CN102370976A - Mixed virus-like particles of swine influenza virus and foot and mouth disease virus, preparation method and application thereof - Google Patents
Mixed virus-like particles of swine influenza virus and foot and mouth disease virus, preparation method and application thereof Download PDFInfo
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- Peptides Or Proteins (AREA)
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
Technical field
The present invention relates to the hybrid virus appearance granule of swine influenza virus and Schweineseuche virus, and preparation and application.
Background technology
Swine flue is by swine influenza virus (Swine influenza virus; The respiratory infectious disease of the different days that SIV) causes, sex and article boar a kind of acute, hot and height contact only, it is a characteristic with burst, hyperpyrexia, cough, dyspnea, depletion and death clinically.Each age, sex, article boar all can infect, and sickness rate is high.The infected pigs influenza virus can cause a pig fertility performance to descend separately, and feedstuff-meat ratio raises, and weightening finish slows down, and causes the certain economic loss to the pig farm.Even more serious is that pig is fowl, people, swine influenza virus reorganization and duplicates " blender " that swine influenza virus need not recombinated just has the ability of infected person to greatest extent.And early stage human influenza virus can also be stored in the pig body, and infected person once more 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 to be seen and other eqpidemic disease; Like pig breeding and concurrent or secondary infection such as respiratory disorder syndrome virus, PRV, contagious pleuropneumonia Actinobacillus, Streptococcus suis, eperythrozoon suis; Epidemic situation is become repeatedly and complicated, be one of modern intensive pig farm ubiquity and porcine respiratory infectious disease of being difficult to eradicate, 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 enormous economic loss, and worldwide people's public health security has been caused a very big challenge.Break out in the Mexican influenza A H1N1 whole world in April, 2009 and attract attention, subsequently, states such as the U.S. and Canada also occur the people in succession and infect the situation of H1N1 swine flue and the part patient death is arranged.Developing effective vaccine in a short time prevents influenza A H1N1 influenza virus inhibition epidemic situation propagation and increases the weight of extremely urgent.The vaccine major part that is used for fowl poultry flu-prevention virus of domestic production at present is to adopt traditional chick embryo method multiplication by culture live virus, after the chemical reagent deactivation, produces vaccine.This technology is divided into two types of different preparation methods again: the one, directly infect the Embryo Gallus domesticus preparation with wild-type virus; Another kind of is the heritability that adopts reverse genetic technological transformation wild virus earlier; The new virus of using " rescue " to transform then infects Embryo Gallus domesticus, and virus of proliferation is produced vaccine.
These influenza vaccines technologies of preparing have its advantage, but also exist very big defective.Especially the unusual characteristic of influenza sense poison; For example: the altofrequency sudden change; Hereditary material reorganization and antigenic drift etc. between the dual and multiple subtype virus---make the long-term safety of these vaccines and effectiveness receive very big challenge; Even produced " invalid vaccine ", be difficult to deal with the infection of new saltant 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: be the leakage diffusion of the live virus that prevents artificial contaminated environment and production, a whole set of production must be undertaken by being defined under three grades of laboratory conditions of bio-safety, and the deactivation of virus must guarantee fully, and is thorough.
3, can't distinguish by the pig of immunity only with the pig that is infected by the virus only.
(foot and mouth disease is a kind of acute, the height contagious disease that is caused by foot and mouth disease virus (FMDV) FMD) to foot and mouth disease, mainly infects the artiodactyl beast, and people and non-artiodactyl also can infect.International Office of Epizootics classifies foot and mouth disease as first of the category-A infectious disease, and China also classifies it as first disease of livestock contagious disease.In a single day foot and mouth disease is broken out and can be caused enormous economic loss, also has influence on the normal trade activity of countries and regions simultaneously.Because the international trade of foot and mouth disease direct threats and national reputation and animal ecology and human food's health, thereby receive people's common concern.In recent years more classified as the biological weapons security pact and organize emphasis inspection object, the country that countries in the world comprise no foot and mouth disease all maintains sharp vigilance to primary disease, and carry out nosetiology energetically, diagnostic techniques and the anti-Study on Technology of making.
FMDV is the animal RNA viruses of known minimum, and virion is spherical in shape, the regular dodecahedron structure, and diameter 20～30nm, molecular weight 6.9 * 106u, sedimentation coefficient 146s, the buoyant density in cesium chloride are 1.43g/mL.Intact virus contains sub-thread positive chain RNA and capsid protein two parts, no cyst membrane.Capsid is by VP1, VP2, VP3, each 60 molecular composition of VP4, and geneome RNA is made up of about 8500 nucleotide, can be directly as messenger RNA.FMDV only contains an ORF (ORFs), the precursor polyprotein of coding virus.The precursor polyprotein forms 4 kinds of structural protein (VP1-VP4) after twice cracking, wherein VP1 (1D) gene is positioned at genomic 2977～3615,213 aminoacid of encoding.Early stage research shows that structural protein are the main components of inducing 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 the VP1; Wherein the G-H at 140-160 amino acids place ring is topmost protective antigen site, and the space conformation more complicated has bigger mobility; Having formed Arg-Gly-Asp (RGD) sequence of a high conservative at its top, is the cell receptor site of FMDV.The 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 a t cell epitope in VP1 the 141st～160 amino acid residue, can induce the narrow spectrum t cell responses of FMDV.
China adopts vaccine immunity and slaughters the policy that combines the anti-system of foot and mouth disease, to the foot and mouth disease employing compulsory immunization of pig.The foot-and-mouth disease vaccine that uses at present mainly is two kinds of totivirus inactivated vaccine and polypeptide vaccines, because of the defective that has potential safety hazard or immune effect difference can not provide ideal immunoprotection.Therefore, efficient, the safe and reliable vaccine of research seems very necessary.
Virus-like particle (VLPs) is the one or more primary structure albumen that contain certain virus; The hollow bead that does not comprise viral nucleic acid that in the vivoexpression system, is assembled into automatically; Its form is same or similar with real virion with size; So can effectively induce body immune system to produce the 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 the genome of H1N1 influenza virus, the 11 kinds of albumen of encoding altogether, the shell of virus be one deck by the film that lipid and lipoprotein constitute, be wrapped in the hereditary material and the nucleoprotein of virus.Have 4 kinds of protein, belong to viral primary structure albumen, they are: matrix prote m1, the main body albumen of formation virus coat; NP forms the virion nucleocapsid, participates in virion and forms; Hemagglutinin HA, the main glycoprotein on the virus coat skin covering of the surface has 16 hypotypes.Be to bring out the major antigen body that host's body produces antibody.Neuraminidase NA also is a kind of glycoprotein on the 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 research proof; The correct expression of matrix prote m1; It is the important step that guarantees that virus coat forms; And one of function of memebrane protein NA is to strip down viral entity from host cell surface, form virion, so structural protein M1, HA and the NA of bird flu virus is the core of synthesizing new anti-avian influenza virus vaccine.3 primary structure albumen such as albumen HA, NA and M1 are expressed simultaneously and just can be assembled into the hollow virus genomic virus-like particle that do not have automatically.But type specific antigen NP albumen effective stimulus CTL (CTL) reaction of the influenza virus of research proof in addition; Suppress viral infection, in virus-like particle, add the cellular immune level that NP albumen will improve this new generation vaccine to a certain extent.
Swine influenza virus and foot and mouth disease virus be influence each other in the 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 simultaneously is those skilled in the art's problem demanding prompt solutions.
Summary of the invention
In view of this, one of the object of the invention is to provide a kind of bivalent vaccine, and this vaccine can prevent H1N1 swine flue and Schweineseuche simultaneously.
The objective of the invention is to realize through content once.
The present invention provides a kind of hybrid virus appearance granule to comprise the matrix prote m1 of influenza virus; The surface antigen hemagglutinin HA of influenza virus; A nexus albumen; It comprises the capsid protein that contains the major antigen epi-position of Schweineseuche virus, and the hemagglutinin HA of influenza virus strides film district and film inner region; The film foreign lands of the roughly the same length of the 5 ' end of the hemagglutinin HA of the capsid protein replacement influenza virus that contains the major antigen epi-position of said Schweineseuche virus; The capsid protein that contains the major antigen epi-position of the surface antigen hemagglutinin HA of while expression of influenza virus and Schweineseuche virus on the particulate surface of said hybrid virus appearance.
The present invention also provides swine flue and Schweineseuche bivalent vaccine, comprises described hybrid virus appearance granule and adjuvant.
The present invention also provides preparation hybrid virus appearance particulate method.
Utilization comprises the vaccine of the virus-like particle formation of said fusion rotein, can prevent H1N1 swine flue and Schweineseuche simultaneously, and have tangible 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 longer duration can prevent H1N1 swine flue and Schweineseuche simultaneously.
(2) because virus-like particle does not contain viral genome, viral gene and host chromosome gene integration just can not take place in the VLPs of this hollow shell structure in the immune animal body, and whole process of production do not contact infectious virus alive, so as safe as a house.For influenza virus and two kinds of viruses of foot and mouth disease virus, the anxiety of all virus-free diffusion.
(3) and general genetic engineering subunit vaccine relatively because virus-like particle can stimulate body to produce better immunoreation more near the form and the size of natural viral, immune effect is better.
(4) can distinguish out by the animal of artificial immunity and by the animal of viral infection.Therefore virus-like particle does not contain albumen such as influenza virus NS, PB1, PB2, when carrying out serologic test, with the immune animal of virus-like particle particle vaccines, with the antibody that can not produce specific anti NS or PB, whether can distinguish wild virus infection.For foot and mouth disease, with any foot and mouth disease structural protein such as VP2 albumen, the N albumen etc. outside the VP1 albumen, and the non-structural protein of foot and mouth disease virus is as detecting antigen, whether can distinguish wild virus infection.
Above-mentioned explanation only is the general introduction of technical scheme of the present invention; Understand technological means of the present invention in order can more to know; And can implement according to the content of description, and for let above-mentioned and other purposes of the present invention, feature and advantage can be more obviously understandable, below in conjunction with preferred embodiment; And conjunction with figs., specify as follows.
Description of drawings
Fig. 1 is the electrophoretogram of the HA (a1) through different Auele Specific Primer pcr amplifications, NA (a2), M1 (b), NP (c), HF (d) genetic fragment;
Fig. 2 is reorganization baculovirus expression plasmid rBacmid-HA/HF-NA, rBacmid-M1, rBacmid-NP sketch map;
Fig. 3 is virus-like particle western blot result.
Fig. 4 is M1 albumen and the proteic indirect immunofluorescence result of S1 in the virus-like particle.A, M1 antibody show fluorescent orange for the RBITC labelling, and C, S1 antibody show green fluorescence, B, the negative contrast of D for the FITC labelling.
Fig. 5 A is the image of virus-like particle under ultramicroscope behind the SDGC purification, and b is the partial enlarged drawing of a.
The specific embodiment
The present invention is the basis with Bac-to-Bac
insect baculovirus expression system; Utilize the stromatin M and the NP of H1N1 swine influenza virus; And skin covering of the surface egg HA and NA albumen, the virus-like particle (VLP) of H1N1 swine influenza virus is constructed in common assembling automatically.On this basis, make up the bivalence VLPs of H1N1 swine influenza virus and Schweineseuche virus.
At first; Two peptide sections 132～159 (A) on the Schweineseuche virus VP 1,200～213 (B) are synthesized with the placed in-line form of A-B-A-B-A-B-A; The N end of A peptide section has extra 5 aminoacid (MVPSR), and the C end of B peptide section has extra 6 aminoacid (QFELEF), adds 2 aminoacid (PG) linker between A and the B peptide section; Form 11 aminoacid (QFELEFMVPSR) linker in the B-A junction; The aminoacid that these two linker comprise has pliability preferably, and the fragment that helps to connect forms certain space structure, and this series connection fragment is named as the F polypeptide.The F peptide sequence is replaced the part of the HA GFP of H1N1 influenza virus; The two constitutes fusion gene HF; The F peptide sequence is positioned at the 5 ' end of fusion gene HF, replaces 5 ' terminal sequence of the HA gene of length about equally, with the length that ensures fusion gene and HA mrna length about equally.
Then; According to the method that forms influenza virus-like particles; With the gene of matrix prote m1, NP and surface membrane protein HA and the NA of H1N1 influenza virus, and the gene order of expressing the fusion rotein HF of Schweineseuche 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, be assembled into the virus-like particle that does not contain the influenza virus hereditary material automatically, will be released in the cell culture fluid after virus-like particle forms.The VLPs that forms had so both had the main surface antigen of H1N1 influenza virus, had the major antigen tandem polypeptide F of Schweineseuche virus again, was H1N1 swine influenza virus and Schweineseuche virus bivalence VLPs.
It is pointed out that Schweineseuche virus is no cyst membrane, its surface antigen protein does not have naturally to stride the film district and can not be integrated directly on the VLP.In addition, its epitope disperses, so surface antigen protein directly is fused to the structure that can destroy HA on the influenza virus HA.In order to overcome these difficult problems, the present invention adopts the mode of series connection epitope, is guaranteeing under the situation that a plurality of epitopes exist, and has reduced the size of series connection antigen epitope polypeptide, can be fused on the HA.Through merging the viral series connection antigen epitope polypeptide of Schweineseuche to HA, the series connection antigen epitope polypeptide of Schweineseuche virus can be expressed on the 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 proteic VLP of being incorporated into of HA is efficiently simultaneously; Can guarantee like this has enough HA albumen on the surface of VLP, can be used as effective vaccine and uses.
Can further be expressly understood the present invention through specific embodiment given below, but following embodiment is not to qualification of the present invention.
Implement the amplification of row 1:M1, NP, HA, NA and fusion gene HF.
(1) Schweineseuche virus F polypeptide and swine influenza virus HA fusion gene HF's is synthetic
The nucleotide sequence of fusion gene HF sees that SEQ ID NO:9, its amino acid sequence coded see SEQ ID NO:10, and fusion gene is according to its nucleic acid sequence SEQ ID NO:9 synthetic.The F polypeptide contains 198 aminoacid, substitutes 5 ' 184 aminoacid of end of HA gene.
(2) 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 250 μ L swine influenza virus H1N1 hypotype virus strain infection's allantoic fluids and 750 μ L TRIzol LS respectively, add in the 1.5mL microcentrifugal tube, blow and beat abundant mixing with suction pipe, room temperature is placed 10min; Add 200 μ L chloroforms, thermal agitation 15s, after room temperature leaves standstill 5 minutes, 4 ℃ of centrifugal 15min of following 12000rpm; Get supernatant in a new sterilization 1.5mL centrifuge tube, add 500 μ L isopropyl alcohols, abundant mixing, room temperature is placed 10min, at 4 ℃ of centrifugal 10min of following 12000rpm; The supernatant that inclines adds 70% ethanol 750 μ L in the deposition, mixing gently, washing once, 4 ℃ of centrifugal 15min of following 12000rpm, supernatant discarded, air-dry; Add the tri-distilled water lytic virus RNA (deposition) of 10 μ L, directly be used for RT-PCR or-80 ℃ of preservations are subsequent use with the no RNA enzyme of DEPC water treatment.Operation instruction with reference to the AMV reverse transcription of TaKaRa is carried out, and in 20 μ L reaction systems, adds following component respectively: RNA:3 μ L; 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.Behind the mixing, room temperature is placed 10min, 42 ℃ of insulation 1h, and ice bath 2min, RT product directly are used for 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, the HA gene that is used to increase, its two ends have added Xho I and Nco I/restriction enzyme site respectively, are positioned under the P10 promoter, primer sequence is following:
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, the NA gene that is used to increase, its two ends have added that respectively the restriction enzyme site of Sal I and Hind III is positioned at P
PHUnder the promoter, these 2 primer sequences are 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, the M1 gene that is used to increase, its two ends have added that respectively the restriction enzyme site of Sal I and Hind III is positioned at P
PHUnder the promoter, these 2 primer sequences are 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, the NP gene that is used to increase, its two ends have added Xho I and Nco I/restriction enzyme site respectively, are positioned under the P10 promoter, primer sequence is following:
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 (sequence table 9 and sequence table 10) of fusion gene HF, 1 pair of primer of design, the fusion gene HF that is used to increase, its two ends have added Xho I and Sph I restriction enzyme site respectively, are positioned under the P10 promoter, primer sequence is following:
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, with the product of reverse transcription or synthetic dna segment directly as the template of pcr amplification M1, NP, HA, NA, HF gene.The PCR reaction condition is 94 ℃ of preparatory degeneration 3min, 94 ℃ of degeneration 40S, and 56 ℃ of annealing 90s, 72 ℃ are extended 90s, circulate 30 times, extend 10min at last, and the PCR product (contains the 0.5ug/ml ethidium bromide, EB) electrophoresis detection with 1.5% agarose gel.The about 1.7Kb of length of the HA gene of pcr amplification (Fig. 1 a), and the about 1.35Kb of the length of NA gene (Fig. 1 a), the about 0.75Kb of the length of M1 gene (Fig. 1 b), the length of NP gene is about 1.5Kb (Fig. 1 c), and the length of fusion gene HF is about 1.7Kb (Fig. 1 d).All PCR product samples are after the 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) after the digestion, are further purified respectively again, use in order to next step construction recombination plasmid.Concrete operations are following: prepare 1% agarose gel and contain the 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; Cutting-out contains the gel strip of sample band, in the little plastic centrifuge tube of packing into.Reclaim test kit (Qiagen Company products) 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 products) with glue, instruct centrifugal post, the postdigestive M1 of purifying and recovering enzyme action, NP, HA, NA, the HF fragment crossed according to explanation.
Embodiment 2: the insect baculovirus of the baculovirus expression plasmid of construction expression M1, NP, HA, NA, HF gene and synthetic reorganization in insect cell
(1) recombiant plasmid of construction expression M1 and NP gene
Insect baculovirus plasmid PFastBac-dual (Invitrogen Company products) after 3 hours, reclaims plasmid PFastBac-dual test kit reclaim purification enzyme action after with glue through restricted enzyme Sal I/37 ℃ of enzyme action of Hind III.Under the effect of T4 dna ligase, the M1 dna fragmentation behind the plasmid behind the enzyme action and the enzyme action is connected in 16 ℃ and spends the night.Reaction system is following: 10 * T4 connects buffer 1ml, the dna fragmentation 3ml that the M1 enzyme action reclaims, and enzyme action PFastBac-dual plasmid reclaims product 1ul, T4DNA ligase 1ul, ddH2O mends to 10ul.Adopting the heat shock method will connect product transduces and joins in a little plastic centrifuge tube in the Top10 competent cell; After mixing gently tubule is placed 30min on ice; Change heat shock 90s in 42 ℃ of water-baths over to, put back to rapidly 5 minutes on ice, think wherein to add the 200ulLB culture fluid.37 ℃ of shaking tables were cultivated 1 hour.Get 100ul bacterium liquid and coat on the LB solid medium (containing two kinds of antibiotic of ammonia benzyl and gentamycin), cultivate 16h for 37 ℃, picking positive colony bacterium colony from the flat board carries out bacterium liquid PCR, carries out single double digestion evaluation with Sal I/Hind III behind the extracting plasmid.Behind the 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 behind restricted enzyme Xho I/Nco I enzyme action; Under the effect of T4DNA ligase; To be inserted into the below of P10 promoter by the postdigestive HA gene DNA fragment of same restriction endonuclease; This connects product with in the Top10 competent cell after hot body gram method is transduceed, cultivate, the recombinant plasmid dna of several positive colony bacterium colonies of extracting, the single double digestion of bacterium liquid PCR and Xho I/Nco I identify and the DNA sequence order-checking definite errorless after; Select one of them recombinant plasmid dna, carry out the enzyme action second time.Used restricted enzyme is Sal I/Hind III.With same endonuclease digestion NA gene DNA fragment, and be inserted into another promoter P of recombiant plasmid
PHThe below, through conversion, screening, cultivation, extracting, obtain the plasmid of two degree reorganization.Behind the dna sequencing, be required recombinant baculovirus expression plasmid PFastBac-dual-HA-NA.Whole experimental implementation Step By Condition is said identical with above-mentioned structure PfastBac-dualM1 plasmid.
The construction method of recombiant plasmid PFastBac-dual-HF-NA of expressing fusion gene HF and NA gene simultaneously is the same.
(3) the genomic synthetic and extraction of recombinant baculovirus
During purified recombinant PfatBac-dual-M1, PfatBac-dual-NP, PFastBac-dual-HA-NA and PFastBac-dual-HF-NA plasmid transduceed E.coli competent cell strain DH 10 Bac cells respectively special (American I nvitrogen Company products).The DH10Bac cell contains a special macromole plasmid Bacmid, includes the full gene group of insect baculovirus AcMNPV in it.After in case the expression plasmid of reorganization is integrated into the special site of macromole plasmid Bacmid; Screening and inducing with the X-Gal substrate reactions of IPIG through 3 kinds of antibiotics (gentamycin, tetracycline and kanamycin) are carried out blue white macula screening; The positive colony bacterium colony is white in color, but not the wild bacterium colony of reorganization is blue color.The laboratory manual that experiment condition all provides according to Invitrogen company instructs and sets.The positive colony of selecting places the LB culture fluid (containing above-mentioned 3 kinds of antibiotic) of 3ml; Cultivated 24 hours through 37 ℃ of shaking tables; Macromole plasmid Bacmid a small amount of preparation method according to laboratory manual is indicated extracts the reorganization macromole plasmid Bacmid that purification has M1 gene, NP gene, HA-NA or HF-NA gene.
(4) preparation of recombinant baculovirus
Insect cell line sf9 cell (American I nvitrogen Company products) is incubated in the sf-900II insect cell culture fluid of serum-free (Invitrogen Company products), temperature is set to 27 ℃, the laboratory manual that provides according to Invitrogen company; Adopt the liposome transfection method; Purified recombinant macromole plasmid Bacmid is mixed with lipid soln cellfectin (Invitrogen Company products), be transfected in the sf9 cell, cultivate after 4 to 5 days for 27 ℃; The collecting cell culture supernatant; 3000rpm collected supernatant in centrifugal 10 minutes, obtained the recombinant baculovirus of low titre, and this supernatant of reuse infects the new sf-9 cell of cultivating; Collecting cell culture supernatant after 3 days is the high concentration recombinant baculovirus after the required amplification culture, called after Bac-M1, Bac-NP, Bac-HA-NA and Bac-HF-NA.The recombinant baculovirus that is used for amplification culture and protein expression that obtains is carried out the plaque experiment, and the plaque forming unit of definite virus (plaque forming units, PFU).
1) with Grace ' the s culture medium that contains 10%FBS the Sf9 passage is inoculated in six well culture plates, cell density is about 1 * 10
6Individual cells/ml, every hole adds 2ml (6 orifice plate), and the mixing room temperature makes cell attachment more than 1 hour gently.
2) it is for use P3 to be done 10 times of doubling dilutions for kind of venom with Grace ' the s culture medium that does not contain FBS.
3) discard culture medium in six orifice plates, clean cell 3 times with the culture medium that does not contain serum, then that above-mentioned dilution is good recombinant virus liquid adds in the hand-hole, and each dilution factor is done two multiple holes, infects 1 hour under the room temperature.
4) preparation covering liquid (below be the amount of one six orifice plate): autoclaving agarose glue+1.4ml FBS of two anti-+ 7ml2% of 7ml 2 * Grace ' s medium+140 μ l; Mixing lightly; Then bottle is placed 42 ℃ of water-baths again; Exhaust the viral liquid in every hole behind the viral infection 1h, and fast with the covering liquid covering cell of above-mentioned preparation.
5) treat to wrap and place 27 ℃ of incubators to cultivate 3-5 days with preservative film six orifice plates after agarose solidifies.
6) adding 1ml concentration is the dimethyl diaminophenazine chloride of 1mg/ml, and incubated at room is inhaled after 2 hours and removed dye liquor, and the approximate transparent point of observing recombinant virus formation is plaque.Counting statistics is observed the formation situation (PFU) of virus plaque.
Be used 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 handled, 4 ℃ centrifugal (13; 000rpm) 10 minutes (perhaps cell being carried out ultrasonic disruption under the situation of ice bath); Collect supernatant, carry out the SDS-PAGE gel electrophoresis, analyze matrix prote m1 (perhaps NP, HA, HF and NA albumen) and whether in insect cell Sf9, express.Briefly; With the 2XSDS sample-loading buffer that adds 10ul in the 10ul supernatant samples; 100 ℃ handle 5min after, centrifugal 5 minutes of 10000rpm adds the supernatant sample of all 20ul in the point sample hole of 4%-12%SDS-PAAG (Invitrogen Company products).It is constant voltage 100V that deposition condition is set, and temperature is 4 ℃, about 3 hours of electrophoresis time.When treating the indicator solution bromjophenol blue, stop electrophoresis near the gel bottom.Gel places 1%R type coomassie brilliant blue staining liquid, rocks dyeing 1 hour, places the destaining solution decolouring to spend the night then.
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
200ml sf-9 cell mixture suspension culture is shaken in the bottle in the triangle of 1 liter of volume, and cell culture fluid is the sf-900II (or Grace insect medium of Invitrigen company) of serum-free, and the speed of shaking of shaking table is 100rpm, and temperature constant is in 27 ℃.When cell concentration reaches 2 * 10
6During 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).Cells transfected is collected all samples through the constant temperature wave and culture after 3 days, 4 ℃ centrifugal 30 minutes, be 3000rpm from speed, the collection supernatant.After the centrifugal cell precipitation thing that gets off is handled with cell pyrolysis liquid, 4 ℃ centrifugal 10 minutes, from speed 10,000rpm.Keep the supernatant after centrifugal.Experiment makes up the sf-9 cell that synthetic wild type insect baculovirus is used for the transfection suspension culture when carrying out, MOI is 5.As the negative control that is provided with, the condition of the cell culture after the transfection, the collection of sample and lysis is the same with above-mentioned experiment with step.The all samples of collecting is used for Western blots and analyzes.Its experimental implementation is following: each sample is got (comprising negative control) lysis extract and the cell culture supernatant of 10ul respectively, adds 2 * SDS sample-loading buffer of 10ul more separately.100 ℃ handle 5min after, the biased sample of all 20ul is added in the point sample hole of 4%-12%SDS PAAG.Constant voltage 120V is set, and temperature is 4 ℃, 3 hours time, when blue indicator bromjophenol blue leans on into the gel bottom fully, stop electrophoresis, and take out gel.Cut nitrocellulose filter (pvdf membrane) and two filter paper big or small together with gel phase; Pvdf membrane immerses in the transfering buffering liquid of pre-cooling more than 2 hours, by filter paper with gel, filter paper after 5 minutes with the methanol immersion, and---order of gel---nitrocellulose filter---filter paper is fit into the transfer printing folder.With in the folder near a side joint negative pole of gel, constant voltage 25V electrophoretic blotting 1h. goes out nitrocellulose filter with the tweezers gripping, washs with the PBS-T rinsing liquid, then is 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 changed in the plastic bag, add 3ml and be diluted in anti-H1N1 swine influenza virus chicken source polyclonal antibody among 5% defatted milk powder/PBS (is anti-), place 4 ℃ of mild shaken over night with 1: 500.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 with the anti-chicken IgG of the donkey that is diluted in the horseradish peroxidase-labeled among 5% defatted milk powder/PBS at 1: 10000 (two is anti-), shake incubation 1h under the room temperature.Abandon two and resist, the plain film of PBS-T rinsing fiber 3 times, each 10 minutes; Nitrocellulose filter after the rinsing is moved in the plate, add DAB the 5th colour developing liquid, visible on corresponding molecular weight position separately; The specific band of M1, NP, HA/HF and NA; M1 is described, NP, HA/HF and NA express in the SF-9 of the suspension culture of transfection insect cell effectively.
Embodiment 4: the purification of virus-like particle and indirect immunofluorescence detect and electron microscopic observation.
The cell conditioned medium liquid of above-mentioned centrifugal collection is packed in the ultracentrifugation pipe of 13ml, weigh, after the balance, tube sealing, put into supercentrifuge (Bechmem Company products); 4 ℃ 100, centrifugal 1 hour of 000rpm takes out centrifuge tube then; Carefully outwell supernatant, the dull thing at the bottom of the reservation centrifuge tube.Add the PBS of 5ml, put into 4 ℃ of refrigerators, dissolved 24 hours.In the ultracentrifugation pipe of another 13ml, add earlier the multitudinous sugar juice of 1ml 60% carefully next day, adds the multitudinous sugar juice of 1ml30% and 3ml20% then successively, at last that the sample liquid after the dissolving of 5ml is placed on it.Accurately weigh, after the balance, supercentrifuge on the tube sealing.4 ℃ 100, centrifugal 1 hour of 000rpm.Take out centrifuge tube, collect two bands that are positioned at 20% and 30% and 30% and 60% concentration intersection, the i.e. virus-like particle of purification respectively.Weatern blots analyzes the virus-like particle sample after purification concentrates.Western blots analyzes equally in its operating procedure and the foregoing description 3, just the sample of being got is diluted, i.e. and the virus-like particle sample of 1ul, the water of adding 9ul adds 2 * SDS sample-loading buffer of 10ul again.Behind 100 ℃ of degeneration 5min, last appearance is gone in the 4%-12% polyacrylamide gel sample groove, and Western blots result shows, the macromolecular particle that is obtained from this two band, the virus-like particle that constitutes by M1, NP, HA/HF and NA really.Especially from 30% and 60% concentration intersection obtain like virion content big, specific band concentration is dark.After transfection was described, virus-like particle is oneself's assembling effectively in host cell, and is released in the cell culture supernatant, and further indirect immunofluorescence experiment and Electronic Speculum result have confirmed this point (Fig. 4, Fig. 5).
The indirect immunofluorescence experiment operating procedure is following: in 24 orifice plates, every hole 100,000 cells infect according to the infection multiplicity of MOI=3 VLP to the kind cell with sf9 cell kind; Infect after 72 hours, with PBS with cell washing 3 times, each 5 minutes; Methanol-20 ° the fixed cell 10 minutes that adds 100% pre-cooling was then adding 0.05% Triton x-100 room temperature treatment 10 minutes again, added 3% BSA four degree sealings and spent the night; Second day with PBS with cell washing 3 times, each 5 minutes, in the hole, add the special M1 monoclonal antibody of A type influenza of RBITC labelling then; The perhaps monoclonal antibody of the anti-foot and mouth disease F of pig of FITC labelling, 37 ° of reactions 1 hour, reaction finish the back with PBS with cell washing 3 times; Each 5 minutes, washing finished the back and under fluorescence microscope, observes.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 the RBITC labelling, shows fluorescent orange (Fig. 4 A), and the antibody of anti-F is the FITC labelling; Show green fluorescence (Fig. 4 C), and contrast there is not fluorescence signal ((Fig. 4 B, D).
Electronic Speculum film making experimental implementation is following: after the seemingly virion sample bearing reason after a spot of purification is concentrated; Place freshly prepd uncharge plastic cement/carbon to encapsulate on the network lattice; After washing several times gently with several distilled water, add that 2% Sodium phosphotungstate solution carries out negative staining.Electronics perspective microscopically is observed and 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 is available from Zhongshan University zoopery center, and each is organized the concrete immune embodiment of immune animal and sees table 1.The SPF BALB/C mice in age in 6-8 week 15 days eyeballs after immunity 3 times are got blood, and each is organized mice serum and is used for detecting IgG antibody horizontal and HI antibody horizontal.H1N1 swine flue IgG adopts the IDEXX H1N1 of company influenza antibodies ELISA test kit to detect, and foot and mouth disease IgG antibody adopts (2005) method detections such as Ma Jingyun, uses OD
450The value representation antibody horizontal.The HI antibody test adopts the HI experiment to carry out with reference to Gan Menghou (second edition, Chinese agriculture publishing house).The 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 the 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 the Schweineseuche bivalence VLPs immune balb/c mice serum
Last institute should be noted that; Above embodiment is only in order to technical scheme of the present invention to be described but not to the restriction of protection domain of the present invention; Although the present invention has been done detailed description with reference to preferred embodiment; Those of ordinary skill in the art should be appreciated that and can make amendment or be equal to replacement technical scheme of the present invention, and do not break away from the essence and the scope of technical scheme of the present invention.
Claims (8)
1. hybrid virus appearance granule is characterized in that, comprises:
The matrix prote m1 of influenza virus;
The surface antigen hemagglutinin HA of influenza virus;
A nexus albumen, it comprises the capsid protein that contains the major antigen epi-position of Schweineseuche virus, and the hemagglutinin HA of influenza virus strides film district and film inner region; The film foreign lands of the roughly the same length of the 5 ' end of the hemagglutinin HA of the capsid protein replacement influenza virus that contains the major antigen epi-position of said Schweineseuche virus;
The capsid protein that contains the major antigen epi-position of the surface antigen hemagglutinin HA of while expression of influenza virus and Schweineseuche virus on the particulate surface of said hybrid virus appearance.
2. hybrid virus appearance granule as claimed in claim 1, wherein said hybrid virus appearance granule also contains the neuraminidase NA of influenza virus.
3. according to claim 1 or claim 2 hybrid virus appearance granule, wherein said hybrid virus appearance granule also contains the NP of influenza virus.
4. hybrid virus appearance granule as claimed in claim 1, the capsid protein that contains the major antigen epi-position of wherein said Schweineseuche virus is a kind of fusion rotein, it contains a plurality of antigenic peptides sections.
5. hybrid virus appearance granule as claimed in claim 1, wherein the proteic sequence of nexus is shown in SEQ ID NO:10.
6. swine flue and Schweineseuche bivalent vaccine is characterized in that, comprise like claim 1-5 described hybrid virus appearance granule and adjuvant.
6. prepare the particulate method of hybrid virus appearance, it is characterized in that said method comprises following steps:
Make up the baculovirus expression carrier; Its expression vector contains the matrix prote m1 of influenza virus; The surface antigen hemagglutinin HA of influenza virus; Or a nexus albumen, it comprises the capsid protein that contains the major antigen epi-position of Schweineseuche virus, and the hemagglutinin HA of influenza virus strides film district and film inner region; The film foreign lands of the roughly the same length of the 5 ' end of the hemagglutinin HA of the said capsid protein replacement influenza virus that contains the major antigen epi-position that contains Schweineseuche virus;
With the baculovirus expression carrier of said structure infected insect cell simultaneously in proportion; Pack out hybrid virus appearance granule, the surface antigen hemagglutinin HA and the viral capsid protein that contains the major antigen epi-position of Schweineseuche of expression of influenza virus are simultaneously gone up in its surface.
7. like the particulate method of claim 6 preparation hybrid virus appearance; It is characterized in that; Wherein said structure baculovirus expression carrier also contains the neuraminidase NA of influenza virus, and it packs out the neuraminidase NA that hybrid virus appearance granule contains influenza virus.
8. like claim 6 or the particulate method of 7 preparation hybrid virus appearance, it is characterized in that wherein said structure baculovirus expression carrier also contains the NP of influenza virus, it packs out the NP that hybrid virus appearance granule contains influenza virus.
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| CN103667196A (en) * | 2012-09-25 | 2014-03-26 | 普莱柯生物工程股份有限公司 | Vaccine composition containing porcine circovirus-2 antigen and swine flu antigen |
| WO2017020570A1 (en) * | 2015-08-06 | 2017-02-09 | Medigen Biotechnology Corp. | Virus-like particle vaccines |
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| CN103667196A (en) * | 2012-09-25 | 2014-03-26 | 普莱柯生物工程股份有限公司 | Vaccine composition containing porcine circovirus-2 antigen and swine flu antigen |
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| WO2017020570A1 (en) * | 2015-08-06 | 2017-02-09 | Medigen Biotechnology Corp. | Virus-like particle vaccines |
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