CN110075290A

CN110075290A - Influenza mucosal vaccine composition and the preparation method and application thereof

Info

Publication number: CN110075290A
Application number: CN201810075119.3A
Authority: CN
Inventors: 吴夙钦; 林师纬; 唐铭甫; 陈廷轩
Original assignee: Individual
Current assignee: Individual
Priority date: 2018-01-25
Filing date: 2018-01-25
Publication date: 2019-08-02

Abstract

The present invention provides a kind of influenza mucosal vaccine composition and the preparation method and application thereof.The composition contains antigen coalescence protein, and it includes influenza antigens and Escherichia coli second type b type to avoid heat type enterotoxin A sub-cell.The inoculation of the antigen coalescence protein can cause the cellularity that one is fought specific influenza virus in vivo and react with humoral immune, react comprising systemic and mucosa-immune, therefore protect the individual from virus infection.

Description

Influenza mucosal vaccine composition and the preparation method and application thereof

Technical field

The present invention relates to a kind of vaccine compositions and the preparation method and application thereof, are melted especially with regard to a kind of using antigen The influenza mucosal vaccine composition of hop protein and the preparation method and application thereof

Background technique

Influenza virus is to belong to ribonucleic acid (RNA) the virus of Orthomyxoviridae family (Orthomyxoviridae) to feel The influenza virus for contaminating the mankind includes A type (Type A), Type B (Type B) and c-type (TyPe C).Influenza A once causes to count It is secondary to be very popular, including spanish influenza caused by the virus of H1N1 in 1918, Asia influenza caused by nineteen fifty-seven H2N2 virus and Global swine flu caused by H1N1 viruses in 2009.

Can the influenza A of infected poultry be divided into highly pathogenic bird flu (highly according to the severity of associated diseases Pathogenic avian influenza, HPAI) virus, such as H5N1 virus, with low pathogenicity bird flu (low Pathogenic avian influenza, LPAI) virus.Highly pathogenic avian influenza virus can connect by the mankind and illness birds It touches and infects the mankind, have high lethality rate through the lower respiratory tract of the infection mankind in birds and the mankind and replicate, height causes a disease Property avian influenza virus cause in patient's pulmonary lesion and serum cytohormone to lack of proper care, eventually lead to serious disease in 1997 years, H5N1 virus causes a large amount of sudden deaths of chicken in Hong Kong chicken farm, also results in ten many cases human infection thereafter, H5N1 bird flu also exists All parts of the world occurs, including Asia, the Middle East, Europe and Africa

To avoid Major health caused by influenza and economic loss, medicine related researcher focuses on research and development influenza epidemic disease Seedling includes nasal cavity, sublingual, mouth including that can cause the mode of bestowing of the mucosal vaccine mucosal vaccine of mucosa-immune reaction in individual Clothes, rectum and vaginal injection, wherein nasal cavity vaccinates the mucous membrane and systemic immunity that can produce for antigen, provides breathing Fight the protection of external cause of disease in road.However, nasal cavity vaccinates it is generally necessary to which the assistance of adjuvant can just cause effective mucous membrane Immunity common mucosal vaccine adjuvant includes cholera toxin (cholera toxin, CT) and avoids heat type enterotoxin (heat- Labile enterotoxin, LT), demethylation dinucleotides (unmethylated CpG dinucleotides), single phosphorus Sour lipid (monophosphoryl lipid A, MPL) and class tongued bell receptor (Toll-like receptor) stimulant.

Avoiding heat type enterotoxin is a kind of bacterio protein toxin, according to its amino acid sequence and to gangliosides (gangliosides) binding force can be divided into the first type (Type I) and second type (Type II) sub-family, and second type is sub Family can be categorized further, as three subgroups, including second type a type (Type IIa), second type b type (Type IIb) and second Type c type (Type IIc) avoids heat type enterotoxin and is made of A sub-cell and B sub-cell, and wherein A sub-cell has adenosine diphosphate (ADP)- Ribose glycosidation (ADP-ribosylation) activity, and the pentamer (pentamer) that B sub-cell is formed is in combination with thin to eukaryon The glycoprotein of cellular surface, so that enterotoxin is entered cell will cause intestines due to avoiding the effect of heat type enterotoxin A sub-cell in the cell Moisture absorption reduces and causes diarrhea in road, usually used to contain A when prior art is to avoid heat type enterotoxin as vaccine adjuvant The holotoxin (holotoxin) of sub-cell mutant, or using avoiding the pentamer of heat type enterotoxin B sub-cell about can be with More easy mode, such as in conjunction with avoid heat type enterotoxin A sub-cell and the influenza antigen of wild type be single fusion protein, Is still unknown in preparing effective influenza mucosal vaccine

Summary of the invention

Edge this, a purpose of the invention is providing a kind of influenza mucosal vaccine composition, include an antigen coalescence protein, In the antigen coalescence protein include that an influenza antigen and an Escherichia coli second type b type avoid heat type enterotoxin A sub-cell (Type IIb heat-labile enterotoxin A subunit).

Another object of the present invention is in the use for providing a kind of antigen coalescence protein and being used to prepare influenza mucosal vaccine composition On the way, wherein the antigen coalescence protein includes that an influenza antigen and an Escherichia coli second type b type avoid heat type enterotoxin A time list First

In one embodiment of this invention, which is for a hemagglutinin extracellular domain (hemagglutinin ectodomain)；The N-terminal of the antigen coalescence protein further includes a histidine tract；And influenza mucosal vaccine combination Object further includes an Escherichia coli second type b type and avoids heat type enterotoxin B sub-cell, be separated with the antigen coalescence protein or In conjunction with the antigen coalescence protein.

In one embodiment of this invention, which includes the extracellular containing the hemagglutinin of about 10 μ g The antigen coalescence protein in domain is to bestow via nasal cavity

In one embodiment of this invention, the antigen coalescence protein containing the H5 type hemagglutinin extracellular domain activates class tongued bell receptor 2/1 (Toll-like receptor 2/1, TLR2/1), and cause the reaction of T cell related immune, such as interferon-γ (interferon- γ, IFN-γ), Jie Bai Su -4 (interleukin-4, IL-4), Jie Bai Su -17A (interleukin- 17A, IL-17A), or any combination thereof secretion

A further object of the present invention is providing a kind of preparation method of aforementioned influenza mucosal vaccine composition, includes: preparation One antigen coalescence protein, it includes an influenza antigens and an Escherichia coli second type b type to avoid heat type enterotoxin A sub-cell； And the antigen coalescence protein and a pharmaceutically acceptable carrier are mixed to obtain the influenza mucosal vaccine composition.

In one embodiment of this invention, the preparation method further include addition one Escherichia coli second type b type avoid heat Type enterotoxin B sub-cell is to influenza mucosal vaccine composition

Influenza mucosal vaccine composition of the invention effectively causes a confrontation influenza in vivo using an antigen coalescence protein The antibody mediated of viral antigen reacts with cellular immunity, comprising in the influenza virus in blood and bronchovesicular mucous membrane and anti- The cytohormone of a variety of regulation nature-nurture immune systems secreted by body and T cell therefore, the influenza mucosal vaccine Composition can be promoted individual to systemic and the mucosa-immune power of resisiting influenza virus, flu-prevention virus via respiratory tract intrusion, And reduce the even death of injury caused by virus infection.

Embodiments of the present invention are further illustrated below in conjunction with schema, and following cited embodiments are to illustrate Invention feature of the invention and application, rather than to limit the scope of the invention, it is any to be familiar with this those skilled in the art, do not departing from the present invention Spirit and scope in, when can do it is a little change and retouch, therefore protection scope of the present invention is when depending on appended applying for a patent model It encloses subject to institute's defender.

Detailed description of the invention

What Figure 1A showed one embodiment of the invention includes a H5 type hemagglutinin extracellular domain (abbreviation H5HA herein) and one Escherichia coli second type b type avoids the H5HA-LTIIb-A fusion protein of heat type enterotoxin A sub-cell (abbreviation LTIIb-A herein) Construction schematic diagram；The H5HA-LTIIb-A fusion protein includes a histidine tract for being located at N-terminal；

Figure 1B shows the construction schematic diagram of the H5HA-LTIIb-A fusion protein for the histidine tract for being located at C-terminal comprising one；

Fig. 1 C is melted with sodium dodecyl sulfate polyacrylamide colloid electrophoresis (SDS-PAGE) analysis H5HA-LTIIb-A Hop protein and H5HA recombinant protein；

Fig. 1 D is to analyze H5HA-LTIIb-A fusion protein and H5HA using anti-H5 type hemagglutinin antibody with Western blot Recombinant protein；

Fig. 1 E is using anti-H5 type hemagglutinin antibody with H5HA- of the Western blot analysis comprising N-terminal histidine tract LTIIb-A fusion protein (a, b) and H5HA-LTIIb-A fusion protein (c, d) comprising C-terminal histidine tract；A, c institute in figure The H5HA-LTIIb-A fusion protein of representative is taken from the cell culture medium after Sf9 insect cell shows protein 48 hours；b,d Representative H5HA-LTIIb-A fusion protein is taken from the cell culture medium after Sf9 insect cell shows protein 72 hours；

Fig. 1 F is with the more anti-histidine tract antibody of Western blot and the H5HA- comprising N-terminal histidine tract The binding force of LTIIb-A fusion protein (a, b) or the H5HA-LTIIb-A fusion protein (c, d) comprising C-terminal histidine tract；Figure H5HA-LTIIb-A fusion protein representated by middle a, c is taken from the cell training after Sf9 insect cell shows protein 48 hours Support base；B, the H5HA-LTIIb-A fusion protein representated by d is taken from the cell after Sf9 insect cell shows protein 72 hours Culture medium；

Fig. 2A is the red blood cell for comparing H5HA recombinant protein and H5HA-LTIIb-A fusion protein with erythrocyte agglutination test It is aggregated effect；

Fig. 2 B is the tire ball for comparing H5HA-LTIIb-A fusion protein and H5HA recombinant protein in conjunction with test with myosin Protein binding capacity；

Fig. 3 is with 2/1 functional trial of class tongued bell receptor analysis multiple protein quality sample activation class tongued bell receptor 2/1 and NF- κ B Ability, which includes that H5HA recombinant protein, H5HA-LTIIb-A fusion protein, Escherichia coli second type b type are avoided Heat type enterotoxin B sub-cell (abbreviation LTIIb-B5 herein) recombinant protein and H5HA-LTIIb-A fusion protein and 10⁴ng/ The combination (being denoted as H5HA-LTIIb-A+10 μ g/ml LTIIb-B) of the LTIIb-B5 recombinant protein of ml；

Fig. 4 A-4B shows that BALB/c mouse after different inoculations, fights exempting from for H5HA recombinant protein in serum The potency of epidemic disease Lysozyme (IgG) and immunoglobulin A (IgA)；Inoculation be bestow phosphate buffered saline solution (PBS solution), H5HA recombinant protein, H5HA-LTIIb-A fusion protein or H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein It combines (being denoted as H5HA-LTIIb-A+LTIIb-B)；

Fig. 4 C-4D shows BALB/c mouse after different inoculations, and BAL fluid (wash by abbreviation lung Liquid) in confrontation H5HA recombinant protein IgG and IgA potency；Inoculation be bestow PBS solution, H5HA recombinant protein, The combination of H5HA-LTIIb-A fusion protein or H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein；

Fig. 5 A-5B shows BALB/c mouse after different inoculations, and H5N1 influenza is false in serum and lung washing lotion The neutralization curve of virucidin；Inoculation is to bestow PBS solution, H5HA recombinant protein, H5HA-LTIIb-A fusion egg White or H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein combination；

Fig. 5 C-5D is calculated in serum and the lung washing lotion of the BALB/c mouse through different inoculations according to Fig. 5 A-5B The potency of the false virucidin of H5N1 influenza；

Fig. 6 A-6B display BALB/c mouse is after different inoculations, the IFN- of spleen and cervical lymph nodal cell γ secretory volume；Inoculation is to bestow PBS solution, H5HA recombinant protein, H5HA-LTIIb-A fusion protein or H5HA- The combination of LTIIb-A fusion protein and LTIIb-B5 recombinant protein；

Fig. 6 C-6D display BALB/c mouse is after different inoculations, the IL-4 of spleen and cervical lymph nodal cell Secretory volume；Inoculation is to bestow PBS solution, H5HA recombinant protein, H5HA-LTIIb-A fusion protein or H5HA-LTIIb-A The combination of fusion protein and LTIIb-B5 recombinant protein；

Fig. 6 E-6F display BALB/c mouse is after different inoculations, the IL- of spleen and cervical lymph nodal cell 17A secretory volume；Inoculation is to bestow PBS solution, H5HA recombinant protein, H5HA-LTIIb-A fusion protein or H5HA- The combination of LTIIb-A fusion protein and LTIIb-B5 recombinant protein；

Fig. 7 A shows the survival curve after the BALB/c mouse of different inoculations is injected H5N1 virus；Immune note Penetrate is to bestow PBS solution, 10 μ gH5HA recombinant proteins, 10 μ gH5HA-LTIIb-A fusion proteins or 10 μ gH5HA-LTIIb-A The combination of fusion protein and 5 μ gLTIIb-B5 recombinant proteins

Fig. 7 B shows the percent weight after the BALB/c mouse of different inoculations is injected H5N1 virus；It is immune Injection is to bestow PBS solution, 10 μ gH5HA recombinant proteins, 10 μ gH5HA-LTIIb-A fusion proteins or 10 μ gH5HA-LTIIb- The combination of A fusion protein and 5 μ gLTIIb-B5 recombinant proteins；

Fig. 7 C shows the survival curve after the BALB/c mouse of different inoculations is injected H5N1 virus；Immune note Penetrate is to bestow PBS solution, 5 μ gH5HA recombinant proteins, 5 μ gH5HA-LTIIb-A fusion proteins or 5 μ gH5HA-LTIIb-A fusion The combination of albumen and 5 μ gLTIIb-B5 recombinant proteins；

Fig. 7 D shows the percent weight after the BALB/c mouse of different inoculations is injected H5N1 virus；It is immune Injection is to bestow PBS solution, 5 μ gH5HA recombinant proteins, 5 μ gH5HA-LTIIb-A fusion proteins or 5 μ gH5HA-LTIIb-A to melt The combination of hop protein and 5 μ gLTIIb-B5 recombinant proteins；

Fig. 7 E shows the survival curve after the BALB/c mouse of different inoculations is injected H5N1 virus；Immune note Penetrate is to bestow PBS solution, 2.5 μ gH5HA recombinant proteins, 2.5 μ gH5HA-LTIIb-A fusion proteins or 2.5 μ gH5HA- The combination of LTIIb-A fusion protein and 5 μ gLTIIb-B5 recombinant proteins；

Fig. 7 F shows the percent weight after the BALB/c mouse of different inoculations is injected H5N1 virus；It is immune Injection is to bestow PBS solution, 2.5 μ gH5HA recombinant proteins, 2.5 μ gH5HA-LTIIb-A fusion proteins or 2.5 μ gH5HA- The combination of LTIIb-A fusion protein and 5 μ gLTIIb-B5 recombinant proteins；

Fig. 8 A-8B shows that chicken after different inoculations, fights the immune globulin of H5HA recombinant protein in serum The potency of white Y (IgY) and IgA；Inoculation be bestow PBS solution, H5HA recombinant protein, H5HA-LTIIb-A fusion protein, Or the combination of H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein；

Fig. 9 is the antiserum effect for inhibiting chicken of test (HIassay) detection through different inoculations with viral hemagglutinin Valence；Inoculation is to bestow PBS solution, H5HA recombinant protein, H5HA-LTIIb-A fusion protein or H5HA-LTIIb-A fusion The combination of albumen and LTIIb-B5 recombinant protein；

Figure 10 A is with H5N1 stream in the serum of molten chicken of spot neutralization test (PRNT) measurement through different inoculations of virus The neutralization curve of Influenza Virus neutralizing antibody；Inoculation be bestow H5HA recombinant protein, H5HA-LTIIb-A fusion protein or The combination of H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein；

Figure 10 B is to calculate H5N1 influenza virus neutralizing antibody in the serum of the chicken through different inoculations according to Figure 10 A Potency.

Specific embodiment

Definition

Numerical value used herein is approximation, and all experimental datas all indicate in the range of 20%, preferably exist In the range of 10%, most preferably in the range of 5%

So-called " H5HA " herein is the abbreviation of H5 type hemagglutinin extracellular domain (H5hemagglutinin ectodomain). The H5 type hemagglutinin extracellular domain is a structural domain (domain) for the aminoterminal (N-terminal) positioned at H5 hemagglutinin.H5 type hemagglutinin is still Comprising position c-terminus (C-terminal) transmembrane domain (transmembrane domain) and tail end (cytoplasmic intracellular tail).

Material and method

The preparation of H5 type hemagglutinin extracellular domain (H5HA) recombinant protein

The performance and purifying of H5 type hemagglutinin extracellular domain recombinant protein are according to Lin et al. (Lin et al., PloS One, 2011；6 (5): e20052) method carry out .H5 hemagglutinin gene from the A/ of influenza A H5N1 hypotype Complementary DNA (cDNA) (the complementarydeoxyribonucleic of Thailand/1 (KAN-1)/2004 Strain Acid, cDNA) cuts by protease in order to avoid H5HA recombinant protein, and it will the corresponding nucleic acid sequence for cutting position in H5HA genetic fragment Column are slightly modified, and the amino acid sequence for cutting position is made to replace with PQRETRG (SEQ ID by PQRERRRKKRG (SEQ ID NO:1) NO:2) thereafter, shows aforementioned H5HA weight using Bac-to-Bac insect baculovirus protein expression system (Invitrogen) Histone is in short, grow aforementioned modified H5HA genetic fragment choosing into pFastbac.1 matter according to manufacturer's operation instructions Body turns shape with plastid progress DH10Bac Escherichia coli, chooses the H5HA genetic fragment via blue and white screening and be embedded in rod-shaped disease The Escherichia coli of poisonous carrier (Bacmid), and baculovirus vector is purified secondly, the baculovirus vector is transfected (transfection) to Sf9 insect cell (Invitrogen) makes cell generate and release with the H5HA genetic fragment It is 2 × 10 that SF900 cell culture medium (Invitrogen) cell density, which is added, in the baculoviral by baculoviral⁶A cell/ The Sf9 insect cell of ml is simultaneously cultivated 48 hours in 27 DEG C, can make cells show and secretes H5HA recombinant protein and is thin to SF900-II Born of the same parents' culture medium

Escherichia coli second type b type avoids the preparation of heat type enterotoxin B sub-cell (LTIIb-B5) recombinant protein

For the LTIIb-B5 recombinant protein of form of expression pentamer, codon is optimized into (codon Optimization enterotoxigenicEscherichia coli (enterotoxigenic Escherichia coli, ETEC)) LTIIb-B5 gene (SEQ ID NO:3) choosing grows (cloning) to pET22b (+) carrier with construction LTIIb-B5-pET22b (+) Plastid is secondly, turn shape (transformation) to e. coli bl21 cell (DE3) for LTIIb-B5-pET22b (+) plastid (Invitrogen), make the bacterium in 37 DEG C of LB culture medium (Luria- overnight being incubated at containing Ampicillin (ampicillin) Bertani medium) the culture solution overnight is inoculated in the LB culture medium containing Ampicillin, in 37 DEG C of cultures to it in 600nm Light absorption value (O.D.600) up to 0.4~0.6, add isopropyl-β-D-thiogalactoside (isopropyl β-D-1- Thiogalactopyranoside, IPTG) and it is further cultured at 37 DEG C the performance to induce LTIIb-B5 recombinant protein in 4 hours. With (10,000rpm, 10 minutes, 4 DEG C) collection cell precipitations (cell pellet) of centrifugation.

When carrying out protein purification, above-mentioned cell precipitation is resuspended in containing phenylmethylsulfonyl fluoride Buffer solution A (the tri- hydrogen aminomethane of 300mM of (phynelmethane-sulfonyl fluoride, PMSF) (Tris), 50mM sodium chloride, 10mM imidazoles (imidazole), 5% glycerol, pH 7.2), and it is thin with high pressure (15Kpsi) cracking Cell pyrolysis liquid is centrifuged 10 minutes at 4 DEG C with 10,000rpm by born of the same parents, and it is mixed with the buffer solution A of the urea containing 8M again to collect sediment Aforementioned mixture is closed at 4 DEG C with 10,000rpm centrifugation 10 minutes, supernatant is collected and is mixed with nickel ion Ao resin (TOSOH) It closes placement overnight and fills the resin compound in tubing string, rinsed with the buffer solution A containing 0.5%Triton X-100, then It is washed using 30~40% buffer solution B (300mM Tris, 50mM sodium chloride, 500mM imidazoles, 5% glycerol, pH 7.2~7.5) Deviate from LTIIb-B5 recombinant protein.The purifying graduation of LTIIb-B5 recombinant protein is placed in the bag filter of molecular cut off 10kDa With phosphate buffered saline solution (phosphate buffered saline, 137mM sodium chloride, 2.7mM potassium chloride, 7.7mM phosphoric acid Disodium hydrogen, 1.47mM potassium dihydrogen phosphate, pH 7.4, abbreviation PBS solution) in 4 DEG C of progress overnight dialysis, then with 10kDa be concentrated from Heart pipe (Millipore) is concentrated and is stored in -20 DEG C of LTIIb-B5 recombinant protein with sodium dodecyl sulfate polyacrylamide Colloid electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) Its status is confirmed with Western blot (western blotting).

Sodium dodecyl sulfate polyacrylamide colloid electrophoresis (SDS-PAGE)

The operation of SDS-PAGE is summarized as follows firstly, protein example and SDS are loaded into buffer solution (tri- hydrogen first of 50mM Base aminomethane-hydrogen chloride (Tris-HCl), pH 6.8,100mM dithiothreitol (DTT) (dithiothreitol, DTT), 2% SDS, 0.1% bromophenol blue (bromophenol blue), 10% glycerol) it mixes and boils 5 minutes according to 3: 1.Meanwhile preparation includes 12% separation colloid (2.5ml 1M Tris, pH 8.8,3.3ml deionized water, 30% acrylamide premixed liquid of 4ml (acrylamide mix), 10% ammonium persulfate of 0.1ml 10%SDS, 0.1ml (ammonium persulfate, APS), 0.01ml tetramethyl is with diamines (TEMED)) and 5% coke collection colloid (0.63ml 1M Tris, pH 6.8,3.4ml deionized water, 0.83ml30% acrylamide premixed liquid, 0.05ml 10%SDS, 0.05ml 10%APS, 0.005ml TEMED) running gel Body.Electrophoresis is to carry out Jiao Ji at voltage 80V and carry out separation at 150V thereafter, and colloid is molten with Coomassie brilliant blue stain Liquid (0.1%coomassie R250,10% acetic acid, 50% methanol) dye 1 hour, then with de-inking solution (10% acetic acid, 50% Methanol) decoloration

Western blot

The operation of Western blot is summarized as follows that the protein example separated through SDS-PAGE is transferred to by with voltage 135V Nitrocellulose membrane (nitrocellulose membrane), then the film is placed in three containing Tween-20 for adding 5% defatted milk Methylol buffer saline (abbreviation TBST solution, 50mM Tris, 150mM sodium chloride, 0.05%Tween-20), and shake Swing at least 1 hour to block non-specific combination film to clean through TBST solution, with anti-histidine tract antibody (Bethyl, A190-114P) or anti-H5 type hemagglutinin antibody (Genetex, GTX41297) according to extension rate 1: 10000 in TBST solution Reason 1 hour, then cleaned through TBST solution, it is small to link resisting for horseradish peroxidase (horseradish peroxidase, HRP) Rat immune globulin G secondary antibody (GeneTex) is handled 1 hour in TBST solution according to extension rate 1: 1500, then with TBST Solution cleaning.When detection, enhanced chemical luminescence reagent (Western Lighting Plus ECL, PerkinElmer) is added The film is added to generate cold light signal, and is developed to medical X-ray egative film (Medical X-ray Film, Fujifilm)

Erythrocyte agglutination tests (hemagglutination assay)

Firstly, the protein example of preparation serial dilution, initial concentration is 70 μ g/ml and with PBS solution (pH 7.4) Two times of serial dilution are carried out secondly, 96 hole V-type disks are added according to 50 holes μ l/ in the protein example of serial dilution, then according to 50 μ l/ The PBS solution containing 0.5% turkey erythrocytes is added in hole, in being stored at room temperature 30 minutes to observe hemagglutination with first observed To potency (titer) as recombinant protein of maximum dilution multiple of erythrocyte sedimentation

Myosin combines test (fetuin binding assay)

By the binding soln (0.05M carbonate buffer solution, pH 9.6) containing 100 μ g/ml myosins according to 100 holes μ l/ 96 porose discs are added, after standing 16~18 hours at 4 DEG C, (abbreviation PBST is molten with the PBS solution containing 0.05%Tween-20 Liquid) cleaning, the 200 μ l PBS solutions containing 1% bovine serum albumin (bovine serum albumin, BSA) are added in room temperature Lower blocking (blocking) 1 hour to avoid non-specific combination secondly, 96 porose disc is cleaned with PBST solution and added in each hole Entering the protein example of serial dilution, (initial concentration is 10 μ g/ml and with the PBS solution containing 1%BSA and 0.05%Tween-20 Carry out two times of serial dilutions), in incubated at room temperature 1 hour, then with the cleaning of PBST solution.Then, 96 porose disc is with anti-H5 hemagglutinin Antibody (extension rate 1: 10000) handles 1 hour according to 100 holes μ l/ and with the cleaning of PBST solution, then to link horseradish peroxidase The sub- immunoglobulin G secondary antibody of anti-rabbit (extension rate 1: 5000) according to 100 holes μ l/ in room temperature handle 1 hour it is final, will Horseradish peroxidase by 3,3 ', 5,5 '-tetramethyl benzidine of matter (3,3 ', 5,5 '-Tetramethyl-benzidine, TMB； Biolegend 96 porose discs) are added according to 100 holes μ l/, after dark place carries out color reaction 15 minutes, 2N sulphur is added according to 100 holes μ l/ Acid terminates reaction, uses ELISA disk-read machine (TECAN SUNRISE^TM) each hole is measured in the light absorption value of wavelength 450nm (O.D.450).

2/1 functional trial of class tongued bell receptor

Human embryonic kidney cells HEK-293A (Invitrogen, article No. R70507) is incubated at addition 5%BSA and 100U/ The DMEM culture medium (Dulbecco ' s Modified Essential Medium, Invitrogen) of ml Pen .- Strep. When carrying out class tongued bell 2/1 functional trial of receptor, HEK-293A cell is according to 6 × 10⁶A cell/disk is inoculated in 10 centimeters of cell culture Disk (SPL) is incited somebody to action in the CMC model of 37 DEG C, 5% carbon dioxide overnight using transfection reagent Turbofect (Fermentas) PDUO-hTLR1/2 carrier (InvivoGen) and the transfection of pGL4.32 [1uc2p/NF- κ B-RE/Hygro] carrier (Promega) are extremely Cell replaces culture medium and in the CMC model of 37 DEG C, 5% carbon dioxide overnight through transfecting cell according to 5 × 10 after a few hours⁴ A cells/well is seeded to 96 porose discs, by the protein example of serial dilution (10ng/ml to 1pg/ml) is added to cell, and in 37 DEG C are cultivated 5 hours.96 porose disc is after PBS solution is cleaned, with cell pyrolysis liquid (Glo-lysis buffer；Promega) Processing cell 5 minutes, then cold light is added by matter (neolite assay, PerkinElmer), after five minutes with more according to 50 holes μ l/ Porose disc analyzer Victor II (PerkinElmer) measures each hole in the relative light unit (relative of wavelength 560nm Luminescence units, RLU).

Animal immune injection and sample collection

Immunization experiment is to be injected using the BALB/c female mice of six week old and every mouse of chicken of seven ages in days with nasal cavity (intranasal) mode bestows the PBS solution or PBS solution itself containing recombinant protein of fixed volume.To be infused convenient for nasal cavity It penetrates, with arcotic Ai Kening (isoflurane；Panion&BF Biotech) suction-type general anesthesia is carried out to mouse, then will Protein example instills its nasal cavity each group mouse and all gives inoculation three times, and interval is about three weeks in third time injection two weeks Mouse blood sample is acquired afterwards；Mouse is sacrificed after three weeks in third time injection and acquires BAL fluid (bronchoalveolar lavage fluid, BALF, abbreviation lung washing lotion) and spleen and cervical lymph node (cervical Lymph nodes, CLNs) according to similar fashion, with nasal cavity injection to every chicken bestow fixed volume containing recombinant protein PBS solution or PBS solution itself.Each group chicken is all injected three times, and injection interval is about two weeks before the injection and third time is injected After two weeks by venous collection chicken blood sample said blood sample under the wing through 56 DEG C heat 2 hours, with 3000rpm from It separates to obtain blood cell and serum within the heart 10 minutes, serum is taken to be stored in -20 DEG C.Aforementioned lung washing lotion is centrifuged after ten minutes with 3000rpm, Supernatant is taken to be stored in -20 DEG C of

Antibody content measurement

It is measured using ferment combination immunoabsorption (enzyme-linked immunosorbent assay, ELISA) small Immunoglobulin G (immunoglobulin G, IgG) and immunoglobulin A (immunoglobulin in mouse serum and lung washing lotion A, IgA) potency and chicken Immunoglobulin in Serum Y (immunoglobulin Y, IgY) and IgA potency.Firstly, 96 porose discs are added according to 100 holes μ l/ in binding soln containing 0.2 μ g/ml H5HA recombinant protein, it is small that 16-18 is stood at 4 DEG C Shi Hou is added the 200 μ l PBS solutions containing 1%BSA and is blocked at room temperature 1 hour with the cleaning of PBST solution.Secondly, this 96 With the cleaning of PBST solution and PBS solution serial dilution of the 100 μ l containing 1%BSA and 0.05%Tween-20 is added in each hole in porose disc Aforementioned serum or lung washing lotion, in incubated at room temperature 1 hour, then with PBST solution cleaning.Then, horseradish peroxidase will be linked Anti-mouse IgG antibody (extension rate 1: 60000), anti-mouse IgA antibody (extension rate 1: 50000), anti-chicken IgY antibody are (dilute Release multiple 1: 10000) or according to 100 holes μ l/ 96 porose disc is added in anti-chicken IgA antibody (extension rate 1: 5000) (BETHYL), in It cultivates 1 hour at room temperature, then final with PBST solution cleaning, 96 porose disc is added according to 100 holes μ l/ in TMB, is carried out in dark place After color reaction 15 minutes, 2N sulfuric acid is added according to 100 holes μ l/ and terminates reaction, measures each hole in 450nm using ELISA disk-read machine Light absorption value.

The preparation of the false virus of H5N1 influenza

The preparation method of H5N1 influenza false viral (H5N1 pseudo-type virus particle) is with reference to previous Paper (Nefkens et al., 2007；Lin et al., PloS One, 2011；6 (5): e20052) is by HEK-293A cell According to 3 × 10⁶10 centimeters of cell cultures are added in a cell/disk, overnight in the CMC model of 37 DEG C, 5% carbon dioxide.Using turn Transfection reagent Turbofect will have the pNL-Luc-E of cold light reporter gene^-R^-Plastid (inhibition of HIV skeleton), pcDNA 3.1 (+)- HA carrier (A/Thailand/1 (KAN-1)/2004) and pcDNA 3.1 (+)-NA carrier (A/Vietnam/1203/2004) turn Dye replaces culture medium to HEK-293A cell, after a few hours after 37 DEG C, CMC model 48 hours of 5% carbon dioxide, will be thin The collection of born of the same parents' culture solution is stored in -20 DEG C of

Neutralizing antibody test

By canine kidney cells strain MDCK (Madin-Darby canine kidney cells；It is ground by Academia Sinica Zheng parasol pine The person of studying carefully provides) according to 1 × 10⁵A cells/well is overnight to be incubated at 96 porose disc thereafter, and mice serum or lung washing lotion are with DMEM culture medium Serial dilution takes the H5N1 influenza with cold light reporter gene of 50 μ l dilutions Yu fixed amount (40000 equivalent cold light intensity) False the isometric mixing of virus 1 hour, then mdck cell is added in the mixed liquor to cultivate in 37 DEG C 2 days 96 porose discs molten through PBS Liquid cleaning after, with cell pyrolysis liquid processing cell 5 minutes, then according to 50 holes μ l/ be added cold light by matter (neolite assay, PerkinElmer), each hole is measured after five minutes with porous disc analyzer Victor II to compare only in the luminous value of wavelength 560nm With the luminous value of the control group cell of false virus treated, the luminous value of reduction can be used for due to serum-virus mixture processing Calculating viral percent neutralization and drawing the potency of neutralization curve neutralization curve and neutralizing antibody is using software Graph Pad Prism version 5 calculates with regression analysis

Viral hemagglutinin inhibits test (viral hemagglutinin inhibition (HI) assay)

Before test, with 30 μ l receptor destroying enzyme (receptor destroy enzyme, RDE at 37 DEG C；Denka Seiken it) handles 10 μ l chicken serum 18~20 hours, removes the non-specific substance for causing erythrocyte agglutination whereby thereafter, it is preceding It states serum mixture and heats 30 minutes in 56 DEG C to remove RDE enzyme activity, add 60 μ l PBS solutions to mixture totality Product is 100 μ l., the 100 μ l serum mixture and 5 μ the l PBS solution that contains 0.5% turkey erythrocytes in 4 DEG C after culture 1 hour, The supernatant serum supernatant serum is collected with centrifugation (400xg, 10 minutes, 4 DEG C) with two times of serial dilutions of PBS solution Afterwards, 25 μ l dilutions and 4 is taken to be aggregated unit (4HA unit) delta H5N1 virus (according to Mariana Baz et al. preparation； Mariana Baz etal., Virus research.2013.H5N1 vaccines in humans) mixing in equal volume, in 37 DEG C culture 30 minutes, 50 μ l, 0.5% turkey erythrocytes are added, in being stored at room temperature 30~60 minutes to observe hemagglutination.

The molten spot neutralization test (plaque reduction neutralization test, PRNT) of virus

Chicken serum is with MEM- α culture medium (Minimum Essential Medium α；Gibco it) after two times of serial dilutions, takes It is isometric that 20 μ l dilutions and the 100 molten spots of virus form unit (plaque forming unit, PFU) delta H5N1 virus Mixing, in 37 DEG C of 1 hour of culture secondly, by the serum-virus mixture together with contain 0.5 μ g/ml through tosyl-L- The pancreas egg of aminobphenyl chloromethyl ketone (N-tosyl-L-phenylalanine chloromethyl ketone, TPCK) processing White enzyme (trypsin；Sigma 960 μ l MEM- α culture mediums) are added to according to 9.5 × 10⁵A cells/well inoculation mdck cell 6 hole culture plates are cultivated 1 hour in 37 DEG C.After the cell is cleaned with PBS solution, the pancreas that will be handled containing 0.5 μ g/ml through TPCK Protease and the 3ml MEM- α culture medium of 0.5% agar are added to each hole, and to cover the cell cell, that 48 are cultivated at 37 DEG C is small Shi Hou, with 4% metaformaldehyde (paraformaldehyde；Sigma 8 hours are fixed, then) to be dissolved in the 1% of 20% formaldehyde crystallization Purple (crystal violet；Sigma it) dyes 8 hours and dye is moved back with water to count the molten spot of virus and to compare only with virus treated Control group the molten spot number of virus, the molten spot number of virus of reduction can be used for calculating neutralization hundred due to serum-virus mixture processing Divide and compares and draw neutralization curve

Cytohormone assay

Gained cell is according to 5 × 10 after spleen and cervical lymph nodal tissue are ground⁵A cells/well is incubated at 24 porose discs.Its Afterwards, which gives the thorn of the H5 type hemagglutinin victory peptide fragment of 1 μ g/ml H5N1 influenza virus (A/Vietnam/1203/2004) Swash, the victory peptide fragment 1-50 (NOs:4~53 SEQ ID) comprising HA1 sub-cell and HA2 sub-cell from the hemagglutinin, in In ELISA set group (Biolegend) analysis cell culture fluid points is used in 72 hours of CMC model of 37 DEG C, 5% carbon dioxide Not by the first type helper T lymphocyte (T helper 1 cells, abbreviation Th1 cell), second type helper T lymphocyte (T helper 17cells, abbreviation Th17 are thin for (Thelper2cells, abbreviation Th2 cell) and the 17th type helper T lymphocyte Born of the same parents) secreted by the cytohormones such as IFN-γ, IL-4 and IL-17A content

Embodiment 1

The preparation of antigen coalescence protein

The present embodiment is the illustration of influenza antigen with H5 type hemagglutinin extracellular domain (abbreviation H5HA), illustrates stream of the present invention The antigen coalescence protein of all embodiments is to wrap herein for the preparation method event of antigen coalescence protein in sense mucosal vaccine composition Heat type enterotoxin A sub-cell (abbreviation LTIIb-A is avoided containing a H5 type hemagglutinin extracellular domain and an Escherichia coli second type b type；Amino Acid sequence is SEQ ID NO:54), abbreviation H5HA-LTIIb-A fusion protein herein

To prepare the H5HA-LTIIb-A fusion protein, construction first is sequentially loaded with H5 type hemagglutinin born of the same parents from 5 ' ends to 3 ' ends Foreign lands genetic fragment and Escherichia coli second type b type avoid the DNA building (DNA of heat type enterotoxin A sub-cell gene construct).A/Thailand/1 (KAN-1)/2004 of the gene of H5 type hemagglutinin from influenza A H5N1 hypotype Strain, through select plantation technology can by the gene obtain H5 type hemagglutinin extracellular domain genetic fragment (SEQ ID NO:55).Large intestine Bacillus second type b type avoid heat type enterotoxin A sub-cell gene (SEQ ID NO:56) can pass through select plantation technology big by enterotoxin type The chromosome of enterobacteria obtains.The Escherichia coli second type b type avoid heat type enterotoxin A sub-cell with SEQ ID NO:54 or with SEQ ID NO:54 has the amino acid sequence of 90% or more sequence identity.

One that the DNA building of aforementioned H5HA-LTIIb-A fusion protein can further include between aforementioned two gene is short Nucleic acid encodes white amino acid zipper (the GCN4leucine zipper of a GCN4；SEQ ID NO:57) and one contain glycine The short victory peptide of (glycine, Gly) and silk amino acid (serine, Ser), such as with Gly-Gly-Ser-Gly-Gly-Gly- The white amino acid zipper of GS connection molecule (GS linker) GCN4 of the amino acid sequence of Ser-Gly (SEQ ID NO:58) is used for H5HA-LTIIb-A fusion protein is set to form tripolymer, and the GS connection molecule is for connecting H5 type hemagglutinin extracellular domain and large intestine Bacillus second type b type avoids heat type enterotoxin A sub-cell

To purify convenient for subsequent protein, the N-terminal the 19th of H5HA-LTIIb-A fusion protein is corresponded in the DNA building DNA sequence dna Figure 1A of position setting one encoding histidine (histidine, His) segment of~No. 24 amino acid is shown comprising one The construction schematic diagram of the H5HA-LTIIb-A fusion protein of N-terminal histidine tract alternatively, the encoding histidine segment DNA sequence Column may be disposed at the white amino acid zipper of GCN4 and GS connection molecule that H5HA-LTIIb-A fusion protein is corresponded in the DNA building Between and neighbouring C-terminal position.Figure 1B shows the construction signal of the H5HA-LTIIb-A fusion protein comprising a C-terminal histidine tract Figure.Herein unless specifically stated otherwise, so-called H5HA-LTIIb-A fusion protein is all comprising N-terminal histidine tract

Secondly, being showed using Bac-to-Bac insect baculovirus protein expression system (Invitrogen) aforementioned H5HA-LTIIb-A fusion protein.In short, the choosing of aforementioned DNA building is grown into pFastbac.1 according to manufacturer's operation instructions Plastid turns shape with plastid progress DH10Bac Escherichia coli, chooses the DNA building via blue and white screening and be embedded in rod-shaped disease The Escherichia coli of poisonous carrier (Bacmid), and purify the baculovirus vector.Secondly, transfecting the baculovirus vector to Sf9 Insect cell makes cell generate and release the baculoviral with the DNA building to SF900 cell culture medium (Invitrogen) it is 2 × 10 that cell density, which is added, in the baculoviral by⁶The Sf9 insect cell of a cell/ml is simultaneously trained in 27 DEG C It supports 48 hours, cells show can be made and secrete H5HA-LTIIb-A fusion protein to SF900-II cell culture medium

When carrying out protein purification, aforementioned cells culture medium is suspended carefully for 10 minutes at 4 DEG C with 10,000rpm centrifugation with removal Born of the same parents, then the concentration concentrate of the filter device through molecular cut off 10kDa adjust pH value extremely with Tris buffer solution (pH 8.0) 7.4 and after being filtered with 0.45 μm of filter membrane, it is overnight mix placement at 4 DEG C with nickel ion chelating resin (TOSOH), reuses 30 ~40% buffer solution B elutes H5HA-LTIIb-A fusion protein.The purifying graduation of the H5HA-LTIIb-A fusion protein with 30kDa concentration centrifuge tube (Millipore) concentrates in PBS solution and is stored in -20 DEG C of

The H5HA-LTIIb-A fusion protein is to confirm its status through SDS-PAGE (Fig. 1 C) and Western blot (Fig. 1 D). As shown in Figure 1 C, H5HA-LTIIb-A fusion protein can correspond to the main protein band that a molecular weight is about 100kDa, substantially It is equivalent to H5HA recombinant protein (about 72kDa) and Escherichia coli second type b type avoids heat type enterotoxin A sub-cell (about 28kDa) As shown in figure iD, anti-H5 type hemagglutinin antibody is combinable and detects H5HA-LTIIb-A fusion protein and H5HA by molecular weight summation Recombinant protein, illustrate H5HA-LTIIb-A fusion protein possess H5 type hemagglutinin extracellular domain epitope (epitope) and Structure feature those can successfully obtain H5HA-LTIIb-A fusion protein according to preceding method as the result is shown

To assess influence of the position of histidine tract to H5HA-LTIIb-A fusion protein purification efficiency, with Western The more anti-histidine tract antibody of method and the H5HA-LTIIb-A fusion protein comprising N-terminal histidine tract include C-terminal group ammonia The binding force of the H5HA-LTIIb-A fusion protein of acid fragment is according to Fig. 1 E, and Western blot is analysis shows that anti-H5 type hemagglutinin Antibody may detect that the H5HA-LTIIb-A fusion protein comprising C-terminal histidine tract of about 100kDa however, such as Fig. 1 F institute Show, when carrying out Western blot analysis with anti-histidine tract antibody, the H5HA-LTIIb-A fusion comprising N-terminal histidine tract Albumen can show between anti-histidine tract antibody preferably compared to the H5HA-LTIIb-A fusion protein comprising C-terminal histidine tract Binding force, display selection comprising N-terminal histidine tract H5HA-LTIIb-A fusion protein carry out nickel ion chelating resin it is pure Purification efficiency can be improved in change, therefore the fusion egg of H5HA-LTIIb-A used in embodiment 2~6 is all comprising N-terminal histidine tract H5HA-LTIIb-A fusion protein in figure 1f, the signal of about 70kDa is from non-for H5HA-LTIIb-A fusion protein Other protein

For verifying H5HA-LTIIb-A fusion protein in H5 type hemagglutinin extracellular domain normal configuration and function, below into The test of row erythrocyte agglutination and myosin combine test in erythrocyte agglutination test, by turkey erythrocytes and serial dilution 30 minutes of H5HA-LTIIb-A fusion protein or H5HA recombinant protein immixture according to Fig. 2A, H5HA-LTIIb-A fusion Albumen and H5HA recombinant protein can not all promote erythrocyte agglutination when concentration is 0.88 μ g/100 μ L or less and lead to red blood cell Precipitating, display H5HA-LTIIb-A fusion protein have the ability for being equal to H5HA recombinant protein to go the saliva in conjunction with erythrocyte surfaces Acid, therefore cause identical hemagglutination effect.

It is combined in test in myosin, quantitative myosin is fixed on 96 porose discs, the H5HA- of serial dilution is added LTIIb-A fusion protein or H5HA recombinant protein add anti-H5 hemagglutinin antibody and shine by matter, and with ELISA disk-read machine The light absorption value that each hole is detected in wavelength 450nm changes.According to Fig. 2 B, the light absorption value of 96 porose discs is with H5HA-LTIIb-A fusion egg The concentration of bletilla H5HA recombinant protein increases and rises to about 0.15, and H5HA-LTIIb-A fusion protein and H5HA recombinant protein Binding curve show similar ascendant trend, show that the two has similar myosin binding ability hemagglutination test Test all proves that Escherichia coli second type b type, which is avoided heat type enterotoxin A sub-cell, is connected to H5 type blood clotting in conjunction with myosin Plain extracellular domain will not significantly change the structure in the region H5HA and function in H5HA-LTIIb-A fusion protein

Embodiment 2

H5HA-LTIIb-A fusion protein activates class tongued bell receptor 2/1

The present embodiment activates class tongued bell receptor 2/ with 2/1 functional trial of class tongued bell receptor test H5HA-LTIIb-A fusion protein The ability of 1 message transmission path.Firstly, 2/1 display carriers of mankind's class tongued bell receptor and NF- κ B cold light reporter vector are transfected simultaneously To HEK-293A cell, class tongued bell receptor 2/1 is made to find expression in HEK-293A cell surface secondly, by the H5HA- of serial dilution LTIIb-A fusion protein, H5HA recombinant protein, Escherichia coli second type b type avoid heat type enterotoxin B sub-cell (abbreviation LTIIb- B5) combination of recombinant protein or H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein be added to cell when class tongued bell by Body 2/1 is activated by foregoing proteins quality sample, transcription factor NF-KB by class tongued bell receptor 2/1 stimulate and with NF- κ B cold light reporter vector On NF- κ B response factor (NF- κ B response element) combine, and induce downstream cold light enzyme (luciferase) base The performance of cause, therefore can be by addition cold light by the work of matter to cell lysate and detecting cold light strength assessment class tongued bell receptor 2/1 Change.

The result of afore-mentioned test as shown in Figure 3 is 10 when protein example concentration⁴The processing of ng/ml, H5HA recombinant protein Cold light intensity is set to be similar to background value；The processing of LTIIb-B5 recombinant protein makes cold light intensity be about 6 times of background value；H5HA- The processing of LTIIb-A fusion protein makes cold light intensity be about 4 times of of background value when with the H5HA-LTIIb-A fusion protein of low concentration (10^-3To 10²) and 10 ng/ml⁴The LTIIb-B5 recombinant protein of ng/ml handles cell simultaneously, and measured cold light intensity is similar to Bestow 10⁴It is all 10 that the LTIIb-B5 recombinant protein of ng/ml, which works as with concentration,⁴The H5HA-LTIIb-A fusion protein of ng/ml and LTIIb-B5 recombinant protein handles cell simultaneously, and measured cold light intensity, which is higher than, individually imposes 10⁴The H5HA-LTIIb-A of ng/ml Fusion protein or 10⁴The LTIIb-B5 recombinant protein of ng/ml.Antigen coalescence protein of the present invention can activate class tongued bell as the result is shown for those Receptor 2/1, facilitate cause individual innate immunity for the present embodiment, by H5HA-LTIIb-A fusion protein with LTIIb-B5 recombinant protein, which merges use, can produce optimal 2/1 activation effect of class tongued bell receptor simultaneously, this result also implies The fusion protein that H5HA-LTIIb-A fusion protein is further formed with LTIIb-B5 protein binding also has similar effect.

Embodiment 3

Systemic and mucosa-immune effect of the H5HA-LTIIb-A fusion protein to mouse

For examine H5HA-LTIIb-A fusion protein can effectively cause mammal to the systemic of resisiting influenza virus with Mucosa-immune reaction merges egg with the H5HA-LTIIb-A that nasal cavity injection system bestows three doses to BALB/c mouse (each group 5) White (10 μ g), H5HA recombinant protein (10 μ g), H5HA-LTIIb-A fusion protein (10 μ g) and LTIIb-B5 recombinant protein (5 μ g) Combination or PBS solution (negative control group), and mice serum and lung washing lotion are detected with ferment combination immunoabsorption (ELISA) In with each secondary inoculation interval IgG and IgA content of H5HA specificity be about three weeks, adopted after third time is injected two weeks Collect mouse blood sample, sacrifice mouse after three weeks in third time injection and acquires lung washing lotion.It is fought in mice serum or lung washing lotion IgG the and IgA potency of H5HA recombinant protein is to be four times in Sample Dilution multiple corresponding to negative control group light absorption value

Fig. 4 A-4B shows IgG the and IgA potency that H5HA recombinant protein is fought in mice serum respectively, shows data in figure Point and its average value, * indicate p < 0.05, * * indicate p < 0.01, * * * indicate p < 0.001, * * * * indicate p < 0.0001. according to According to Fig. 4 A, IgG is not detected in the antiserum of PBS solution processing, in contrast H5HA recombinant protein then can cause a small amount of IgG., The combination of H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein can cause the IgG of maximum amount, and H5HA-LTIIb-A melts Hop protein takes second place according to Fig. 4 B, IgA is not detected in the antiserum of PBS solution processing, H5HA recombinant protein can then cause on a small quantity IgA. relatively, the combination of H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein can cause the IgA of maximum amount, H5HA-LTIIb-A fusion protein take second place this as the result is shown antigen coalescence protein of the present invention can cause in serum it is significant more anti- Former specificity IgG and IgA.

Fig. 4 C-4D shows IgG the and IgA potency that H5HA recombinant protein is fought in mouse lung washing lotion respectively, shows number in figure Strong point and its average value, * indicate that p < 0.05, * * indicate that p < 0.01, * * * * indicate p < 0.0001.It is molten through PBS according to Fig. 4 C IgG is not detected in lung washing lotion after liquid or H5HA recombinant protein are immune；But H5HA-LTIIb-A fusion protein and LTIIb-B5 The combination of recombinant protein or H5HA-LTIIb-A fusion protein can cause the IgG of significantly higher amount, and significant with the former gained IgG Higher than the latter according to Fig. 4 D, relative to PBS solution or the group of H5HA recombinant protein, H5HA-LTIIb-A fusion protein with The combination of LTIIb-B5 recombinant protein or H5HA-LTIIb-A fusion protein can cause the IgA of significantly higher amount, and with the former institute It is more to obtain IgA.Bestowing for antigen coalescence protein of the present invention can be obviously improved that have antigen in the mammalian body special as the result is shown for those IgA content in IgG and the IgA content of one property, especially lung washing lotion, therefore, antigen coalescence protein of the present invention is to mammal With systemic and mucosa-immune effect for the present embodiment, LTIIb-B5 recombinant protein and H5HA-LTIIb-A are merged Albumen merges using the antibody content that can further increase a antigenic specificity of tool in vivo

Embodiment 4

The influenza virus neutralizing antibody of H5HA-LTIIb-A fusion protein initiation mouse

After the present embodiment inquires into mouse according to the different inoculations of 3 the method for embodiment progress, in serum and lung washing lotion The serum of serial dilution or lung washing lotion are reported base with cold light by the potency for fighting the neutralizing antibody of the false virus of H5N1 influenza The false virus of the H5N1 influenza of cause is mixed 1 hour secondly, the serum or lung washing lotion and the mixture of virus are added in 37 DEG C 96 porose discs of inoculation mdck cell are added to carry out neutralizing antibody test and obtain neutralization curve.The potency of neutralizing antibody is defined as Virus infection is reduced up to serum needed for 50% or the extension rate of lung washing lotion.

Fig. 5 A-5B shows the neutralization curve of the false virucidin of H5N1 influenza in mice serum and lung washing lotion respectively. According to Fig. 5 A, the antiserum of PBS solution or H5HA recombinant protein any concentration all can not in and 50% or more false virus.Phase For, the antiserum of H5HA-LTIIb-A fusion protein maximum concentration can in and 90% false virus, and H5HA-LTIIb- The combined antiserum of A fusion protein and LTIIb-B5 recombinant protein can the false virus foundation figure of complete neutralization in maximum concentration 5B, lung washing lotion after PBS solution or H5HA recombinant protein are immune any concentration all can not in and 60% or more false virus Relatively, the lung washing lotion after H5HA-LTIIb-A fusion protein immunization maximum concentration can in and 90% false virus, and pass through Lung washing lotion after the combination of H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein is immune maximum concentration can completely in With false virus.

Fig. 5 C-5D shows that by the potency of the calculated neutralizing antibody of Fig. 5 A-5B, * * * indicates p < 0.001, N.D in figure Expression is not detected.According to Fig. 5 C, the potency of PBS solution and the neutralizing antibody in the caused serum of H5HA recombinant protein is about respectively 155 and 34；The potency of the caused neutralizing antibody of H5HA-LTIIb-A fusion protein is about 1541；H5HA-LTIIb-A fusion protein Potency with the caused neutralizing antibody of combination of LTIIb-B5 recombinant protein is about 3662. according to Fig. 5 D, PBS solution and H5HA weight The potency of neutralizing antibody in the caused lung washing lotion of histone is about respectively 57 and 32；H5HA-LTIIb-A fusion protein is caused The potency of neutralizing antibody be about the combination of 282, H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein also cause it is identical The neutralizing antibody of potency those as the result is shown antigen coalescence protein of the present invention can cause in the mammalian body it is obvious it is more in And antibody helps to inhibit influenza infection cell for the present embodiment comprising the neutralizing antibody in lung washing lotion, it will LTIIb-B5 recombinant protein and H5HA-LTIIb-A fusion protein, which merge use, can further increase an influenza virus neutralization in vivo The content of antibody

Embodiment 5

H5HA-LTIIb-A fusion protein causes the T cell related immune reaction of mouse

The present embodiment assesses mouse, and according to 3 the method for embodiment to carry out the T cell related immune after different inoculations anti- It answers.After mouse is sacrificed, spleen and cervical lymph nodal tissue are acquired and ground, gained cell is incubated at 24 porose disc according to fixed amount Thereafter, it with the T cell in H5 type hemagglutinin victory peptide mixer stimulation spleen and cervical lymph node, and detects in cell culture fluid and divides Not from Th1 cell, the IFN-γ of Th2 cell and Th17 cell, the secretory volume of IL-4, IL-17A.

Fig. 6 A-6B shows the IFN-γ secretion amount of mouse spleen and cervical lymph nodal cell respectively, and * * * * indicates p < in figure 0.0001, * * * indicates p < 0.001. foundation Fig. 6 A, the non-secretion of gamma-IFN of spleen cell after PBS solution is immune, through H5HA Spleen cell after recombinant protein is immune then secretes micro IFN-γ relative to this micro IFN-γ, is melted with H5HA-LTIIb-A The combination of hop protein and LTIIb-B5 recombinant protein, which be immunized, can make the secretory volume of IFN-γ rise to 3 times, and with H5HA- LTIIb-A fusion protein, which carries out immunity energy, makes the secretory volume of IFN-γ rise to 6 times of according to Fig. 6 B, through PBS solution or H5HA weight The non-secretion of gamma-IFN of cervical lymph nodal cell after histone is immune, is recombinated with H5HA-LTIIb-A fusion protein and LTIIb-B5 The combination of albumen, which be immunized, can cause a small amount of IFN-γ secretion, and be immunized with H5HA-LTIIb-A fusion protein can then make IFN-γ secretion amount reaches highest

Fig. 6 C-6D shows the IL-4 secretory volume of mouse spleen and cervical lymph nodal cell respectively.It is molten through PBS according to Fig. 6 C Spleen cell after liquid is immune does not secrete IL-4, and the spleen cell after H5HA recombinant protein is immune can secrete micro IL-4.Phase Over the ground, be immunized with the combination of H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein can be such that IL-4 secretory volume reaches Highest, with H5HA-LTIIb-A fusion protein carry out it is immune time high IL-4 secretory volume can be caused according to Fig. 6 D, with PBS solution, The combination of H5HA recombinant protein or H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein carries out after being immunized, neck leaching It fawns on cell and does not secrete IL-4, only be immunized with H5HA-LTIIb-A fusion protein can make IL-4 secretory volume reach 0.5pg/ ml.

Fig. 6 E-6F shows the IL-17A secretory volume of mouse spleen and cervical lymph nodal cell respectively, and * * * * indicates p < in figure 0.0001, * * indicates the IL-17A that p < 0.01. does not secrete according to Fig. 6 E, the spleen cell after PBS solution is immune, through H5HA Spleen cell after recombinant protein is immune then secretes micro IL-17A. relatively, with H5HA-LTIIb-A fusion protein with The combination of LTIIb-B5 recombinant protein or H5HA-LTIIb-A fusion protein itself, which be immunized, can make IL-17A secretory volume significant Increase the IL-17A that does not secrete according to Fig. 6 F, the cervical lymph nodal cell after PBS solution or H5HA recombinant protein are immune, with The combination of H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein, which be immunized, can cause a small amount of IL-17A to secrete, but with H5HA-LTIIb-A fusion protein, which be immunized, can dramatically increase IL-17A secretory volume.Those Antigen Fusions of the present invention as the result is shown Albumen can be reacted in a internal initiation T cell related immune, and especially the generation of IFN-γ is for the present embodiment, H5HA- The inoculation of LTIIb-A fusion protein can make cervical lymph nodal cell secrete most IFN-γ, IL-4 and IL-17A, and LTIIb-B5 recombinant protein and H5HA-LTIIb-A fusion protein are merged using the generation that can interfere those cytohormones.

Embodiment 6

The inoculation protection mouse of H5HA-LTIIb-A fusion protein fights H5N1 influenza infection

Make individual from H5N1 influenza infection for vaccine composition of the verifying containing H5HA-LTIIb-A fusion protein Effect, three doses of H5HA-LTIIb-A fusion protein (10 is first bestowed to BALB/c mouse (each group 5) with nasal cavity injection system μ g, 5 μ g or 2.5 μ g), H5HA recombinant protein (10 μ g, 5 μ g or 2.5 μ g), H5HA-LTIIb-A fusion protein (10 μ g, 5 μ g, Or 2.5 μ g) combination or PBS solution with LTIIb-B5 recombinant protein (5 μ g), each secondary inoculation interval is about three weeks thirds Inject the H5N1 virus (NIBRG-14 of 20 times of lethal doses after secondary inoculation two weeks for mouse again；By Academia Sinica's gene Body center Zhan Jiacong researcher provides), mouse weight and survival condition are recorded daily, and calculates survival rate and weight within continuous 14 days Percentage

Fig. 7 A, 7C, 7E show that the immune mouse for receiving various dose immunogene is injected the survival curve after H5N1 virus, * indicates p < 0.05. according to the experiment (Fig. 7 A and 7C) that commercial weight is 10 μ g or 5 μ g is immunized, through the mouse that PBS solution is immune in figure The total death in 5 or 6 days after virus injection, but with H5HA recombinant protein, H5HA-LTIIb-A fusion protein or H5HA- When the group of LTIIb-A fusion protein and LTIIb-B5 recombinant protein is combined into immunogene, mouse is immunized 14 after virus injection in each group It survival rate is greater than 80%, and to each other without significant difference however, H5HA is recombinated when immune commercial weight is down to 2.5 μ g (Fig. 7 E) Protein immunization mouse 6 days survival rates after virus injection are 25%；H5HA-LTIIb-A fusion protein immunization mouse is in virus 14 days survival rates are 100% after injection；Mouse is immunized in disease in H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein 13 days survival rates are 80% after poison injection；Mouse and the H5HA-LTIIb-A fusion protein immunization is immunized in the H5HA recombinant protein The survival rate of mouse have statistically significant difference this compare influenza antigen as the result is shown, antigen coalescence protein of the present invention The best protection power of individual confrontation influenza infection can be provided by bestowing.

Fig. 7 B, 7D, 7F show the weight percentage that the immune mouse for receiving various dose immunogene is injected after H5N1 virus Than the 0th day mouse weight being set as 100%. according to the experiment (Fig. 7 B) that commercial weight is 10 μ g is immunized, H5HA recombinant protein is exempted from 80% is dropped within the weight of epidemic disease mouse 4 days after virus injection, but was gone up at the 14th day to 95%；H5HA-LTIIb-A merges egg The weight of white immune mouse is not below 95% after virus injection during 14 days, and returns back to initial value at any time；H5HA- The weight that mouse is immunized in LTIIb-A fusion protein and LTIIb-B5 recombinant protein drops to 90% on day 4, then returns at any time It is multiple that the weight of mouse is immunized in virus injection according to the experiment (Fig. 7 D) that commercial weight is 5 μ g, H5HA recombinant protein is immunized to initial value It is down within 6 days 84% afterwards, but went up at the 14th day to 92%；The weight of H5HA-LTIIb-A fusion protein immunization mouse is infused in virus It is down within 4 days 85% after penetrating, but went up at the 14th day to 92%；H5HA-LTIIb-A fusion protein is exempted from LTIIb-B5 recombinant protein It is down within the weight of epidemic disease mouse 5 days after virus injection 89%, but was gone up at the 14th day to 97%.It is 2.5 μ g according to commercial weight is immunized Experiment (Fig. 7 F), what H5HA recombinant protein was immunized mouse is down to 79% for weight 6 days after virus injection, but gos up at the 14th day To 94%；It is down within the weight of H5HA-LTIIb-A fusion protein immunization mouse 5 days after virus injection 82%, but was returned at the 14th day Rise to 93%；The weight of mouse is immunized 5 days after virus injection in H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein It is down to 86%, but this compares influenza antigen as the result is shown in rise in the 14th day to 98%., antigen coalescence protein of the present invention The influence bestowed to whose body weight is less, so have quite safe property.

Embodiment 7

Systemic immunity effect of the H5HA-LTIIb-A fusion protein to chicken

To examine H5HA-LTIIb-A fusion protein that can effectively cause birds to the systemic and mucous membrane of resisiting influenza virus Immune response, with nasal cavity injection system to chicken (each group 3) bestow three doses H5HA-LTIIb-A fusion protein (10 μ g), H5HA recombinant protein (10 μ g), H5HA-LTIIb-A fusion protein (10 μ g) and LTIIb-B5 recombinant protein (5 μ g) combination or PBS solution (negative control group), and detect in small chicken serum to have IgY and IgA content each time of H5HA specificity with ELISA and exempt from Epidemic disease injection interval is about two weeks, acquires chicken blood sample before the injection and after third time injection two weeks

Fig. 8 A-8B shows that IgY the and IgA potency that H5HA recombinant protein is fought in small chicken serum according to Fig. 8 A, is compared respectively IgY content before inoculation in serum, the immune of H5HA recombinant protein increases IgY slightly, but H5HA-LTIIb-A melts The immune of hop protein makes IgY obviously increase similarly, the combination of H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein Also cause the IgY. of a large amount according to Fig. 8 B, compared to the IgA content in serum before inoculation, H5HA recombinant protein or H5HA- LTIIb-A fusion protein it is immune all make IgA slightly increase this bestowing for antigen coalescence protein of the present invention can be significant as the result is shown The IgG content of tool antigenic specificity in birds body is promoted, therefore, antigen coalescence protein of the present invention has systemic immunity to birds Effect

Embodiment 8

The influenza virus neutralizing antibody of H5HA-LTIIb-A fusion protein initiation chicken

The present embodiment inhibits test (HI aasay) detection chicken according to 7 the method for embodiment using influenza virus hemagglutinin After carrying out different inoculations, inhibit the presence of the antibody of the hamegglution of H5N1 influenza virus in serum, and utilizes The molten spot neutralization test (PRNT) of virus measures the potency that the neutralizing antibody of H5N1 influenza virus is fought in the serum.Neutralizing antibody Potency is defined as reduction virus infection up to the extension rate of serum needed for 50%

Fig. 9 shows that viral hemagglutinin inhibits the potency for testing medium and small chicken antiserum according to the figure, and H5HA recombinant protein is exempted from Epidemic disease can not make chicken generate the antibody that can inhibit hemagglutination.However, through H5HA-LTIIb-A fusion protein or H5HA-LTIIb- Gained serum, which is diluted 40 times or more, after the combination of A fusion protein and LTIIb-B5 recombinant protein is immune still can inhibit H5N1 influenza Hemagglutination caused by virus shows that antigen coalescence protein of the present invention can effectively cause in birds body and inhibits H5N1 influenza virus The antibody of effect

Figure 10 A shows the neutralization curve of H5N1 virus neutralizing antibody in small chicken serum, and Figure 10 B, which is shown, to be calculated by Fig. 8 A and obtained Neutralizing antibody potency, * indicates that p < 0.05. according to Figure 10 A, is immunized compared to H5HA recombinant protein in figure, passes through Gained after the combination of H5HA-LTIIb-A fusion protein or H5HA-LTIIb-A fusion protein and LTIIb-B5 recombinant protein is immune Antiserum has preferable H5N1 virus neutralising capacity foundation Figure 10 B, the neutralizing antibody in the caused serum of H5HA recombinant protein Potency is about that the potency of the caused neutralizing antibody of 6.367, H5HA-LTIIb-A fusion protein is about 11.09, H5HA-LTIIb-A The potency of the caused neutralizing antibody of combination of fusion protein and LTIIb-B5 recombinant protein is about 12.55.This result illustrates this hair Bright antigen coalescence protein can cause the neutralizing antibody of obvious more confrontation H5N1 influenza virus in birds body

In conclusion bestowing (such as nasal cavity injection) influenza mucosal vaccine composition of the invention to an individual can effectively draw Sending out this, confrontation the antibody mediated of influenza virus reacts with cellular immunity in vivo, comprising having in blood and bronchovesicular mucous membrane Antigenic specificity and can neutralize influenza virus IgG (or IgY) and IgA and T cell secreted by IFN-γ, IL-4 and The cytohormones such as IL-17A.Therefore, which can provide the guarantor that individual effectively antagonizes influenza infection Protect power, such as the infection of confrontation highly pathogenic bird flu virus H 5 N 1.

Claims

1. a kind of influenza mucosal vaccine composition includes antigen coalescence protein, wherein the antigen coalescence protein includes influenza virus Antigen and Escherichia coli second type b type avoid heat type enterotoxin A sub-cell.

2. influenza mucosal vaccine composition according to claim 1, which is characterized in that the influenza antigen is blood clotting Plain extracellular domain.

3. influenza mucosal vaccine composition according to claim 1, which is characterized in that the N-terminal of the antigen coalescence protein Further include a histidine tract.

4. described in any item influenza mucosal vaccine compositions according to claim 1~3, which is characterized in that further include one Escherichia coli second type b type avoids heat type enterotoxin B sub-cell.

5. influenza mucosal vaccine composition according to claim 2, is characterized in that, include at least Antigen Fusion of 10 μ g Albumen.

6. a kind of antigen coalescence protein is used to prepare the purposes of influenza mucosal vaccine composition, wherein the antigen coalescence protein includes One influenza antigen and an Escherichia coli second type b type avoid heat type enterotoxin A sub-cell.

7. purposes according to claim 6, which is characterized in that the influenza antigen is for a hemagglutinin extracellular domain.

8. purposes according to claim 6, which is characterized in that the N-terminal of the antigen coalescence protein further includes one group Propylhomoserin segment.

9. purposes according to claim 6, which is characterized in that the antigen coalescence protein activates class tongued bell receptor 2/1.

10. purposes according to claim 6, which is characterized in that it is anti-that the antigen coalescence protein causes T cell related immune It answers.

11. purposes according to claim 10, which is characterized in that the T cell related immune reaction includes interferon- γ, Jie Bai Su -4, Jie Bai Su -17A, or any combination thereof secretion.

12. purposes according to claim 6, which is characterized in that the influenza mucosal vaccine composition further includes one Escherichia coli second type b type avoids heat type enterotoxin B sub-cell.

13. purposes according to claim 6, which is characterized in that the influenza mucosal vaccine composition is applied via nasal cavity It gives.

14. a kind of preparation method of influenza mucosal vaccine composition, includes:

An antigen coalescence protein is prepared, it includes an influenza antigens and an Escherichia coli second type b type to avoid heat type enterotoxin A Sub-cell；And

The antigen coalescence protein and a pharmaceutically acceptable carrier are mixed to obtain the influenza mucosal vaccine composition.

15. preparation method according to claim 14, which is characterized in that the influenza antigen is that hemagglutinin is extracellular Domain.

16. preparation method according to claim 14, which is characterized in that the N-terminal of the antigen coalescence protein further wraps Containing a histidine tract.

17. preparation method according to claim 14, is characterized in that, one Escherichia coli second type b of addition is further included Type avoids heat type enterotoxin B sub-cell to the influenza mucosal vaccine composition.