CN102373184A - Avian influenza and infectious bronchitis hybrid virus-like particle as well as preparation method and application thereof - Google Patents

Avian influenza and infectious bronchitis hybrid virus-like particle as well as preparation method and application thereof Download PDF

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CN102373184A
CN102373184A CN2010102487076A CN201010248707A CN102373184A CN 102373184 A CN102373184 A CN 102373184A CN 2010102487076 A CN2010102487076 A CN 2010102487076A CN 201010248707 A CN201010248707 A CN 201010248707A CN 102373184 A CN102373184 A CN 102373184A
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influenza virus
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ibv
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曹永长
刘大才
薛春宜
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Sun Yat Sen University
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Sun Yat Sen University
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Abstract

The invention provides a hybrid virus-like particle. The particle comprises a matrix protein M1 of influenza virus, surface antigen hemagglutinin (HA) of the influenza virus and fused membrane protein, wherein the fused membrane protein comprises an extracellular domain of major epitope of infectious bronchitis virus S protein, and a transmembrane domain and an intracellular domain of the HA of the influenza virus; the extracellular domain of the main surface antigen of the infectious bronchitis virus S protein replaces the extracellular domain with approximately same length of the 5'-terminal of the HA of the influenza virus; and the surface antigen HA of the influenza virus and the surface antigen S protein of the infectious bronchitis virus are expressed simultaneously on the surface of the hybrid virus-like particle. The invention also provides a bivalent vaccine of avian influenza and infectious bronchitis, and the bivalent vaccine comprises the hybrid virus-like particle and adjuvants. The invention also provides a preparation method of the hybrid virus-like particle.

Description

Bird flu and infectious bronchitis hybrid virus appearance particle, preparation method and application
Technical field
The present invention relates to bird flu and infectious bronchitis hybrid virus appearance particle, and preparation and application.
Background technology
Bird flu (Avian Influenza; AI) be a kind of infection and/or the disease syndrome of the bird (poultry and wild fowl) that causes by the A of orthomyxovirus section type influenza virus; Occur on one's body birds such as chicken, duck, goose, pigeon and the birds; But mainly encroach on turkey and chicken, Mammals such as pig, horse, sea dog, people etc. also can infect.According to bird flu virus (Avian Influenza Virus; AIV) factors such as kind, environment, feeding and management condition and concurrent disease of pathogenic, the fowl of strain; Metainfective fowl only shows as symptomless infection, slight upper airway symptoms, egg productivity drops to acute various ways such as pathogenic death; Directly influence health and products thereof the quality of chicken body; Be one of modernized aviculture important fowl upper transmissible disease of being difficult to tackle, the same with Marek, infectious bursal disease, white blood disease, RE hyperplasia disease etc., also be the important immunosuppressive disease of serious threat poultry.Classified as the category-A animal epidemic by OIE, China classifies it as type of animal epidemic.
Since nineteen fifty-five confirmed the checken pest broken out of Italy in 1878 be the influenza that causes by A type influenza virus to come calendar year 2001,19 H5N1 avian flu cases have been reported in the whole world altogether, wherein turkey is taken place 5,14 of chickens.Got into since this century, H5N1 avian flu cases also constantly takes place.Calendar year 2001 Hong-Kong generation H5N1 subtype influenza; At the beginning of 2003, the Continent of Europe is broken out bird flu in succession, Dutch generation of in February, 2003 H7N7 hypotype, in April, 2003 Belgium's generation H7N7 hypotype, in May, 2003 Germany's generation H7N7 subtype avian influenza.At the beginning of 2003 year ends 2004, countries in Asia break out bird flu in succession, and epidemic situation relates to 10 countries altogether.In December, 2003; Bird flu takes place in Korea S; In January, 2004; Bird flu successively takes place in countries and regions such as Japan, Vietnam, Taiwan, Thailand, the match of card Pu, Indonesia, Pakistan, Laos and China, and wherein the H5N2 subtype avian influenza has taken place for Japan, Taiwan, and the H5N1 subtype avian influenza has taken place for other countries and area.Breaking out of these epidemic situations caused crushing blow not only for countries in Asia's aviculture, and badly influenced people health, and countries such as Vietnam, Thailand bird flu have successively taken place infected the mankind, murderous serious consequence.In February, 2004, bird flu takes place at American continent again, and wherein the H7N7 bird flu has taken place in the U.S., and April, bird flu also took place in Canada.At present, high pathogenic avian influenza particularly the H5N1 bird flu also spread in Asia and part countries and regions, North America.
The present popular high pathogenic avian influenza of China mainly is the H5N1 subtype avian influenza.The vaccine major part that is used for birds flu-preventing 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 prepare with wild-type virus infected chicken embryo; Another kind of is the heredity that adopts reverse genetic technological transformation wild virus earlier; The new virus of using " rescue " to transform then comes the infected chicken embryo, 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 high frequency sudden change; Genetic material reorganization and antigenic drift etc. between the dual and multiple subtype virus---make the long-term safety of these vaccines and validity receive very big challenge; Even produced " invalid vaccine ", be difficult to deal with the infection of new mutant influenza virus.In addition, these vaccine preparations also exist following deficiency: (1) 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 contaminate environment and production, a whole set of production must be undertaken by being defined under three grades of laboratory conditions of Biosafety, and the deactivation of virus must guarantee fully, and is thorough.(3) can't distinguish by the chicken of immunity and the chicken that is infected by the virus.
Infectious bronchitis (Avian Infectious Bronchitis) is a kind of acute, the height social disease of the chicken that caused by avian infectious bronchitis virus; It is characterized by disease chicken respiratory symptom, the quantity of laying eggs and quality decline, ephrosis and become, young hen infects can cause the permanent degeneration of uterine tube.(Infectious Bronchitis Virus IBV) can infect the chicken of all ages in days to avian infectious bronchitis virus, causes growth retardation, death, weightening finish and the price of deed to reduce; Also usually cause secondary infection such as Mycoplasma polyinfection and intestinal bacteria and improve chicken crowd's mortality ratio that giving raises chickens produces and bring massive losses.At present, this disease is distributed widely in each foster fowl countries and regions, the world, like NA, South America, West Europe, Africa, China, Australia and New Zealand etc., is one of serious infectious diseases of serious harm world aviculture.Infection rate at China's infectious bronchitis of fowl can reach 80-100%, the laying rate 50-70% that can descend, and mortality ratio can reach 10-36.7%, and the financial loss that causes every year can be above 1,000,000,000 yuan.This disease is after newcastle disease, chicken Marek's disease, infectious bursal disease, the deadly infectious disease of another serious harm China aviculture.
IBV is the representative species of coronaviridae coronavirus genus, and genome is the sub-thread positive chain RNA.Morphology of virus is subsphaeroidal, and diameter 80-120nm has cyst membrane, and the cyst membrane surface has bar-shaped fibre prominent, is about 20nm.The IBV genome is divided into 6 zones, has 10 open reading frames (ORF) at least.In the IBV cells infected, can detect 6 kinds of mRNA, be respectively mRNA1 from small to large to mRNA6, all mRNA have common 3 ' end structure, and 5 ' end is then different in size.Encode respectively three primary structure albumen of IBV of mRNA A, m RNA C, m RNAE have been confirmed now: nucleocapsid protein (nucleocapsidprotein; N albumen), stromatin (membrane glycoprotein; M albumen), spike protein (spike glycoprotein, S albumen); M RNA B, D 7.5KD, 9.5KD and 6.7KD, 7.4KD, the 12.4KD totally 5 kinds of small proteins that function it be unclear that of encoding respectively; The m RNA F RNA polymerase of dependenc RNA of then possibly encoding, but still lack direct evidence.The structure of IBV mRNA is identical with the structure of eukaryotic mrna.The proteic molecule mol ratio of primary structure Protein S albumen, M albumen and N of IBV is 1: 6: 15.The structure of its virus particle of I comprises that by genome and N protein binding curl, the outside of inside one deck constitutes cyst membrane by M albumen, and petal-shaped or the riveting of pyriform S albumen fix on cyst membrane and stretch and come out to form projection.What research was maximum at present is S albumen.
S albumen (180KD) is positioned at the outermost of virus, and the fibre that constitutes virus is prominent.It can be cracked into equimolar S1 (90KD), S2 (84KD) subunit in host cell.Connect by disulfide linkage between S1 after the cracking and the S2.S1 forms a balloon-shaped structure outside, and S2 embeds in the viromembrane through a little hydrophobic transmembrane fragment, has formed the handle of S1.S1 and S2 have glycosylation site.Research shows that the proteic amino acid variation of different strain S mostly betides on the S1 and concentrates on 3 zone: N holds the 37th ~ 81 amino acid place (including two hydrophilic areas); The 117th ~ 160 amino acid place (being a hydrophobic region); The 269th ~ 298 amino acid place (including a strong hydrophilicity district).Research at present shows, compares with M, N albumen, and S albumen is evolved active, and variation mainly occurs on the S1.S albumen is the most important protective antigen of IBV, serotype and the tissue tropism of decision IBV, and its effect shows as stimulates body to produce neutralizing antibody, in the virus absorption onto cell process, plays a role, and on serological classification, plays a decisive role.Wherein, S1 albumen plays a significant role in viral adsorption process, its with host cell membrane on glycoprotein receptor to combine be the precondition of IBV adherent cell; S1 brings out hemagglutination inhibition antibody and most neutralizing antibody; And the proteic aminoterminal of S1 has certain effect aspect tissue affinity that determines the IBV strain and the virulence thereof.Teri Hodgson (2004) still can induce protective immunity with morbific M41 strain S protein gene reorganization IB Beaudette perform toxic attenuation; Soonjeon Youn (2005) identifies that with the infectious c dna clone of IBV S albumen transports the importance of signal infective virus, shows that the proteic endocytosis signal of IBV S plays an important role to producing viral infection.Kuster etc. have compared the S1 protein sequence of the IBV strain of 4 kinds of different serotypes; Find that most of IBV serotype antigen determinants are positioned at preceding 300 amino-acid residues of S1; And find that through amino acid sequence analysis the change of serotype possibly be because the change (being point mutation) of the few amino acids of S1 gene causes.Reports such as Song with the IBV S1 protein immunization chicken of recombinant baculovirus expression, can be induced higher immunoreactivity.Johnson etc. (2003) report, behind the IBV S1 protein immunization chicken with reorganization fowl gland shape expressing viral, to homology or not the homologous strain attack the protection ratio that can produce 90-100%.
A little less than malicious seedling and deactivation vaccine be the main vaccine of using at present.IB seedling alive is mainly used in fryer, the first immunisation of planting chicken and laying hen and reinforced immunological inoculation; Oil emulsion inactivated vaccine can effectively prevent to infect caused egg drop reduction by IBV after breeding the hen booster immunization.
The S1 gene is the main immunogens gene of IBV; Through S1 encoding sox in the clone IBV genome, pass through vitro recombination and expression then, obtain and the external protein expressioning product of S1 homologous; As the IBV gene engineering vaccine, have advantages such as efficient, wide spectrum, nontoxic and high specificity.But the S1 subunit vaccine must can form certain immunoprotection through 3-4 booster immunization.Therefore, be necessary to develop the immune efficacy improved vaccine.
Virus-like particle (VLP) 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 virus particle with size; So can effectively induce body immune system to produce the immunoprotection reaction, demonstrate good prospects for application as a kind of new generation vaccine.Typical H5N1 SIV particle is spherical in shape or oval; Diameter 80~120nm; 8 gene fragments of the genome of H5N1 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 genetic 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 virus particle nucleocapsid, participates in virus particle and forms; Phytolectin HA, the main gp on the virus coat surface film has 16 hypotypes.Be to bring out the major antigen body that host's body produces antibody.Neuraminidase NA also is a kind of gp on the virus coat surface film, has 9 hypotypes, is the major antigen body that induce antibody produces equally.
Surface membrane protein HA of influenza virus and NA cause that body produces the main inducer of immunne response, and matrix prote m1 is the main body that forms virus coat, also can be used as the inducer of tissue-type immunne response.Relevant research proves; The correct expression of matrix prote m1; It is the important step that guarantees that virus coat forms; And one of function of membranin 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 the conservative protein of the influenza virus of research proof in addition--NP albumen effective stimulus CTL (CTL) reaction suppresses virus infection, in virus-like particle, adds the cellular immune level that NP albumen will improve this new generation vaccine to a certain extent.
Bird flu virus and IBV be influence each other in the chicken body, and infection simultaneously can increase the weight of the state of an illness.So be necessary to provide a kind of bivalent vaccine to prevent H5N1 bird flu and newcastle disease infectious bronchitis simultaneously.
Summary of the invention
One of the object of the invention is to provide a kind of vaccine, and this vaccine can prevent H5N1 bird flu and infectious bronchitis simultaneously.
The objective of the invention is to realize through following content.
The present invention provides a kind of hybrid virus appearance particle to comprise the matrix prote m1 of influenza virus; The surface antigen Phytolectin HA of influenza virus; A nexus albumen; It comprises the film foreign lands of the proteic major antigen epi-position of IBV S, and the Phytolectin 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 Phytolectin HA of the said film foreign lands replacement influenza virus that contains the proteic main surface antigen of IBV S; The surface antigen Phytolectin HA of while expression of influenza virus and the surface antigen S albumen of IBV on said hybrid virus appearance particulate surface.
The present invention also provides a kind of bird flu and infectious bronchitis bivalent vaccine, comprises described hybrid virus appearance particle and adjuvant.
The present invention also provides the said hybrid virus appearance of a kind of preparation particulate method.
Utilization comprises the vaccine that the proteic hybrid virus appearance of said nexus particle constitutes, and can prevent H5N1 bird flu and infectious bronchitis simultaneously, and have tangible advantage and effect:
(1) H5N1 bird flu and infectious bronchitis VLP bivalent vaccine can cause that body immune system produces strong type and replys, and immunizing power is strong, and longer duration can prevent H5N1 bird flu and infectious bronchitis simultaneously.
(2) because virus-like particle does not contain viral genome; Virogene and host chromosome gene integration just can not take place in the VLP of this hollow shell structure in the immune animal body; And whole process of production does not contact infectious virus alive; Therefore as safe as a house, for influenza virus and two kinds of viruses of IBV, 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) animal that can distinguish out by artificial immunization with by the animal of virus infection.Virus-like particle does not contain albumen such as influenza virus NS, PB1, PB2, and when therefore carrying out serologic test, with the animal of VLP vaccine immunity, whether with the antibody that can not produce specific anti NS or PB, can distinguish is wild virus infection.For infectious bronchitis, whether as detecting antigen, can distinguish is wild virus infection with any virus structural protein outside the S albumen such as M albumen, E albumen etc.
Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail, but the present invention is not limited to these embodiments, any on essence spirit of the present invention improvement or substitute, still belong to scope required for protection in claims of the present invention.
Description of drawings
Fig. 1 is the electrophoresis picture of the HA (a) that goes out with Auele Specific Primer pcr amplification separately, NA (a), M1 (b), NP (c), S/HA (d) gene fragment.
Fig. 2 be recombinant baculovirus expression plasmid rBacmid-HA (/S/HA)-NA, rBacmid-M1, rBacmid-NP synoptic diagram.
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 mark, and C, S1 antibody show green fluorescence, B, the negative contrast of D for the FITC mark.
Fig. 5 A is the image of virus-like particle under electron microscope behind the SDGC purifying, and Fig. 5 B is an enlarged view.
Embodiment
The present invention is the basis with Bac-to-Bac
Figure BSA00000222213100041
insect baculovirus expression system; The matrix prote m1, surface film egg HA and the NA albumen that utilize the H5N1 bird flu virus are (and randomly; NP), the virus-like particle (VLP) of H5N1 bird flu virus is constructed in common assembling automatically.On this basis, make up the divalence VLP of H5N1 bird flu virus and IBV.
At first, with a part of film foreign lands that the main surface antigen gene S1 of IBV replaces the HA protein gene of H5N1 bird flu virus, the two constitutes fusion gene (S/HA), and the S1 gene is positioned at the 5 ' end of fusion gene S/HA.Generally speaking, S1 gene replacement equates 5 ' terminal sequence of the HA gene of substantial distance, with the length that ensures fusion gene S/HA 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 the surface membrane protein HA and the NA of H5N1 bird flu virus, and the gene of expressing the fusion rotein S/HA of IBV S1 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 cell expressing, be assembled into the virus-like particle that does not contain the bird flu virus genetic material automatically, will be released in the cell culture fluid after virus-like particle forms.The VLP that forms had so both had the main surface antigen HA of H5N1 bird flu virus, had the main surface antigen S 1 of IBV again, was H5N1 bird flu virus and IBV divalence VLP.
It is pointed out that and there are some researches show that different membranins can be transported to the different film micro areas of cytolemma behind cell inner expression, what the distribution on cytolemma depended primarily on membranin strides film district and film internal area.Might after expression, be distributed in the different film micro areas of cytolemma without the S that transforms and HA albumen, thereby when expressing simultaneously without the S of transformation and HA albumen, they might be incorporated into same VLP simultaneously, but their content and the ratio in VLP there is very big-difference.The present invention is fused to the proteic part film of S foreign lands on the HA of disappearance part film foreign lands, and S/HA fusion rotein and HA albumen contain the part film foreign lands of same HA like this, stride film district and film internal area; Guarantee that like this S/HA fusion rotein and HA albumen are distributed in the film micro area of same cytolemma; Therefore in the forming process of VLP, the S/HA fusion rotein is incorporated on the VLP by the same with HA albumen very possibly effectively, and they are guaranteed at content and the ratio in VLP.
In addition, the structural protein of influenza virus are because we find influenza virus VLP high yield, stable as the skeleton of divalence 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.
Implement the amplification of row 1:M1, NP, HA, NA and fusion gene S/HA
(1) H5N1 subtype avian influenza virus RNA extracting and RT-PCR
The working instructions (method) that extract test kit by GibcoBRL company's T RIzol LS Reagent RNA carry out.Get 250 μ L bird flue virus H 5 N 1 subtype virus strain infection's allantoic fluids and 750 μ L TRIzol LS respectively, add in the 1.5mL Eppendorf 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 Virahols, 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 ThermoScript II of TaKaRa is carried out, and in 20 μ L reaction systems, adds following component respectively: RNA:3 μ L; 5 * RTbuffer:4 μ L; DNTP:4 μ L; RNA enzyme inhibitors: 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.
(2) pcr amplification of M1, NP, HA, NA gene
The RT-PCR product that derives from (1) can be used for the amplification of M1, NP, HA, NA gene.
According to 1 pair of primer of HA gene order (SEQ ID NO:1) 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 promotor, primer sequence is following:
HA?Xho?I: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 (SEQ ID NO:3) 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 promotor, these 2 primer sequences are respectively:
NA?Sal?I:5’-GCCGTCGACATGAATCCAAACCAAAG-3’(SEQ?ID?NO:13)
NA?Hind?III:5’-GCCAAGCTTCTACTTGTCAATGGTG-3’(SEQ?ID?NO:14)
According to 1 pair of primer of M1 gene order (SEQ ID NO:5) design, the M1 gene that is used to increase, its two ends have added the restriction enzyme site of Sal I and Hind III respectively, are positioned at P PHUnder the promotor, these 2 primer sequences are respectively:
M1?Sal?I:5’-GCCGTCGACATGAGTCTTCTAACCGAG-3’(SEQ?ID?NO:15)
M1?Hind?III:5’-GCCAAGCTTTCACTTGAATCGTTG-3’(SEQ?ID?NO:16)
According to 1 pair of primer of NP gene order (SEQ ID NO:7) 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 promotor, 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)
(3) fusion gene S/HA's is synthetic
The nucleotide sequence of S/HA gene is seen SEQ ID NO:9; The proteinic aminoacid sequence of S/HA is seen SEQ ID NO:10.Fusion gene is synthetic according to its nucleotide sequence.Fusion gene S/HA comprises 3 ' end 185-568 (totally 384) amino acid of HA gene of 1-200 amino acid and the H5N1 of IBV S gene 5 ' end.Fusion gene S/HA 584 amino acid of encoding, its gene order is shown in SEQ ID NO:9.Fusion gene S/HA is by synthetic.According to the sequence (SEQ ID NO:9) of fusion gene F/HA, 1 pair of primer of design, the fusion gene S/HA that is used to increase, its two ends have added Xho I and Sph I restriction enzyme site respectively, are positioned under the P10 promotor, primer sequence is following:
S/HA?XhoI:5’-CCGCTCGAG?ATGTTGGGGAAGTCACTG-3’(SEQ?ID?NO:19)
S/HA?SphI:5’-ACATGCATGCTTAAATGCAAATTCTGCAT-3’(SEQ?ID?NO:20)
Adopt M1, NP, HA, NA, S/HA 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, S/HA gene.The PCR reaction conditions is 94 ℃ of preparatory sex change 3min, 94 ℃ of sex change 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% sepharose.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 S/HA is about 1.7Kb (Fig. 1 d).All PCR product samples are after the running gel extracting; Obtain M1, NP, HA, NA, the S/HA gene DNA fragment of purifying; Through restriction enzyme Xho I/NcoI (digestion HA fragment), Sal/Hind III (digestion NA fragment); 37 ℃ of Xho I/NcoI (digestion NP fragment), Sal I/Hind III (digestion M1 fragment), Xho I and SphI (digestion S/HA segment) after the digestion, are further purified respectively again, use in order to next step construction recombination plasmid.Concrete operations are following: prepare 1% sepharose 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) specification sheets, extracting and purifying M1, NP, HA, S/HA and NA gene DNA fragment with reference to glue.After 37 ℃ of digested overnight of restriction enzyme, reclaim test kit (Qiagen Company products) with glue, instruct according to explanation, the centrifugal post of crossing, the purifying and recovering enzyme is cut postdigestive M1, NP, HA, NA, S/HA fragment.
Embodiment 2: the baculovirus expression plasmid of construction expression M1, NP, HA, NA, S/HA gene reaches in insect cell The insect baculovirus of synthetic reorganization
(1) recombinant plasmid of construction expression M1 and NP gene
Insect baculovirus plasmid PFastBac (Invitrogen Company products) reclaims test kit with glue and reclaims the plasmid PFastBac after purifying enzyme is cut after 37 ℃ of enzymes of restriction enzyme Sal I/Hind III are cut 3 hours.Under the effect of T4DNA ligase enzyme, the plasmid after enzyme is cut is connected in 16 ℃ with M1DNA fragment after enzyme is cut and spends the night.Reaction system is following: 10 * T4 connects damping fluid 1ml, and the dna fragmentation 3ml that the switchback of M1 enzyme is received, enzyme cut the PFastBac plasmid and reclaim product 1 μ l, T4DNA ligase enzyme 1 μ l, ddH 2O mends to 10 μ l.Adopting the heat-shocked 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-shocked 90s in 42 ℃ of water-baths over to, put back to rapidly 5 minutes on ice, to wherein adding 200 μ 1LB nutrient solutions.37 ℃ of shaking tables were cultivated 1 hour.Get 100 μ l bacterium liquid and coat on the LB solid medium (containing two kinds of microbiotic of ammonia benzyl and qingfengmeisu qiong), 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 recombinant plasmid PFastBac-M1.
The construction process of the recombinant plasmid PFastBac-NP of expression NP gene is the same.
(2) recombinant plasmid of construction expression HA or S/HA and NA gene
Baculovirus expression plasmid PFastBac-dual (Invitrogen Company products) is after restriction enzyme Xho I/Nco I enzyme is cut; Under the effect of T4 dna ligase; To be inserted into the below of P10 promotor 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, bacterium liquid PCR and Xho I/Nco I enzyme cut identify and the dna sequence dna order-checking definite errorless after; Select 1 recombinant plasmid dna wherein, carry out second time enzyme and cut.Used restriction enzyme is Sal I/Hind III.With same endonuclease digestion NA gene DNA fragment, and be inserted into another promotor P of recombinant 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.
The construction process of recombinant plasmid PFastBac-dual-S/HA-NA of expressing fusion gene S/HA and NA gene simultaneously is the same.
(3) the genomic synthetic and extraction of recombinant baculovirus
During purified recombinant PfatBac-M1, PfatBac-NP, PFastBac-dual-HA-NA and PFastBac-dual-S/HA-NA plasmid transduceed E.coli competent cell strain DH 10Bac cell 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 microbiotic (qingfengmeisu qiong, tsiklomitsin and kantlex) are carried out blue hickie screening; The positive colony bacterium colony is white in color, but not the wild bacterium colony of reorganization is blue look.The laboratory manual that experiment condition all provides according to Invitrogen company instructs and sets.The positive colony of selecting places the LB nutrient solution (containing above-mentioned 3 kinds of microbiotic) 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 purifying has M1 gene, NP gene, HA-NA or S/HA-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 nutrient solution 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 liquid; 3000rpm collected supernatant in centrifugal 10 minutes, obtained the recombinant baculovirus of low titre, infected the new sf-9 cell of cultivating with this supernatant again; Collecting cell culture supernatant liquid after 3 days is the high density recombinant baculovirus after the required amplification culture, called after Bac-M1, Bac-NP, Bac-HA-NA and Bac-S/HA-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 the GraceShi substratum that contains 10%FBS the Sf9 passage is inoculated in six well culture plates, cell density is about 1 * 10 6Individual cell/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 the GraceShi substratum that does not contain FBS.
3) discard substratum in six orifice plates, clean cell 3 times with the substratum that does not contain serum, then that above-mentioned dilution is good recombinant virus liquid adds in the hand-hole, and each extent of dilution 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 sepharose+1.4ml FBS of two anti-+ 7ml 2% of 7ml 2 * GraceShi substratum+140 μ l; Mixing lightly; Then bottle is placed 42 ℃ of water-baths again; Exhaust the viral liquid in every hole behind the virus 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 toluylene red 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 throw out of Bac-NP, Bac-HA-NA and Bac-S/HA-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, S/HA and NA albumen) and whether in insect cell Sf9, express.Briefly; With the 2 * SDS sample-loading buffer that adds 10 μ l in the 10 μ l supernatant samples; 100 ℃ handle 5min after, centrifugal 5 minutes of 10000rpm adds the supernatant sample of all 20 μ l in the point sample hole of 4%-12%SDS-polyacrylamide gel (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 indicating liquid bromjophenol blue, stop electrophoresis near the gel bottom.Gel places 1%R type coomassie brilliant blue staining liquid, rocks dyeing 1 hour, places the destainer 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, S/HA 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 Kun Chongshi substratum of Invitrigen company) of serum-free, and the speed of shaking of shaking table is 100rpm, and homo(io)thermism is in 27 ℃.When cell concn reaches 2 * 10 6During cell/ml, with Bac-M1, Bac-NP, Bac-HA-NA, the common transfection sf9 of Bac-S/HA-NA insect baculovirus cell.The MOI ratio of virus is 3 (Bac-M1 and Bac-NP): 1 (Bac-HA-NA): 1 (Bac-S/HA-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.Make up the sf-9 cell that synthetic wild-type insect baculovirus is used for the transfection suspension culture when experiment is carried out, MOI is 5.As the negative control that is provided with, the condition of the cell cultures 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 (comprising negative control) is got lysis extract and the cell culture supernatant of 10 μ l respectively, adds 2 * SDS sample-loading buffer of 10 μ l more separately.100 ℃ handle 5min after, the biased sample of all 20 μ l is added in the point sample hole of 4%-12%SDS polyacrylamide gel.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 precooling more than 2 hours, by filter paper with gel, filter paper after 5 minutes with the methyl alcohol 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.Go out nitrocellulose filter with the tweezers gripping,, then be transferred in skim-milk/PBS solution of 5%, sway sealing 1 hour with PBS-T rinsing liquid washing.With PBS-T rinsing liquid washing 3 times, each 3 minutes; Then nitrocellulose filter is changed in the plastics bag, add 3ml and be diluted in anti-H5N1 bird flu virus chicken source polyclonal antibody among 5% skim-milk/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 plastics bag; Add 5ml with the anti-chicken IgG of the donkey that is diluted in the horseradish peroxidase-labeled among 5% skim-milk/PBS at 1: 10000 (two is anti-), shake incubation 1h under the room temperature.Abandon two anti-, the plain film of PBS-T rinsing fiber 3 times, each 10 minutes, the 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/S/HA and NA.M1 is described, NP, HA/S/HA and NA have expressed in the SF-9 of the suspension culture of transfection insect cell effectively.
embodiment 4: the purifying 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 ultracentrifuge (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 soln of 1ml 60% carefully next day, adds the multitudinous sugar soln of 1ml 30% and 3ml 20% then successively, at last that the sample liquid after the dissolving of 5ml is placed on it.Accurately weigh, after the balance, ultracentrifuge 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 purifying respectively.Weatern blots analyzes the virus-like particle sample after purifying concentrates.Western blots analyzes equally in its operation steps and the foregoing description 3, just the sample of being got is diluted, i.e. the virus-like particle sample of 1 μ l adds the water of 9 μ l, adds 2 * SDS sample-loading buffer of 10 μ l again.Behind 100 ℃ of sex change 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/S/HA 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 operation steps is following: with sf9 cell kind in 24 orifice plates, every hole 100,000 cells, the operation steps of embodiment 3; The kind cell is infected, infects after 72 hours, with PBS with cell washing 3 times; Each 5 minutes, add the methyl alcohol-20 ° fixed cell 10 minutes of 100% precooling then, added 0.05% Triton x-100 room temperature treatment again 10 minutes; Add 3% BSA four degree sealings and spend the night, second day with PBS with cell washing 3 times, each 5 minutes; The special M1 monoclonal antibody of A type influenza that in the hole, adds the RBITC mark then, the perhaps monoclonal antibody of the infectivity resistant bronchitis virus S1 of FITC mark, 37 ° of reactions 1 hour; Reaction finish the back with PBS with cell washing 3 times, each 5 minutes, washing finished back observation under fluorescent microscope.Carry out immune electron microscopy with the monoclonal antibody of anti-M1 and the monoclonal antibody of S1; Can see fluorescently-labeled virus-like particle; Wherein the antibody of anti-M1 is the RBITC mark, shows fluorescent orange (Fig. 4 A), and the antibody of anti-S1 is the FITC mark; Show green fluorescence (Fig. 4 C), and contrast there is not fluorescent signal ((Fig. 4 B, D).
Electronic Speculum film making experimental implementation is following: after the seemingly virion sample bearing reason after a spot of purifying is concentrated; Place freshly prepd uncharge plastic cement/carbon to encapsulate on the network lattice; After washing several times gently with several zero(ppm) water, add that 2% Tungstophosphoric acid, sodium salt 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:H5N1 bird flu and gumboro's disease divalence VLP immunity SPF chicken
60 SPF chickens are available from the Guangdong SPF of Wen Shi food group chicken house, and each is organized the concrete immune embodiment of immune animal and sees table 1.The SPF chicken in 2 ages in week is at back 21 days wing venous blood collections of immunity, conventional separation of serum.Adopt the IDEXX IBD of company antibody ELISA detection kit to detect IBD antibody in the chicken serum, adopt hemagglutination-inhibition test (HI) to detect H5N1 avian influenza antibody in the serum, carry out with reference to Gan Menghou (2002) method.The result shows, H5N1 bird flu and gumboro's disease divalence VLP immunity SPF chicken in 2 age in week, and the antibody (table 2) of existing anti-H5N1 bird flu in the immune chicken serum has also produced the antibody (table 3) of IBV.
Table 1 H5N1 bird flu and gumboro's disease divalence VLPs immunity SPF chicken experimental program
Figure BSA00000222213100101
Table 2 H5N1 bird flu and infectious bronchitis divalence VLPs immunity SPF chicken serum HI detected result
Figure BSA00000222213100102
Table 3 H5N1 bird flu and infectious bronchitis divalence VLP immunity SPF chicken serum IBV antibody test result
Figure BSA00000222213100103
Figure ISA00000222213300011
Figure ISA00000222213300021
Figure ISA00000222213300031
Figure ISA00000222213300041
Figure ISA00000222213300071
Figure ISA00000222213300081
Figure ISA00000222213300091
Figure ISA00000222213300101
Figure ISA00000222213300111
Figure ISA00000222213300121
Figure ISA00000222213300131
Figure ISA00000222213300141
Figure ISA00000222213300161
Figure ISA00000222213300181

Claims (8)

1. hybrid virus appearance particle is characterized in that, comprises:
The matrix prote m1 of influenza virus;
The surface antigen Phytolectin HA of influenza virus;
A nexus albumen, it comprises the film foreign lands of the proteic major antigen epi-position of IBV S, and the Phytolectin 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 Phytolectin HA of the said film foreign lands replacement influenza virus that contains the proteic main surface antigen of IBV S;
The surface antigen Phytolectin HA of while expression of influenza virus and the surface antigen S albumen of IBV on said hybrid virus appearance particulate surface.
2. hybrid virus appearance particle as claimed in claim 1, wherein said hybrid virus appearance particle also contains the neuraminidase NA of influenza virus.
3. according to claim 1 or claim 2 hybrid virus appearance particle, wherein said hybrid virus appearance particle also contains the NP of influenza virus.
4. hybrid virus appearance particle as claimed in claim 1, wherein the proteic sequence of nexus is shown in SEQ ID NO:10.
5. bird flu and infectious bronchitis bivalent vaccine is characterized in that, comprise like claim 1-4 described hybrid virus appearance particle and adjuvant.
6. prepare hybrid virus appearance particulate method, 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 Phytolectin HA of influenza virus; Or a nexus albumen, it comprises the film foreign lands of the proteic major antigen epi-position of IBV S, and the Phytolectin 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 Phytolectin HA of the said film foreign lands replacement influenza virus that contains the proteic main surface antigen of IBV S;
With the baculovirus expression carrier of said structure infected insect cell simultaneously in proportion; Pack out hybrid virus appearance particle, the surface antigen Phytolectin HA of while expression of influenza virus and the surface antigen S albumen of IBV are gone up in its surface.
7. like claim 6 preparation hybrid virus appearance particulate method; 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 particle contains influenza virus.
8. like claim 6 or 7 preparation hybrid virus appearance particulate methods, 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 particle contains influenza virus.
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CN104353070A (en) * 2014-11-05 2015-02-18 中山大学 Genetic engineering subunit vaccine of chicken infectious bronchitis virus and preparation method thereof
CN104797594A (en) * 2012-11-27 2015-07-22 高等教育联邦系统-匹兹堡大学 Computationally optimized broadly reactive antigens for H1N1 influenza
CN105949321A (en) * 2016-05-04 2016-09-21 中国农业科学院上海兽医研究所 B cell antigen epitope polypeptide of infectious bronchitis virus isolate S1 protein, and vaccine thereof
CN106119213A (en) * 2016-06-20 2016-11-16 中国疾病预防控制中心病毒病预防控制所 MERS CoV mosaic type VLPs vaccine with influenza virus M1 as skeleton

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CN1903363A (en) * 2006-08-02 2007-01-31 中国农业大学 Mosaic type virus-like particle DNA vaccine

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CN1437655A (en) * 2000-06-23 2003-08-20 美国氰胺公司 Assembly of wild-type and chimeric influenza virus-like particles (VLPS)
CN1849134A (en) * 2003-07-11 2006-10-18 诺瓦瓦克斯股份有限公司 Functional influenza virus-like particles(VLPs)
CN1903363A (en) * 2006-08-02 2007-01-31 中国农业大学 Mosaic type virus-like particle DNA vaccine

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CN104797594A (en) * 2012-11-27 2015-07-22 高等教育联邦系统-匹兹堡大学 Computationally optimized broadly reactive antigens for H1N1 influenza
US10017544B2 (en) 2012-11-27 2018-07-10 University of Pittsburgh—of the Commonwealth System of Higher Education Computationally optimized broadly reactive antigens for H1N1 influenza
CN104353070A (en) * 2014-11-05 2015-02-18 中山大学 Genetic engineering subunit vaccine of chicken infectious bronchitis virus and preparation method thereof
CN104353070B (en) * 2014-11-05 2017-04-19 中山大学 Genetic engineering subunit vaccine of chicken infectious bronchitis virus and preparation method thereof
CN105949321A (en) * 2016-05-04 2016-09-21 中国农业科学院上海兽医研究所 B cell antigen epitope polypeptide of infectious bronchitis virus isolate S1 protein, and vaccine thereof
CN105949321B (en) * 2016-05-04 2019-08-23 中国农业科学院上海兽医研究所 The B cell antigen epi-position polypeptide and its vaccine of infectious bronchitis virus separation strains S1 albumen
CN106119213A (en) * 2016-06-20 2016-11-16 中国疾病预防控制中心病毒病预防控制所 MERS CoV mosaic type VLPs vaccine with influenza virus M1 as skeleton

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