CN106279431A

CN106279431A - A kind of pig circular ring virus subunit inactivated vaccine

Info

Publication number: CN106279431A
Application number: CN201610608443.8A
Authority: CN
Inventors: 张晓丹; 李殿明; 蒲勤; 齐春梅; 田春辉; 刘甜甜; 任百亮; 张导春; 党将将; 吴启凡
Original assignee: Qingdao Mingqin Biological Technology Co Ltd
Current assignee: Qingdao Mingqin Biological Technology Co Ltd
Priority date: 2016-07-13
Filing date: 2016-07-13
Publication date: 2017-01-04
Anticipated expiration: 2036-07-13
Also published as: CN106279431B

Abstract

The present invention relates to the preparation of a kind of pig circular ring virus subunit inactivated vaccine.Described vaccine comprises PCV1 major structural protein Cap protein subunit, PCV2 major structural protein Cap protein subunit, pig interleukin 2 and 6 and purification tag.This vaccine stable preparation process, is suitable for large-scale production.Animal experiment shows, pig circular ring virus subunit inactivated vaccine of the present invention has good safety, and can produce significant humoral immunization and cellular immunization by induced animal body, effectively prevents the infection of pig circular ring virus.

Description

A kind of pig circular ring virus subunit inactivated vaccine

Technical field

The invention belongs to biological technical field, specifically, the present invention relates to a kind of for immunity fusion protein, its by The subunit of major structural protein Cap protein of PCV1 and PCV2, pig interleukin-2 and purification tag sequence are in series, Then be cloned into carrier, convert Host Strains, by fermentation, purification, emulsifying process be prepared as pig circular ring virus subunit inactivation epidemic disease Seedling, this vaccine can be induced body to produce notable humoral immunization and cellular immunization, effectively be prevented the infection of pig circular ring virus.

Background technology

Infection of Porcine circovirus is a kind of infectious disease caused by pig circular ring virus (Porcine circovirus, PCV). Main clinical manifestation be body constitution decline, become thin, anemia, jaundice, retarded growth, diarrhoea, dyspnea, sow breeding barrier Hinder, internal organs and the heavy swelling of the extensive pathological change of skin, particularly kidney, spleen and lymph nodes of body as a whole, hemorrhage and Downright bad.

Pig circular ring virus is porcine circovirus section Circovirus, by Tischer in 1974 first at PK-15 (ATCC- CCL) cell finds.PCV has 2 kinds of serotypes, i.e. PCV1 and PCV2.Pathogenic relatively low to pig of known PCV1, can draw once in a while Play the fetal infection of farrowing sow, cause breeding difficulty, but pollution rate is high in normal swinery and pig source cell.PCV2 is to pig Very harmful, the main immune system encroaching on pig body, cause immunity of organism suppression and resistance to decline, thus other disease of secondary Sick.Have turned out to be the necessary cause of disease that piglet multisystemic exhaustion syndrome occurs, additionally, Corii Sus domestica inflammation nephrotic syndrome, porcine respiratory The generation of the diseases such as syndrome, sow breeding difficulty is also relevant with the infection of PCV2.PCV gene element is two kinds of genotype, i.e. PCV1 type genome and PCV2 type genome.PCV1 full-length genome about 1.7kb, PCV2 about 1.768kb.The base of PCV1 and PCV2 Because structure is very much like, all contain 11 ORFs, wherein ORF1 and ORF2 be topmost two ORF, ORF1 coding Rep and Rep ' albumen, is primarily involved in virus replication；ORF2 encodes Cap protein, size about 27.8KD, is the unique structural protein of PCV2, Also it is main immunogenic protein, body can be induced to produce neutralizing antibody.Gene sequencing finds, each in same serotype Nucleotide sequence homology between strain is more than 96%, and different serotypes nucleotide sequence homology is less than 80%. DNA replication dna sintering relevant with duplication between PCV1 and PCV2 and Rep protein gene sequence homology be respectively 79.5% and 82%；And Cap protein gene exists larger difference, homology only has about 62%.

From PCV find since, PCV1 and PCV2 have been found to into worldwide popular and exist virus, China is by Lang Hongwu Being separated to PCV2 first from swinery Deng calendar year 2001, current each pig farm the most generally exists.Serosurvey finds, domestic swinery sun Property rate is up to 52.8%-100%.This disease sickness rate is up to 50%, and mortality rate is because of situations such as pig farm condition, secondary infection not With, typically at 5%-70%, it is one of Infectious Diseases causing pig industry heavy economic losses at present.At present, commercialization epidemic disease Seedling mainly has subunit vaccine and the PCV2 inactivated vaccine of baculovirus expression, and antigen purity is the highest and expensive, adds upper body Outer cultivation PCV is relatively difficult, and virus titer is relatively low, thus be badly in need of develop safely and effectively new generation vaccine to prevent pig circular ring virus Infection.

Interleukin-2 (Interleukin-2, IL-2), also known as SCIF, is by helper T lymphocyte (THC) point A kind of glycoprotein secreted.IL-2 can promote T cell differentiation and maturation and amplification in vitro, promotes the growth of THC, stimulates NK cell raw Grow and strengthen its molten cell function, strengthen B cell and the effect of macrophage, be one have extensive bioactive cell because of Son.IL-2 plays a significant role in specific immune response reacts, and with protective antigen gene, IL-2 is connected and composed fusion Albumen, the antibody that can strengthen subunit vaccine produces and cellular immune level.The invention provides a kind of new for immunity Fusion protein and vaccine combination, can effectively prevent the infection of pig circular ring virus.

Summary of the invention

The present invention according to different structure albumen PCV virus infect in effect, select PCV1 and PCV2 primary structure egg White Cap protein subunit, as vaccine frame structure, is connected with porcine interleukin-2 after being connected by flexible Linker again, is cloned into Escherichia coli are converted, by fermentation, purification, the technique such as emulsifying, it is thus achieved that there is the pig circle of Desirable immunogenic after pRSETB carrier Circovirus virus subunit vaccine.The vaccine utilizing the present invention to prepare can effectively prevent the infection of pig circular ring virus.

At first aspect, the invention provides a kind of fusion protein for preparing immunity, its contain PCV1 type and PCV2 type virus major structural protein Cap protein subunit and porcine interleukin 2 (IL-2).Described " PCV1 type and PCV2 type Virus major structural protein Cap protein subunit " refer to the major structural protein Cap with immunogenic PCV1 and PCV2 A part of polypeptide of albumen or its there is essentially identical immunogenic function equivalent, be preferably selected from SEQ ID No.4,6 Aminoacid sequence or its function equivalent.Described " porcine interleukin-2 " refers to pig interleukin-2 (IL-2) subunit Or it has the function equivalent of essentially identical adjuvant function, preferably aminoacid sequence as shown in SEQ ID No.8 or its Function equivalent.Load required for described fusion protein or polypeptide or pharmaceutically acceptable salt and antigen expressed epi-position Body.Carrier can also include the sequence of separately encoded each epitope, and series connection can be carried out by genetic engineering method.Described epidemic disease Seedling also includes nonimmune active substance, is the coupling part of each polypeptide, does not have the immunogenicity of epitope, do not have Any adjuvanticity, mainly has purification tag, joint peptide, chemical modification part, N end signal peptide and C end polyadenylic acid etc..Institute State pharmaceutically acceptable salt and refer to avirulence, stimulation and allergy, it is adaptable to the salt of human or animal tissues.Inert matter It is well known to those skilled in the art with pharmaceutically acceptable salt.

At second aspect, the invention provides a kind of nucleic acid molecule, its coding pig described in first aspect present invention Porcine circovirus subunit vaccine polypeptide.Nucleotide of the present invention can be rna form, DNA form, is closed by synthetic mode Become epitope tandem sequence, then connect rear clone by genetic engineering operation and enter carrier, be transformed into escherichia coli, through sieving The techniques such as choosing, fermentation, purification obtain pig circular ring virus subunit vaccine polypeptide.This nucleic acid can be carried out routine in the present invention Molecular biology manipulations, such as: PCR, digestion with restriction enzyme, connection etc..The preferably such as SEQ of the nucleotide sequence in the present invention Shown in ID No.1.

At the 3rd aspect, the invention provides a kind of carrier, it contains the nucleic acid molecules described in second aspect present invention. Term " recombinant expression carrier " herein, " expression vector ", be only called " carrier " sometimes, can replace use alternately at this, Refer to bacterial plasmid commonly used in the art, cosmid, phasmid, yeast plasmid, plant cell virus, animal virus and other are each Plant viral vector.The carrier being suitable in the present invention includes but not limited to: the carrier (prokaryotic expression carrier) expressed in antibacterial, The carrier (such as pichia vector, Hansenula vectors etc.) expressed in yeast, the shaft-like disease expressed in insect cell Poisonous carrier, the carrier expressed in mammalian cell (vaccinia virus vector, retroviral vector, adenovirus vector Deng), the plant viral vector expressed in plant and the various carriers expressed in mammal galactophore.In a word, only Wanting stable in host cell to replicate, any plasmid and carrier can use.Preferred expression carrier comprises selected marker base Cause, as the ampicillin resistance gene of antibacterial, tetracycline resistance gene, kalamycin resistance gene, streptomycin resistance gene, Chloramphenicol resistance gene；Saccharomycetic neomycin resistance gene, Zeocin resistant gene, saccharomycetic defect selection marker, as His, Leu, Trp etc.；The neomycin resistance gene of eukaryotic cell, Zeocin resistant gene, dihydrofolate reductase gene and glimmering Photoprotein marker gene etc..In a preferred embodiment, described expression vector is coli expression carrier, art technology Personnel may utilize a series of technology such as DNA recombinant technique, builds the DNA sequence containing encoding fusion protein of the present invention, suitable The expression vector of the particular element such as transcription and translation regulating and controlling sequence, promoter and selected marker.Above-mentioned carrier can be used Convert, transfect suitable host cell, the fusion protein required for obtaining.

At the 4th aspect, the invention provides a kind of host cell, it is characterised in that described cell has used the present invention Nucleic acid molecules described in the third aspect converts or transfection.Host cell can be prokaryotic cell, it is also possible to be eukaryotic cell, as carefully Bacterium cell, yeast cells, plant cell, insect cell, mammalian cell etc..Host cell is converting or is transfecting containing the present invention After the gene order of described encoding fusion protein, i.e. constitute through engineering approaches cell or cell strain, can be used for producing required fusion protein. Those skilled in the art can select suitable carrier, host cell rightly, and know how by carrier high-efficiency convert or Being transfected in host cell, method therefor includes but not limited to: Calcium Chloride Method, electroporation are used for bacterial cell, electroporation Use for yeast cells, liposome, coprecipitation of calcium phosphate, electro fusion method and microinjection with protoplast fusion method In eukaryotic cells such as mammalian cells.

At the 5th aspect, the invention provides the method for fusion protein described in preparation first aspect of the present invention, its bag Include following steps: use the fusion protein of first aspect of the host cell expression present invention of the 4th aspect of the present invention, and separate Described fusion protein.The engineering cell obtained can be cultivated by conventional method, be induced and express required fusion protein, bag Include sweat and purifying process.The albumen of above-mentioned expression can be on intracellular, cell membrane or be secreted into periplasmic, cell Outward.As required, the physics of available fusion protein, chemistry and other biological characteristic, carry out isolated and purified.Method Include but not limited to: split bacterium (ultrasound wave splits bacterium, infiltration pressure break bacterium), centrifugal, saltout, molecular sieve chromatography, ion exchange chromatography, inhale Attached chromatograph (affinity chromatograph, metal chelate chromatography), reverse chromatograms, high performance liquid chromatography, capillary electrophoresis, the point focusing such as preparative And the degeneration of routine, renaturation process etc., these methods are all well-known to those skilled in the art.

At the 6th aspect, the invention provides a kind of pharmaceutical composition for immunity, it includes the present invention first Fusion protein described in aspect and pharmaceutically acceptable carrier, can prevent the infection of pig circular ring virus.General for this area For logical technical staff, there is a lot of known method can be by protein or polypeptide active composition and pharmaceutically acceptable load Body prepares into pharmaceutical composition.Pharmaceutically acceptable carrier used herein refer to nontoxic filler, diluent, adjuvant or Other pharmaceutical adjuncts.According to techniques known, can be according to therapeutic purposes, the needing pharmaceutical composition of route of administration Making various dosage form, preferably said composition is unit dosage form, such as tablet, capsule, powder, emulsion agent, injection, spray Type or the dosage form as feed additive.Preferably this pharmaceutical composition is injection type, spray-type or as feed additive Dosage form, these compositionss include different buffer content (such as phosphate buffer, Tris-HCl buffer), corresponding ion Intensity and pH value, and other materials (such as polylactic acid, mannitol etc.).It is also preferred that this pharmaceutical composition is vaccine combination, excellent Choosing contains adjuvant, further such as Freund's complete adjuvant, incomplete Freund's adjuvant, CpG sequence etc..

At the 7th aspect, the invention provides the fusion protein described in first aspect of the present invention at preparation prevention pig circle Application in the medicine that circovirus virus infects.At the 8th aspect, the invention provides the 5th medicine described in aspect of the present invention Compositions application in the medicine of preparation prevention Infection of Porcine circovirus.In a preferred embodiment of the present invention, logical Cross laboratory animal with given dose administered intramuscular, be effectively protected laboratory animal.

Accompanying drawing explanation

Drawings below is used for illustrating specific embodiments of the present invention, rather than limits and to be defined by the claims The scope of the invention.

Fig. 1 is the schematic diagram of the expression vector pRSETB-PCV-IL-2 containing fusion protein encoding gene.

Fig. 2 shows the electrophoresis result after recombinant expression carrier pRSETB-PCV-IL-2 HamH I+HindIII enzyme action, Wherein M is DNAMarker, and swimming lane 1 is plasmid enzyme restriction figure, and swimming lane 2 is the comparison of non-enzyme action, and swimming lane 3 is empty plasmid.

Fig. 3 is fusion protein encoding gene expression product SDS-PAGE qualification result, and wherein M is molecular weight Marker, from Being followed successively by 94KD, 66KD, 45KD, 33KD, 26KD, 20KD, 14KD down, 1 swimming lane is induction purification of samples.

Fig. 4 is Sample Purification on Single Western Blot Blot results, and wherein M is pre-dyed Marker, is followed successively by from top to bottom 94KD, 62KD, 40KD, 30KD, 24KD, 16KD, swimming lane 1 is sample, and swimming lane 2 is negative control.

Fig. 5 is immunity white mice ELISA detection serum specific antibody result, wherein (-◆-) it is 20150302 groups；(- ■-) it is 20150304 groups；(-▲-) it is 20150306 groups；(-●-) it is commercialization inactivated vaccine group；(-+-) it is PBS control group.

Fig. 6 is that PCV stimulates mice spleen cell T lymphproliferation response testing result.

Detailed description of the invention

It is only exemplary description that concrete test method described in embodiment describes, and is used for elaborating the present invention, but also It is not meant to limit the scope of the invention, the following stated experimental technique, does not illustrate, all according to " Molecular Cloning: A Laboratory Guide " (2002, the third edition, Science Press) described method carries out.

The source of embodiment one antigen-4 fusion protein gene

The present invention comprehensively analyzes the gene order of domestic porcine circovirus types 1 and 2 Major Epidemic strain, antigenic structure, stream Row disease learns progress, according to the aminoacid sequence of its major structural protein Cap protein, utilizes relevant biological information software pair Its hydrophilic, antigenicity, plasticity, surface accessibility and secondary structure are analyzed, it was predicted that possible epitope, and comprehensively Relevant report, so that it is determined that PCV1 and Porcine circovirus type 2 Cap subunit.Connected by flexible Linker after forming vaccine framing structure Connecting with porcine interleukin 2, this vaccine population structure is again:

PCV1 Cap-PCV2 Cap-IL-2-Tag

Embodiment two coli expression carrier and the structure of expression strain

The polypeptide-coding nucleotide designed in embodiment one is served the synthesis of extra large handsome biotech company, nucleotide sheet Section two ends have separately designed BamH I (5 ' end) and HindIII (3 ' end) restriction enzyme site, by synthetic fragment clone On pMD18T carrier, sequencing confirms to insert genetic fragment consistent with relating to sequence (see sequence table).Recombiant plasmid is ordered Entitled pMD18T-PCV-IL-2, carries out enzyme action process with corresponding restricted enzyme to plasmid, and coli expression carrier selects With the pRSETB plasmid of Invitrogen company, also use identical restriction enzyme ferment treatment, enzyme action condition: 10 μ L reactants System, adds plasmid 2 μ L, 5 active units of restricted enzyme (New England biolabs), 10 × buffer 1 in system μ L, deionized water polishing, 37.0 DEG C of enzyme action 1.5 hours.Enzyme action adds 1 μ L 200mM EDTA and terminates reaction after terminating.1% Electrophoresis 30 minutes in agarose gel electrophoresis.Under uviol lamp, pRSETB plasmid and purpose fragment are cut, according to Qiagen company Gel reclaims test kit description and carries out glue recovery.According to carrier: fragment be 1: 2-3 ratio by multi-epitope nucleotide fragments with Expression vector mixes, and reaction system 15 μ L is attached by T4DNA ligase, and 16 DEG C connect overnight, it is thus achieved that recombiant plasmid is named For pRSETB-PCV-IL-2 (see Fig. 1), transformed competence colibacillus e. coli bl21 (DE3) pLysS.

Convert: pRSETB-PCV-IL-2 is put thawed on ice, add 1mL coupled reaction liquid, again mix, ice-water bath 30 Minute, 42 DEG C 30 seconds, put back to the most rapidly ice-water bath 90 seconds, add 1mL LB culture fluid, 37.0 DEG C of quiescent culture 1 are little Time, 4000g low-temperature centrifugation is abandoned supernatant in 10 seconds, with the 200 μ L resuspended thalline of LB culture medium, is spread evenly across bacterium solution containing 100 On the LB agar plate of μ L/mL ampicillin, it is inverted in 37 DEG C of calorstats cultivation 12-16 hour, until Clone formation.

Identifying: in the monoclonal on picking flat board to LB culture medium, 37 DEG C, 200rpm concussion is cultivated 12 hours, extracts matter Grain, uses restricted enzyme BamH I and HindIII to carry out double digestion, can cut out corresponding pig circular ring virus subunit vaccine The clone of gene size fragment is about 1700bp, can primarily determine that for positive colony (see Fig. 2), positive colony carries out DNA sequence Row measure verifies its correctness (see sequence table) further.

Abduction delivering: by positive colony incubated overnight, morning next day cultivates 3 hours according to 1: 100 switching, 37 DEG C of concussions Wait, add 0.5mM IPTG induction, continue to cultivate 3 hours, prepare sample.The expression feelings of conventional SDS-PAGE testing goal albumen Condition, can see specific band for correct clone (see Fig. 3) at 66KD molecular weight.Take correct clone, amplification culture, SDS- After PAGE confirms to express correctly, use conventional Western-blot to be further characterized by it and express accuracy (see Fig. 4).Finally screen The engineering bacteria of the efficient secretory expression fusion protein obtained, named pRSETB-PCV-IL-2/BL21 (DE3, PLys).

Fermentation, purification and the emulsifying of embodiment three engineering bacteria

Ferment and be inoculated into producing strain in the LB fluid medium that 2mL contains 100 μ L/mL ampicillin, 37 DEG C, 12 hours activated spawn are cultivated in 180rpm concussion.The strain activated is accessed shaking flask, 37 DEG C of concussion trainings with the inoculum concentration of 1: 100 Support to OD600=3, can inoculate into fermentation tank in 10% ratio.Fermentation culture medium is semisynthetic medium, joins with distilled water System, does not wherein contain any antibiotic.Correction dissolved oxygen and pH value electrode, open tank body stirring, and revolution is 300rpm, and tank body is online Sterilizing, when culture-liquid temp is down to 37 DEG C in tank, demarcates pH and dissolved oxygen (OD) zero point.Fermentation temperature is 37.0 DEG C ± 0.1 DEG C, Dissolved oxygen controls about 20%, and pH controls 7.0, flow feeding 500mL when cultivating thalline OD600=1.0-1.2 after inoculation, mends Expecting latter 1 hour to add IPTG (final concentration of 0.5mM) abduction delivering, continuous induction after fermentation in 6 hours terminates, and SDS-is in sampling PAGE detects expression.

The thalline that purification will be collected, with inclusion body washing liquid I (1%Triton X-100,20Mm Tris-cl pH 8.0) Carry out ultrasonic after suspendible, 2000W ultrasonic degradation 1 hour.4 DEG C, the centrifugal inclusion body of collecting of 12000rpm, and by inclusion body washing liquid II (1%DOC, 4M carbamide, 20mM Tris-cl pH 8.0) suspendible twice ultrasonic washing inclusion body, secondary low-temperature centrifugation collects bag Contain body.Inclusion body precipitation 8M carbamide, 0.3% β-ME, 20mMTris-cl (pH=8.0) mixing, it is stirred at room temperature 4 hours, 8000rpm low-temperature centrifugation 30 minutes, discards precipitation.Denatured protein 1: 100 dilutes, renaturation solution Tris (pH=8.0) buffer body System, adds 0.3M arginine, and 4 DEG C are stirred renaturation 24 hours.The 20mM phosphate buffer of renaturation solution pH=8.0,0.5M chlorination Sodium, 20mM imidazoles, affinity column in balance, with the 20mM phosphate buffer of pH=8.0,0.5M sodium chloride, 0.5M imidazoles is washed De-.Again with 1.5M ammonium sulfate, the upper hydrophobic chromatography post of 10mM disodium hydrogen phosphate balance of 100mM EDTA, pH=8.5, reequilibrate, With the 10mM disodium hydrogen phosphate eluting of pH=8.5, obtain pig circular ring virus subunit vaccine semi-finished product stock solution, carry out SDS-PAGE And Western-blot marking calibrating purified product whether for the purpose of albumen.

The semi-finished product stock solution sterilizing PBS of purification is diluted to 200 μ g/mL by emulsifying.Take import white oil mineral oil adjuvant DUOPRIME (pharmaceutical grade) was through 121 DEG C of sterilizings 15 minutes, standby.By oil phase: aqueous phase=50: the proportions of 50, first by oil phase Add in emulsion tank, start blender and be slowly stirred with the speed of 80-100r/min, be slowly added into aqueous phase, after adding, be stirred for 2 Minute, then with 5500r/min high-speed circulating emulsifying 9 minutes, make the single-phase vaccine of Water-In-Oil.

Embodiment four pig circular ring virus subunit inactivated vaccine safety experiment

Material

Vaccine: pig circular ring virus subunit inactivated vaccine, lot number 20150302,20150304,20150306, company research and develop Center provides.

Experimental animal: 8 week old BaIb/c white mice are pleased laboratory animal purchased from Jinan friend and breed company limited.28 ages in days are healthy Three way cross ablactational baby pig is provided by the pharmacy of Yongshun, Guangdong.

Method

The vaccine safety to white mice

40 8 week old BaIb/c white mice are randomly divided into 3 sets of batches and 1 matched group, 10/group.Batch component The pig circular ring virus subunit inactivated vaccine of other 3 different batches of subcutaneous injection, 0.5mL/ is only.Matched group subcutaneous injection physiology salt Water white oil Emulsion 0.5mL/ is only.Continuous Observation 14 days, the health status of observation white mice.

The vaccine safety to piglet

20 28 age in days health three way cross ablactational baby pig are randomly divided into 3 sets of batches and 1 matched group, 5/group. The pig circular ring virus subunit inactivated vaccine of sets of batches 3 different batches of auricularis posterior meat injection respectively, 2mL/ head.Matched group is injected Normal saline white oil Emulsion 2mL/ is only.Continuous Observation 14 days, the health status of observation piglet.

Result of the test

The vaccine safety testing to white mice

The results are shown in Table 1, after immunity, all there is not any anaphylaxis or poisoning symptom, the mental status in all test mices Well, body temperature, search for food, drinking-water etc. all goes well, and obvious local inflammation equivalent damage clinical side reaction does not occurs, send out without dead Raw, consistent with matched group, show that pig circular ring virus subunit inactivated vaccine is safe to white mice.

Table 1 vaccine safety testing result to white mice

Group

Size of animal

Body temperature

Appetite

Spirit

Health status

Dead quantity

20150302

10

Normally

Well

0

20150304

10

Normally

Well

0

20150306

10

Normally

Well

0

Matched group

10

Normally

Well

0

The vaccine safety testing to piglet

Result such as table 2, in the whole observation period, the piglet body temperature of all immunity and appetite are all normal, and the mental status is good, not Any clinical abnormal phenomena occur, immunity position does not finds allergy or inflammation, occurs without dead, and sets of batches is consistent with matched group, Show that pig circular ring virus subunit inactivated vaccine is safe to ablactational baby pig.

Table 2 vaccine safety testing result to piglet

Group

Size of animal

Body temperature

Appetite

Spirit

Health status

Dead quantity

20150302

5

Normally

Well

0

20150304

5

Normally

Well

0

20150306

5

Normally

Well

0

Matched group

5

Normally

Well

0

Embodiment five subunit vaccine and traditional vaccine immune efficacy contrast test

Vaccine: pig circular ring virus subunit inactivated vaccine, lot number 20150302,20150304,20150306, company research and develop Center provides.Commercialization inactivated vaccine is provided by the pharmacy of Yongshun, Guangdong.

Experimental animal: 8 week old BaIb/c white mice are pleased laboratory animal purchased from Jinan friend and breed company limited.

Method

25 8 week old BaIb/c white mice are randomly divided into 5 groups, 3 vaccine immunity groups, 1 commercialization inactivated vaccine comparison Group and 1 blank group, 5/group.According to packet situation, test white mice being carried out immunity, subcutaneous injection 0.2ml/ is only, first After exempting from 21 days, Isodose same method booster immunization is once.Respectively before exempting from and head exempt from after 7,14,21,28,35,42,49, Docking blood sampling in 56 days, separates serum and is used for antibody test, and after last blood sampling, slaughter mice, separate mice spleen cell and use In T lymphocyte proliferation assay.

Embodiment six indirect ELISA detection specific antibody

The CBS (pH9.6) of the PCV recombinant protein antigen 50mmol/L of purification is diluted to 1 μ g/mL, adds ELISA Plate In, 100 μ L/ holes, 4 DEG C of standings are coated overnight；Remove liquid, wash plate three times with PBST (containing 0.05%Tween-20, pH7.4), Add confining liquid (containing the PBST of 5% little horse serum), 100 μ L/ holes, close 1 hour for 37 DEG C；Wash plate three times, use serum dilution (containing the PBST of 5% little horse serum) is by measuring samples 1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1 : 12800 times of dilutions, add ELISA Plate, 100 μ L/ holes, do negative control with the serum of immunity PBS simultaneously, hatch 1 hour for 37 DEG C； Wash plate three times, add the HRP labelling sheep anti-mouse igg of 1: 5000 dilution, 100 μ L/ holes, hatch 1 hour for 37 DEG C；Wash plate four times, add Entering tmb substrate 100 μ L/ hole, lucifuge develops the color 15 minutes；Add 2MH₂SO₄Terminate reaction, under 450nm wavelength, detect absorbance (BIORAD 680 microplate reader).

Result of the test:

Such as Fig. 5, exempt from latter 14 days, the mouse serum of vaccine immunity group and commercialization inactivated vaccine matched group starts to detect Specific antibody, then antibody horizontal constantly raises, and 35 days after head exempts from peak, and the most stably decline, are continued for To off-test.Immune group and blank group are pole significant difference (P ＜ 0.01).Result shows the pig annulus that the present invention provides Viral sub-units inactivated vaccine can stimulate and produces stronger humoral immune reaction in Mice, and antibody horizontal and existing goods Change inactivated vaccine suitable, without significant difference (P ＞ 0.05).

Embodiment seven T lymphopoiesis detects

Head exempts from latter 56 days aseptic collection spleens and carries out T lymphocyte proliferation assay, after detecting subunit vaccine immunity Cell immune response.Mouse boosting cell is prepared as cell suspension (2 × 10⁵Individual/100 μ L), add in 96 porocyte versions, 100 μ L/ hole；It is subsequently added culture medium mixing, 100 μ L/ holes；With canavaline (5 μ g/mL, sigma) as positive control, each The repetition of 3, sample；37 DEG C, 5%CO₂Hatch 72 hours；Every hole adds 20 μ L MTS, hatches 5 hours；Enzyme-linked immunosorbent assay instrument Reading under (BIORAD 680 microplate reader) 490nm wavelength.Stimulation index (SI) meansigma methods in antigen stimulated cells hole compares cell The meansigma methods in (stimulation) hole calculates.

Result of the test:

Such as Fig. 6, subunit vaccine immune group observed the proliferation of splenocytes of higher level, hence it is evident that higher than commercialization Inactivated vaccine group and matched group (P ＜ 0.05), and no significant difference (P ＞ 0.05) between three batches.Show what the present invention provided Subunit vaccine can strengthen cell immune response at experimental animal Immune inducing in vivo.

Embodiment eight subunit vaccine protest test

Experimental animal: 28 Yongshun, Guangdong, age in days health three way cross ablactational baby pig mountain pharmacy provide.

Method

25 28 age in days piglets are randomly divided into 5 groups, 3 vaccine immunity groups, 1 commercialization inactivated vaccine matched group and 1 Matched group, 5/group.According to packet situation, test piglet being carried out immunity, auricularis posterior meat injection 2ml/ is only, after head exempts from 21 days, same Deng dosage same method booster immunization once, within the 35th day, PCV2-LG strain (10 is used^4.0TCID₅₀/ ml) carry out counteracting toxic substances.Observe and The morbidity of record test piglet and death condition.Within after counteracting toxic substances continuous 14 days, observing the mental status of swinery, perineal position and extremity is No there is symptom, death condition, and statistics death toll also calculates mortality rate.

Protective rate=[(counteracting toxic substances matched group mortality rate-vaccine immunity group mortality rate)/counteracting toxic substances matched group mortality rate] × 100%.

Result of the test:

Result such as table 3,5 the whole morbidities of counteracting toxic substances matched group, wherein 4 death；Commercialized vaccine group 2 hair is sick, wherein One death；In subunit vaccine group 20150302 groups and 20150306 groups to be respectively arranged with a hair sick, wherein 20150306 groups one Death, 20150304 groups without morbidity, without dead, protective rate is not less than commercialization inactivated vaccine.

The table 3 pig annulus subunit inactivated vaccine protective effect to piglet

Group	Morbidity number	Death toll	Protective rate (n=5)
				20150302	1	0	100%
20150304	0	0	100%
				20150306	1	1	75%
Commercialization inactivated vaccine	2	1	75%
				Matched group	5	4	-

Claims

1., for a fusion protein for immunity, its aminoacid sequence is as shown in SEQ ID No.2.

2. a nucleic acid molecules, its coding fusion protein described in claim 1, nucleotide sequence is as shown in SEQ ID No.1.

3. a carrier, it contains the nucleic acid molecules described in claim 2.

4. a host cell, it contains the carrier described in claim 3.

5. the method preparing fusion protein described in claim 1, it includes with the host cell expression described in claim 4 Fusion protein described in claim 1, and the fusion protein described in separation.

6. for preventing the pharmaceutical composition of Infection of Porcine circovirus, it include fusion protein described in claim 1 with And pharmaceutically acceptable carrier.

7. the application in preparing pig circular ring virus subunit inactivated vaccine of the fusion protein described in claim 1.