Micallef, 1996), enhancing it is immune, in terms of have Have great potential using value (Kanda, 2000；Marshall, 2006), in immune response, especially cellular immunity One of response important cytokine, can be used as vaccine immunologic adjuvant (Degen, 2005；Puehler, 2003).

Summary of the invention

The present invention is with H9N2 subtype avian influenza virus major structural protein hemagglutinin (HA), neuraminidase (NA), core clothing The neutralizing epitope of glutelin (NP) and stromatin 2 (M2), Th epitope, CTL epitope and B cell epitope are as vaccine frame knot Structure by protein purification, emulsifies after expression in escherichia coli with avian cytokines interleukin 18 (chIL-18) gene tandem Etc. techniques, obtain have Desirable immunogenic the reinforced polyepitope vaccines of recombination H9N2 subtype avian influenza.This vaccine immunity target It can induce effective humoral immunity and cell immune response after animal.

One of the objects of the present invention is to provide a kind of new recombination reinforcements that can be used to prevent H9N2 subtype avian influenza Type polyepitope vaccines polypeptide and its vaccine composition；The second object of the present invention is the provision of the reinforced multilist of the bird flu The building of position vaccine and preparation method；The third object of the present invention, which is the provision of, can express the reinforced multilist of the bird flu The engineering strain of position vaccine；The fourth object of the present invention is the provision of the preparation method of the polyepitope vaccines；This hair The bright fifth purpose is the provision of purposes of the reinforced polyepitope vaccines of the bird flu in prevention H9N2 subtype avian influenza.

In a first aspect, the present invention provides a kind of reinforced multi-epitope epidemic diseases of recombination H9N2 subtype avian influenza for prevention Seedling polypeptide and combinations thereof.Its contain major structural protein hemagglutinin (HA), neuraminidase (NA), nucleocapsid protein (NP) and The neutralizing epitope of stromatin 2 (M2), Th epitope, CTL epitope are with, B cell epitope and avian cytokines interleukin 18 (chIL- 18).The reinforced polyepitope vaccines albumen of the bird flu or polypeptide or pharmaceutically acceptable salt and expression epitope Carrier required for albumen.Carrier also may include the sequence of separately encoded each epitope, and series connection can pass through genetic engineering Method carries out.The vaccine also includes nonimmune active material, and the coupling part of as each polypeptide is exempted from without epitope Epidemic focus does not have any adjuvanticity yet, mainly there is purification tag, joint peptide, chemical modification part, N-terminal signal peptide and C-terminal Polyadenylic acid etc..The pharmaceutically acceptable salt refers to non-toxic, stimulation and allergy, is suitable for human or animal tissues Salt.Inert matter and pharmaceutically acceptable salt are well known to those skilled in the art.Recombination H9N2 subtype avian influenza adds Strong type polyepitope vaccines polypeptid acid sequence is as follows:

SCEEIAVCAVRLRENLCLYFEDDELECDAFCKDKTIKRFFRNVNSQLLVVRPDLNVAAFEDVTDQEVKS GSGMYFDIHCYKTTAPSAGMPVASSVQVEDKSYYMCCEKEHGKMVVRFREGEVPKDIPGESNIIFFKKTFTSCSSKA FKFEYSLEQGMFLAFEEEDSLRKLILKKLPREDEVDETTKFVTSHNERHNLGDIPGCKVAEYKNWSKPGGLNNKHSN GTTHDRIPGSGTPRDDGSSSSSNCIDPNNECAAYLTQKNNAYPTQDAQYTNNQEGSGNGTYNRRKYQEESKLERQGD IPGCKQVRESRNPGGSGRSNENPAHKGSGNTEGRTSDMGGPSCKRGPSTEGVPESMREEYRQEQ

In second aspect, the present invention provides a kind of nucleic acid molecules, encode the stream of fowl described in first aspect present invention Feel reinforced polyepitope vaccines polypeptide.Nucleotide of the present invention can be rna form, and DNA form is closed by artificial synthesized mode At more epitope tandem sequences and fowl interleukin 18 sequence, then enters carrier by genetic engineering operation connection rear clone, turn Escherichia coli are dissolved into, screening, obtains bird flu polyepitope vaccines polypeptide at fermentation after purification.It in the present invention can be to the nucleic acid Conventional molecular biology manipulations are carried out, such as: PCR, digestion with restriction enzyme, connection, 5 ' end of nucleic acid design and 3 ' ends are equal Restriction enzyme site is added.It is preferred that the nucleotide sequence in 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 present invention provides a kind of carriers, in addition to containing coding described in second aspect of the present invention The reinforced polyepitope vaccines nucleic acid molecule of H9N2 subtype avian influenza, also containing with the operable connection of the nucleotide sequence, Expression control element needed for procaryotic cell expression (transcription and translation).Most basic expression control element includes promoter, turns Terminator, enhancer, selected marker etc. are recorded, these controlling elements are known in the art.Preferred Escherichia coli in the present invention BL21 (DE3, Plys) is used as expression vector.

In fourth aspect, the present invention provides a kind of host cells, contain carrier described in third aspect present invention.Place Chief cell it is inverted or transfection containing it is of the present invention coding albumen gene order, and after through detection have good heredity After expression stability, the reinforced polyepitope vaccines polypeptide of H9N2 subtype avian influenza needed for can be used for fermentation expression production.

At the 5th aspect, the present invention provides a kind of preparation method of reinforced polyepitope vaccines of H9N2 subtype avian influenza, It is the following steps are included: engineering bacterium fermentation expresses H9N2 subtype avian influenza vaccine polypeptide, by slightly purifying and polishing purification technique And subsequent emulsifying process, polypeptide required for obtaining.The method being directed to includes but is not limited to bacterial cell disruption, inclusion body Washing, centrifugation, denaturation, affinity chromatography, hydrophobic chromatography, anion-exchange chromatography, reverse-phase chromatography, renaturation, emulsification etc..In the present invention The preparation method being related to is well known to those skilled in the art.

At the 6th aspect, the recombinant fowl influenza that the present invention provides a kind of for preventing H9N2 subtype avian influenza is reinforced more Epiposition vaccine comprising polypeptide described in first aspect present invention and pharmaceutically acceptable carrier.The polyepitope vaccines It can prevent the outburst of H9N2 subtype avian influenza.Pharmaceutically acceptable carrier of the present invention is immunopotentiator or is immunized Adjuvant, preferably immunologic adjuvant are import white-oil adjuvant.

At the 7th aspect, the present invention provides answering for recombination H9N2 subtype avian influenza polyepitope vaccines described in the 6th aspect With.Vaccine centainly effective dose intramuscular injection, intradermal or inoculated with subcutaneous injections animal can generate body fluid effective enough Immune and cell immune response (see embodiment five, six, seven, eight, nine, ten), stimulation neutralizing antibody generate, and induce peripheral blood and spleen CD4+ and CD8+T lymphopoiesis is organized, while antiviral activity being provided, substantially reduces toxin expelling, protects animal from H9N2 The attack of subtype avian influenza virus prevalence strain.In addition, in embodiments of the invention, by carrying out laboratory peace to vaccine Full property test, shows that recombination H9N2 subtype avian influenza polyepitope vaccines of the present invention are safe (see example IVs).

In addition, it is necessary to which, it is noted that based on the disclosure in the context of this application, other of the invention have The aspect of substantive distinguishing features is obvious for those of ordinary skill in the art.In addition, the present invention which also uses disclosure Document, their entire contents are included in be referred to herein.

Detailed description of the invention

Following drawings is for illustrating specific embodiments of the present invention, rather than limits and be defined by the claims The scope of the invention.Fig. 1 recombinates the reinforced polyepitope vaccines expression plasmid pRSETB-chIL18-AIV of H9N2 subtype avian influenza (H9N2) structure figures；Fig. 2 pRSETB-chIL18-AIV (H9N2) vector plasmid cleavage map, wherein swimming lane 1 is DNAmarker, from It is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp up to lower molecular weight, swimming lane 2 is digested plasmid, swimming lane 3 For non-digested plasmid, swimming lane 4 is empty plasmid；Fig. 3 SDS-PAGE detection figure, wherein swimming lane 1 is not induce control sample, swimming lane 2 For albumen Marker, it is followed successively by 97KD, 66KD, 43KD, 31KD, 20KD, 14KD from top to bottom, swimming lane 3,4 is induced samples, arrow The signified destination protein for expression of head；Fig. 4 Westernblot detection figure, wherein swimming lane 1 be pre-dyed marker, from top to bottom according to Secondary is 200KD, 140KD, 100KD, 80KD, 60KD, 50KD, 40KD, 30KD, 20KD, and swimming lane 2 is purpose albumen, and swimming lane 3 is yin Property control.Fig. 5 be fermented sample SDSPAGE figure: wherein swimming lane 1 is albumen Marker, be followed successively by from top to bottom 97KD, 66KD, 43KD, 31KD, 20KD, 14KD, swimming lane 2 are non-induced samples, and swimming lane 3 is positive control, 4 fermentation inducement samples of swimming lane；Fig. 6 For fermented sample Westernblot detect scheme, wherein swimming lane 1 be pre-dyed marker, be followed successively by from top to bottom 200KD, 140KD, 100KD, 80KD, 60KD, 50KD, 40KD, 30KD, 20KD, swimming lane 2 are positive control, and swimming lane 3 is fermentation purification of samples, swimming lane 4 For negative control.Fig. 7 is that ELISA method detects M2 protein I gG antibody titre results in serum；Fig. 8 is that each experimental group lymph is thin Born of the same parents' stimulus index testing result；

Specific embodiment

Specific test method description as described in the examples is only exemplary description, for elaborating the present invention, but simultaneously It is not meant to limit the scope of the invention, many variations according to the present invention are well known to those skilled in the art.

The mentality of designing of one H9N2 subtype avian influenza polyepitope vaccines albumen of embodiment

The present invention is according to current country H9N2 subtype avian influenza Major Epidemic strain structural proteins hemagglutinin (HA), neuraminic acid Enzyme (NA), nucleocapsid protein (NP) and stromatin 2 (M2) amino acid sequence, using relevant bioinformatics software DNASTAR, BIMAS and SYFPEITHI right pop strain carries out neutralizing epitope, Th epitope, CTL epitope and B cell epitope analysis, introduces simultaneously Avian cytokines interleukin 18 (chIL-18) is used as adjuvant molecules.Designed epitope and interleukin 18 molecular polypeptide are connected It is co-expressed in Escherichia coli afterwards, through techniques such as everfermentation, purifying, emulsifications, obtains the H9N2 hypotype with Desirable immunogenic The reinforced polyepitope vaccines of bird flu.Prepared by the method vaccine can effectively prevent H9N2 subtype avian influenza.

Comprehensive analysis country H9N2 subtype avian influenza virus epidemic strain genome sequence, antigenic structure, epidemiological study Design is optimized in recombinant fowl influenza polyepitope vaccines by progress.The present invention is using bioinformatics software to its structure egg The hydrophily of white progress, antigenicity, plasticity, the secondary structure of surface accessibility and Garnier-Robson are analyzed, in advance It surveys on the basis of possible B cell antigen epi-position, CTL epitope and t cell epitope, according to epitope position and amino acid sequence Similitude analyzes each popular strain and shares epitope, and with reference to the sequence information in GenBank, to the epitope of prediction It is compared, conservative of the epitope in different virus strain is further analyzed, so that it is determined that relevant to NA albumen 3 sections of Thelper antigen epitope polypeptide, 2 sections of the relevant neutralization cell antigen epitope polypeptide of HA albumen, the relevant CTL of NP albumen resists 3 sections of former epitope polypeptide, 1 section of the relevant B cell antigen epi-position polypeptide of M2 albumen will form the skeleton of vaccine after the series connection of all epitopes Structure, while molecule adjuvant is added in skeleton nitrogen end.The overall structure of the vaccine are as follows:

Molecular Adjuvant(chIL 18)-NA Thelper Epitopel-NA Thelper Epitope2- NA Thelper 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 building of two coli expression carrier of embodiment and expression bacterial strain

Designed polypeptide-coding nucleotide is served into the handsome biotech company's synthesis in sea, nucleotide fragments both ends difference BamH I (5 ' end) and HindIII (3 ' end) restriction enzyme site are devised, is cloned into respectively after this segment is synthesized On pMD18T carrier, sequencing confirms that insertion genetic fragment is consistent with implementation sequence (see sequence table).Recombinant plasmid is distinguished It is named as pMD18T-chIL18-AIV (H9N2).Plasmid is subjected to digestion processing, Escherichia coli with corresponding restriction enzyme Expression vector selects the pRSETB plasmid of Invitrogen company, also uses identical restriction enzyme enzymatic treatment, digestion condition: 10 μ l reaction systems, system is interior to be added 2 μ l plasmids, and restriction enzyme is 5 active unit (New England Biolabs), be added 10 × buffer, 1 μ l, deionized water polishing, 37 DEG C digestion 1.5 hours.1 μ l is added after digestion 200mM EDTA terminates reaction.In 1% agarose gel electrophoresis, electrophoresis 30 minutes.By 2.86kb pRSETB matter under ultraviolet lamp Grain and 1100bp chIL18-AIV (H9N2) segment are cut, and carry out glue according to Qiagen company gel reclaims kit specification Recycling.According to carrier: the ratio of segment 1:2~3 individually mixes multi-epitope nucleotide fragments with expression vector, reaction system 15 μ l is attached by T4DNA ligase, and 16 DEG C of connections overnight, obtain recombinant plasmid and are respectively designated as pRSETB-chIL18-AIV (H9N2), (see Fig. 1), transformed competence colibacillus e. coli bl21 (DE3) pLysS.

Conversion: pRSETB-chIL18-AIV (H9N2) being set and is melted on ice, and 2 μ l connection reaction solutions are added, mix again, Ice-water bath 30 minutes, 42 DEG C 30 seconds, then put back to rapidly ice bath 1.5 minutes, be added 1mL LB culture solution, 37 DEG C, stationary culture 1 Hour, thallus is resuspended with 200 μ lLB culture mediums in 10 seconds abandoning supernatants of 4000g low-temperature centrifugation；By bacterium solution be spread evenly across containing It on the LB agar plate of 100 μ g/mL ampicillins, is inverted in 37 DEG C of insulating boxs and cultivates 12~16 hours, until clone It is formed.

Identification: the monoclonal on picking plate is into LB culture medium, 37 DEG C, 200rpm shake culture 12 hours, extracts matter Grain carries out double digestion using restriction endonuclease BamH I and HindIII respectively, can cut out corresponding avian influenza vaccine gene size segment Clone, 1100bp can primarily determine as positive colony (see Fig. 2)；Positive colony carries out determined dna sequence and further verifies it Correctness (see sequence table).

Inducing expression.Positive colony is incubated overnight, morning next day is added after culture 3 hours by 1: 100 switching 0.2mM IPTG continues culture 4 hours, prepares sample；Conventional SDS-PAGE testing goal protein expression situation --- in 45KD (see Fig. 3), seeing specific band is correct clone；Correct clone, amplification culture are taken, SDS-PAGE is confirmed after expressing correctly, Further confirm that it expresses accuracy using conventional Western-blot (see Fig. 4)；It, can after above-mentioned building and evaluation program The foundation of original species word bank is carried out using the positive colony selected as engineering bacteria, strain names pRSETB-chIL18-AIV (H9N2)/BL21 (DE3, Plys).

Fermentation, purifying and the emulsification of three engineering bacteria of embodiment

Fermentation takes production strain, is inoculated in 2mL LB liquid medium (containing 100 μ g/mL ampicillins), 37 DEG C, 12 hours activated spawns of 200rpm shaken cultivation.Shaking flask, 37 DEG C of shaken cultivations to OD600=are accessed with 1: 100 inoculum concentration again 3, it can be inoculated in 10% ratio into fermentor.Fermentation is semisynthetic medium with culture medium, is prepared with distilled water.Correct dissolved oxygen and PH value electrode opens tank body stirring, and revolution 300rpm, tank body sterilizes online, when culture-liquid temp in tank is down to 37.0 DEG C, Demarcate pH and dissolved oxygen (OD) zero point.Fermentation temperature is 37.0 ± 0.1 DEG C, and dissolved oxygen control is controlled in 40% or so, pH 7.0, is connect Flow feeding 500mL, 1 hour addition IPTG (final concentration of 0.2mM) after feed supplement when cultivating thallus OD600=1.0~1.2 after kind Inducing expression, 5 hour post-fermentations of continuous induction terminate, and SDS-PAGE calibrating expression is done in sampling (see Fig. 5).

The thallus that will be collected into is purified, it is mixed with occlusion body washing lotion I (1%Triton X-100,20mMTris-cl PH8.0) Ultrasound is carried out after outstanding, 2000W ultrasound cracks 1 hour.4 DEG C, 12000rpm is collected by centrifugation occlusion body, and with occlusion body washing lotion II (1%DOC, 4M urea, 20mMTris-cl PH8.0) suspension twice ultrasonic washs occlusion body, and secondary low-temperature centrifugation collection includes Body.Occlusion body precipitating 8M urea, 0.3% β-ME, 20mM Tris-cl (pH=8.00) are mixed, are stirred at room temperature 4 hours, 8000rpm low-temperature centrifugation 30min, discards precipitating.Albuminate 1: 100 dilutes, renaturation solution Tris (PH8.0) buffer system, Be added 0.3M arginine, 4 DEG C stirring renaturation 24 hours.The 20mM phosphate buffer of renaturation solution pH=8.0,0.5M sodium chloride, 20mM imidazoles, affinity column in balance, with the 20mM phosphate buffer of pH=8.0,0.5M sodium chloride, the elution of 0.5M imidazoles； Up to recombination H9N2 subtype avian influenza polyepitope vaccines semi-finished product stoste.

The PBS that the semi-finished product of purifying sterilize is diluted to 100 μ g/mL by emulsification.Follow the example of Guo Sai BIC Corp Montanide ISA 50V2 adjuvant sterilizes 15 minutes by 121 DEG C, spare.In oily phase: water phase=50: 50 ratio is matched Oil, is first added in emulsion tank, starts blender and be slowly stirred with the speed of 80~100r/min, be slowly added into water phase by system, It is stirred for 2min after adding, 9min is then emulsified with 5500r/min high-speed circulating, the single-phase vaccine of Water-In-Oil is made.

Example IV recombinates 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, lot number 140321,140323,140326.

Test uses the experimental design of single-factor completely random, and 20 30 age in days experimental animals are randomly divided into 4 at random Group, 3 lot number groups and control group, every group 5, without weight differences between group.Immune group is in every chicken leg portion intramuscular injection vaccine 0.3mL/ head, to 10 days, control group used physiological saline emulsion 0.3mL for observation.After immune, adopting for each experimental animal is observed It raises, whether drinking-water, whether spirit is normal, whether have poisoning symptom, whether generate allergic reaction, is dead or other abnormal feelings occur Condition.Before immune and after the test, all animals weighing.Weighing results carry out statistical analysis.

3 results

3.1 clinical observation

Do not observe any allergic reaction or poisoning symptom after 3 immune group animal immunes, the spirit of SPF chicken, adopt feeding, Drinking-water, activity, excrement etc. are all gone well, and do not occur obvious local inflammation equivalent damage clinical side reaction, and occur without death.

3.2 changes of weight

It the results are shown in Table 1, compared with the control, three immune group the weight of animals increase and not significant (the P > of control group difference 0.05), show that the immune recombination reinforced polyepitope vaccines of H9N2 subtype avian influenza have no adverse effects to the weight of animal.

Table 1 recombinates influence of the reinforced polyepitope vaccines of H9N2 subtype avian influenza to SPF chicken changes of weight

Lot number	Weight (g) before immune	10 days weight (g) after immune	Daily 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	-

Five animal experiment of embodiment is grouped and is immunized

It 1 vaccine and attacks malicious viral

Recombinant vaccine is provided by Hongqiao Ming Qin research and development centre, lot number 140321,140323,140326, H9N2 inactivation epidemic disease Seedling (Re-2 plants) is given by Guangdong Yongshun pharmaceutical development center, lot number 2014011.

2 experimental animals

One age in days SPF chicken 50, it is pre- to raise two weeks, adapt to environment.

3 groupings are divided into 5 groups, every group 10 with immune.

Two week old SPF chickens are divided into 5 groups, polyepitope vaccines group, inactivated vaccine group and PBS control group.Immune group distinguishes flesh Meat injection polyepitope vaccines and inactivated vaccine (Re-2 plants), 0.15mL/, 14 days same dose booster immunizations after head exempts from, PBS pairs According to immune PBS emulsion is organized, 0.15mL/ is only.7 days after immune, take a blood sample and be used for M2 protein I gG within 14 days, 21 days, 28 days Antibody test, the detection of HI antibody titer and the measurement of lymphopoiesis stimulus index, and carry out within 28 days attacking malicious examination after immune It tests, is detected for toxin expelling.

Six M2 protein I gG antibody test of embodiment

1,2,3,4 week serum after acquiring first immunisation detects specific IgG antibodies with end dilution ELISA.Micropore The vaccine protein coating of plate (Nunc Maxisorp, Nalge Nunc International, Denmark) purifying, 2~8 DEG C Overnight；5% skim milk makees 1: 10,1: 10 in 37 DEG C of closing 1h, by antiserum², 1: 10³, 1: 10⁴, 1: 10⁵, 1: 10⁶, 1: 10⁷, 1: 10⁸It dilutes again, while using the serum of immune PBS as negative control, microwell plate is added, 100 holes μ L/ are incubated in 37 DEG C 1h, then use rabbit-anti chicken IgG-HRP ELIAS secondary antibody (1: 10000, Sigma, St louis), be added microwell plate, 100 holes μ L/, In 37 DEG C of incubation 1h.TAB substrate is protected from light colour developing 10min, 2M H₂S0₄Reaction is terminated, detects absorbance value under 450nm wavelength. Using the inverse of highest serum dilution as antibody titer, average absorbance value (>=0.2) is higher than the average suction of preimmune serum Light value+2SD, as cutoff value.

Can as seen from Figure 2, after the first exemption the 1st week, antibody male rotary, and average titer point just had occurred in immune group animal It Wei 1: 10²With 1: 10³, it is successively increased in subsequent 2,3,4 weeks antibody levels, until the 4th week, four are immunized final average antibody Level has reached 1: 10⁵More than, wherein reach for 140323 groups of recombinant vaccine: 1: 10⁶More than, the serum of opposite PBS control group mouse In can't detect antibody.The above result shows that the recombination H9N2 bird flu polyepitope vaccines that the present invention designs being capable of induced high levels Specific M2 protein I gG antibody.

The detection of seven blood clotting of embodiment inhibition (HI) antibody

The preparation of 11% red cell suspension

It is anticoagulant with 2% sodium citrate salt water to acquire experimental group chicken blood, adds the brine red blood cell of 5 times of volumes, 1000rpm is centrifuged 10min, the physiological saline resuspension red blood cell that 100 times of volumes of cell pack are added afterwards three times is continuously washed, after shaking up Set 4 DEG C it is spare.

2 Microhemagglutinations test (HA)

Viral hemoagglutination titration: taking 96 hole V-arrangement micro-reaction plates, adds 25 μ L in the every hole in 1~12 hole with micropipettor PBS, drips 8 rows altogether, and 25 μ L PBS are added in the 1st column hole of rear four row again.Draw 25 μ L standard avian influenza antigens (Harbin dimension section) It is added in the 1st column hole, blows and beats 3~5 times and mix well.The antigen liquid after 25 μ L are mixed, which is drawn, from the 1st column hole is added to the 2nd column hole In, 25 μ L are drawn after mixing and are added in the 3rd column hole, successively carry out serial doubling dilution to the 11st column hole, finally from the 11st column Hole respectively draws 25 μ L and abandons it, if the 12nd column hole is red blood cell control.The chicken that successively 25 μ L 1% are added to each hole in right-to-left is red Cell suspension.Reaction plate is placed on micro oscillator and vibrates 1min, after (20~25 DEG C) standing 30min of room temperature observation as a result, The viral highest dilution for 100% erythrocyte agglutination occur is the viral agglutination valence of the sample.

3 microdose cytopathogenic effect assays (HI)

25 μ L PBS solutions are added in the 1st to the 11st hole of the 96 every row of hole blood clotting suppressing plate of V-shaped, 50 μ L are added in the 12nd hole PBS solution is as negative control；25 μ L are added in the 1st hole and are detected serum, 25 μ L are removed after mixing well and add to the 2nd hole, successively Analogize, doubling dilution to the 10th hole, the 10th hole discards 25 μ L, if the 11st hole is virus control, the 12nd hole is red blood cell control.? 25 μ L, 4 unit antigen is respectively added in 1st~11 hole, and tapping reaction plate is uniformly mixed reactant, stands 30min at room temperature.From The chicken erythrocyte suspension of 25 μ L 1% is successively added in a dextrad left side to each hole.Reaction plate is placed on micro oscillator and vibrates 1min, Observation is as a result, red blood cell control wells can be sentenced when sinking to bottom hole at apparent button shape after (20~25 DEG C) standing 40min of room temperature Determine result.

4 results

The HI antibody level of all immune groups gradually rises with the time, until 28 days after immune, recombinant multi-epitope vaccine group HI antibody titer more than 10, inactivated vaccine group 9.9 does not form significant difference (P > 0.05) between immune group.It is all immune Group forms extremely significant difference (P < 0.01) (being shown in Table 2) with PBS control group.

HI antibody level detection in different time periods after 2 SPF chicken immune of table

Note: extremely significant (P < 0.01) with difference is designated as on the different letters of column data.

The measurement of eight lymphopoiesis stimulus index of embodiment

It is sterile to take each group SPF chicken periphery anticoagulation 2mL 28 days after immune, and gently mixed with isometric Hank ' s liquid It is even, the anticoagulation diluted is slowly added to the centrifuge tube containing 4mL lymphocyte separation medium.Under room temperature, 2000rpm centrifugation 15min.The white cellular layer being located among centrifuge tube is carefully shifted with pipettor, and the H9N2 inactivated through ultraviolet irradiation is added (Re-2 plants), are centrifuged 10min by 4 DEG C, 2000rpm, in triplicate, after cell count, adjust the number of every solencyte to 1 × 10⁶cells/mL.The cell diluent for taking 100 μ L to adjust concentration concurrently sets concanavalin A into 96 orifice plates (ConA) positive control, negative control and blank group, every group of three repetitions, every hole final volume are 200 μ L.It is placed in 5%CO₂Culture Case, after 37 DEG C of culture 44h, 10 μ L 5.0mg/mL MTT solution are added in every hole, are continued after cultivating 4h, under room temperature, 2000rpm from Heart 5min discards 100 μ L supernatants, and 100mL DMSO lysate is added, and 37 DEG C are protected from light vibration 15min.Measure the extinction at 570nm Degree, is calculated by formula lymphopoiesis stimulus index (stimulation index, SI), and (test class value-feminine gender is right by SI= According to value)/negative control value.

As a result

It is similar with hemagglutination inhibition reaction, it is thin in the lymph that epitope polypeptide vaccine group and inactivated vaccine group observe highest level Born of the same parents' proliferation activity, is shown in Fig. 8.The T lymphocyte proliferation of control group is significantly lower than other immune groups (P < 0.05), recombination epitope epidemic disease The stimulus index average level of seedling group is slightly above inactivated vaccine group, but not shown significant difference (P > 0.05).

Toxin expelling rate detects after embodiment nine attacks poison

1RNA is extracted from attack poison after acquire throat and cloacal swabs respectively within the 3rd day, 5 days, 7 days, use RT-PCR method It detects the toxin expelling situation of each group test chicken: brush,throat and cloacal swabs being respectively put into and fill 1mL sterilizing It in 0.01moL/L pH7.0~7.4PBS (including penicillin 2000IU/mL, streptomysin 2mg/mL), covers, number.According to Trizol kit specification extracts each sample virus total RNA.

Random primer (10 μm of ol/L) 2 μ L, 2 μ of dNTP (10mmol/Leach) are added in the 6 μ L of synthetic system RNA of 2cDNA L, 0.5 μ L of RNase inhibitor (RNasin) (40U/ μ L), 0.5 μ L of AMV reverse transcriptase (5U/ μ L) and 5 × RT buffer 4ml, most It is mended afterwards with DEPC water to 20 μ L.42 DEG C reverse transcription 1 hour, then 94 DEG C of 10min.

3PCR amplification

According to the gene order for the H9N2 strain delivered on Genebank, with 5.0 software design 2 of Primer to H9N2 HAl gene-specific primer it is a pair of, 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), 1 μ L (5U/ μ of Taq DNA polymerase L), cDNA3 μ L, 10 × PCR buffer, 5 μ L, are finally mended with distilled water to 50 μ L.After being slightly centrifuged, PCR amplification is carried out.PCR is most Good reaction condition: 95 DEG C of initial denaturations 5min, 95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s recycle 30cycles；Most 72 DEG C of extension 10min afterwards.

5 identify: carry out conventional agarose gel electrophoresis, 80~100v, observe under 30min gel imaging system as a result, Visible obvious amplified fragments are positive findings, i.e. toxin expelling at 938bp.

6 results

The 3rd day toxin expelling rate highest after poison is attacked as can be seen from Table 3, and larynx tracheae toxin expelling rate is higher than cloaca toxin expelling rate；Weight Group vaccine immunity group toxin expelling rate is minimum, and except 140323 groups in 2 toxin expellings of appearance in 3 days, other recombinant vaccine lot number groups do not occur Toxin expelling situation, inactivated vaccine group are apparently higher than recombinant vaccine group in third day toxin expelling rate, do not find toxin expelling after 7 days.

Table 3 attacks the virus detection result of each group cotton swab after poison

Note: toxin expelling rate=toxin expelling size of animal/this test group of animals total amount

The detection of mouse T lymphocyte subclass quantity is immunized in embodiment ten

90 mouse of the grouping of 1 experimental animal and sampling are randomly divided into 5 groups, and every group 18, every subcutaneous injection epidemic disease of immune group Only, 200 μ L/ of PBS emulsion is subcutaneously injected only in immune group to 200 μ L/ of seedling.Two weeks booster immunizations are primary after head exempts from, immunization method and agent Amount is exempted from unanimously with head.25th day, 32 days, 39 days, 46 days each periods after head exempts from, each group slaughters 3 mouse, plucks eyeball and take blood. Anticoagulation separation lymphocyte does T lymphocyte subclass analysis, while sterile its spleen being taken to do T lymphocyte subclass analysis.

The separation blood sampling 0.5ml of 2 peripheral blood lymphocytes, adds Hank ' s liquid 0.5ml, mixes, it is thin to be gently added in 1ml lymph On born of the same parents' laminated fluid level.2000r/min is centrifuged 15 minutes, lymphocyte among careful collection, with Hank ' s liquid centrifuge washing cell Precipitating 2 times, then with RPMI1640 culture medium (1mmol/L containing Sodium Pyruvate, beta -mercaptoethanol 2 × 10^-6Mol/L, penicillin 100U/ml, 100 μ g/ml of streptomysin, calf serum 10%), cell is diluted to 1 × 10⁷A/ml, is made single cell suspension.

3 spleen list lymphocyte suspensions prepare it is sterile take mouse spleen, add 3ml Hank ' s, (200 on small copper mesh Mesh) on shred, squeeze, by copper mesh cell suspension collect sterile centrifugation tube, 1500r/min be centrifuged 5 minutes, abandon supernatant.With Hank ' s liquid centrifuge washing tube bottom cell precipitation 2 times, cell is diluted to 2 × 10 by (2) in the same way⁷A/ml is made slender Born of the same parents' suspension.

The detection of 4T lymphocyte call subtype quantity

The pretreatment of 4.1 peripheral blood lymphocytes takes anticoagulation 0.1ml, adds 8ml erythrocyte cracked liquid, and room temperature acts on 10 points Clock, 1500r/min are centrifuged 10 minutes, are abandoned supernatant, are added 5ml PBS, are suspended, and 1500r/min is centrifuged 10 minutes, are repeated 2 times.

The pretreatment extracting spleen cell suspension 0.1ml of 4.2 splenic lymphocytes adds 5ml PBS, 1500r/min to be centrifuged 10 minutes, It is repeated 2 times with 0.5ml PBS suspension liquid.

The fluorescent marker FITC of 4.3T lymphocyte marks rat anti-mouse monoclonal antibody (0.1mg/ml), dilutes 10 times (0.01mg/ml).Every pipe takes cell suspension 0.5ml, dilutes 10 μ l of monoclonal antibody (0.1 μ g) respectively, and 4 DEG C act on 1 hour, PBS buffering Liquid is washed 1 time, by tube bottom cell 1ml PBS suspension and test sample.

4.4FACS detection and data processing FACS detect 3000 cells, and the data obtained carries out statistical procedures, calculates it Average value.

5 results

5.1CD4+T the variation of lymphocyte call subtype quantity

5.1.1 the variation recombinant multi-epitope vaccine immunity group peripheral blood CD4+T lymph of peripheral blood CD4+T lymphocyte quantity Cell number overall trend is to gradually increase with immunization time, starts to be declined slightly behind arrival peak by 32 days, and inactivated vaccine group The growth trend for closing the not shown variation with immunization time of peripheral blood CD4+T lymphocyte of PBS control group and changing.Multi-epitope Vaccine group peripheral blood CD4+T lymphocyte number is significantly higher than inactivated vaccine group cell number (P < 0.05) and PBS control group cell number (P < 0.05), inactivated vaccine group peripheral blood CD4+T lymphocyte number and PBS control group are without significant difference (P > 0.05) (being shown in Table 4).

4 vaccine immune mouse peripheral blood CD4+T cellular change of table

Grouping	25 days	32 days	39 days	46 days
					Recombination 140321	31.9±2.04	42.5±1.81	33.6±2.93	35.8±3.34
Recombination 140323	33.1±3.72	42.8±3.15	35.2±2.62	36.5±1.92
					Recombination 140326	32.6±1.69	43.4±3.27	34.9±2.54	35.2±2.88
Inactivation 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 variation recombinant vaccine spleen CD4+T lymphocyte quantity overall trend of spleen CD4+T lymphocyte quantity To be gradually increased with immunization time, begun to decline behind arrival peak by 39 days, until 46 days, without significant difference (P > between immune group 0.05), but inactivated vaccine group and PBS control group do not change this and peripheral blood lymphocytes quantity with the variation of immunization time Testing result is consistent (being shown in Table 5).After immune during 25~39 days, recombination epitope vaccine group spleen CD4+T lymphocyte number is significant Higher than inactivated vaccine group cell number (P < 0.05) and PBS control group (P < 0.01), and inactivated vaccine group is integrally higher than PBS control group But not shown significant difference (P > 0.05), this is consistent with peripheral blood CD4+T lymphocyte number testing result.

5 vaccine immune mouse spleen CD4+T cellular change of table

Grouping	25 days	32 days	39 days	46 days
					Recombination 140321	22.3±2.52	38.4±1.75	31.5±2.18	18.7±1.49
Recombination 140323	23.1±1.83	37.9±3.35	30.6±2.86	17.3±1.91
					Recombination 140326	21.5±2.08	38.1±2.17	30.3±3.84	18.2±3.42
Inactivation 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 variation of lymphocyte call subtype quantity

5.2.1 the variation recombinant multi-epitope vaccine immunity group peripheral blood CD8+T lymph of peripheral blood CD8+T lymphocyte quantity Cell number increases, not shown downward trend as immunization time increases, until 46 days, quantity and 39 days not shown significant difference (P > 0.05), and inactivated vaccine group and the not shown apparent increase and decrease of PBS control group CD8+T lymphocyte quantity.25 after immune It, recombinant multi-epitope vaccine group CD8+T lymphocyte quantity and inactivated vaccine group are without significant difference (P > 0.05), but obvious height In control group (P < 0.05)；32~46 days after immune, recombinant multi-epitope vaccine group CD8+T lymphocyte quantity, which is apparently higher than, to go out Live vaccine group and PBS control group, and difference is extremely significant (P < 0.01) (being shown in Table 6).

The variation of 6 vaccine immune mouse peripheral blood CD8+T lymphocyte of table

Grouping	25 days	32 days	39 days	46 days
					Recombination 140321	11.09±1.62	17.85±2.44	23.64±2.18	24.36±3.02
Recombination 140323	12.11±2.85	18.93±1.73	24.72±2.09	25.07±2.16
					Recombination 140326	11.76±1.17	17.52±2.38	24.33±1.95	24.15±2.63
Inactivation 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 variation recombinant multi-epitope vaccine immunity group spleen CD8+T lymphocyte of spleen CD8+T lymphocyte quantity Number is higher than inactivated vaccine group and PBS control group, and extremely significant difference (P < 0.01) is presented, although inactivated vaccine group is higher than PBS pairs According to group, but not shown significant difference (P > 0.05), and the CD8+T lymphocyte quantity of inactivated vaccine group and PBS control group not with Time change and change, this is consistent with peripheral blood CD8+T lymphocyte number testing result.Rising is presented always with peripheral blood to become Gesture is different, and spleen tissue CD8+T lymphocyte quantity peaked at 32 days, hereafter falls after rise rapidly, until 46 days, slightly above inactivation epidemic disease Seedling group, but without significant difference (P > 0.05).(being shown in Table 7).

The variation of 7 vaccine immune mouse spleen tissue CD8+T lymphocyte of table

Grouping	25 days	32 days	39 days	46 days
					Recombination 140321	15.82±1.33	23.15±2.64	15.29±1.92	7.08±1.48
Recombination 140323	13.38±2.54	22.47±3.08	14.82±2.29	6.55±2.03
					Recombination 140326	15.35±1.62	22.81±2.83	14.93±1.25	6.74±0.81
Inactivation 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 (6)

1. a kind of reinforced polyepitope vaccines fusion protein of H9N2 subtype avian influenza, amino acid sequence is SEQ ID No.2.

2. a kind of nucleic acid molecules, nucleic acid sequence is SEQ ID No.1, encodes claim 1 fusion protein.

3. a kind of carrier contains nucleic acid molecules as claimed in claim 2.

4. a kind of host cell contains carrier as claimed in claim 3.

5. a kind of for preventing the vaccine of H9N2 subtype avian influenza, it includes fusion protein described in claim 1 and pharmacy Upper acceptable carrier.

6. fusion protein described in claim 1 answering in the reinforced polyepitope vaccines of preparation and reorganization H9N2 subtype avian influenza With.