CN104693310A - Chimeric protein, virus-like particle and application thereof - Google Patents
Chimeric protein, virus-like particle and application thereof Download PDFInfo
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- CN104693310A CN104693310A CN201510086266.7A CN201510086266A CN104693310A CN 104693310 A CN104693310 A CN 104693310A CN 201510086266 A CN201510086266 A CN 201510086266A CN 104693310 A CN104693310 A CN 104693310A
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Abstract
The invention provides a chimeric protein, a virus-like particle and application thereof and belongs to the field of molecular biology. The chimeric protein is prepared by the following steps: replacing one, two or three of four sites in a porcine circovirus 2 type Cap protein by a main B cell epitope of a porcine O-shaped foot-and-mouth disease virus VP1 protein to obtain 58th-66th amino acid residues, 72nd-94th amino acid residues, 122nd-147th amino acid residues and 162nd-197th amino acid residues. The chimeric protein can form the chimeric virus-like particle after soluble expression, shows the main B cell epitope of the porcine O-shaped foot-and-mouth disease virus VP1 protein on the surface of the chimeric virus-like particle, has very good immunogenicity to both PCV2 and FMDV and can generate a high antibody to the PCV2 and the FMDV, by once immune.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of chimeric protein, virus-like particle and application thereof.
Background technology
Porcine circovirus 2 type (
porcine circovirus type 2pCV2) be the main pathogen causing pmws (PWMS), it not only can cause weanling pig generation exhaustion, death, also miscarry with piglet A2 type congenital tremors (AII, CT), farrowing sow, Adult Pig dermatitis nephritic syndrome (PDNS) and prdc (PRDS) closely related.Since Canadian reported first PMWS in 1991, this disease has now involved all over the world, popular very serious in China swinery, causes great financial loss to whole world pig industry.In international hyothere medical university in 2010 meeting (IPVS), porcine circovirus type 2 infection becomes the highest disease of attention rate, is one of Important Infectious Diseases of the harm pig industry that the whole world is generally acknowledged.In recent years, domestic porcine circovirus type 2 infection is in rising trend.
Pig O type foot and mouth disease (FMD) is that the one of the pig caused by O type foot and mouth disease virus (FMDV) is acute, hot, high degree in contact sexually transmitted disease.Except infected pigs, also can infected cattle, sheep etc.
artiodactyl.This disease is propagated rapidly, and sickness rate is high, is
oIE(OIE) regulation must be reported
transmissible diseaseone of,
chinastatutory regulation is
one class animal epidemic.This disease not only causes cub dead, and production performance declines, and has a strong impact on domestic animal and livestock product circulation and trade activity.The artiodactyls such as foot and mouth disease harm ox, pig, sheep, to propagate rapidly, infection rate is high and famous, and International Office of Epizootics is classified as first of category-A transmissible disease.The Main Antigenic of the GH loop of the VP1 protein surface of O type foot and mouth disease virus (FMDV) to be VP1 albumen be also FMDV, it comprises continuity B cell antigen epi-position, this epi-position can induce body to produce the neutralizing antibody of high-titer, is the first-selected target of development foot and mouth disease epiposition vaccine.
Both there is no the bigeminy conventional vaccine of commercial porcine circovirus 2 type and O type foot and mouth disease in the market, do not had about two sick recombinant vaccines yet.Therefore, in order to prevent pig to infect above-mentioned two kinds of diseases, needing to inoculate corresponding vaccine respectively, at least needing inoculation twice, workload is large, and labor cost is higher, and efficiency is low.
Summary of the invention
The object of the invention is to provide a kind of chimeric protein, Hybrid virus like particles can be formed after solubility expression, by the major B-cell epitope display of pig O type FMDV VP1 albumen to the surface of Hybrid virus like particles, to PCV2 and FMDV, all there is good immunogenicity.
Another object of the present invention is to provide the encoding gene of described chimeric protein.
Another object of the present invention is to provide Hybrid virus like particles, by the major B-cell epitope display of pig O type FMDV VP1 albumen to the surface of virus-like particle, all has good immunogenicity to PCV2 and FMDV.
Another object of the present invention is to provide porcine circovirus 2 type and O type foot and mouth disease bigeminy vaccine, to PCV2 and FMDV, all there is good immunogenicity, only to after pig inoculation once, just can produce the higher antibody horizontal for PCV2 and FMDV simultaneously, greatly reduce workload, cost is low.
Object of the present invention adopts following technical scheme to realize.
The invention provides a kind of chimeric protein, is obtain after selecting in carrying Cap gene of porcine circovirus type 2 in following four sites one, two or three to replace with pig O type FMDV VP1 albumen major B-cell epi-position:
(1) 58-66 amino acids residue;
(2) 72-94 amino acids residues;
(3) 122-147 amino acids residues;
(4) 162-197 amino acids residues.
In the present invention, the aminoacid sequence of described carrying Cap gene of porcine circovirus type 2 is as shown in SEQ ID NO:9; The aminoacid sequence of described pig O type FMDV VP1 albumen major B-cell epi-position is as shown in SEQ ID NO:10.
The invention provides the encoding gene of described chimeric protein, nucleotide sequence is as shown in one of SEQ ID NO:3-8.
The invention provides the recombinant vectors containing described gene; In preferred technical scheme, described recombinant vectors is that described gene inserts expression vector gained; Described expression vector is preferably pTXB1.
The present invention also provides the recombinant bacterium containing described gene, is the recombinant vectors containing described gene is imported the recombinant bacterium obtained in Host Strains; Described recombinant vectors is that described gene is inserted expression vector pTXB1 gained.
The present invention also provides a kind of Hybrid virus like particles, is formed by described chimeric protein self assembly, and described Hybrid virus like particles surface display has pig O type FMDV VP1 albumen major B-cell epi-position.
In the present invention, described Hybrid virus like particles is adopted and is prepared with the following method: induce described recombinant bacterium to express chimeric protein, cracking recombinant bacterium, gets lysate supernatant, adopts ammonium sulfate precipitation method purifying, obtains described Hybrid virus like particles.
The present invention also provides porcine circovirus 2 type and O type foot and mouth disease virus bigeminy vaccine, and activeconstituents is described Hybrid virus like particles.
In the present invention, term " 206 adjuvant " refers to the adjuvant of Montanide ISA 206 series that match Bick (SEPPIC) company of France produces, comprise Montanide ISA 206, ISA 206VG etc., this adjuvant belongs to oil emulsion adjuvant, is water-in-oil-in water (w/o/w) immunological adjuvant that a class contains octadecanoic acid and anhydrous Nitranitol.
The present invention select pig O type foot and mouth disease VP1 albumen major B-cell epitope gene (GH loop) take the mode of gene substitution replace in 4 loop rings (corresponding to four sites) of PCV2 Cap protein encoding gene inside one, two or three, make restructuring chimer protein molecule inside containing 1,2 or 3 FMDV VP1 albumen major B-cell epi-positions, namely obtain chimeric protein of the present invention.The chimeric protein of several solubility expressions, can become Hybrid virus like particles by self assembly in vitro, by the surface of B cell epitope display main for pig O type FMDV VP1 albumen to Hybrid virus like particles.Hybrid virus like particles of the present invention all has good immunogenicity to PCV2 and FMDV.The preparation method of Hybrid virus like particles of the present invention is simple, and cost is low.
The porcine circovirus 2 type being activeconstituents with Hybrid virus like particles of the present invention and O type foot and mouth disease bigeminy vaccine, to PCV2 and FMDV, all there is good immunogenicity, only need primary immune response, just can produce in pig body higher for PCV2 and FMDV antibody horizontal, especially by replacing the Hybrid virus like particles inserting chimeric protein that two pig O type FMDV VP1 albumen major B-cell epi-positions obtain and formed in PCV2 Cap protein, create the antibody horizontal being significantly higher than commercialization PCV2 and FMDV vaccine after immune swine, achieve beyond thought technique effect.In addition, can find from the present invention, when in PCV2 Cap protein, 3 sites replace with other epitope simultaneously, still Hybrid virus like particles can be formed, although poor to the immunogenicity of PCV2 Cap protein, but have extraordinary immunogenicity to other epitope, therefore, PCV2 Cap protein can as a kind of carrier efficiently carrying epitope.
Accompanying drawing explanation
Fig. 1 is that recombinant plasmid enzyme cuts qualification figure, 1:Marker; 2-5: recombinant plasmid pTXB-Cap-loop1, pTXB-Cap-loop2, pTXB-Cap-loop3 and pTXB-Cap-loop4 double digestion product.
Fig. 2 is that recombinant plasmid pTXB-Cap-loop2-loop4 enzyme cuts qualification figure, 1:Marker; 2,3: recombinant plasmid pTXB-Cap-loop2-loop4 double digestion product.
Fig. 3 is that recombinant plasmid pTXB-Cap-loop2-loop3-loop4 enzyme cuts qualification figure, 1:Marker; 2,3: recombinant plasmid pTXB-Cap-loop2-loop3-loop4 double digestion product.
Fig. 4 is chimeric protein Cap-loop1 soluble analysis result, 1:marker; 2-4 is respectively the rear full bacterium of pTXB-Cap-loop1/BL21 induction, the rear lysate supernatant of induction, the rear lysate precipitation of induction.
Fig. 5 is chimeric protein Cap-loop2 soluble analysis result, 1:marker; 2-4 is respectively: lysate precipitation after full bacterium, the rear lysate supernatant of induction, induction after pTXB-Cap-loop2/BL21 induction; 5: control strain pTXB1/BL21 after induction.
Fig. 6 is chimeric protein Cap-loop3 soluble analysis result, 1:marker; 2-4 is respectively: lysate precipitation after full bacterium, the rear lysate supernatant of induction, induction after pTXB-Cap-loop3/BL21 induction; 5: control strain pTXB1/BL21 after induction.
Fig. 7 is chimeric protein Cap-loop4 soluble analysis result, 1:marker; 2-4 is respectively: lysate precipitation after full bacterium, the rear lysate supernatant of induction, induction after pTXB-Cap-loop4/BL21 induction; 5: control strain pTXB1/BL21 after induction.
Fig. 8 is chimeric protein Cap-loop2-loop4 soluble analysis result, 1:marker; 2-4 is respectively: lysate precipitation after full bacterium, the rear lysate supernatant of induction, induction after pTXB-Cap-loop2-loop4/BL21 induction.
Fig. 9 is chimeric protein Cap-loop2-loop3-loop4 soluble analysis, 1:marker; 2-4 is respectively: lysate precipitation after full bacterium, the rear lysate supernatant of induction, induction after pTXB-Cap-loop2-loop3-loop4/BL21 induces; 5: control strain pTXB1/BL21 after induction.
To be chimeric protein Cap-loop1 identify for the Western-blot of PCV2 Figure 10,1: control strain pTXB/BL21; 2-4 is respectively: full bacterium after lysate precipitation, the rear lysate supernatant of induction, induction after pTXB-Cap-loop1/BL21 induction; 5:marker.
To be chimeric protein Cap-loop2 identify for the Western-blot of PCV2 Figure 11,1:Marker; 2: control strain pTXB/BL21; 3-4 is respectively: full bacterium lysate supernatant after full bacterium, induction after pTXB-Cap-loop2/BL21 induces.
To be chimeric protein Cap-loop3 identify for the Western-blot of PCV2 antibody Figure 12,1:Marker; 2-3 is respectively: lysate supernatant after full bacterium, induction after pTXB-Cap-loop3/BL21 induction.
To be chimeric protein Cap-loop4 identify for the Western-blot of PCV2 antibody Figure 13,1:Marker; After 2-3 is respectively pTXB-Cap-loop4/BL21 induction, after lysate supernatant, induction, lysate precipitates.
To be chimeric protein Cap-loop2-loop4 identify for the Western-blot of PCV2 Figure 14, and 1-2 is respectively lysate supernatant after lysate precipitation after pTXB-Cap-loop2-loop4/BL21 induction, induction; 3:Marker.
Figure 15 is that chimeric protein Cap-loop2-loop3-loop4 albumen is identified for PCV2 Western-blot, 1:Marker; After 2-3 is respectively pTXB-Cap-loop2-loop3-loop4/BL21 induction, after lysate supernatant, induction, lysate precipitates.
To be chimeric protein Cap-loop1 identify for the Western-blot of FMDV antibody Figure 16,1:Marker; 2-3 is full bacterium, the rear lysate supernatant of induction after being respectively pTXB-Cap-loop1/BL21 induction.
To be chimeric protein Cap-loop2 identify for the Western-blot of FMDV antibody Figure 17,1:Marker; 2: control strain pTXB1/BL21 after induction; After 3-5 is respectively pTXB-Cap-loop2/BL21 induction, lysate precipitates, induces rear full bacterium, the rear lysate supernatant of induction.
To be chimeric protein Cap-loop3 identify for the Western-blot of FMDV antibody Figure 18,1:Marker; 2-4 is respectively the rear full bacterium of pTXB-Cap-loop3/BL21 induction, the rear lysate supernatant of induction, the rear lysate precipitation of induction.
To be chimeric protein Cap-loop4 identify for the Western-blot of FMDV antibody Figure 19,1:Marker; 2-4 is respectively the rear full bacterium of pTXB-Cap-loop4/BL21 induction, the rear lysate supernatant of induction, the rear lysate precipitation of induction.
To be chimeric protein Cap-loop2-loop4 identify for the Western-blot of FMDV antibody Figure 20,1:Marker; 2-4 is respectively the rear full bacterium of pTXB-Cap-loop2-loop4/BL21 induction, the rear lysate supernatant of induction, the rear lysate precipitation of induction.
To be chimeric recombinant protein Cap-loop2-lop3-loop4 identify for the Western-blot of FMDV antibody Figure 21,1:marker; 2-4 is respectively pTXB-Cap-loop2-lop3-loop4/BL21 and induces rear full bacterium, the rear full bacterium lysate supernatant of induction, induces full bacterium lysate to precipitate.
Figure 22-25 is the Electronic Speculum figure of the chimeric VLP that chimeric protein Cap-loop1, Cap-loop2, Cap-loop3, Cap-loop4 are formed respectively.
Figure 26-27 is the Electronic Speculum figure of the chimeric VLP that chimeric protein Cap-loop2-loop4, Cap-loop2-loop3-loop4 are formed respectively.
Figure 28 is the mean antibody levels for PCV2 Cap protein after each group of pig immunity.
Figure 29 is the mean antibody levels for FMDV VP1 albumen after each group of pig immunity.
Embodiment
Embodiment 1 contains chimeric protein and the virus-like particle of 1 FMDV VP1 albumen major B-cell epi-position
1, the design of chimeric protein and the structure of recombinant plasmid
The aminoacid sequence of carrying Cap gene of porcine circovirus type 2 is as shown in SEQ ID NO:9.The aminoacid sequence of FMDV VP1 albumen major B-cell epi-position is as shown in SEQ ID NO:10.The encoding gene of carrying Cap gene of porcine circovirus type 2 is as shown in SEQ ID NO:1.Pig O type FMDV VP1 albumen major B-cell epi-position encoding gene is as shown in SEQ ID NO:2.Contriver utilizes the higher structure of software analysis PCV2 Cap protein, devises following 4 chimeric proteins and encoding gene thereof respectively:
(1) chimeric protein Cap-loop1: replace with 1 FMDV VP1 albumen major B-cell epi-position to the 66th amino acids residue by the 58th of carrying Cap gene of porcine circovirus type 2 the.The sequence of chimeric protein Cap-loop1 encoding gene is as shown in SEQ ID NO:3.
(2) chimeric protein Cap-loop2: replace with 1 FMDV VP1 albumen major B-cell epi-position to the 94th amino acids residue by the 72nd of carrying Cap gene of porcine circovirus type 2 the.The sequence of chimeric protein Cap-loop2 encoding gene is as shown in SEQ ID NO:4.
(3) chimeric protein Cap-loop3: replace with 1 FMDV VP1 albumen major B-cell epi-position to the 147th amino acids residue by the 122nd of carrying Cap gene of porcine circovirus type 2 the.The sequence of chimeric protein Cap-loop3 encoding gene is as shown in SEQ ID NO:5.
(4) chimeric protein Cap-loop4: replace with 1 FMDV VP1 albumen major B-cell epi-position to the 197th amino acids residue by the 162nd of carrying Cap gene of porcine circovirus type 2 the.The sequence of chimeric protein Cap-loop4 encoding gene is as shown in SEQ ID NO:6.
The encoded chimeric protein gene of sequence as shown in SEQ ID NO:3 ~ SEQ ID NO:6 is synthesized by Ying Jun Bioisystech Co., Ltd and is cloned into prokaryotic expression carrier pTXB1's respectively
ndei with
xhobetween I restriction enzyme site, obtain the recombinant plasmid inserting each gene.
2, the qualification of recombinant expression vector
The recombinant plasmid that the present embodiment title 1 obtains is transformed respectively
e.colidH5 α competent cell, coating is dull and stereotyped containing the LB of penbritin, 37 DEG C of cultivations.Single bacterium colony on picking flat board, cultivates in the LB substratum containing penbritin, extracts plasmid, warp
ndei with
xhoi double digestion qualification.Double digestion system: 10 × H buffer 2 μ l,
ndei 0.5 μ l,
xhoi 0.5 μ l, recombinant plasmid 3 μ l, dH
2o supplies volume to 20 μ l.Double digestion reaction system is acted on 45min under 37 DEG C of conditions, then identifies digestion products with agarose gel electrophoresis.
As shown in Figure 1, positive recombinant plasmid enzyme cut after electrophorogram in there is the object band that size is about about 5000bp and 700bp.The name of positive recombinant plasmid is as table 1.
The name of table 1 recombinant plasmid
| Recombinant plasmid is numbered | Insert gene order |
| pTXB-Cap-loop1 | SEQ ID NO:3 |
| pTXB-Cap-loop2 | SEQ ID NO:4 |
| pTXB-Cap-loop3 | SEQ ID NO:5 |
| pTXB-Cap-loop4 | SEQ ID NO:6 |
3, the structure of recombinant expressed bacterium
By the transform competent E. coli BL21 respectively of recombinant plasmid in table 1, coating is dull and stereotyped containing amicillin resistance LB, picking list bacterium colony, cultivation, upgrading grain, enzyme cut qualification, and positive recombinant bacterium respectively called after pTXB-Cap-loop1/BL21(contains pTXB-Cap-loop1), pTXB-Cap-loop2/BL21(contains pTXB-Cap-loop2), pTXB-Cap-loop3/BL21(contains pTXB-Cap-loop3) contain pTXB-Cap-loop4 with pTXB-Cap-loop4/BL21(); By empty carrier pTXB1(purchased from American NEB company) transform competent E. coli BL21, obtain positive recombinant bacterium, called after control strain pTXB1/BL21.
4, the abduction delivering of recombinant protein
Induce the recombinant bacterium carrying goal gene in the present embodiment title 3 to express chimeric protein respectively, negative control is control strain pTXB1/BL21.
(1) single bacterium colony of picking recombinant bacterium, is inoculated in the LB liquid nutrient medium (containing 10g/L Tryptones, 5g/L yeast powder, 10g/LNaCl) containing penbritin (100 μ g/mL), 37 DEG C of incubated overnight.
(2) get the recombinant bacterium nutrient solution 100 μ l that step (1) obtains, be inoculated in 5ml and contain in the LB liquid nutrient medium of penbritin (100 μ g/mL), 37 DEG C, shaking culture is about 2h under 220rpm condition, makes OD
600reach 1.0 ~ 2.0.
(3) to OD
600reach in the bacterium liquid of 1.0 ~ 2.0 and add the IPTG that final concentration is 0.1mmol/L, carry out abduction delivering, inducing temperature is 15 DEG C, and induction time is 24h, obtains fermented liquid.
(4) fermented liquid step (3) obtained, at 4 DEG C, centrifugal 10min under 12000rpm condition, collects thalline; Carry out resuspended after thalline is washed 2 times with PBS damping fluid (0.05M, pH7.2).
(5) thalline suspension is placed in ice bath, uses ultrasonic treatment thalline, power is 200W, ultrasonic degradation 5s, pause 5s, until the clarification of bacterium liquid, obtains cellular lysate liquid.
(6) by cellular lysate liquid, at 4 DEG C, centrifugal 15min under 12000rpm condition, cleer and peaceful lysate precipitation on lysate is collected respectively.By lysate precipitation with resuspended with supernatant isopyknic PBS damping fluid (0.05M, pH7.2).
5, the qualification of chimeric protein
After getting the recombinant bacterium induction of carrying goal gene respectively, after full bacterium, induction, after lysate supernatant, induction, lysate precipitation and control strain pTXB1/BL21 carry out SDS-PAGE electroresis appraisal.As can be seen from Fig. 4, Fig. 5, Fig. 6, after carrying the recombinant bacterium induction of Cap-loop1, Cap-loop2, Cap-loop3 encoding gene, full bacterium and lysate supernatant swimming lane are located all to deposit an obvious band at about 27kDa, and after the induction of corresponding recombinant bacterium, lysate precipitation swimming lane and control strain pTXB1/BL21 swimming lane do not have band at 27kDa place; As can be seen from Figure 7, after carrying the recombinant bacterium induction of Cap-loop4 encoding gene, full bacterium and lysate supernatant swimming lane are located all to deposit an obvious band at about 27kDa, and after the induction of this recombinant bacterium, lysate precipitation swimming lane only exists very faint band at 27kDa place.The above results further demonstrates, and chimeric protein Cap-loop1 ~ Cap-loop4 successfully obtains expression, and is present in recombination bacillus coli kytoplasm with solubility or almost soluble form.
6. Western-blot qualification restructuring chimeric protein is for the reactionogenicity of PCV2
1. after getting recombinant bacterium pTXB-Cap-loop1/BL21, pTXB-Cap-loop2/BL21, pTXB-Cap-loop3/BL21 and pTXB-Cap-loop4/BL21 induction respectively, after lysate supernatant, induction, lysate precipitation and control strain pTXB1/BL21 carry out SDS-PAGE electrophoresis.
2. transfer printing: after SDS-PAGE electrophoresis, adopts half-dried transfer printing Western blot to be gone to pvdf membrane (PVDF membrane).
3. by the aqueous solution of pvdf membrane confining liquid (being containing 6.05g/L Tris-base(Tutofusin tris), 8.79g/L NaCl, 0.5g/LTween-20 and 50g/L skimming milk) close, be placed in 40min under room temperature condition.
4. by close after pvdf membrane immerse 1:200 dilution porcine circovirus 2 type positive serum (purchased from Veterinary Medical Research & Development, American), 4 DEG C of overnight incubation.
5. TBST damping fluid (being the aqueous solution containing 6.05g/L Tris-base, 8.79g/L NaCl and 0.5g/L Tween-20) is used to wash pvdf membrane 5 times, 8min/ time.
6. pvdf membrane is immersed the goat-anti pig antibody (Nanjing Sheng Xing Bioisystech Co., Ltd) of the horseradish peroxidase-labeled of 1:5000 dilution, incubated at room 1h.
7. TBST buffer solution pvdf membrane 5 times are used, 5min/ time.
8. horseradish peroxidase DAB colouring reagents box (Wuhan doctor's moral company) is used to develop the color.
The results are shown in Figure 10, Figure 11, Figure 12, Figure 13.As can be seen from Figure 10, Figure 11, Figure 12, Figure 13, after only carrying the recombinant bacterium induction of goal gene, full bacterium and lysate supernatant swimming lane all deposit an obvious band at 27kDa place, 4 restructuring chimeric proteins that the recombinant bacterium showing to carry goal gene is expressed all can react with porcine circovirus 2 type positive serum, have good antigenicity.
7. Western-blot identifies the reactionogenicity of chimeric protein for FMDV
Adopt Western-blot qualification carry the recombinant bacterium pTXB-Cap-loop1/BL21 of goal gene, pTXB-Cap-loop2/BL21, pTXB-Cap-loop3/BL21 and pTXB-Cap-loop4/BL21 expression chimeric protein for the reactionogenicity of FMDV.Western-blot authentication method is with the present embodiment title 6 herein, only by step 4. in " 1:200 dilution porcine circovirus 2 type positive serum " change into " the foot and mouth disease polypeptide vaccine positive serum (purchased from Veterinary Medical Research & Development, American) of 1:200 dilution ".
The results are shown in Figure 16, Figure 17, Figure 18, Figure 19.As can be seen from Figure 16, Figure 17, Figure 18, Figure 19, after only carrying the recombinant bacterium induction of goal gene, full bacterium and lysate supernatant swimming lane all deposit an obvious band at about 27kDa, show that 4 restructuring chimeric protein Cap-loop1 ~ Cap-loop4 that recombinant bacterium is expressed all can react with pig O type foot and mouth disease virus positive serum, there is good antigenicity.
8, the electron microscopic observation of restructuring chimeric protein Cap-loop1 ~ Cap-loop4
Lysate supernatant after recombinant bacterium pTXB-Cap-loop1/BL21, pTXB-Cap-loop2/BL21, pTXB-Cap-loop3/BL21 and pTXB-Cap-loop4/BL21 induction is adopted PBS damping fluid respectively, and (concentration is 0.05M, pH7.2) dialyse, then adopt transmission electron microscope observing (dyeing of phosphoric acid tungsten).Result, as Figure 22, Figure 23, Figure 24 and Figure 25, can see that multiple restructuring chimeric protein can become diameter to be the Hybrid virus like particles of about 20nm by self assembly.
Embodiment 2: the chimeric protein containing 2 FMDV VP1 albumen major B-cell epi-positions and the preparation of virus-like particle
The design of the chimeric protein 1, containing 2 FMDV VP1 albumen major B-cell epi-positions and construction of recombinant vector
The aminoacid sequence of carrying Cap gene of porcine circovirus type 2 is as shown in SEQ ID NO:9.The aminoacid sequence of FMDV VP1 protein B cell epitope is as shown in SEQ ID NO:10.On the basis of embodiment 1 result of study, contriver utilizes the higher structure of software analysis PCV2 Cap protein, devises chimeric protein Cap-loop2-loop4: by the 72nd of carrying Cap gene of porcine circovirus type 2 the to the 94th amino acids residue, the 162nd adopt 1 FMDV VP1 albumen major B-cell epi-position to replace respectively to the 197th amino acids residue.The sequence of chimeric protein Cap-loop2-loop4 encoding gene, as shown in SEQ ID NO:7, is synthesized by Ying Jun Bioisystech Co., Ltd and is cloned into prokaryotic expression carrier pTXB1's
ndei with
xhobetween I restriction enzyme site, obtain the recombinant expression plasmid inserting chimeric protein Cap-loop2-loop4 encoding gene.
2, the qualification of recombinant expression vector
Recombinant expression plasmid in the present embodiment title 1 is transformed
e.colidH5 α competent cell, coating is dull and stereotyped containing the LB of penbritin, 37 DEG C of cultivations.Single bacterium colony on picking flat board, cultivates in the LB substratum containing ammonia benzyl paraxin, extracts plasmid, warp
ndei with
xhoi double digestion qualification.Double digestion system: 10 × H buffer 2 μ l,
ndei 0.5 μ l,
xhoi 0.5 μ l, recombinant plasmid 3 μ l, ddH
2o supplies volume to 20 μ l.Double digestion reaction system is acted on 45min under 37 DEG C of conditions, then identifies digestion products with agarose gel electrophoresis.As shown in Figure 2, positive recombinant plasmid enzyme cut after electrophorogram in there is the object band that size is about about 5000bp and 700bp.Positive recombinant plasmid called after pTXB-Cap-loop2-loop4.
3, the structure of recombinant expressed bacterium
By recombinant plasmid pTXB-Cap-loop2-loop4 transform competent E. coli BL21, coating is dull and stereotyped containing ammonia benzyl chlorampenicol resistant LB, and picking list bacterium colony, obtains recombinant bacterium pTXB-Cap-loop2-loop4/BL21; By empty plasmid pTXB1 transform competent E. coli BL21, obtain positive recombinant bacterium, called after control strain pTXB1/BL21.
4, the abduction delivering of recombinant protein
Title 4 method induction recombinant bacterium pTXB-Cap-loop2-loop4/BL21 in embodiment 1 is adopted to express chimeric protein, lysate precipitation after full bacterium, the rear lysate supernatant of induction, induction after acquisition recombinant bacterium pTXB-Cap-loop2-loop4/BL21 induction.Meanwhile, same procedure is adopted to cultivate to control strain pTXB1/BL21.
5, the expression identification of chimeric protein Cap-loop2-loop4
Getting recombinant bacterium pTXB-Cap-loop2-loop4/BL21 respectively induces rear full bacterium, the rear lysate supernatant of induction, the rear lysate precipitation of induction to carry out SDS-PAGE electrophoresis, obtains Fig. 8.As can be seen from Figure 8, after recombinant bacterium pTXB-Cap-loop2-loop4/BL21 induces, after full bacterium, induction, lysate supernatant swimming lane all deposits an obvious band at about 27kDa, and lysate precipitation swimming lane does not have band after recombinant bacterium induction at about 27kDa place, this result further demonstrates chimeric protein Cap-loop2-loop4 and successfully obtains expression, and is present in recombination bacillus coli with the form of solubility.
6. Western-blot identifies the reactionogenicity of chimeric protein Cap-loop2-loop4 for PCV2
After being induced by recombinant bacterium pTXB-Cap-loop2-loop4/BL21, SDS-PAGE electrophoresis is carried out in the empty bacterium contrast of lysate supernatant, lysate precipitation and pTXB1/BL21, then Western-blot qualification is carried out, concrete grammar, with title 6 in embodiment 1, the results are shown in Figure 14.As can be seen from Figure 14, after only having recombinant bacterium pTXB-Cap-loop2-loop4/BL21 to induce, lysate supernatant swimming lane all deposits an obviously band at 27kDa place, show that the chimeric protein that pTXB-Cap-loop2-loop4/BL21 expresses can react with porcine circovirus 2 type positive serum, there is good antigenicity.
7. Western-blot identifies that chimeric protein Cap-loop2-loop4 identifies for the reactionogenicity of FMDV
Recombinant bacterium pTXB-Cap-loop2-loop4/BL21 induced rear full bacterium and lysate supernatant, induce rear lysate precipitation to carry out SDS-PAGE electrophoresis, then carry out Western-blot qualification, concrete grammar is with title 7 in embodiment 1.The results are shown in Figure 20, as can be seen from Figure 20, only have recombinant bacterium pTXB-Cap-loop2-loop4
/after BL21 induction, full bacterium and lysate supernatant swimming lane all deposit an obvious band at 27kDa place, show that the chimeric protein that pTXB-Cap-loop2-loop4/BL21 expresses can react with pig O type foot and mouth disease virus positive serum, have good antigenicity.
8, the Hybrid virus like particles (chimeric VLP) that assembled by chimeric protein Cap-loop2-loop4 of electron microscopic observation
After recombinant bacterium pTXB-Cap-loop2-loop4/BL21 induces, lysate supernatant is after PBS damping fluid (concentration is 0.05M, pH7.2) dialysis, adopts transmission electron microscope observing (dyeing of phosphoric acid tungsten).There is the Hybrid virus like particles be assembled into by multiple chimeric protein in a large number as can be seen from Figure 26 after induction in lysate supernatant, virus-like particle diameter is about 20nm.
Embodiment 3: the chimeric protein containing 3 FMDV VP1 albumen major B-cell epi-positions and the preparation of virus-like particle
1, the mosaic gene containing 3 FMDV VP1 albumen major B-cell epi-positions and qualification
The aminoacid sequence of carrying Cap gene of porcine circovirus type 2 is as shown in SEQ ID NO:9.The aminoacid sequence of FMDV VP1 albumen major B-cell epi-position is as shown in SEQ ID NO:10.In embodiment 1 with on the basis of embodiment 2 result, contriver utilizes the higher structure of software analysis PCV2 Cap protein, devise chimeric protein Cap-loop2-loop3-loop4, by the 72nd of carrying Cap gene of porcine circovirus type 2 the to the 94th amino acids residue, the 122nd adopt 1 FMDV VP1 albumen major B-cell epi-position to replace to the 147th amino acids residue and the 162nd respectively to the 197th amino acids residue.The sequence of chimeric protein Cap-loop2-loop3-loop4 encoding gene, as shown in SEQ ID NO:8, is synthesized by Ying Jun Bioisystech Co., Ltd and is cloned into prokaryotic expression carrier pTXB1's
ndei with
xhobetween I restriction enzyme site, obtain the recombinant expression plasmid inserting chimeric protein Cap-loop2-loop3-loop4 encoding gene.
2, the qualification of recombinant expression vector
Recombinant expression plasmid in the present embodiment title 1 is transformed
e.colidH5 α competent cell, coating is dull and stereotyped containing the LB of ammonia benzyl, 37 DEG C of cultivations.Single bacterium colony on picking flat board, cultivates in the LB substratum containing ammonia benzyl, extracts plasmid, warp
ndei with
xhoi double digestion qualification.Double digestion system: 10 × H buffer 2 μ l,
ndei 0.5 μ l,
xhoi 0.5 μ l, recombinant plasmid 3 μ l, ddH
2o supplies volume to 20 μ l.Double digestion reaction system is acted on 45min under 37 DEG C of conditions, then identifies digestion products with agarose gel electrophoresis.
As shown in Figure 3, positive recombinant plasmid enzyme cut after electrophorogram in there is the band that size is about 5000bp and 850bp two entry.Positive recombinant plasmid called after pTXB-Cap-loop2-loop3-loop4.
3, the acquisition of recombinant expressed bacterium
By recombinant plasmid pTXB-Cap-loop2-loop3-loop4 transform competent E. coli BL21, coating is dull and stereotyped containing the LB of ammonia benzyl chlorampenicol resistant, and picking list bacterium colony, obtains positive recombinant bacterium, called after pTXB-Cap-loop2-loop3-loop4/BL21; By plasmid pTXB1(purchased from American NEB company) transform competent E. coli BL21, obtain control strain pTXB1/BL21.
4, the abduction delivering of chimeric protein
Title 4 method induction recombinant bacterium pTXB-Cap-loop2-loop3-loop4/BL21 in embodiment 1 is adopted to express chimeric protein, lysate precipitation after full bacterium, the rear lysate supernatant of induction, induction after acquisition recombinant bacterium pTXB-Cap-loop2-loop3-loop4/BL21 induction.Meanwhile, same procedure is adopted to cultivate to control strain pTXB1/BL21.
5, the expression identification of restructuring chimeric protein
Getting recombinant bacterium pTXB-Cap-loop2-loop3-loop4/BL21 respectively induces rear full bacterium, the rear lysate supernatant of induction, the rear lysate precipitation of induction to carry out SDS-PAGE electrophoresis, obtains Fig. 9.As can be seen from Figure 9, after recombinant bacterium pTXB-Cap-loop2-loop3-loop4/BL21 induces, full bacterium and the rear lysate supernatant swimming lane of induction all deposit an obvious band at about 27kDa, and recombinant bacterium pTXB-Cap-loop2-loop3-loop4/BL21 induces rear lysate precipitation swimming lane and control strain pTXB1/BL21 swimming lane not to have band at about 27kDa place, the results show that chimeric protein Cap-loop2-loop3-loop4 successfully obtains expression, and be present in recombination bacillus coli with the form of solubility.
6. Western-blot identifies the antigenicity of chimeric protein Cap-loop2-loop3-loop4 for PCV2
After being induced by recombinant bacterium pTXB-Cap-loop2-loop3-loop4/BL21, after lysate supernatant, induction, lysate precipitation carries out SDS-PAGE electrophoresis, and then carry out Western-blot qualification, concrete grammar, with title 6 in embodiment 1, the results are shown in Figure 15.As can be seen from Figure 15, after only having recombinant bacterium pTXB-Cap-loop2-loop3-loop4/BL21 to induce, lysate supernatant swimming lane deposits an obvious band at about 27kDa place, show that the chimeric protein Cap-loop2-loop3-loop4 that pTXB-Cap-loop2-loop3-loop4/BL21 expresses can react with porcine circovirus 2 type positive serum, prompting chimeric protein Cap-loop2-loop3-loop4 has good antigenicity.
7. Western-blot identifies that chimeric protein Cap-loop2-loop3-loop4 identifies for the reactionogenicity of FMDV
After recombinant bacterium pTXB-Cap-loop2-loop3-loop4/BL21 being induced rear full bacterium and induction, lysate supernatant, the rear lysate precipitation of induction carry out SDS-PAGE electrophoresis, then Western-blot qualification is carried out, concrete grammar, with title 7 in embodiment 1, the results are shown in Figure 21.As can be seen from Figure 21, recombinant bacterium pTXB-Cap-loop2-loop3-loop4 is only had
/after BL21 induction, after full bacterium, induction, lysate supernatant deposits an obvious band at 27kDa place, show that the chimeric protein that pTXB-Cap-loop2-loop3-loop4/BL21 expresses can react with pig O type foot and mouth disease virus positive serum, point out this chimeric protein to have good antigenicity.
8, the virus-like particle of electron microscopic observation chimeric protein Cap-loop2-loop3-loop4 self assembly
Recombinant bacterium pTXB-Cap-loop2-loop3-loop4
/after BL21 induction, lysate supernatant is after PBS damping fluid (concentration is 0.05M, pH7.2) dialysis, adopts transmission electron microscope observing (dyeing of phosphoric acid tungsten).As can be seen from Figure 27, there is the Hybrid virus like particles of multiple chimeric protein Cap-loop2-loop3-loop4 self assembly after induction in lysate supernatant, the diameter of Hybrid virus like particles is about 20nm.
Embodiment 4: the preparation of porcine circovirus 2 type and O type foot and mouth disease virus bigeminy vaccine and immune effect
1, the preparation of Hybrid virus like particles
Adopt the rear lysate supernatant of method preparation restructuring pTXB-Cap-loop1/BL21, pTXB-Cap-loop2/BL21, pTXB-Cap-loop3/BL21, pTXB-Cap-loop4/BL21, pTXB-Cap-loop2-loop4/BL21 and pTXB-Cap-loop2-loop3-loop4/BL21 induction in embodiment 1-3.
After getting each recombinant bacterium induction of step acquisition, lysate supernatant carries out purifying: in lysate supernatant, add ammonium sulfate, the saturation ratio of ammonium sulfate in solution is made to reach 18-22%, place l h for 4 DEG C, centrifugal 30 min under l0 000 r/min condition, abandon supernatant, get precipitation PBS damping fluid (concentration is 0.05M, pH7.2) and dissolve, obtain Hybrid virus like particles, the concentration of adjustment chimeric protein is 1mg/mL.
2, porcine circovirus 2 type and O type foot and mouth disease virus bigeminy vaccine are to the immunogenic qualification of PCV2 and FMDV
(1), the preparation of test vaccine
1.. the preparation of test vaccine: each Hybrid virus like particles obtained in adjustment the present embodiment title 1, make its protein concentration be 100 μ g/ml, then respectively with 206 adjuvants according to 1:1(V/V) mix, emulsification, prepare self-control vaccine, name is as table 2.
The name of vaccine respectively made by oneself by table 2
| Self-control vaccine title | Activeconstituents |
| Self-control Cap-loop1 vaccine | The chimeric VLP that chimeric protein Cap-loop1 is formed |
| Self-control Cap-loop2 vaccine | The chimeric VLP that chimeric protein Cap-loop2 is formed |
| Self-control Cap-loop3 vaccine | The chimeric VLP that chimeric protein Cap-loop3 is formed |
| Self-control Cap-loop4 vaccine | The chimeric VLP that chimeric protein Cap-loop4 is formed |
| Self-control Cap-loop2-loop4 vaccine | The chimeric VLP that chimeric protein Cap-loop2-loop4 is formed |
| Self-control Cap-loop2-loop3-loop4 vaccine | The chimeric VLP that chimeric protein Cap-loop2-loop3-loop4 is formed |
2.. setting up of control vaccine: carrying Cap gene of porcine circovirus type 2 (aminoacid sequence is as shown in SEQ ID NO:9) encoding gene is inserted expression vector pTXB1, then transformation of E. coli BL21 competent cell, obtain positive recombinant bacterium, abduction delivering Cap protein.Prepare Cap protein according to the purification process of chimeric protein, then mix with 206 adjuvants, emulsification, obtain self-control PCV2 Cap vaccine.The PCV2 recombinant vaccine (for preventing porcine circovirus type 2 infection) that PCV2 comparable product adopts Bo Linge company baculovirus expression to prepare; Foot and mouth disease comparable product is the Schweineseuche synthetic peptide vaccine (for preventing pig O type mouth disease virus infection) that UNT bio tech ltd, Shanghai (UBI) produces; Set up negative control simultaneously.
(2) vaccine, is made by oneself to the immunogenicity of experiment pig
1.. the screening of test pig: select 2-3 sodium selenite in age in week 50, porcine circovirus 2 type ELISA antibody assay kit (Wuhan Ke Qian Bioisystech Co., Ltd) is utilized to detect PCV2 antibody before test, utilize the PCR antigen detection kit of PCV2 (Beijing Century Yuan Heng Bioisystech Co., Ltd) to detect PCV2 specific nucleic acid simultaneously, guarantee that every test pig is that PCV2 antigen, antibody are double-negative.
2.. vaccine immunity: test pig double-negative to 50 PCV2 antigens, antibody is divided into 10 groups at random, is labeled as H1-H10, often organize 5.H1 group immunity self-control Cap-loop1 vaccine; H2 group immunity self-control Cap-loop2 vaccine; H3 group immunity self-control Cap-loop3 vaccine; H4 group immunity self-control Cap-loop4 vaccine; H5 group immunity self-control Cap-loop2-loop4 vaccine; H6 group immunity self-control Cap-loop2-loop3-loop4 vaccine; H7 group immunity self-control PCV2 Cap vaccine; The PCV2 recombinant vaccine of the immune Bo Linge company of H8 group; The aftosa synthetic peptide vaccine of H9 immunity UBI; H10 injects the PBS of equivalent as negative control.Each group of immunizing dose is 1ml/ head.
3.. antibody test after immunity: after first immunisation 28 days, by all test pig blood sampling separation of serum, porcine circovirus 2 type ELISA antibody assay kit (Wuhan Ke Qian Bioisystech Co., Ltd) is utilized to detect the specific antibody level for PCV2 Cap protein in serum, evaluate the antibody horizontal of each group of experiment pig for FMDV VP1 albumen by the Schweineseuche VP1 protein antibodies detection kit that UBI company produces, the results are shown in Figure 28,29.
As can be seen from Figure 28, Figure 29, compared with H7 group (the Cap protein vaccine that immunity is complete) and H8 group, H1-H4 group (vaccine activity composition is the chimeric VLP of insertion 1 FMDV major B-cell epi-position) test pig all creates significantly for the antibody of FMDV VP1 albumen, and H7 and H8 group does not have to produce the antibody for FMDV VP1 albumen.H1-H4 group and H7 group test pig all create significantly for the specific antibody of PCV2 Cap protein, and difference is not obvious between each group, illustrate that the present invention selects to be adopted in one of four sites in PCV2 Cap protein FMDV VP1 albumen major B-cell epi-position to replace the chimeric VLP of the chimeric protein formation obtained, not only remain the immunogenicity for PCV2 Cap protein but also to FMDV VP1 albumen, there is immunogenicity.Antibody horizontal relatively for FMDV VP1 albumen after the immunity of H1-H6 group finds, along with increasing of the FMDV VP1 albumen major B-cell epitope number of replacing in PCV2 Cap protein, after the vaccine immunity being activeconstituents with the chimeric VLP of correspondence, the antibody horizontal for FMDV VP1 albumen also significantly increases, when replacing insertion 3 FMDV VP1 albumen major B-cell epi-positions in PCV2 Cap protein, obviously reduce for the antibody horizontal of PCV2 Cap protein after the vaccine immunity being activeconstituents with the Hybrid virus like particles of correspondence, and when replacing insertion 2 FMDV VP1 albumen major B-cell epi-positions in PCV2 Cap protein, after the vaccine immunity being activeconstituents with the Hybrid virus like particles of correspondence, antibody horizontal for PCV2 Cap protein and FMDV VP1 albumen is significantly higher than commercialized vaccine (the PCV2 recombinant vaccine of Bo Linge company and the aftosa synthetic peptide vaccine of UBI).The above results explanation, 4 each sites that applicant selects in PCV2 Cap protein, a site, 2 wherein can be replaced with FMDV VP1 albumen major B-cell epi-position at most simultaneously, if the site of replacing too much (>=3), then can affect the immunogenicity of chimeric VLP to PCV2.
In sum, 6 kinds of chimeric proteins that the present invention obtains all can form chimeric VLP, after particularly replacing the chimeric VLP immunity piglet of 2 sites acquisitions, do not affect the generation for PCV2 VP1 protein antibodies simultaneously, simultaneously high for FMDV antibody horizontal, reguarity is good.Adopt the inventive method, only need abduction delivering recombinant bacterium, just can obtain and there are two kinds of immunogenic Hybrid virus like particles simultaneously, Hybrid virus like particles of the present invention only needs primary immune response, just can produce the higher antibody for PCV2 and FMDV by effective stimulus body, save a large amount of labor forces, significantly reduce the cost of vaccine preparation and raising pig.
SEQUENCE LISTING
<110> Jiangsu Province Agriculture Science Institute
<120> a chimeric protein, virus-like particle and application thereof
<130> 20150211
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 702
<212> DNA
<213> artificial
<220>
<223> Cap protein encoding gene
<400> 1
atgacatatc ctcgtcgtcg ttttcgtcgt cgtcgttatc gtcctcgttc tcatcttggc 60
caaattcttc gtcgccgtcc ttggcttgta catcctcgtc atcgttaccg ctggcgtcgc 120
aagaatggta ttttcaatac tcgtcttagt cgtactattg gctacactgt caagaaaacc 180
actgttcgta ctcctagttg gaatgttgat atgatgcgtt tcaatattaa tgactttctt 240
ccgcctggcg gtggtagtaa tcctcttaca gttccgttcg aatactatcg tattcgtaaa 300
gttaaggttg agttttggcc gtgtagtcct attacacaag gtgaccgtgg cgttggcagt 360
acagctgtca ttcttgatga taatttcgtt actaaagcaa atgctcttac atacgaccct 420
tatgtgaatt actctagccg tcacactatc acccaaccgt tctcctacca cagccgttac 480
tttacaccta aacctgtctt agatcgtaca attgactatt tccagcctaa taataaacgt 540
aatcagcttt ggcttcgtct tcaaaccaca ggcaatgttg atcatgtagg tcttggtact 600
gcatttgaaa atagtattta cgaccaggat tacaatattc gtattacaat gtacgtgcaa 660
ttccgtgaat ttaatcttaa agatcctcca ttgaatccgt aa 702
<210> 2
<211> 108
<212> DNA
<213> artificial
<220>
<223> pig O type FMDV VP1 albumen major B-cell epi-position encoding gene
<400> 2
ggcaactgca aatatggcga aaacgcggtg accaacgtgc gcggcgatct gcaggtgctg 60
gcgcagaaag cggcgcgctg cctgccgacc agctttaact atggcgcg 108
<210> 3
<211> 783
<212> DNA
<213> artificial
<220>
<223> Cap-loop1 encoding gene
<400> 3
atgacatatc ctcgtcgtcg ttttcgtcgt cgtcgttatc gtcctcgttc tcatcttggc 60
caaattcttc gtcgccgtcc ttggcttgta catcctcgtc atcgttaccg ctggcgtcgc 120
aagaatggta ttttcaatac tcgtcttagt cgtactattg gctacactgt cggcaactgc 180
aaatatggcg aaaacgcggt gaccaacgtg cgcggcgatc tgcaggtgct ggcgcagaaa 240
gcggcgcgct gcctgccgac cagctttaac tatggcgcgt ggaatgttga tatgatgcgt 300
ttcaatatta atgactttct tccgcctggc ggtggtagta atcctcttac agttccgttc 360
gaatactatc gtattcgtaa agttaaggtt gagttttggc cgtgtagtcc tattacacaa 420
ggtgaccgtg gcgttggcag tacagctgtc attcttgatg ataatttcgt tactaaagca 480
aatgctctta catacgaccc ttatgtgaat tactctagcc gtcacactat cacccaaccg 540
ttctcctacc acagccgtta ctttacacct aaacctgtct tagatcgtac aattgactat 600
ttccagccta ataataaacg taatcagctt tggcttcgtc ttcaaaccac aggcaatgtt 660
gatcatgtag gtcttggtac tgcatttgaa aatagtattt acgaccagga ttacaatatt 720
cgtattacaa tgtacgtgca attccgtgaa tttaatctta aagatcctcc attgaatccg 780
taa 783
<210> 4
<211> 741
<212> DNA
<213> artificial
<220>
<223> Cap-loop2 encoding gene
<400> 4
atgacatatc ctcgtcgtcg ttttcgtcgt cgtcgttatc gtcctcgttc tcatcttggc 60
caaattcttc gtcgccgtcc ttggcttgta catcctcgtc atcgttaccg ctggcgtcgc 120
aagaatggta ttttcaatac tcgtcttagt cgtactattg gctacactgt caagaaaacc 180
actgttcgta ctcctagttg gaatgttgat atgggcaact gcaaatatgg cgaaaacgcg 240
gtgaccaacg tgcgcggcga tctgcaggtg ctggcgcaga aagcggcgcg ctgcctgccg 300
accagcttta actatggcgc gtactatcgt attcgtaaag ttaaggttga gttttggccg 360
tgtagtccta ttacacaagg tgaccgtggc gttggcagta cagctgtcat tcttgatgat 420
aatttcgtta ctaaagcaaa tgctcttaca tacgaccctt atgtgaatta ctctagccgt 480
cacactatca cccaaccgtt ctcctaccac agccgttact ttacacctaa acctgtctta 540
gatcgtacaa ttgactattt ccagcctaat aataaacgta atcagctttg gcttcgtctt 600
caaaccacag gcaatgttga tcatgtaggt cttggtactg catttgaaaa tagtatttac 660
gaccaggatt acaatattcg tattacaatg tacgtgcaat tccgtgaatt taatcttaaa 720
gatcctccat tgaatccgta a 741
<210> 5
<211> 732
<212> DNA
<213> artificial
<220>
<223> Cap-loop3 encoding gene
<400> 5
atgacatatc ctcgtcgtcg ttttcgtcgt cgtcgttatc gtcctcgttc tcatcttggc 60
caaattcttc gtcgccgtcc ttggcttgta catcctcgtc atcgttaccg ctggcgtcgc 120
aagaatggta ttttcaatac tcgtcttagt cgtactattg gctacactgt caagaaaacc 180
actgttcgta ctcctagttg gaatgttgat atgatgcgtt tcaatattaa tgactttctt 240
ccgcctggcg gtggtagtaa tcctcttaca gttccgttcg aatactatcg tattcgtaaa 300
gttaaggttg agttttggcc gtgtagtcct attacacaag gtgaccgtgg cgttggcagt 360
acaggcaact gcaaatatgg cgaaaacgcg gtgaccaacg tgcgcggcga tctgcaggtg 420
ctggcgcaga aagcggcgcg ctgcctgccg accagcttta actatggcgc gcacactatc 480
acccaaccgt tctcctacca cagccgttac tttacaccta aacctgtctt agatcgtaca 540
attgactatt tccagcctaa taataaacgt aatcagcttt ggcttcgtct tcaaaccaca 600
ggcaatgttg atcatgtagg tcttggtact gcatttgaaa atagtattta cgaccaggat 660
tacaatattc gtattacaat gtacgtgcaa ttccgtgaat ttaatcttaa agatcctcca 720
ttgaatccgt aa 732
<210> 6
<211> 702
<212> DNA
<213> artificial
<220>
<223> Cap-loop4 encoding gene
<400> 6
atgacatatc ctcgtcgtcg ttttcgtcgt cgtcgttatc gtcctcgttc tcatcttggc 60
caaattcttc gtcgccgtcc ttggcttgta catcctcgtc atcgttaccg ctggcgtcgc 120
aagaatggta ttttcaatac tcgtcttagt cgtactattg gctacactgt caagaaaacc 180
actgttcgta ctcctagttg gaatgttgat atgatgcgtt tcaatattaa tgactttctt 240
ccgcctggcg gtggtagtaa tcctcttaca gttccgttcg aatactatcg tattcgtaaa 300
gttaaggttg agttttggcc gtgtagtcct attacacaag gtgaccgtgg cgttggcagt 360
acagctgtca ttcttgatga taatttcgtt actaaagcaa atgctcttac atacgaccct 420
tatgtgaatt actctagccg tcacactatc acccaaccgt tctcctacca cagccgttac 480
tttggcaact gcaaatatgg cgaaaacgcg gtgaccaacg tgcgcggcga tctgcaggtg 540
ctggcgcaga aagcggcgcg ctgcctgccg accagcttta actatggcgc gcttggtact 600
gcatttgaaa atagtattta cgaccaggat tacaatattc gtattacaat gtacgtgcaa 660
ttccgtgaat ttaatcttaa agatcctcca ttgaatccgt aa 702
<210> 7
<211> 717
<212> DNA
<213> artificial
<220>
<223> Cap-loop2-loop4 encoding gene
<400> 7
atgcctcgtt ctcatcttgg ccaaattctt cgtcgccgtc cttggcttgt acatcctcgt 60
catcgttacc gctggcgtcg caagaatggt attttcaata ctcgtcttag tcgtactatt 120
ggctacactg tcaagaaaac cactgttcgt actcctagtt ggaatgttga tatggtgtat 180
aacggcaact gcaaatatgg cgaaaacgcg gtgaccaacg tgcgcggcga tctgcaggtg 240
ctggcgcaga aagcggcgcg ctgcctgccg accagcttta actatggcgc gattaaatac 300
tatcgtattc gtaaagttaa ggttgagttt tggccgtgta gtcctattac acaaggtgac 360
cgtggcgttg gcagtacagc tgtcattctt gatgataatt tcgttactaa agcaaatgct 420
cttacatacg acccttatgt gaattactct agccgtcaca ctatcaccca accgttctcc 480
taccacagcc gttactttgg caactgcaaa tatggcgaaa acgcggtgac caacgtgcgc 540
ggcgatctgc aggtgctggc gcagaaagcg gcgcgctgcc tgccgaccag ctttaactat 600
ggcgcgcttg gtactgcatt tgaaaatagt atttacgacc aggattacaa tattcgtatt 660
acaatgtacg tgcaattccg tgaatttaat cttaaagatc ctccattgaa tccgtaa 717
<210> 8
<211> 762
<212> DNA
<213> artificial
<220>
<223> Cap-loop2-loop3-loop4 encoding gene
<400> 8
atgcctcgtt ctcatcttgg ccaaattctt cgtcgccgtc cttggcttgt acatcctcgt 60
catcgttacc gctggcgtcg caagaatggt attttcaata ctcgtcttag tcgtactatt 120
ggctacactg tcaagaaaac cactgttcgt actcctagtt ggaatgttga tatggtgtat 180
aacggcaact gcaaatatgg cgaaaacgcg gtgaccaacg tgcgcggcga tctgcaggtg 240
ctggcgcaga aagcggcgcg ctgcctgccg accagcttta actatggcgc gattaaatac 300
tatcgtattc gtaaagttaa ggttgagttt tggccgtgta gtcctattac acaaggtgac 360
cgtggcgttg gcagtacagt gtataacggc aactgcaaat atggcgaaaa cgcggtgacc 420
aacgtgcgcg gcgatctgca ggtgctggcg cagaaagcgg cgcgctgcct gccgaccagc 480
tttaactatg gcgcgattaa acacactatc acccaaccgt tctcctacca cagccgttac 540
tttggcaact gcaaatatgg cgaaaacgcg gtgaccaacg tgcgcggcga tctgcaggtg 600
ctggcgcaga aagcggcgcg ctgcctgccg accagcttta actatggcgc gcttggtact 660
gcatttgaaa atagtattta cgaccaggat tacaatattc gtattacaat gtacgtgcaa 720
ttccgtgaat ttaatcttaa agatcctcca ttgaatccgt aa 762
<210> 9
<211> 233
<212> PRT
<213> porcine circovirus 2 type
<400> 9
Met Thr Tyr Pro Arg Arg Arg Phe Arg Arg Arg Arg Tyr Arg Pro Arg
1 5 10 15
Ser His Leu Gly Gln Ile Leu Arg Arg Arg Pro Trp Leu Val His Pro
20 25 30
Arg His Arg Tyr Arg Trp Arg Arg Lys Asn Gly Ile Phe Asn Thr Arg
35 40 45
Leu Ser Arg Thr Ile Gly Tyr Thr Val Lys Lys Thr Thr Val Arg Thr
50 55 60
Pro Ser Trp Asn Val Asp Met Met Arg Phe Asn Ile Asn Asp Phe Leu
65 70 75 80
Pro Pro Gly Gly Gly Ser Asn Pro Leu Thr Val Pro Phe Glu Tyr Tyr
85 90 95
Arg Ile Arg Lys Val Lys Val Glu Phe Trp Pro Cys Ser Pro Ile Thr
100 105 110
Gln Gly Asp Arg Gly Val Gly Ser Thr Ala Val Ile Leu Asp Asp Asn
115 120 125
Phe Val Thr Lys Ala Asn Ala Leu Thr Tyr Asp Pro Tyr Val Asn Tyr
130 135 140
Ser Ser Arg His Thr Ile Thr Gln Pro Phe Ser Tyr His Ser Arg Tyr
145 150 155 160
Phe Thr Pro Lys Pro Val Leu Asp Arg Thr Ile Asp Tyr Phe Gln Pro
165 170 175
Asn Asn Lys Arg Asn Gln Leu Trp Leu Arg Leu Gln Thr Thr Gly Asn
180 185 190
Val Asp His Val Gly Leu Gly Thr Ala Phe Glu Asn Ser Ile Tyr Asp
195 200 205
Gln Asp Tyr Asn Ile Arg Ile Thr Met Tyr Val Gln Phe Arg Glu Phe
210 215 220
Asn Leu Lys Asp Pro Pro Leu Asn Pro
225 230
<210> 10
<211> 36
<212> PRT
<213> FMDV
<400> 10
Gly Asn Cys Lys Tyr Gly Glu Asn Ala Val Thr Asn Val Arg Gly Asp
1 5 10 15
Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Cys Leu Pro Thr Ser Phe
20 25 30
Asn Tyr Gly Ala
35
Claims (8)
1. a chimeric protein obtains after selecting in carrying Cap gene of porcine circovirus type 2 in following four sites one, two or three to replace with pig O type FMDV VP1 albumen major B-cell epi-position:
(1) 58-66 amino acids residue;
(2) 72-94 amino acids residues;
(3) 122-147 amino acids residues;
162-197 amino acids residue.
2. chimeric protein according to claim 1, is characterized in that the aminoacid sequence of described carrying Cap gene of porcine circovirus type 2 is as shown in SEQ ID NO:9; The aminoacid sequence of described pig O type FMDV VP1 albumen major B-cell epi-position is as shown in SEQ ID NO:10.
3. the encoding gene of the described chimeric protein of one of claim 1-2, is characterized in that the nucleotide sequence of described encoding gene is as shown in one of SEQ ID NO:3-8.
4. the recombinant vectors containing gene described in claim 3; In preferred technical scheme, described recombinant vectors is that gene described in claim 3 is inserted expression vector gained; Described expression vector is preferably pTXB1.
5. the recombinant bacterium containing gene described in claim 4, is characterized in that: be that the recombinant vectors containing gene described in claim 3 is imported the recombinant bacterium obtained in Host Strains; Described recombinant vectors is that described gene is inserted expression vector pTXB1 gained.
6. a Hybrid virus like particles, is formed by the described chimeric protein self assembly of one of claim 1-2, and described Hybrid virus like particles surface display has pig O type FMDV VP1 albumen major B-cell epi-position.
7. Hybrid virus like particles according to claim 6, it is characterized in that described Hybrid virus like particles is adopted to prepare with the following method: described in induction claim 5, recombinant bacterium expresses chimeric protein, cracking recombinant bacterium, get lysate supernatant, adopt ammonium sulfate precipitation method purifying, obtain described Hybrid virus like particles.
8. porcine circovirus 2 type and O type foot and mouth disease virus bigeminy vaccine, activeconstituents is Hybrid virus like particles described in claim 6 or 7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510086266.7A CN104693310B (en) | 2015-02-17 | 2015-02-17 | A kind of chimeric protein, virus-like particle and its application |
Applications Claiming Priority (1)
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Cited By (6)
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| WO2016160761A3 (en) * | 2015-03-30 | 2016-11-24 | Boehringer Ingelheim Vetmedica, Inc. | Pcv2 orf2 carrier platform |
| CN109134667A (en) * | 2018-09-19 | 2019-01-04 | 天康生物股份有限公司 | Fusion protein and preparation method thereof, application, expression system and vaccine |
| CN109187993A (en) * | 2018-09-13 | 2019-01-11 | 中国农业科学院兰州兽医研究所 | A kind of foot and mouth disease A-type virus sIgA antibody ELISA detection kit and its application |
| CN109265521A (en) * | 2018-09-13 | 2019-01-25 | 湖南农业大学 | PCV2Loop EF region mutant, primer, preparation method and application thereof |
| CN110305225A (en) * | 2019-08-02 | 2019-10-08 | 天康生物(上海)有限公司 | SVA-PCV2 fusion protein and preparation method thereof, gene, biomaterial, application and vaccine |
| CN113336861A (en) * | 2021-06-16 | 2021-09-03 | 郑州市第六人民医院 | Chimeric protein using norovirus VP1 protein as carrier, preparation method thereof and virus-like particle |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016160761A3 (en) * | 2015-03-30 | 2016-11-24 | Boehringer Ingelheim Vetmedica, Inc. | Pcv2 orf2 carrier platform |
| US10555994B2 (en) | 2015-03-30 | 2020-02-11 | Boehringer Ingelheim Animal Health USA Inc. | PCV2 ORF2 carrier platform |
| CN109187993A (en) * | 2018-09-13 | 2019-01-11 | 中国农业科学院兰州兽医研究所 | A kind of foot and mouth disease A-type virus sIgA antibody ELISA detection kit and its application |
| CN109265521A (en) * | 2018-09-13 | 2019-01-25 | 湖南农业大学 | PCV2Loop EF region mutant, primer, preparation method and application thereof |
| CN109187993B (en) * | 2018-09-13 | 2020-06-16 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease type A virus sIgA antibody ELISA detection kit and application thereof |
| CN109265521B (en) * | 2018-09-13 | 2021-05-18 | 湖南农业大学 | PCV2Loop EF region mutant, primer, preparation method and application thereof |
| CN109134667A (en) * | 2018-09-19 | 2019-01-04 | 天康生物股份有限公司 | Fusion protein and preparation method thereof, application, expression system and vaccine |
| CN110305225A (en) * | 2019-08-02 | 2019-10-08 | 天康生物(上海)有限公司 | SVA-PCV2 fusion protein and preparation method thereof, gene, biomaterial, application and vaccine |
| CN113336861A (en) * | 2021-06-16 | 2021-09-03 | 郑州市第六人民医院 | Chimeric protein using norovirus VP1 protein as carrier, preparation method thereof and virus-like particle |
| CN113336861B (en) * | 2021-06-16 | 2023-09-12 | 郑州市第六人民医院 | Chimeric protein taking norovirus VP1 protein as carrier, preparation method thereof and virus-like particle |
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