CN109385435B

CN109385435B - A kind of recombination PRRSV virus-like particle and its preparation with immunogenicity

Info

Publication number: CN109385435B
Application number: CN201811114020.6A
Authority: CN
Inventors: 陈瑞; 杜恩岐; 董剑辉; 张满义
Original assignee: Shaanxi Lihua Norwich Biotechnology Co Ltd
Current assignee: Shaanxi Lihua Norwich Biotechnology Co Ltd
Priority date: 2018-09-25
Filing date: 2018-09-25
Publication date: 2019-10-25
Anticipated expiration: 2038-09-25
Also published as: CN109385435A

Abstract

The invention discloses a kind of recombination porcine reproductive and respiratory syndrome virus (PRRSV) virus-like particles (VLP) and the preparation method and application thereof.Comparison analysis based on GP5 and the M gene order to PRRSV prevalence strain, artificial synthesized GP5 and M tandem sequence GP5M, using pBAC5 plasmid as skeleton, the GP5M gene order of synthesis is cloned into the carrier, obtain baculovirus transfer vector pBAC-PRRSVGP5M, and then restructuring rod granule rBacmid-GP5M is obtained, rod granule is transfected into sf9 cell, obtains recombinant baculovirus Ac-PRRSVGP5M.GP5 and the M albumen of recombinant baculovirus high efficient expression PRRSV simultaneously forms virus-like particle.Albumen expressed by recombinant baculovirus of the invention prepares subunit vaccine, and the attack that can induce body generation specific immune response after animal, and pig body confrontation porcine reproductive and respiratory syndrome virus can be protected virulent is immunized.

Description

A kind of recombination PRRSV virus-like particle and its preparation with immunogenicity

Technical field

The invention belongs to technical field of agricultural veterinary biology, and in particular to a kind of recombination PRRSV disease with immunogenicity Malicious sample particle and its preparation and application.

Background technique

Porcine reproductive and respiratory syndrome (porcine reproductive and respiratory syndrome, It PRRS) is a kind of highly contagious disease caused by porcine reproductive and respiratory syndrome virus (PRRS virus, PRRSV), because It clinically often results in ear's cyanosis, also known as blue otopathy, mainly cause sow premature labor, miscarriage, produce stillborn foetus or the mummification of fetus, There is respiratory disease in piglet and bred pigs, and incidence and mortality is high.The disease is initially first in the U.S. the 1980s It first reports, finds the first clinical virus purification report in China from 1996, the big face of highly pathogenic PRRSV occurred in the end of the year 2005 Product outburst.PRRSV is the RNA virus in single-stranded positive, belongs to Arteriviridae, not according to antigenic and gene order Together, by the PRRSV point in the whole world for using VR-2332 as representative american type (North American, NA) and with Lelystad virus (LV) is the Europe class (European, EU) of representative.The PRRSV of China's prevalence is mainly classical America Strain and the strain of anomaly America, but European strain is also found repeatly in recent years.In addition, PRRSV variability and recombination ability are strong, frequently You Xin branch occurs, for example the state that fashion trend is gradually increasing is presented in the NADC30-Like hypotype and GM2 hypotype reported recently Gesture.PRRS is huge threat to global pig breeding industry, and PRRS economic loss caused by U.S.'s pig-breeding is within 2005 5.6 hundred million dollars, wherein sow reproductive stage loses 67,000,000 dollars, and weanling pig is 2.01 hundred million dollars, and growing-finishing pig damages It loses at most, has reached 2.92 hundred million dollars.2013, Holtkampra estimated PRRS economic loss caused by U.S.'s pig-breeding 6.63 hundred million dollars are reached, this was higher by 10% than 2005.Europe data in 2012 show that economic loss caused by PRRS is 126 Euros/head, this is more taller than the data in the U.S. in 2005 (121 dollar /).

PRRSV is single-stranded positive, non-segmented negative, the RNA virus for having cyst membrane, and virion size is about 50-70 nanometers (nanometer, nm), full-length genome are 14.9-15.5 kb, contain at least 10 open reading frame (open reading Frame, ORF).NSP1 α/β is extremely for ORF1a regional code non-structural protein (non-structural protein, NSP) The region NSP8, ORF1b then encodes NSP9 to NSP12；ORF2a, ORF2b and ORFs3-7 are encoded in conjunction with virion Structural proteins (Structural protein, SP) GP2, E, GP3, GP4, ORF5a, GP5, M and N, they are total by nido 3 ' A series of subgenomic mRNA s translation of end；It is that 5 ' end noncoding regions and 3 ' ends are non-respectively 10 two sides ORFs Code area.The structural proteins of PRRSV include GP5-M dimer protein, GP2-GP3-GP4 complex protein and have hydrophobic E protein and ORF5a albumen, they are closely related with the intrusion and budding of virus.GP5 albumen (contain about 200 amino acid) is With the structural proteins of high variation property in PRRSV, pass through disulfide-bonded shape with M albumen (containing about 175 amino acid) At GP5-M dimer protein viruses adsorption can be made to host cell table in conjunction with the sialoadhesin receptor of host cell Face, and the internalization step of virus is also closely related with GP5, if there is great expression in the host cell of research discovery recently When GP5 albumen, the proliferation for generating beta interferon to inhibit PRRSV can be mediated；Existing research report GP5 and M can form virus-like Grain, virus-like particle (virus-like particles, VLPs) are that one or more structural proteins of certain virus are assembled into Hollow bead, the stability and immunogenicity of its existing similar natural viral, but also with carrying the latent of foreign protein or polypeptide Can, virulent nucleic acid is free of, without infectivity, it is made to be expected to the good carrier as building polyvaccine.

PRRS vaccine currently on the market is all traditional inactivated vaccine and attenuated live vaccines, main development & production enterprise packet Containing German Bo Linge, Spain Hai Bolai, French Cimmeria, Dutch Intervet, Shandong Shandong, Guangdong Yongshun, Zhong Mu group etc. Unit.However there is no a kind of vaccine that pig breeding industry can be allowed thoroughly to get rid of damage of the PRRSV to live pig, market need it is safer more For efficient PRRS vaccine, and novel gene engineered vaccine is the developing direction of the following animal vaccine.Nam in 2010 etc. is open Virus-like particle containing PPRSV GP5 and M albumen, above two albumen by recombinant baculovirus expression, then assemble and At virus-like particle, it can be used for preparing vaccine, while thinking that GP5 the and M albumen of PPRSV is to form virion institute not by research The part that can or lack.The gene of the M albumen for encoding PRRSV, N protein and GP5 albumen is cloned by 2011 Nian Lvfeng woodss etc. respectively Same cell is transfected in carrier for expression of eukaryon (such as pHWD2000, pOPI3CAT, pCAGGS, pcDNA6/TR, pCMV-HA) simultaneously System's 3 kinds of albumen of expression are capable of forming virus-like particle, and can generate good cellular immunity and humoral immunity.2012 Nian Liyang By tri- genes of two genes of the ORF5 of PRRSV and ORF6 or ORF5, ORF6 and ORF7 or ORF5, ORF6, ORF7 and NSP2a tetra- Virus-like particle can be formed after a assortment of genes in rhabdovirus expression vector.Rodriguez in 2016 etc. is by PRRSV GP2 GP3 GP4 E GP5 M expression after be prepared as virus-like particle.

Summary of the invention

Based on current country's PRRSV Vaccine Development the case where, the present invention is intended to provide a kind of with good immunogenicity Recombination PRRSV virus-like particle and its preparation and application, one side can enrich the type of PRRSV vaccine, be PRRSV vaccine Research and development effective immunogene is provided on the other hand improves the immunogenicity of virus-like particle by genetic modification, compare Existing GP5-M virus-like particle has higher immunogenicity and animal protection rate, can preferably realize animal protection.

In order to solve the above technical problems, the present invention uses following technological means:

A kind of porcine reproductive and respiratory syndrome virus PRRSV GP5-M gene order of manually modified synthesis, sequence is such as Shown in SEQ ID NO.1, the protein of coding is shown in SEQ ID NO.4.

A kind of baculovirus transfer vector pBAC-PRRSVGP5-M, carrier contain above-mentioned PRRSV GP5-M gene order and Marker gene GFP.

A kind of recombinant baculovirus for expressing porcine reproductive and respiratory syndrome virus PRRSV GP5 and M albumen, strain are ordered Entitled recombinant baculovirus Ac-PRRSVGP5-M.

A kind of preparation side of expression porcine reproductive and respiratory syndrome virus PRRSV GP5 and the recombinant baculovirus of M albumen Method, which comprises the steps of:

The building of the recombinant baculovirus of step 1, expression PRRSV GP5 and M albumen, is prepared by following step:

(1) the manually modified synthesis of the tandem gene GP5M of the immunogenic gene GP5 and M of PRRSV: analysis is compared The gene order of PRRSV prevalence strain between 2006-2016, according to Field epidemic advantage strain amino acid characteristics and codon Inclined preferendum, and according to the needs of immunogenicity, after having carried out genetic modification to it, artificial synthesized GP5-M gene order, nucleosides Acid sequence is shown in SEQ ID NO.1；

(2) building of baculovirus transfer vector: using pBAC5 plasmid as skeleton, pass through Xba I and BamH I digestion carrier With Insert Fragment, then by T4 ligase in 16 DEG C of connections overnight, transformation and selection positive colony extracts recombinant plasmid, digestion Identification obtains baculovirus transfer vector pBAC-PRRSVGP5-M；

Step 2, the building of recombinant baculovirus Ac-PRRSVGP5-M:

(1) acquisition and identification of restructuring rod granule rBac-PRRSVGP5-M: sequencing is taken to identify that correct baculoviral transfer carries Body pBAC-PRRSVGP5-M Plasmid DNA is mixed with DH10 Bac competent cell, after ice bath 30 minutes, is carried out 45 seconds in 42 DEG C Water-bath heat shock, then ice bath 5 minutes are added SOC fluid nutrient medium, 37 DEG C shaken cultivation 2 hours, be serially diluted it by 10 times Afterwards, each gradient bacterium solution is coated with LB plate, purifies positive bacterium colony using the screening reagent box screening of life company, extracts restructuring rod granule rBac-PRRSVGP5-M。

(2) acquisition of recombinant baculovirus Ac-PRRSVGP5-M: the restructuring rod granule rBac- that previous step is extracted PRRSVGP5-M is transfected using lipofectamine Lipofectamine3000 into sf9 cell, in 28 DEG C of lasting cultures Observation 24 hours after transfection, utilized fluorescence microscope recombinant baculovirus green florescent signal with 72 hours in 48 hours, And 72 hours collection cell conditioned mediums after transfection, recombinant baculovirus is obtained, then it is inoculated with to health sf9 cell expansion again Culture, collection virus liquid are placed in -70 DEG C as seed culture of viruses and save backup.

The recombination of expression the porcine reproductive and respiratory syndrome virus PRRSV GP5 and M albumen is also claimed in the present invention Baculoviral includes PRRSV virus-like particle and adjuvant preparing the application in subunit vaccine, the subunit vaccine.

Based on above technical scheme, the invention has the advantages that and effect:

First, Field epidemic PRRSV strain gene order 2006-2016 is compared and heredity in the present invention Evolutionary analysis filters out Field epidemic advantage strain gene order, manually modified rear gene chemical synthesis is carried out, with more wide spectrum Cross immunogenicity.

Second, porcine reproductive and respiratory syndrome virus virus-like particle of the invention has high immunogenicity, the disease of preparation For malicious sample particle vaccines relative to existing inactivated vaccine, immune protective rate is higher, can generate effective guarantor to the virulent infection of PRRSV Shield.Porcine reproductive and respiratory syndrome virus virus sample particle vaccines of the invention have more safely relative to existing live vaccine Effect, there is no virulence to return risk that is strong and causing recombinant virus to occur.

Third, relative to the GP5 albumen of most of reports, is clipped by the way that PRRSV GP5 and M albumen is optimized The segments such as " TFVIFPVLTHIVSYGALTTSHFL ", " YVLSSIYAVCALAALICFVI ", will be in amino acid fragment " VVLDGS " sports " EELDGS ", and " EKGGKV " of amino acid fragment is sported " EKEEKV ", by the study found that transformation Sequence afterwards can effectively raise the stability and protein yield of virus-like particle expression, improve exempting from for virus-like particle Epidemic focus can preferably stimulate immune animal to generate protection antibody, greatly improve antibody level and animal protection rate.

4th, the present invention uses rigidity linker (EAAAK) by bacterial flagellin Tflg segment and PRRSV GP5 egg It is white to be attached, compare flexibility linker(GGGGS), it can be improved the titre of the virus under same condition of culture and be immunized Originality is enabled flagellin to be preferably exposed to virion surface, have stimulated machine using rigid linker connection Tflg The generation antibody of body earlier, and antibody titer of the invention will be apparently higher than the potency of flexible linker, up to its twice, say Bright vaccine of the invention has better immune protective effect.

In conclusion means of the present invention by genetic engineering, have been transformed PRRSV GP5-M gene, it is rod-shaped by constructing The PRRSV virus-like particle with high immunogenicity is prepared in virus expression carrier, and is used for the system of subunit vaccine It is standby, by animal experiments show that, preparation-obtained PRRSV virus-like particle compared to transformation before greatly improve antibody level With animal protection rate, there is good vaccine prospect.

Detailed description of the invention

Fig. 1: being pBAC-GP5-M rhabdovirus expression vector structural schematic diagram.

Fig. 2: being destination protein after indirect immunofluorescence analysis recombinant baculovirus Ac-PRRSVGP5-M infection sf9 cell Expression schematic diagram.

The electron microscope of the virus-like particle formed after Fig. 3: recombinant baculovirus Ac-PRRSVGP5-M infection sf9 cell.

Specific embodiment

According to following embodiments, the present invention can be more clearly understood.Technical step of the present invention, such as not special theory It is bright, it is ordinary skill in the art means, or be commercialization or published reagent material.

Embodiment 1:

Express the building of the PRRSV GP5 of manually modified synthesis and the recombinant baculovirus Ac-PRRSVGP5-M of M albumen

1. the synthesis of PRRSV GP5 and M gene order:

Based on the complete genome sequence of Field epidemic PRRSV Strain between 2006-2016, DNAMAN, MEGA5.1 etc. are used Software is compared, and screens the gene order of Field epidemic advantage strain, after manually modified and codon optimization, It send gene chemical synthesis company to carry out gene order synthesis, obtains the tandem sequence GP5-M of GP5 and M.

2. the building and identification of recombinant transfer vector:

Plasmid containing objective gene sequence GP5-M and carrier pBAC5 carry out Xba I/BamHI double digestion respectively, return After receiving purifying, reaction is attached using T4 ligase, and chemical method is converted to DH5 α competent cell, after screening positive clone Plasmid is extracted, is identified by bacterium colony PCR and plasmid enzyme restriction, the recombinant transfer vector pBAC-PRRSVGP5-M for the building that succeeds (the visible Fig. 2 of carrier schematic diagram).

3. recombinating the building and identification of Bacmid

The Plasmid DNA of recombinant transfer vector pBAC-PRRSVGP5-M is converted respectively to DH10Bac competent cell, is turned Skeleton carrier in transfer body and competent cell obtains the restructuring rod granule comprising PRRSV target gene by homologous recombination rBac-PRRSVGP5-M。

4. the preparation of recombinant baculovirus

4.1 restructuring rod granule transfection insect cells: sf9 plating cells to six orifice plates are used when its cell confluency degree is to 80% Lipofectamine Lipofectamine3000 is transfected, specific as follows:

4.1.1 2 μ g restructuring rod granule rBac-PRRSVGP5-M and 2 μ L p3000 are added to 250 μ L without serum and resisted In the opti-MEM culture solution of raw element, mix gently；

4.1.2 4 μ l Lipofectamine3000 liposomes are added to the opti- that 250 μ l are free of serum and antibiotic In MEM culture solution, mix gently；

4.1.3 the liquid of step 4.1.1 and step 4.1.2 are stored at room temperature 10-15 minutes after mixing；

4.1.4 step 4.1.3 mixed liquor is instilled dropwise in six orifice plate sf9 cells, then sets six orifice plate sf9 cells 27 DEG C of constant temperature incubations and continuous observation.

The harvest and amplification of 4.2 recombinant baculovirus

4.2.1 the harvest of recombinant baculovirus:

Careful observation is carried out daily to the cell after transfection, when cell occurs becoming larger, become irregular or even starting upper float extremely When liquid level, cell and supernatant are collected, extract sample RNA and carries out RT-PCR detection, whether identification target gene GP5 deposits , generally after transfection 4-5 days visible obvious cytopathies when, multigelation collect cell and supernatant, pressed in 4 DEG C 12000r/min is centrifuged 10 minutes, collects supernatant and labeled as P1 for recombinant baculovirus；

4.2.2 the amplification of recombinant baculovirus:

P1 is inoculated with sf9 cell according to 1:10 for recombinant baculovirus, is cultivated 4-5 days in 27 DEG C, it is obvious to cytopathy When generation, P2 is harvested for recombinant baculovirus；P3 generation and higher generation recombinant baculovirus are obtained in the same way, collection Recombinant baculovirus is saved backup in -70 DEG C.

4.2.3 the titer determination of recombinant baculovirus

P1, P2 and P3 generation virus are carried out 10 times respectively to be serially diluted, are then inoculated with 96 orifice plate sf9 cells, each dilution 8 holes of degree inoculation are placed in 27 DEG C of constant temperature incubations and observe cytopathy situation daily, then calculate virus TCID by Karber method₅₀ Value obtains P1, P2, P3 of recombinant baculovirus Ac-PRRSVGP5-M for virus titer 10^4.875TCID₅₀/ ml~ 10^6.175TCID₅₀/ml。

5. the expression and identification of recombinant protein

The Immunofluorescence test of 5.1 recombinant proteins:

Recombinant baculovirus infects health sf9 cell in 10% ratio, while blank control wells are arranged, 27 DEG C of culture 48-72 Hour, simultaneously continuous observation fixed cell 2 hours with 80% acetone of pre-cooling, then PBST when cytopathy reaches 80% or more Washing 3 times, 5% skim milk (being prepared using TBST) is in 4 DEG C of closings overnight, and then PBST is washed 3 times, and 1:1000 dilution is added GP5 monoclonal antibody, 37 DEG C be protected from light incubation 1 hour, then PBST wash 3 times, be added the diluted FITC fluorescence mark of 1:1000 Remember sheep anti mouse secondary antibody, 37 DEG C are incubated for 1 hour, and fluorescence microscopy is under the microscope after PBST is washed 3 times and judgement is as a result, recombinate rod-shaped disease Poison infection the visible obvious green fluorescence of cell and control group unstressed configuration, it is confirmed that target gene can be in sf9 insect cell It expresses (Fig. 2).

The observation of 5.2 recombinant protein Electronic Speculum

By the recombinant baculovirus liquid power transmission sem observation of fresh collection, form and PRRSV virion are detected as the result is shown The similar virus-like particle of son, diameter about 30-50nm, it was demonstrated that GP5 and M albumen can form virus-like particle (Fig. 3) in vitro.

In R&D process of the present invention, inventor attempts several genes modification scheme, for example, using flexible Linker(GGGGS rigid linker(EAAAK of the invention) is substituted) obtain control group 1, nucleotide sequence such as SEQ ID Shown in No:2, amino acid sequence such as SEQ ID No:5；The control group 2 such as obtained using different genetic modification means, nucleosides Acid sequence is as shown in SEQ ID No:3, and amino acid sequence is as shown in SEQ ID No:6, in comparison, control group 2 and traditional PRRSV amino acid sequence is closer, and amino acid sequence of the invention is clipped compared to the GP5 albumen of most of reports The segments such as " TFVIFPVLTHIVSYGALTTSHFL ", " YVLSSIYAVCALAALICFVI ", compared to the control group 2 traditional piece " VVLDGS " in amino acid fragment is sported " EELDGS ", " EKGGKV " of amino acid fragment is sported by section, the present invention “EKEEKV”。

For the method that embodiment 1 is respectively adopted in control group 1 and control group 2, corresponding recombinant baculovirus is prepared, And corresponding virus-like particle is prepared in expression in sf9 insect cell, and for subsequent corresponding virus sample particle vaccines Preparation.

After measured, the same condition of culture of embodiment 1, P1, P2, P3 generation virus of 1 recombinant baculovirus of control group are based on Titre is 10^3.135TCID₅₀/ ml~10^5.125TCID₅₀/ml；P1, P2, P3 of 2 recombinant baculovirus of control group exist for virus titer 10^4.175TCID₅₀/ ml~10^5.375TCID₅₀/ml。

Embodiment 2:

(1) preparation of porcine reproductive and respiratory syndrome virus virus sample particle vaccines is recombinated:

The recombinant baculovirus Ac-PRRSVGP5-M that embodiment 1 is prepared is inoculated with health sf9 cell in 10% ratio, After 27 DEG C are cultivated 4-5 days, multigelation collects cell and supernatant, and 12000r/min centrifugation is collected after twenty minutes under the conditions of 4 DEG C Then supernatant is precipitated destination protein using ammonium sulfate precipitation method, after resuspension using binary ethylenimine (BEI) to protein solution into It inactivates within row 36-48 hours, is neutralized using equivalent sodium thiosulfate, finally matched with 206 adjuvant mixing and emulsifying of Seppic ISA later Vaccine processed, be placed in 2-8 DEG C it is spare.

During vaccine preparation, the recombinant baculovirus of the embodiment of the present invention 1, control group 1 and control group 2 is through cultivating Afterwards, virus titer is adjusted to 10⁵TCID₅₀/ ml then continues the operation such as subsequent freeze thawing.During vaccine preparation, control Protein concentration processed is 100 μ g/mL.

(2) vaccine test method and result:

Method prepares two batches vaccine in accordance with the above-mentioned embodiment 1, and lot number is respectively 20170501,20170601.

2.1 characters are examined: two batches inactivated vaccine appearance pinkiness emulsion state.

2.2 steriling tests: two batches inactivated vaccine is attached according to existing " Republic of China Veterinary Pharmacopoeia " version third portion in 2010 Record is tested, and T.G, G.P pipe and G.A slant medium do not observe bacterium colony.

2.3 exogenous virus are examined: two batches inactivated vaccine is according to " Republic of China Veterinary Pharmacopoeia " version third portion in 2010 Annex is tested, without swine fever virus, bovine viral diarrhea virus, pig parvoviral, porcine pseudorabies virus, rotavirus, Transmissible gastro-enteritis virus etc. pollution, it was demonstrated that recombination porcine reproductive and respiratory syndrome virus virus sample particle vaccines seed culture of viruses be Pure.

2.4 mycoplasmas are examined: two batches inactivated vaccine is attached according to " Republic of China Veterinary Pharmacopoeia " version third portion in 2010 Record is tested, and does not find that significant change occur in bottle and tubule culture color, the liquid culture of transplanting is in solid culture Without " fried egg " shape mycoplasma bacterium colony on base.

2.5 safety verifications: taking the 30-40 age in days sodium selenite 20 that PRRSV antigen-antibody is double-negative, be randomly divided into 2 groups, Every group 10,10 part vaccines of every intramuscular injection, clinical observation 14 days, equal 100% strong work had no adverse reaction.

Table 1: two batches recombinate porcine reproductive and respiratory syndrome virus virus sample particle vaccines inspection result

Inspection project	20131201	20131202
			Character is examined	Pink emulsion	Pink emulsion
Steriling test	Asepsis growth	Asepsis growth
			Mycoplasma is examined	No mycoplasma growth	No mycoplasma growth
Exogenous virus is examined	No exogenous virus pollution	No exogenous virus pollution
			Safety detection	100% strong work, has no adverse reaction	100% strong work, has no adverse reaction

Embodiment 3: vaccine safety experiment

(1) piglet safety testing:

Virus sample particle vaccines prepared by 5 batches of embodiments 2 of laboratory preparation are randomly selected, every batch of takes 3 bottles at random, so Afterwards after evenly mixing, 2 multiple dose intramuscular injection inoculation is carried out to 30 age in days piglets, while sets blank control group test pig.It is all Piglet is before inoculation after 3 days and inoculation during 14 days, and measurement body temperature 2 times, and observe the feelings such as the spirit of test pig, appetite daily Condition.The result shows that the body temperature of immune group test pig and control group test pig, the state of mind and appetite are normal.Show the virus-like Particle vaccines are safe to piglet.

(2) pregnant sow safety testing

5 batches of virus sample particle vaccines of laboratory preparation are randomly selected, every batch of takes 3 bottles at random, then after evenly mixing, right Antenatal 70 days pregnant sows carry out 2 multiple dose intramuscular injection inoculation, while setting blank control group test pig.All test sows Before inoculation 3 days with inoculation after during 14 days, daily measure 2 body temperature, to the state of mind of test pig, appetite, breeding produce As a result continuous observation is carried out.The result shows that pregnant sow is acted normally after production after inoculation, breeding difficulty feelings are had no Condition and respiratory disease situation.Show that the virus sample particle vaccines are safe to pregnant sow.

Embodiment 4: piglet immunological protest test

The double-negative 30-40 age in days sodium selenite 50 of PRRSV antigen-antibody is screened, is divided into five groups by 10 every group, the Ac-PRRSVGP5-M vaccine is immunized in battery of tests pig, and the immune Ac-PRRSVGP5-M(of second group of test pig is unmutated, control group 1) Vaccine, third group test pig immune Ac-PRRSVGP5-M(control group 2) vaccine, the 4th group of test pig be immunized that commercially available certain is public JXA1-R plants of attenuated live vaccines are taken charge of, the 5th group of test pig is blank control group.Use highly pathogenic PRRSV within 28th day after immune Malicious infection is attacked in velogen strain intramuscular injection, 2 days and is attacked after poison before attacking poison and is continued to monitor within 21 days experimental animal body temperature and clinical table It is existing, and cut open within the 21st day after attacking poison and kill experimental animal, dissect lesion is observed, emphasis records lung lesion situation.

PRRSV attacks after poison standard of falling ill in bibliography, it may be assumed that (1) continuous body temperature on the 3rd is higher than 41 DEG C or cough and breathing is tired Difficult symptom；(2) lung's consolidation of the visible sheet of dissect.The result shows that virus sample particle vaccines prepared by the embodiment of the present invention 2 The immune protective rate highest (table 1) of Ac-PRRSVGP5-M vaccine compares JXA1-R plants of attenuated live vaccines of PRRSV, system of the present invention Standby virus sample particle vaccines are more safe and effective, return risk that is strong or causing virus to recombinate without virulence.

The Immunoprotection test result of 2 piglet of table

Grouping

Immunogene

Clinical symptoms

High fever symptom (>=41 DEG C)

Lung's consolidation

Morbidity determines

Protective rate

First group

Ac-PRRSVGP5-M virus sample particle vaccines (embodiment 2)

1/10 is abnormal

0/10

1/10

90%:

Second group

Virus sample particle vaccines (control group 1)

4/10 is abnormal

2/10

4/10

5/10

60%

Third group

Virus sample particle vaccines (control group 2)

3/10 is abnormal

1/10

3/10

70%:

4th group

PRRSV JXA1-R attenuated live vaccines

1/10 is abnormal

1/10

90%

5th group

Physiological saline

10/10 is abnormal

9/10

10/10

0%

Embodiment 5: the antibody of vaccine immunity piglet disappears the rule that rises

(1) according to the method preparation and reorganization porcine reproductive and respiratory syndrome virus virus sample particle vaccines of Examples 1 and 2.

(2) the double-negative 30-40 age in days sodium selenite 15 of screening PRRSV antigen-antibody, is randomly divided into 5 groups, every group 3 Head.First group of immune Ac-PRRSVGP5-M vaccine, second group of immune Ac-PRRSVGP5-M(control group 1) vaccine, third group exempts from Epidemic disease Ac-PRRSVGP5-M(control group 2) vaccine, JXA1-R plants of attenuated live vaccines of certain commercially available company are immunized in the 4th group of test pig, and Five groups of test pigs are blank control group.It 14 days after immune, takes a blood sample within 28,60,90,120,150, uses commodity Change PRRSV antibody Elisa kit (BioChek) and carries out antibody test.

4 difference PRRSV vaccine immunity piglet antibody test result of table

Note: "-" indicates that antibody test result is feminine gender, with the antibody effect that OD value is positive serum maximum dilution multiple Valence.

From experimental result as can be seen that the preparation-obtained recombination porcine reproductive and respiratory syndrome disease of the embodiment of the present invention 1 Malicious virus sample particle vaccines and PRRSV JXA1-R attenuated live vaccines have similar antibody titer, the equal energy after immunity inoculation Reach the antibody titer of 1:3200, and by antibody generation time comparatively, the preparation-obtained epidemic disease of 1-2 of the embodiment of the present invention A higher antibody titer level, up to 1:400 can be reached by comparing within seedling 14th day after immune, and attenuated vaccine is only capable of Reach highest 1:200, illustrates that vaccine of the invention can preferably stimulate body to generate antibody animal immune early stage, pass through Result on the 150th compares it is found that vaccine of the invention is still able to maintain a relatively high antibody water after inoculation 150 days It is flat, illustrate that vaccine of the invention has the relatively more longlasting immunoprotection period compared to Attenuate vaccine.1-2 through the embodiment of the present invention Vaccine compared with control group it is found that compared to the control group 1, the present invention uses rigidity linker connection Tflg, so that flagellum egg It is white to be preferably exposed to virion surface, it have stimulated the generation antibody of body earlier, and antibody titer of the invention It is apparently higher than the potency of control group 1, up to its twice, illustrates that vaccine of the invention has better immune protective effect；Phase Than control group 2, the present invention, can be more preferable by improving the immunogenicity of virus sample particle vaccines to GP5 progress genetic modification Ground stimulates body to generate antibody, forms preferably protection.

Although the content of present invention is illustrated in conjunction with above-described embodiment, embodiments of the present invention are not by above-mentioned reality The limitation of example is applied, it is intended that the scope of the present invention be defined by the claims appended hereto, other any made changes in restriction range Or modification, it should be equivalent substitute mode, be all included in the scope of protection of the present invention.

Sequence table

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ggctccggcc acttcgagtc caccaaccgc gtggccggag gttccacctc ccgctgccgc 660

ctgtgcctgc tgggccgcaa gtacatcctg gcccccgccc accacgtgga gtccgccgcc 720

ggcttccacc ccatcgccgc caacgacaac cacgccttcg tggtgcgccg ccccggctcc 780

accaccgtga acggcaccct ggtgcccggc ctgaagtccc tggtgctggg cggccgcaag 840

gccgtgaagc agggcgtggt gaacctggtg aagtacgcca ag 882

<210> 2

<211> 882

<212> DNA

<213>artificial synthesized (procine)

<400> 2

ggagtcgagg tcagactggg agttggtgct gttagcagac tgaaccgcca gttcacgcac 60

acgctgcaag ttgttgttga tggaggagga ggttccgcca agttcgtggc cgcctggacc 120

ctgaaggccg ccgcctccaa cgccgcctcc tcccacatcc agctgatcta caacctgacc 180

ctgtgcgagc tggccggcac cgactggctg gcccagaagt tcgactgggc cgtggaggga 240

ggttccgaca ccgtgggcct ggccaccgtg tccaccgccg gctactacgg ctcgggtcgc 300

ctggccaaga actgcatgtc ctggcgctac tcctgcaccc gctacaccaa cttcctgctg 360

gacaccaagg gccgcctgta ccgctggcgc tcccccgtga tcgtggagaa ggaggagaag 420

gtggaggtgg agggccacct gatcgacctg aagcgcgagg agctggacgg ctccgccgcc 480

acccccctga cccgcgtgtc cgccgagctg tggggccgcc tggggcccgg acctggctcc 540

tccctggacg acttctgcaa cgactccacc gccggcggct ccaaggtgtc ccgcggccgc 600

ggctccggcc acttcgagtc caccaaccgc gtggccggag gttccacctc ccgctgccgc 660

ctgtgcctgc tgggccgcaa gtacatcctg gcccccgccc accacgtgga gtccgccgcc 720

ggcttccacc ccatcgccgc caacgacaac cacgccttcg tggtgcgccg ccccggctcc 780

accaccgtga acggcaccct ggtgcccggc ctgaagtccc tggtgctggg cggccgcaag 840

gccgtgaagc agggcgtggt gaacctggtg aagtacgcca ag 882

<210> 3

<211> 882

<212> DNA

<213>artificial synthesized (procine)

<400> 3

ggagtcgagg tcagactggg agttggtgct gttagcagac tgaaccgcca gttcacgcac 60

acgctgcaag ttgttgttga tgaggccgcc gccaaggcca agttcgtggc cgcctggacc 120

ctgaaggccg ccgcctccaa cgccgcctcc tcccacatcc agctgatcta caacctgacc 180

ctgtgcgagc tggccggcac cgactggctg gcccagaagt tcgactgggc cgtggaggga 240

ggttccgaca ccgtgggcct ggccaccgtg tccaccgccg gctactacgg ctcgggtcgc 300

ctggccaaga actgcatgtc ctggcgctac tcctgcaccc gctacaccaa cttcctgctg 360

gacaccaagg gccgcctgta ccgctggcgc tcccccgtga tcgtggagaa gggcggcaag 420

gtggaggtgg agggccacct gatcgacctg aagcgcgtgg tgctggacgg ctccgccgcc 480

acccccctga cccgcgtgtc cgccgagctg tggggccgcc tggggcccgg acctggctcc 540

tccctggacg acttctgcaa cgactccacc gccggcggct ccaaggtgtc ccgcggccgc 600

ggctccggcc acttcgagtc caccaaccgc gtggccggag gttccacctc ccgctgccgc 660

ctgtgcctgc tgggccgcaa gtacatcctg gcccccgccc accacgtgga gtccgccgcc 720

ggcttccacc ccatcgccgc caacgacaac cacgccttcg tggtgcgccg ccccggctcc 780

accaccgtga acggcaccct ggtgcccggc ctgaagtccc tggtgctggg cggccgcaag 840

gccgtgaagc agggcgtggt gaacctggtg aagtacgcca ag 882

<210> 4

<211> 294

<212> PRT

<213>artificial synthesized (procine)

<400> 4

Gly Val Glu Val Arg Leu Gly Val Gly Ala Val Ser Arg Leu Asn Arg

1 5 10 15

Gln Phe Thr His Thr Leu Gln Val Val Val Asp Glu Ala Ala Ala Lys

20 25 30

Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Ser Asn Ala

35 40 45

Ala Ser Ser His Ile Gln Leu Ile Tyr Asn Leu Thr Leu Cys Glu Leu

50 55 60

Ala Gly Thr Asp Trp Leu Ala Gln Lys Phe Asp Trp Ala Val Glu Gly

65 70 75 80

Gly Ser Asp Thr Val Gly Leu Ala Thr Val Ser Thr Ala Gly Tyr Tyr

85 90 95

Gly Ser Gly Arg Leu Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys

100 105 110

Thr Arg Tyr Thr Asn Phe Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg

115 120 125

Trp Arg Ser Pro Val Ile Val Glu Lys Glu Glu Lys Val Glu Val Glu

130 135 140

Gly His Leu Ile Asp Leu Lys Arg Glu Glu Leu Asp Gly Ser Ala Ala

145 150 155 160

Thr Pro Leu Thr Arg Val Ser Ala Glu Leu Trp Gly Arg Leu Gly Pro

165 170 175

Gly Pro Gly Ser Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Gly

180 185 190

Gly Ser Lys Val Ser Arg Gly Arg Gly Ser Gly His Phe Glu Ser Thr

195 200 205

Asn Arg Val Ala Gly Gly Ser Thr Ser Arg Cys Arg Leu Cys Leu Leu

210 215 220

Gly Arg Lys Tyr Ile Leu Ala Pro Ala His His Val Glu Ser Ala Ala

225 230 235 240

Gly Phe His Pro Ile Ala Ala Asn Asp Asn His Ala Phe Val Val Arg

245 250 255

Arg Pro Gly Ser Thr Thr Val Asn Gly Thr Leu Val Pro Gly Leu Lys

260 265 270

Ser Leu Val Leu Gly Gly Arg Lys Ala Val Lys Gln Gly Val Val Asn

275 280 285

Leu Val Lys Tyr Ala Lys

290

<210> 5

<211> 294

<212> PRT

<213>artificial synthesized (procine)

<400> 5

Gly Val Glu Val Arg Leu Gly Val Gly Ala Val Ser Arg Leu Asn Arg

1 5 10 15

Gln Phe Thr His Thr Leu Gln Val Val Val Asp Gly Gly Gly Gly Ser

20 25 30

Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Ser Asn Ala

35 40 45

Ala Ser Ser His Ile Gln Leu Ile Tyr Asn Leu Thr Leu Cys Glu Leu

50 55 60

Ala Gly Thr Asp Trp Leu Ala Gln Lys Phe Asp Trp Ala Val Glu Gly

65 70 75 80

Gly Ser Asp Thr Val Gly Leu Ala Thr Val Ser Thr Ala Gly Tyr Tyr

85 90 95

Gly Ser Gly Arg Leu Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys

100 105 110

Thr Arg Tyr Thr Asn Phe Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg

115 120 125

Trp Arg Ser Pro Val Ile Val Glu Lys Glu Glu Lys Val Glu Val Glu

130 135 140

Gly His Leu Ile Asp Leu Lys Arg Glu Glu Leu Asp Gly Ser Ala Ala

145 150 155 160

Thr Pro Leu Thr Arg Val Ser Ala Glu Leu Trp Gly Arg Leu Gly Pro

165 170 175

Gly Pro Gly Ser Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Gly

180 185 190

Gly Ser Lys Val Ser Arg Gly Arg Gly Ser Gly His Phe Glu Ser Thr

195 200 205

Asn Arg Val Ala Gly Gly Ser Thr Ser Arg Cys Arg Leu Cys Leu Leu

210 215 220

Gly Arg Lys Tyr Ile Leu Ala Pro Ala His His Val Glu Ser Ala Ala

225 230 235 240

Gly Phe His Pro Ile Ala Ala Asn Asp Asn His Ala Phe Val Val Arg

245 250 255

Arg Pro Gly Ser Thr Thr Val Asn Gly Thr Leu Val Pro Gly Leu Lys

260 265 270

Ser Leu Val Leu Gly Gly Arg Lys Ala Val Lys Gln Gly Val Val Asn

275 280 285

Leu Val Lys Tyr Ala Lys

290

<210> 6

<211> 294

<212> PRT

<213>artificial synthesized (procine)

<400> 6

Gly Val Glu Val Arg Leu Gly Val Gly Ala Val Ser Arg Leu Asn Arg

1 5 10 15

Gln Phe Thr His Thr Leu Gln Val Val Val Asp Glu Ala Ala Ala Lys

20 25 30

Ala Lys Phe Val Ala Ala Trp Thr Leu Lys Ala Ala Ala Ser Asn Ala

35 40 45

Ala Ser Ser His Ile Gln Leu Ile Tyr Asn Leu Thr Leu Cys Glu Leu

50 55 60

Ala Gly Thr Asp Trp Leu Ala Gln Lys Phe Asp Trp Ala Val Glu Gly

65 70 75 80

Gly Ser Asp Thr Val Gly Leu Ala Thr Val Ser Thr Ala Gly Tyr Tyr

85 90 95

Gly Ser Gly Arg Leu Ala Lys Asn Cys Met Ser Trp Arg Tyr Ser Cys

100 105 110

Thr Arg Tyr Thr Asn Phe Leu Leu Asp Thr Lys Gly Arg Leu Tyr Arg

115 120 125

Trp Arg Ser Pro Val Ile Val Glu Lys Gly Gly Lys Val Glu Val Glu

130 135 140

Gly His Leu Ile Asp Leu Lys Arg Val Val Leu Asp Gly Ser Ala Ala

145 150 155 160

Thr Pro Leu Thr Arg Val Ser Ala Glu Leu Trp Gly Arg Leu Gly Pro

165 170 175

Gly Pro Gly Ser Ser Leu Asp Asp Phe Cys Asn Asp Ser Thr Ala Gly

180 185 190

Gly Ser Lys Val Ser Arg Gly Arg Gly Ser Gly His Phe Glu Ser Thr

195 200 205

Asn Arg Val Ala Gly Gly Ser Thr Ser Arg Cys Arg Leu Cys Leu Leu

210 215 220

Gly Arg Lys Tyr Ile Leu Ala Pro Ala His His Val Glu Ser Ala Ala

225 230 235 240

Gly Phe His Pro Ile Ala Ala Asn Asp Asn His Ala Phe Val Val Arg

245 250 255

Arg Pro Gly Ser Thr Thr Val Asn Gly Thr Leu Val Pro Gly Leu Lys

260 265 270

Ser Leu Val Leu Gly Gly Arg Lys Ala Val Lys Gln Gly Val Val Asn

275 280 285

Leu Val Lys Tyr Ala Lys

290

Claims

1. a kind of porcine reproductive and respiratory syndrome virus PRRSV GP5-M gene of manually modified synthesis, sequence such as SEQ ID Shown in NO.1, the protein of coding is shown in SEQ ID NO.4.

2. a kind of baculovirus transfer vector pBAC-PRRSVGP5-M, carrier contains manually modified synthesis described in claim 1 Porcine reproductive and respiratory syndrome virus PRRSV GP5-M gene and marker gene GFP.

3. a kind of recombinant baculovirus for expressing porcine reproductive and respiratory syndrome virus PRRSV GP5 and M albumen, strain are named For recombinant baculovirus Ac-PRRSVGP5-M, the baculoviral is prepared by transfer vector as claimed in claim 2.

4. a kind of preparation method of the recombinant baculovirus of expression porcine reproductive and respiratory syndrome virus PRRSV GP5 and M albumen, It is characterized by comprising the following steps:

(1) the manually modified synthesis of the tandem gene GP5-M of the immunogenic gene GP5 and M of PRRSV, nucleotides sequence are classified as Shown in SEQ ID NO.1；

(2) it the building of baculovirus transfer vector: using pBAC5 plasmid as skeleton, by Xba I and BamH I digestion carrier and inserts Enter segment, then by T4 ligase in 16 DEG C of connections overnight, transformation and selection positive colony extracts recombinant plasmid, and digestion is identified, Obtain baculovirus transfer vector pBAC-PRRSV GP5-M；

Step 2, the building of recombinant baculovirus Ac-PRRSV GP5-M:

(1) acquisition and identification of restructuring rod granule rBac-PRRSVGP5-M: sequencing is taken to identify correct baculovirus transfer vector PBAC-PRRSVGP5-M Plasmid DNA is mixed with DH10 Bac competent cell, after ice bath 30 minutes, in 42 DEG C of progress, 45 seconds water Bathe heat shock, then ice bath 5 minutes are added SOC fluid nutrient medium, 37 DEG C shaken cultivation 2 hours, after being serially diluted by 10 times, Each gradient bacterium solution is coated with LB plate, purifies positive bacterium colony using the screening reagent box screening of life company, extracts restructuring rod granule rBac-PRRSVGP5-M；

(2) acquisition of recombinant baculovirus Ac-PRRSVGP5-M: the restructuring rod granule rBac-PRRSVGP5-M that previous step is extracted, It using lipofectamine Lipofectamine3000, transfects into sf9 cell, in 28 DEG C of lasting culture observations, in transfection Fluorescence microscope recombinant baculovirus green florescent signal was utilized with 72 hours within 24 hours afterwards, 48 hours, and after transfection 72 hours collection cell conditioned mediums, obtain recombinant baculovirus, then it is inoculated with to health sf9 cell expansion culture again, collect Virus liquid is placed in -70 DEG C as seed culture of viruses and saves backup.

5. expressing the rod-shaped disease of recombination of porcine reproductive and respiratory syndrome virus PRRSV GP5 and M albumen described in claim 4 Poison includes PRRSV virus-like particle and adjuvant, the PRRSV preparing the application in subunit vaccine, the subunit vaccine Virus-like particle expresses the recombination of porcine reproductive and respiratory syndrome virus PRRSV GP5 and M albumen as described in claim 4 It expresses and obtains after baculoviral inoculation health sf9 cell.

6. application according to claim 5, which is characterized in that the adjuvant is 206 adjuvant of Seppic ISA.

7. a kind of recombination porcine reproductive and respiratory syndrome virus virus sample particle vaccines, which is characterized in that by PRRSV virus-like Grain and adjuvant composition, PRRSV virus-like particle expression porcine reproductive and respiratory syndrome virus as described in claim 4 It expresses and obtains after the recombinant baculovirus inoculation health sf9 cell of PRRSV GP5 and M albumen.

8. the preparation method of recombination porcine reproductive and respiratory syndrome virus virus sample particle vaccines according to claim 7, It is characterized in that, the recombinant baculovirus Ac-PRRSVGP5-M that claim 4 is prepared is inoculated with health sf9 in 10% ratio Cell, after 27 DEG C are cultivated 4-5 days, multigelation collects cell and supernatant, and 12000r/min is centrifuged after twenty minutes under the conditions of 4 DEG C Collect supernatant, then precipitated destination protein using ammonium sulfate precipitation method, after resuspension using binary ethylenimine to protein solution into It inactivates within row 36-48 hours, is neutralized using equivalent sodium thiosulfate, finally matched with 206 adjuvant mixing and emulsifying of Seppic ISA later Vaccine processed, be placed in 2-8 DEG C it is spare.