CN102370976B - Mixed virus-like particles of swine influenza virus and foot and mouth disease virus, preparation method and application thereof - Google Patents

Abstract

The invention provides a mixed virus-like particles, containing stromatin M1 of influenza virus, surface antigen erythrohemagglutinin HA of influenza virus and a nexus protein containing main antigenic epitope contained capsid protein of foot and mouth disease virus and a transmembrane zone and an inner membrane zone of the erythrohemagglutinin HA of influenza virus. The main antigenic epitope contained capsid protein of foot and mouth disease virus substitutes a basically same length outer membrane zone of a 5' terminal of the erythrohemagglutinin HA of influenza virus. Surface antigen erythrohemagglutinin HA of influenza virus and the main antigenic epitope contained capsid protein of foot and mouth disease virus are expressed simultaneously on surfaces of the mixed virus-like particles. The invention also provides a divalent vaccine of swine influenza and foot and mouth disease; and the vaccine contains the mixed virus-like particles and adjuvants. The invention also provides a method for preparing the mixed virus-like particles.

Description

The hybrid virus sample granule of swine influenza virus and swine foot-and-mouth disease virus, preparation method and application

Technical field

The present invention relates to the hybrid virus sample granule of swine influenza virus and swine foot-and-mouth disease virus, and preparation and application.

Background technology

Swine flue is by swine influenza virus (Swine influenza virus, the respiratory infectious disease of a kind of acute, the hot and height contact of different days, sex and the kind pig SIV) causing, it is clinically take burst, high heat, cough, dyspnea, exhaustion and death as feature.Each age, sex, product boar all can infect, and sickness rate is high.Infected pigs influenza virus can cause swine growth hydraulic performance decline separately, and feedstuff-meat ratio raises, and weightening finish slows down, and causes certain economic loss to pig farm.More seriously, pig is fowl, people, swine influenza virus restructuring and copies " blender ", and swine influenza virus does not need restructuring just to have the ability that infects to greatest extent people.And early stage human influenza virus can also be stored in pig body, and again infect people over a period to come.At present, common swine flue mainly contains 3 kinds of hypotypes such as H1N1, H1N2 and H3N2.Significant be, SIV more common with other epidemic disease, as concurrent or secondary infection such as pig breeding and respiratory disorder syndrome virus, PRV (Pseudorabies virus), actinobacillus pleuropneumoniae, Streptococcus suis, eperythrozoon suises, epidemic situation is become repeatedly and complexity, be modern intensive pig farm ubiquity and be difficult to one of porcine respiratory infectious disease of eradicating, very harmful to pig industry.

The popular aquaculture of a lot of countries of not only giving of influenza A H1N1 influenza virus has caused huge economic loss, and worldwide people's public health security has been caused to a very large challenge.Break out in the Mexican influenza A H1N1 whole world in April, 2009 and attract attention, subsequently, the states such as America & Canada also occur that people infects the situation of H1N1 swine flue and has part patient death in succession.Developing in a short time effective vaccine prevents influenza A H1N1 influenza virus suppress epidemic situation propagation and increase the weight of extremely urgent.The vaccine major part for fowl poultry flu-prevention virus of domestic production is at present to adopt traditional chick embryo method to cultivate propagation live virus, after chemical reagent deactivation, is produced into vaccine.This technology is divided into again the preparation method that two classes are different: the one, directly infect Embryo Gallus domesticus preparation with wild-type virus, another kind of is the heritability that first adopts Reverse Genetics transformation wild virus, then use the new virus of " rescue " transformation to infect Embryo Gallus domesticus, virus of proliferation, produces vaccine.

These influenza vaccines technologies of preparing have its advantage, but also exist very large defect.Especially the unusual characteristic of influenza sense poison, for example: altofrequency sudden change, hereditary material restructuring and antigenic drift etc. between dual and multiple subtype virus---make the long-term safety of these vaccines and effectiveness be subject to very big challenge, even produce " invalid vaccine ", be difficult to deal with the infection of new saltant type H1N1 influenza virus.In addition, these bacterin preparations also exist following deficiency:

1, the production cycle is long: go on the market to production of vaccine from obtaining new subtype virus strain, 5-8 consuming time month, be difficult to contain new epidemic situation great outburst.

2, working condition is high: for preventing the leakage diffusion of live virus of artificial contaminated environment and production, a whole set of production must be carried out in accordance with regulations under three grades of laboratory conditions of bio-safety, and the deactivation of virus must guarantee completely, thorough.

3, cannot distinguish by the pig of immunity and the pig being infected by the virus.

Foot and mouth disease (foot and mouth disease, FMD) is that the one being caused by foot and mouth disease virus (FMDV) is acute, height contagious disease, main infection artiodactyl beast, and people and non-artiodactyl also can infect.International Office of Epizootics classifies foot and mouth disease as first of category-A infectious disease, and China also classifies it as first disease of livestock contagious disease.Can cause huge economic loss once foot and mouth disease is broken out, also have influence on the normal trade activity of countries and regions simultaneously.Because foot and mouth disease directly threatens international trade and national reputation and animal ecology and human food's health, thereby be subject to people's common concern.In recent years be more listed in biological weapons security pact and organize emphasis to check object, countries in the world comprise the country without foot and mouth disease, all primary disease is maintained sharp vigilance, and the research of Efforts To Develop nosetiology, diagnostic techniques and anti-technology processed.

FMDV is known minimum animal RNA viruses, and virion is spherical in shape, regular dodecahedron structure, and diameter 20～30nm, molecular weight 6.9 × 106u, sedimentation coefficient 146s, the buoyant density in cesium chloride is 1.43g/mL.Intact virus is containing sub-thread positive chain RNA and capsid protein two parts, without cyst membrane.Capsid is by VP1, VP2, each 60 molecular compositions of VP3, VP4, and geneome RNA is made up of approximately 8500 nucleotide, can be directly as messenger RNA.FMDV only contains an ORF (open reading frame), the precursor polyprotein of encoding viral.Precursor polyprotein forms 4 kinds of structural protein (VP1-VP4) after twice cracking, and wherein VP1 (1D) gene is positioned at genomic 2977～3615,213 aminoacid of encoding.Early stage research shows, structural protein are main components of induction neutralizing antibody, and it is exposed to the surface of virion.In five antigen sites of the O type of having found; there are three to be positioned on VP1; wherein the G-H at 140-160 amino acids place ring is topmost protective antigen site; space conformation more complicated; there is larger mobility; Arg-Gly-Asp (RGD) sequence that has formed a high conservative at its top is the cell receptor site of FMDV.140th～160 and 200～213 amino acid residues of VP1 are two main B cell epitopes, and the neutralizing antibody that can induce anti-FMDV, also contains at least one t cell epitope in VP1 141st～160 amino acid residues, can induce the narrow spectrum t cell responses of FMDV.

China adopts vaccine immunity and slaughters the policy combining the anti-system of foot and mouth disease, and the foot and mouth disease of pig is adopted to compulsory immunization.The foot-and-mouth disease vaccine using is at present mainly two kinds of inactivated virus vaccine and polypeptide vaccines, because existing the poor defect of potential safety hazard or immune effect that desirable immunoprotection can not be provided.Therefore, study efficient, safe and reliable vaccine and seem very necessary.

Virus-like particle (VLPs) is the one or more major structural proteins that contain certain virus; the hollow bead that does not comprise viral nucleic acid that in expression system, automatic Composition becomes in vitro; its form and size are same or similar with real virion; so can effectively induce body immune system to produce immunoprotection reaction, demonstrate good good immune efficacy and application prospect as a kind of new generation vaccine virus-like particle (VLPs).Typical H1N1 SIV particle is spherical in shape or oval, diameter 80～120nm, 8 genetic fragments of genome of H1N1 influenza virus, the 11 kinds of albumen of encoding altogether, the shell of virus is the film that one deck is made up of lipid and lipoprotein, is wrapped in viral hereditary material and nucleoprotein.Have 4 kinds of protein, belong to viral major structural protein, they are: matrix prote m1, the main body albumen of formation virus coat; NP, forms virion nucleocapsid, participates in virion and forms; Hemagglutinin HA, the main glycoprotein on virus coat skin covering of the surface, has 16 hypotypes.It is the major antigen body that brings out host's body generation antibody.Neuraminidase NA, is also a kind of glycoprotein on virus coat skin covering of the surface, has 9 hypotypes, is the major antigen body that induce antibody produces equally.

The surface membrane protein HA of influenza poison and NA cause that body produces the major antigen of immunne response, and matrix prote m1 is the main component that forms virus coat, also can be used as the antigen of tectotype immunne response.Relevant studies have shown that, the correction of matrix prote m1, it is the important step that guarantees that virus coat forms, and one of function of memebrane protein NA is that viral entity is stripped down from host cell surface, form virion, therefore structural protein M1, the HA of bird flu virus and NA are the cores of synthesizing new anti-avian influenza virus vaccine.Albumen HA, NA just express simultaneously and can become the hollow virus genomic virus-like particle that do not have by automatic Composition with 3 major structural proteins such as M1.The type specific antigen NP albumen that studies have shown that in addition influenza virus can react by effective stimulus CTL (cytotoxic T cell), suppress viral infection, in virus-like particle, add NP albumen will improve to a certain extent the cellular immune level of this new generation vaccine.

Swine influenza virus and foot and mouth disease virus be impact each other in pig body, and infection simultaneously can increase the weight of the state of an illness.Therefore developing the VLPs that a kind of bivalent vaccine prevents H1N1 swine flue and Schweineseuche is simultaneously those skilled in the art's problem demanding prompt solutions.

Summary of the invention

In view of this, one of object of the present invention is to provide a kind of bivalent vaccine, and this vaccine can prevent H1N1 swine flue and Schweineseuche simultaneously.

The object of the invention is to realize by content once.

The invention provides the matrix prote m1 that a kind of hybrid virus sample granule comprises influenza virus, the surface antigen hemagglutinin HA of influenza virus, a nexus albumen, the capsid protein that contains Main Antigenic that it comprises swine foot-and-mouth disease virus, the cross-film district of the hemagglutinin HA of influenza virus and film inner region; The capsid protein that contains Main Antigenic of described swine foot-and-mouth disease virus is replaced the film foreign lands of the roughly the same length of the 5 ' end of the hemagglutinin HA of influenza virus; The surface antigen hemagglutinin HA of while expression of influenza virus and the capsid protein that contains Main Antigenic of swine foot-and-mouth disease virus on the surface of described hybrid virus sample granule.

The present invention also provides swine flue and Schweineseuche bivalent vaccine, comprises described hybrid virus sample granule and adjuvant.

The present invention also provides the method for preparing hybrid virus sample granule.

The vaccine that the virus-like particle that utilization comprises described fusion rotein forms, can prevent H1N1 swine flue and Schweineseuche simultaneously, and have obvious advantage and effect:

(1) H1N1 swine flue and Schweineseuche VLP bivalent vaccine can cause that body immune system produces strong type and replys, and immunity is strong, and the persistent period is long, can prevent H1N1 swine flue and Schweineseuche simultaneously.

(2) because virus-like particle is not containing viral genome, in immune animal body, just can there is not viral gene and host chromosome gene integration in the VLPs of this hollow shell structure, and whole production process do not contact infectious virus alive, therefore very safe.For influenza virus and foot and mouth disease virus two-strain, all anxiety of virus-free diffusion.

And general genetic engineering subunit vaccine comparison (3), because virus-like particle more approaches form and the size of natural viral, can stimulate body to produce better immunoreation, immune effect is better.

(4) can distinguish by the animal of artificial immunity and by the animal of viral infection.Virus-like particle, containing albumen such as influenza virus NS, PB1, PB2, while therefore carrying out serologic test, with the animal of virus-like particle particle vaccines immunity, will can not produce the antibody of the anti-NS of specificity or PB, whether can distinguish wild virus infection.For foot and mouth disease, with any foot and mouth disease structural protein outside VP1 albumen, as VP2 albumen, N albumen etc., and the non-structural protein of foot and mouth disease virus is as detectable antigens, whether can distinguish wild virus infection.

Above-mentioned explanation is only the general introduction of technical solution of the present invention, in order to better understand technological means of the present invention, and can be implemented according to the content of description, and for above and other object of the present invention, feature and advantage can be become apparent, below in conjunction with preferred embodiment, and coordinate accompanying drawing, be described in detail as follows.

Accompanying drawing explanation

Fig. 1 is by the electrophoretogram of the HA of different Auele Specific Primer pcr amplifications (a1), NA (a2), M1 (b), NP (c), HF (d) genetic fragment;

Fig. 2 is restructuring baculovirus expression plasmid rBacmid-HA/HF-NA, rBacmid-M1, rBacmid-NP schematic diagram;

Fig. 3 is virus-like particle western blot result.

Fig. 4 is M1 albumen in virus-like particle and the indirect immunofluorescence result of S1 albumen.A, M1 antibody are that RBITC labelling shows fluorescent orange, and C, S1 antibody are that FITC labelling shows green fluorescence, the negative contrast of B, D.

Fig. 5 is virus-like particle after the sucrose density gradient centrifugation purification image under ultramicroscope, the partial enlarged drawing that b is a.

The specific embodiment

The present invention is with Bac-to-Bac

insect baculovirus expression system is basis, utilizes matrix protein and the NP of H1N1 swine influenza virus, and skin covering of the surface egg HA and NA albumen, and common automatic Composition constructs the virus-like particle (VLP) of H1N1 swine influenza virus.On this basis, build the bivalence VLPs of H1N1 swine influenza virus and swine foot-and-mouth disease virus.

First, by two the peptide sections 132～159 (A) on swine foot-and-mouth disease virus VP1, 200～213 (B) are synthetic with the form of A-B-A-B-A-B-A series connection, the N end of A peptide section has extra 5 aminoacid (MVPSR), the C end of B peptide section has extra 6 aminoacid (QFELEF), between A and B peptide section, add 2 aminoacid (PG) linker, form 11 aminoacid (QFELEFMVPSR) linker in B-A junction, the aminoacid that these two linker comprise has good pliability, the fragment that contributes to connect forms certain space structure, this series connection fragment is named as F polypeptide.F peptide sequence is replaced to a part for the HA protein gene of H1N1 influenza virus, the two forms fusion gene HF, F peptide sequence is positioned at the 5 ' end of fusion gene HF, replaces 5 ' terminal sequence of the HA gene of roughly equal length, roughly equal to ensure length and the HA mrna length of fusion gene.

Then, according to the method that forms influenza virus-like particles, by the matrix prote m1 of H1N1 influenza virus, the gene of NP and surface membrane protein HA and NA, and the gene order of the fusion rotein HF of expression swine foot-and-mouth disease virus antigen tandem polypeptide F and influenza virus HA, be implemented in the vector plasmid of insect baculovirus, and make in its hereditary material DNA that is recombined into insect baculovirus, utilize these foreign proteins of host insect cellular expression, the virus-like particle of automatic Composition Cheng Buhan influenza virus hereditary material, after forming, virus-like particle will be released in cell culture fluid.The VLPs forming so had both had the major surface antigen of H1N1 influenza virus, had again the major antigen tandem polypeptide F of swine foot-and-mouth disease virus, was H1N1 swine influenza virus and swine foot-and-mouth disease virus bivalence VLPs.

It is pointed out that swine foot-and-mouth disease virus is without cyst membrane, its surface antigen protein does not have natural cross-film district and can not be integrated directly on VLP.In addition, its epitope disperses, and therefore surface antigen protein is directly fused to the structure that can destroy HA on influenza virus HA.In order to overcome these difficult problems, the present invention adopts the mode of series connection epitope, in the situation that guaranteeing that multiple epitopes exist, has reduced the size of series connection antigen epitope polypeptide, can be fused on HA.Series connection antigen epitope polypeptide by fusion swine foot-and-mouth disease virus is upper to HA, and the series connection antigen epitope polypeptide of swine foot-and-mouth disease virus can be expressed on VLP film.

In addition, the structural protein of influenza virus are because we find influenza virus VLP high yield, stable as the skeleton of bivalence VLP; The VLP that is incorporated into of HA albumen is efficient simultaneously; Can guarantee like this has enough HA albumen on the surface of VLP, can be used as effective vaccine and use.

Can further be expressly understood the present invention by specific embodiment given below, but following embodiment is not limitation of the invention.

The amplification of implementation column 1:M1, NP, HA, NA and fusion gene HF.

(1) swine foot-and-mouth disease virus F polypeptide and swine influenza virus HA fusion gene HF's is synthetic

The nucleotide sequence of fusion gene HF is shown in that the aminoacid sequence of SEQ ID NO:9, its coding is shown in SEQ ID NO:10, and fusion gene is according to its nucleic acid sequence SEQ ID NO:9 synthetic.F polypeptide contains 198 aminoacid, substitutes 5 ' 184 aminoacid of end of HA gene.

(2) the RNA extracting of H1N1 hypotype swine influenza virus and RT-PCR

The operation instructions (method) that extract test kit by GibcoBRL company's T RIzol LS Reagent RNA carry out.Get respectively 250 μ L swine influenza virus H1N1 hypotype virus strain infection's allantoic fluids and 750 μ L TRIzol LS, add in 1.5mL microcentrifugal tube, blow and beat and fully mix with suction pipe, room temperature is placed 10min; Add 200 μ L chloroforms, thermal agitation 15s, room temperature left standstill after 5 minutes, the centrifugal 15min of 12000rpm at 4 ℃; Get supernatant in a new sterilizing 1.5mL centrifuge tube, add 500 μ L isopropyl alcohols, fully mix, room temperature is placed 10min, the centrifugal 10min of 12000rpm at 4 ℃; The supernatant that inclines, adds 70% ethanol 750 μ L in precipitation, mix gently, washing once, the centrifugal 15min of 12000rpm at 4 ℃, supernatant discarded, air-dry; Add the tri-distilled water lytic virus RNA (precipitation) without RNA enzyme of 10 μ L DEPC water treatments, be directly used in RT-PCR or-80 ℃ and save backup.Operation instruction with reference to the AMV reverse transcription of TaKaRa is carried out, and adds respectively following component: RNA:3 μ L in 20 μ L reaction systems; 5 × RT buffer:4 μ L; DNTPs:4 μ L; RNA enzyme inhibitor: 0.5 μ L; Primer UP:1 μ L; Primer DN:1 μ L; AMV:2 μ L; DEPC water: mend to 20 μ L.After mixing, room temperature is placed 10min, 42 ℃ of insulation 1h, and ice bath 2min, RT product is directly used in pcr amplification or-20 ℃ of preservations.

(3) pcr amplification of M1, NP, HA, NA, HF gene

According to 1 pair of primer of HA gene order (sequence table 1 and sequence table 2) design, for the HA gene that increases, its two ends have added respectively Xho I and Nco I/restriction enzyme site, are positioned under P10 promoter, and primer sequence is as follows:

HA?Xho?Ⅰ：5’-5’GCCCTCGAGATGAAGGCAATACTAGTG-3’(SEQ?ID?NO：11)

HA?Nco?Ⅰ：5’-GCCCCATGGTTAAATACATATTCTGCACTG-3’(SEQ?ID?NO：12)

According to 1 pair of primer of NA gene order (sequence table 3 and sequence table 4) design, for the NA gene that increases, its two ends have added that respectively the restriction enzyme site of Sal I and Hind III is positioned at P _pHunder promoter, these 2 primer sequences respectively:

NA?Sal?Ⅰ：5’-GCCGTCGACATGAATCCAAACCAAAG-3’(SEQ?ID?NO：13)

NA?HindⅢ：5’-GCCAAGCTTCTACTTGTCAATGGTG-3’(SEQ?ID?NO：14)

According to 1 pair of primer of M1 gene order (sequence table 5 and sequence table 6) design, for the M1 gene that increases, its two ends have added that respectively the restriction enzyme site of Sal I and Hind III is positioned at P _pHunder promoter, these 2 primer sequences respectively:

M1?Sal?Ⅰ：5’-GCCGTCGACATGAGTCTTCTAACCGAG-3’(SEQ?ID?NO：15)

M1?HindⅢ：5’-GCCAAGCTTTCACTTGAATCGTTG-3’(SEQ?ID?NO：16)

According to 1 pair of primer of NP gene order (sequence table 7 and sequence table 8) design, for the NP gene that increases, its two ends have added respectively Xho I and Nco I/restriction enzyme site, are positioned under P10 promoter, and primer sequence is as follows:

NP?Xho?Ⅰ：，5’-AGTCTCGAG?ATGAGTGACATCGGAGCCAT-3’(SEQ?ID?NO：17)

NP?Nco?Ⅰ：5’-CATGCCATGGTTAATTGTCATACTCCTCTGCATTG-3’(SEQ?ID?NO：18)

According to the sequence of fusion gene HF (sequence table 9 and sequence table 10), 1 pair of primer of design, for the fusion gene HF that increases, its two ends have added respectively Xho I and Sph I restriction enzyme site, are positioned under P10 promoter, primer sequence is as follows:

HF?Xho?Ⅰ：5’-CCGCTCGAGATGGTTCCGTCTCGTG-3’(SEQ?ID?NO：19)

HF?Sph?Ⅰ：5’-ACATGCATGCTTAAATACATATTCTGCACTG-3’(SEQ?ID?NO：20)

Adopt M1, NP, HA, NA, HF Auele Specific Primer separately, by the product of reverse transcription or synthetic DNA segment directly as the template of pcr amplification M1, NP, HA, NA, HF gene.PCR reaction condition is 94 ℃ of denaturation 3min, 94 ℃ of degeneration 40S, and 56 ℃ of annealing 90s, 72 ℃ are extended 90s, circulate 30 times, finally extend 10min, 1.5% agarose gel for PCR product (containing 0.5ug/ml ethidium bromide, EB) electrophoresis detection.(Fig. 1 a) for the about 1.7Kb of length of the HA gene of pcr amplification, (Fig. 1 a) for the about 1.35Kb of length of NA gene, (Fig. 1 b) for the about 0.75Kb of length of M1 gene, the length of NP gene is about 1.5Kb, and (c), the length of fusion gene HF is about 1.7Kb, and (Fig. 1 d) for Fig. 1.All PCR product samples are after running gel extracting, obtain M1, NP, HA, NA, the HF gene DNA fragment of purification, through restricted enzyme Xho I/Nco I (digestion HA fragment), Sal/Hind III (digestion NA fragment), 37 ℃ of Xho I/Nco I (digestion NP fragment), Sal I/Hind III (digestion M1 fragment), Xho I/Sph I (digestion HF segment) are respectively after digestion, be further purified again, use in order to next step construction recombination plasmid.Concrete operations are as follows: preparation 1% agarose gel is containing 0.5ug/ml ethidium bromide, all PCR samples are added in the sample cell of gel, voltage 100V is set, electrophoresis time 40min is under long-wave ultra violet lamp, cut the gel strip that contains sample band, pack in little plastic centrifuge tube.Reclaim test kit (Qiagen company product) description, extracting and purifying M1, NP, HA, HF and NA gene DNA fragment with reference to glue.After 37 ℃ of digested overnight of restricted enzyme, reclaim test kit (Qiagen company product) with glue, instruct according to explanation, the centrifugal post of crossing, purification reclaims the postdigestive M1 of enzyme action, NP, HA, NA, HF fragment.

Embodiment 2: the insect baculovirus of the baculovirus expression plasmid of construction expression M1, NP, HA, NA, HF gene and synthetic restructuring in insect cell

(1) recombiant plasmid of construction expression M1 and NP gene

Insect baculovirus plasmid PFastBac-dual (Invitrogen company product), reclaims test kit with glue and reclaims the plasmid PFastBac-dual after purification enzyme action after 3 hours through 37 ℃ of enzyme action of restricted enzyme Sal I/Hind III.Under the effect of T4 DNA ligase, the plasmid after enzyme action is connected and spends the night in 16 ℃ with the M1 DNA fragmentation after enzyme action.Reaction system is as follows: 10 × T4 connects buffer 1ml, the DNA fragmentation 3ml that M1 enzyme action reclaims, and enzyme action PFastBac-dual plasmid reclaims product 1ul, T4DNA ligase 1ul, ddH2O mends to 10ul.Adopt heat shock method that connection product is transduceed in Top10 competent cell and joined in a little plastic centrifuge tube, after mixing gently, tubule is placed in to 30min on ice, proceed to heat shock 90s in 42 ℃ of water-baths, put back to rapidly 5 minutes on ice, think wherein to add 200ulLB culture fluid.37 ℃ of shaking tables are cultivated 1 hour.Getting 100ul bacterium liquid coats on LB solid medium (containing two kinds of antibiotic of ammonia benzyl and gentamycin), cultivate 16h for 37 ℃, picking positive colony bacterium colony from flat board, carries out bacterium liquid PCR, after extracting plasmid, carries out single double digestion evaluation by Sal I/Hind III.After determined dna sequence, obtain recombiant plasmid PFastBac-dualM1.

The construction method of the recombiant plasmid PFastBac-dualNP of expression NP gene is the same.

(2) recombiant plasmid of construction expression HA/HF and NA gene

Baculovirus expression plasmid PFastBac-dual is after restricted enzyme Xho I/Nco I enzyme action, under the effect of T4DNA ligase, the below of P10 promoter will be inserted into by the postdigestive HA gene DNA fragment of same restriction endonuclease, this connect product with after transduce in Top10 competent cell through a hot body gram method, the recombinant plasmid dna of cultivation, the several positive colony bacterium colonies of extracting, bacterium liquid PCR and Xho I/Nco I list double digestion are identified and DNA sequence order-checking determine errorless after, select one of them recombinant plasmid dna, carry out enzyme action for the second time.Restricted enzyme used is Sal I/Hind III.With same endonuclease digestion NA gene DNA fragment, and be inserted into another promoter P of recombiant plasmid _pHbelow, through conversion, screening, cultivation, extracting, obtain the plasmid of two degree restructuring.After DNA sequencing, be required recombinant baculovirus expression plasmid PFastBac-dual-HA-NA.Whole experimental implementation Step By Condition is with identical described in above-mentioned structure PfastBac-dualM1 plasmid.

The construction method of recombiant plasmid PFastBac-dual-HF-NA of simultaneously expressing fusion gene HF and NA gene is the same.

(3) the genomic synthetic and extraction of recombinant baculovirus

In the E.coli competent cell strain DH 10 Bac cells that restructuring PfatBac-dual-M1, the PfatBac-dual-NP of purification, PFastBac-dual-HA-NA and PFastBac-dual-HF-NA plasmid are transduceed respectively special (American I nvitrogen company product).DH10Bac cell contains a special macromole plasmid Bacmid, it contains the full gene group of insect baculovirus AcMNPV.Once the expression plasmid of restructuring is integrated into behind the special site of macromole plasmid Bacmid, carry out blue white macula screening through the screening of 3 kinds of antibiotics (gentamycin, tetracycline and kanamycin) and the induction of IPIG and X-Gal substrate reactions, positive colony bacterium colony is white in color, and nonrecombinant wild bacterium colony is blue color.The laboratory manual that experiment condition all provides according to Invitrogen company instructs and sets.The positive colony of selecting is placed in the LB culture fluid (containing above-mentioned 3 kinds of antibiotic) of 3ml, cultivate 24 hours through 37 ℃ of shaking tables, the macromole plasmid Bacmid indicating according to laboratory manual is preparation method in a small amount, extracts the restructuring macromole plasmid Bacmid of purification with M1 gene, NP gene, HA-NA or HF-NA gene.

(4) preparation of recombinant baculovirus

Insect cell line sf9 cell (American I nvitrogen company product) is incubated in the sf-900II insect cell culture fluid of serum-free (Invitrogen company product), temperature setting is set to 27 ℃, the laboratory manual providing according to Invitrogen company, adopt liposome transfection method, the restructuring macromole plasmid Bacmid of purification is mixed with lipid soln cellfectin (Invitrogen company product), be transfected in sf9 cell, cultivate after 4 to 5 days for 27 ℃, collecting cell culture supernatant, 3000rpm collects supernatant for centrifugal 10 minutes, obtain the recombinant baculovirus of low titre, infect the new sf-9 cell of cultivating with this supernatant again, collecting cell culture supernatant after 3 days, is the high concentration recombinant baculovirus after required amplification culture, called after Bac-M1, Bac-NP, Bac-HA-NA and Bac-HF-NA.The recombinant baculovirus for amplification culture and protein expression obtaining is carried out to plaque experiment, determine viral plaque forming unit (plaque forming units, PFU).

1) by Grace ' the s culture medium containing 10%FBS, Sf9 passage is inoculated in six well culture plates, cell density is approximately 1 × 10 ⁶individual cells/ml, every hole adds 2ml (6 orifice plate), mixes gently room temperature and makes cell attachment more than 1 hour.

2) P3 is done to 10 times of doubling dilutions for kind of venom by Grace ' the s culture medium that does not contain FBS stand-by.

3) discard the culture medium in six orifice plates, clean cell 3 times by the culture medium that does not contain serum, then recombinant virus liquid good above-mentioned dilution is added in hand-hole, each dilution factor does two multiple holes, infects 1 hour under room temperature.

4) prepare covering liquid (being the amount of six orifice plates below): 7ml 2 × Grace ' s medium+140 μ l dual anti-+ autoclaving agarose glue+1.4ml FBS of 7ml2%, mix lightly, then bottle is placed again to 42 ℃ of water-baths, after viral infection 1h, exhaust the virus liquid in every hole, and cover cell by the covering liquid of above-mentioned preparation fast.

5) six orifice plates are wrapped and are placed in 27 ℃ of incubators after agarose solidifies with preservative film and cultivate 3-5 days.

6) adding 1ml concentration is the dimethyl diaminophenazine chloride of 1mg/ml, and incubated at room sucked dye liquor after 2 hours, and the approximate transparent point of observing recombinant virus formation is plaque.Counting statistics is observed the formational situation (PFU) of virus plaque.

For amplification culture recombinant baculovirus Bac-M1, the cell centrifugation precipitate of Bac-NP, Bac-HA-NA and Bac-HF-NA, after cell lysis buffer solution is processed, 4 ℃ centrifugal (13,000rpm) 10 minutes (or cell being carried out to ultrasonic disruption the ice bath in the situation that), collect supernatant, carry out SDS-PAGE gel electrophoresis, analyze matrix prote m1 (or NP, HA, HF and NA albumen) and whether in Insect cells Sf9, express.Briefly, the 2XSDS sample-loading buffer of 10ul will be added in 10ul supernatant samples, process after 5min for 100 ℃, centrifugal 5 minutes of 10000rpm, adds the supernatant sample of all 20ul in the point sample hole of 4%-12%SDS-polyacrylamide gel (Invitrogen company product).It is constant voltage 100V that deposition condition is set, and temperature is 4 ℃, electrophoresis time approximately 3 hours.When liquid bromjophenol blue to be instructed approaches gel bottom, stop electrophoresis.Gel is placed in 1%R type coomassie brilliant blue staining liquid, rocks dyeing 1 hour, is then placed in destaining solution decolouring and spends the night.

The expression in the insect cell sf-9 of the suspension culture of common transfection of embodiment 3:M1, NP, HA, HF and NA gene

By 200ml sf-9 cell mixture suspension culture in the triangle shaking flask of 1 liter of volume, cell culture fluid is the sf-900II (or Grace insect medium of Invitrigen company) of serum-free, the speed of shaking of shaking table is 100rpm, and temperature constant is in 27 ℃.When cell concentration reaches 2 × 10 ⁶when cell/ml, with Bac-M1, Bac-NP, Bac-HA-NA, the common transfection sf9 of Bac-HF-NA insect baculovirus cell.The MOI ratio of virus is 3 (Bac-M1 and Bac-NP): 1 (Bac-HA-NA): 1 (Bac-HF-NA).The cell of transfection through constant temperature wave and culture after 3 days, is collected all samples, 4 ℃ centrifugal 30 minutes, be 3000rpm from speed, collect supernatant.After the centrifugal cell precipitation thing cell pyrolysis liquid that gets off is processed, 4 ℃ centrifugal 10 minutes, from speed 10,000rpm.Retain the supernatant after centrifugal.Experiment builds the sf-9 cell of synthetic wild type insect baculovirus for transfection suspension culture when carrying out, and MOI is 5.As the negative control arranging, the condition of the cell culture after transfection, the collection of sample and lysis is the same with above-mentioned experiment with step.The all samples of collecting, analyzes for Western blots.Its experimental implementation is as follows: each sample is got respectively (comprising negative control) lysis extract and the cell culture supernatant of 10ul, then adds separately 2 × SDS sample-loading buffer of 10ul.Process after 5min, the biased sample of all 20ul is added in the point sample hole of 4%-12%SDS polyacrylamide gel for 100 ℃.Constant voltage 120V is set, and temperature is 4 ℃, 3 hours time, in the time that blue indicator bromjophenol blue leans on into gel bottom completely, stop electrophoresis, and take out gel.Cut one with nitrocellulose filter (pvdf membrane) and two filter paper of gel formed objects, pvdf membrane soaks after 5 minutes and immerses together with gel, filter paper in the more than 2 hours transfering buffering liquid of pre-cooling, by filter paper with methanol, and---order of gel---nitrocellulose filter---filter paper is fit into transfer printing and presss from both sides.By in folder near a side joint negative pole of gel, constant voltage 25V electrophoretic blotting 1h. goes out nitrocellulose filter with tweezers gripping, with the washing of PBS-T rinsing liquid, is then transferred in defatted milk powder/PBS solution of 5%, sways and seals 1 hour.With PBS-T rinsing liquid washing 3 times, each 3 minutes; Then nitrocellulose filter is proceeded in a plastic bag, add 3ml to be diluted in anti-H1N1 swine influenza virus chicken source polyclonal antibody (primary antibodie) in 5% defatted milk powder/PBS with 1: 500, be placed in 4 ℃ of mild shaken over night.Next day, take out cellulose membrane, with PBS-T rinsing liquid washing 3 times, each 10 minutes, this nitrocellulose filter is reinstalled in another new plastic bag, add 5ml to be diluted in the anti-chicken IgG of donkey (two is anti-) of the horseradish peroxidase-labeled in 5% defatted milk powder/PBS at 1: 10000, under room temperature, shake incubation 1h.Abandoning two resists, PBS-T rinsing fiber element film 3 times, each 10 minutes, nitrocellulose filter after rinsing is moved in a plate, add DAB the 5th nitrite ion, visible on corresponding separately molecular weight position, the specific band of M1, NP, HA/HF and NA, M1 is described, NP, HA/HF and NA effectively express in the SF-9 of the suspension culture of transfection insect cell.

Embodiment 4: the purification of virus-like particle and indirect immunofluorescene assay and electron microscopic observation.

The cell conditioned medium liquid of above-mentioned centrifugal collection is packed in the ultracentrifugation pipe of 13ml, weigh, after balance, tube sealing, put into supercentrifuge (Bechmem company product), 4 ℃ 100, centrifugal 1 hour of 000rpm, then take out centrifuge tube, carefully outwell supernatant, the deep thing at the bottom of reservation centrifuge tube.Add the PBS of 5ml, put into 4 ℃ of refrigerators, dissolve 24 hours.Next day, in the ultracentrifugation pipe of another 13ml, first adds the multitudinous sugar juice of 1ml 60% carefully, then adds successively the multitudinous sugar juice of 1ml30% and 3ml20%, finally by placed on it the sample liquid after the dissolving of 5ml.Accurately weigh, after balance, supercentrifuge on tube sealing.4 ℃ 100, centrifugal 1 hour of 000rpm.Take out centrifuge tube, collect respectively two bands that are positioned at 20% and 30% and 30% and 60% concentration intersection, the i.e. virus-like particle of purification.Weatern blots analyzes the virus-like particle sample after purification concentrates.Its operating procedure is the same with Western blots analysis in above-described embodiment 3, just got sample is diluted, and the virus-like particle sample of 1ul, adds the water of 9ul, then adds 2 × SDS sample-loading buffer of 10ul.After 100 ℃ of degeneration 5min, loading enters in 4%-12% polyacrylamide gel sample groove, Western blots result show, the macromolecular particle obtaining from this two band, the virus-like particle being formed by M1, NP, HA/HF and NA really.That especially obtains from 30% and 60% concentration intersection is large like virion content, and specific band concentration is dark.Illustrate after transfection, virus-like particle self assembly effectively in host cell, and be released in cell culture supernatant, further indirect immunofluorescence experiment and Electronic Speculum result have confirmed this point (Fig. 4, Fig. 5).

Indirect immunofluorescence experiment operating procedure is as follows: by sf9 cell kind in 24 orifice plates, every hole 100,000 cells, VLP is infected planted cell according to the infection multiplicity of MOI=3, infect after 72 hours, with PBS by cell washing 3 times, each 5 minutes, then add the methanol-20 ° fixed cell 10 minutes of 100% pre-cooling, adding again 0.05% Triton x-100 room temperature treatment 10 minutes, add 3% BSA, tetra-degree sealings to spend the night, second day with PBS by cell washing 3 times, each 5 minutes, then to the special M1 monoclonal antibody of A type influenza that adds RBITC labelling in hole, or the monoclonal antibody of the anti-foot and mouth disease F of pig of FITC labelling, 37 ° are reacted 1 hour, reaction finish rear with PBS by cell washing 3 times, each 5 minutes, washing finish after at fluorescence microscopy Microscopic observation.Carry out immune electron microscopy with the monoclonal antibody of anti-M1 and the monoclonal antibody of F, can see fluorescently-labeled virus-like particle, wherein the antibody of anti-M1 is RBITC labelling, show fluorescent orange (Fig. 4 A), the antibody of anti-F is FITC labelling, show green fluorescence (Fig. 4 C), and contrast do not have fluorescence signal ((Fig. 4 B, D).

Electronic Speculum film making experimental implementation is as follows: after the seemingly virion sample bearing reason after a small amount of purification is concentrated, be placed on the coated network lattice of freshly prepd uncharge plastic cement/carbon, after rinsing gently several times with several distilled water, add that 2% Sodium phosphotungstate solution carries out negative staining.Electronics is had an X-rayed micro-Microscopic observation film making, and as shown in Figure 5, the outward appearance of virus-like particle and volume size are close.

Embodiment 5:H1N1 swine flue and Schweineseuche bivalence VLPs immune mouse

BALB/C mice, purchased from Zhongshan University's zoopery center, is respectively organized the concrete immune embodiment of immune animal in table 1.The SPF BALB/C mice in age in 6-8 week 15 days eyeballs after immunity 3 times are got blood, respectively organize mice serum and are used for detecting IgG antibody horizontal and HI antibody horizontal.H1N1 swine flue IgG adopts the H1N1 of IDEXX company influenza antibodies ELISA test kit to detect, and foot and mouth disease IgG antibody adopts (2005) methods such as Ma Jingyun to detect, and uses OD ₄₅₀value representation antibody horizontal.HI antibody test adopts HI experiment to carry out with reference to Gan Menghou (second edition, Chinese agriculture publishing house).Result shows, H1N1 swine flue and foot and mouth disease bivalence VLPs immunity 6-8 BALB/C mice in age in week, and the antibody of existing anti-H1N1 swine flue (table 2, table 3) in immune serum, has also produced the antibody (table 3) of foot-and-mouth disease virus resistant.

Table 1H1N1 swine flue and Schweineseuche bivalence VLPs immune balb/c mice experimental program

Table 2H1N1 swine flue and Schweineseuche bivalence VLPs immune balb/c mice serum HI testing result

Specific IgG testing result in table 3H1N1 swine flue and Schweineseuche bivalence VLPs immune balb/c mice serum

Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although the present invention is explained in detail with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify or be equal to replacement technical scheme of the present invention, and not depart from essence and the scope of technical solution of the present invention.

Claims (7)

1. hybrid virus sample granule, is characterized in that, comprises:

The matrix prote m1 of influenza virus;

The surface antigen hemagglutinin HA of influenza virus;

A nexus albumen, the capsid protein that contains Main Antigenic that it comprises swine foot-and-mouth disease virus, the cross-film district of the hemagglutinin HA of influenza virus and film inner region; The capsid protein that contains Main Antigenic of described swine foot-and-mouth disease virus is replaced the film foreign lands of the roughly the same length of the 5 ' end of the hemagglutinin HA of influenza virus; The sequence of described nexus albumen is the sequence shown in SEQ ID NO:10;

The surface antigen hemagglutinin HA of while expression of influenza virus and the capsid protein that contains Main Antigenic of swine foot-and-mouth disease virus on the surface of described hybrid virus sample granule.

2. hybrid virus sample granule as claimed in claim 1, wherein said hybrid virus sample granule also contains the neuraminidase NA of influenza virus.

3. hybrid virus sample granule as claimed in claim 1 or 2, wherein said hybrid virus sample granule also contains the NP of influenza virus.

4. swine flue and Schweineseuche bivalent vaccine, is characterized in that, comprises hybrid virus sample granule and adjuvant as described in claim 1-3 any one.

5. the method for preparing hybrid virus sample granule, is characterized in that, described method comprises following steps:

Build baculovirus transfer vector, the matrix prote m1 that its expression vector contains influenza virus, the surface antigen hemagglutinin HA of influenza virus, an or nexus albumen, the capsid protein that contains Main Antigenic that it comprises swine foot-and-mouth disease virus, the cross-film district of the hemagglutinin HA of influenza virus and film inner region;

The described capsid protein that contains Main Antigenic containing swine foot-and-mouth disease virus is replaced the film foreign lands of the roughly the same length of the 5 ' end of the hemagglutinin HA of influenza virus; The sequence of described nexus albumen is the sequence shown in SEQ ID NO:10;

With the baculovirus transfer vector while infected insect cell in proportion of described structure, pack out hybrid virus sample granule, the upper surface antigen hemagglutinin HA of expression of influenza virus simultaneously in its surface and the capsid protein that contains Main Antigenic of swine foot-and-mouth disease virus.

6. prepare the method for hybrid virus sample granule as claim 5, it is characterized in that, wherein said structure baculovirus transfer vector also contains the neuraminidase NA of influenza virus, and it packs out the neuraminidase NA that hybrid virus sample granule contains influenza virus.

7. the method for preparing hybrid virus sample granule as claim 5 or 6, is characterized in that, wherein said structure baculovirus transfer vector also contains the NP of influenza virus, and it packs out the NP that hybrid virus sample granule contains influenza virus.

Images