CN101215576A - Rabbit viral haemorrhagic virus capsid protein gene recombination baculovirus and bacterin - Google Patents
Rabbit viral haemorrhagic virus capsid protein gene recombination baculovirus and bacterin Download PDFInfo
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- CN101215576A CN101215576A CNA2008100192699A CN200810019269A CN101215576A CN 101215576 A CN101215576 A CN 101215576A CN A2008100192699 A CNA2008100192699 A CN A2008100192699A CN 200810019269 A CN200810019269 A CN 200810019269A CN 101215576 A CN101215576 A CN 101215576A
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Abstract
The invention relates to recombinant baculoviral of capsid protein of rabbit viral hemorrhagic disease viruses and gene engineering vaccines, which belong to the bio-pharmaceutical field. A capsid protein VP60 whole gene of the rabbit viral hemorrhagic disease viruses is amplified through RT-PCR, a baculoviral recombinant vector is constructed through the molecular biotechnology, Sf9 cells are transfected, which enables the VP60 gene to obtain expression, and then gene engineering vaccines of the rabbit viral hemorrhagic disease are prepared through taking the capsid protein VP60 of the rabbit viral hemorrhagic disease viruses of the recombinant adenovirus expression as antigen and adding corresponding adjuvant. The invention can provide the capsid protein gene engineering vaccines of the rabbit viral hemorrhagic disease with good immune effects and simple technology, which is used for preventing and controlling the rabbit viral hemorrhagic disease.
Description
Technical field the present invention relates to rabbit viral haemorrhagic virus capsid protein recombinant baculovirus and recombinant vaccine, belongs to field of biological pharmacy.
Background technology
Rabbit hemorrhagic disease, claim the rabbit pest again, be by rabbit hemorrhagic disease virus (RHDV) cause a kind of be the rabbit transmissible disease of feature with acute, hyperinfection, big area death, common 48～72h the death of infected rabbits, principal character is that infectivity is extremely strong, respiratory system is hemorrhage, hepatic necrosis, internal organs oedema, extravasated blood and variation such as hemorrhage, has caused enormous economic loss for whole world rabbit keeping.World Organization for Animal Health classifies this disease as the category-B transmissible disease, and the 96 commands promulgation of China Ministry of Agriculture is two class eqpidemic diseases.RHD occurs relatively suddenly and epidemic rate is exceedingly fast, and existing result of study shows that worldwide all RHDV strain isolateds are same serotype, is that hereditary property or antigenicity are all very stable.Organizing the inactivated vaccine immunity with rabbit hemorrhagic disease at present is this sick major measure of prevention, and must infect lethal rabbit viscera tissue with RHDV in the preparation process of vaccine is raw material.In recent years; along with the increase to the demand of rabbit such as the rise of industries such as rabbit product and other animal vaccines; market has increased many to the demand of vaccine used for virus hemorrhagic disease of rabbit; but the supply that is used to prepare the non-immunize rabbit of organizing inactivated vaccine is but very unstable; cause the cost of traditional tissue inactivation seedling constantly to increase; there are the various shortcoming such as potentially dangerous of disseminating strong poison in tissue inactivation seedling in addition; and RHDV does not go down to posterity on cell successfully so far; seriously restricted safer; the development of stable vaccine used for virus hemorrhagic disease of rabbit and production; make people to the research steering of vaccine used for virus hemorrhagic disease of rabbit can play a protective role, can solve the development of the recombinant vaccine of Biosafety problem again.
Research is in recent years thought, RHDV belongs to Calicivirus, contain a sub-thread positive chain RNA, form by 7437 Nucleotide, different with general Calicivirus, RHDV has only 2 open reading frames (ORF), and by 3 ' end parts sequence encoding RHDV capsid protein VP60 of ORFI, its gene fragment length is 1740bp, 580 amino acid of encoding altogether, VP60 is the unique structural protein of RHDV, and is directly related with the immune response that the inducing anti-disease poison infects.
Rhabdovirus system has the posttranslational modification function that is similar to mammalian cell, make recombinant protein on 26S Proteasome Structure and Function more near native protein.Because baculovirus can only infect non-vertebrates, and the host insect cell of its infection can not parasitize in the rabbit body, so do not have in the rabbit body at baculovirus protein or the proteic antibody of insect cell, therefore, thus utilize the VP60 albumen of this system expression to can be used as the effect that the vaccine antigen protein immunizing rabbit plays the prevention rabbit hemorrhagic disease.
Summary of the invention
Technical problem the objective of the invention is to make up the baculovirus recombinant vectors by Protocols in Molecular Biology, and a kind of good immune effect, rabbit viral haemorrhagic virus capsid protein recombinant vaccine that technology is easy and preparation method thereof are provided.
Technical scheme the present invention is by the full gene of RT-PCR amplification rabbit viral haemorrhagic virus capsid protein VP60, make up the baculovirus recombinant vectors by Protocols in Molecular Biology, transfection Sf9 cell, make the VP60 gene obtain to express, then with the rabbit viral haemorrhagic virus capsid protein VP60 of recombinant baculovirus expression as antigen, add that corresponding adjuvant makes the recombinant vaccine of prevention rabbit hemorrhagic disease.
One, expresses the acquisition of the recombinant baculovirus of rabbit viral haemorrhagic virus capsid protein VP60
1) extraction of the total RNA of rabbit hemorrhagic disease virus
Add in the rabbit liver tissue that rabbit hemorrhagic disease takes place with DEPC treated water 1: 10 by volume, grind to form suspension, in the 1.5mL EP pipe of handling with DEPC of packing into, in-20 ℃ of multigelations 3 times, on high speed freezing centrifuge, with 7200r/min, centrifugal 20min gets supernatant 200 μ L, adds Trizol 800 μ L, behind the vibration mixing, room temperature is placed 10min, adds chloroform 210 μ L, behind the thermal agitation, room temperature is placed 1min, 4 ℃, the centrifugal 15min of 10000r/min, get upper strata water 750 μ L, add the 0.5mL Virahol, mixing is placed 15min for-20 ℃, 4 ℃ of centrifugal 10min of 10000r/min, remove supernatant, slowly add 75% ethanol 1mL washing, 4 ℃ of centrifugal 5min of 8000r/min, remove supernatant, drying at room temperature 20min adds DEPC water 10 μ L, makes abundant dissolving;
2) the synthetic following primer of design:
P1：5’-ATA
GTCGACATGGAGGGCAAAG-3’ Sal I
P2：5’-GC
CTCGAGTCAGACATAAGAAAAGCCA-3’ Xho I
3) amplification capsid protein VP60 gene:
The reverse transcription system is: 10 * Buffer, 2 μ l, 10mM dNTPs 2 μ l, downstream primer 1 μ l, RNA enzyme inhibitors 0.5 μ l, RNA template 12 μ l, AMV ThermoScript II 0.5 μ l, DEPC water 2 μ l, cumulative volume is 20 μ l, moment centrifugal mixing, 65 ℃ the reaction 15min, hatch 1h for 42 ℃, 95 ℃ of 5min ,-20 ℃ of preservations are standby;
The PCR reaction system is: 10 * Reaction buffer, 2.5 μ l, 25mM MgCl
21.5 μ l, 2.5mM dNTPS0.5 μ l, 50mM P1 0.5 μ l, 50mM P2 0.5 μ l, template ribonucleic acid 4.0 μ l, ddH
2O 15 μ l, EX TaqTMpolymerase 0.5 μ l, cumulative volume 25 μ l, moment centrifugal mixing, adopt the heat lid to carry out pcr amplification reaction: 94 ℃ of sex change 3min, 94 ℃ of sex change 1min, 57 ℃ of annealing 1min, 72 ℃ are extended 2min, after 30 circulations, 72 ℃ are extended 10min, finish reaction; The PCR product is carried out agarose gel electrophoresis identify that the capsid protein VP60 gene amplification segment size of acquisition is 1740bp;
4) structure of recombinant baculovirus plasmid Bacmid-VP60:
The VP60 gene of amplification is cloned into transfer vector pFastBac earlier
TMIn 1, obtain recombinant transfer vector pFastBac
TM1-VP60 is then with pFastBac
TMThe 1-VP60 plasmid transforms the E.coliDH10Bac competent cell that contains shuttle vectors Bacmid, converted product is applied to the LB flat board that contains three kinds of microbiotic, X-gal and IPTG, behind 37 ℃ of cultivation 48h, select white colony and cultivate and extract plasmid, as template, carry out PCR with general pUCM13 upstream primer and specificity VP60 downstream primer and identify male recombinant plasmid called after Bacmid-VP60;
5) transfection: with recombinant baculovirus plasmid Bacmid-VP60 transfection Sf9 cell, obtain recombinant virus through purification Identification, and frozen in 4 ℃ ,-20 ℃ and liquid nitrogen;
6) evaluation of recombinant virus
(1) RT-PCR identifies
Collect the cell that infects recombinant virus, carry out RT-PCR with goal gene upstream and downstream primer, 1% agarose gel electrophoresis identifies that the result shows that electrophoretic band is 1740bp, consistent with the goal gene size, the illustration purpose gene is integrated in the cell with viral genome.
(2) evaluation of expression product
Indirect immunofluorescence experiment (IFA)
Recombinant virus is inoculated the cell of logarithmic phase, be cultured to when pathology takes place and detect Recombinant Protein Expression with indirect immunofluorescence (IFA), the result shows, the cell that reorganization is infected has very strong specificity fluorescent, and cellular control unit does not have special fluorescence, illustrate that VP60 albumen obtains expressing, and expression product is present in the cell.
Hemagglutination test (HA) and hemagglutination-inhibition test
Cleer and peaceful cells and supernatant all can aggegation 1% people " O " type red cell suspension in the recombinant virus-infected cell cracking.The proteic blood clotting characteristic of the VP60 of Biao Daing can be suppressed by the RHDV hyper-immune serum simultaneously, shows that the VP60 albumen of expression has the hemagglutination activity similar to RHDV.The viral hemorrhagic disease viral capsid proteins of recombinant virus eukaryotic expression can be used as the diagnostic antigen aspect of rabbit hemorrhagic disease.
Two, the animal immune of express recombinant rabbit viral haemorrhagic virus capsid protein protection effect
The HA that measures the reorganization rabbit viral haemorrhagic virus capsid protein tires, and is diluted to HA with sterile saline and tires 2
6-2
8Between, add the aluminium hydroxide gel adjuvant in proportion, by the various dose immune non-immunizing rabbit that divides into groups, and establish control group, and after immunity, tired with HA in the 22nd day and attack greater than the strong malicious liver suspension 1mL of 10 RHDV, observed 14 days, and the record result, the result shows that immune HA tires 2
6The rabbit that recombinant protein is 1 milliliter just can be resisted the attack of the strong poison of rabbit hemorrhagic disease.
Three, vaccine production: cultivate the Sf9 cell to logarithmic phase, inoculate corresponding recombinant virus, there are after the pathology 5 days and receive poison in cell, surveying blood clotting HA tires, adjust antigenic solutions with sterilization phosphate buffer soln PBS PH 7.2, after 2 ‰ formaldehyde solution deactivations, by antigen: the aluminium glue adjuvant is 9: 1 ratio adding adjuvants, mixing is the rabbit hemorrhagic disease recombinant vaccine of acquisition.
Beneficial effect characteristics of the present invention and advantage are as follows: the present invention chooses the full gene of rabbit viral haemorrhagic virus capsid protein VP60 and expresses as goal gene, reason is that this albumen can react by inducing anti-disease hemorrhagic disease virus infection immunity, and this albumen is unique structural protein of viral hemorrhagic syndrome virus, has good immunogenicity; Eukaryotic expression system has the posttranslational modification function that is similar to mammalian cell, make recombinant capsid protein on 26S Proteasome Structure and Function more near native protein, the result of test shows, expressed proteins has the biological activity identical with the viral hemorrhagic syndrome virus, the carrier for expression of eukaryon system is the effective system of a kind of expression, submission exogenous antigen simultaneously, and recombinant virus can excite people and multiple animal specificity humoral and cellular immunization; Baculovirus expression system can only infect non-vertebrates, and the host insect cell of its infection can not parasitize in the rabbit body.Therefore, utilize the VP60 protein Preparation of this system expression to become recombinant vaccine to have good biological safety; The recombinant protein of expressing need not the special processing purifying and just can be used for the vaccine preparation, and is easy and simple to handle.
Fact proved that the recombinant protein that the present invention expresses has good antigenicity and the hemagglutination activity similar to rabbit hemorrhagic disease virus, is good vaccine used for virus hemorrhagic disease of rabbit antigen therefore.The animal immune test-results shows; the non-immunizing rabbit made from the reorganization rabbit viral haemorrhagic virus capsid protein of vaccine immunity; the immunity back is with the RHD strong virus attack; immunizing rabbit obtains 100% provide protection; illustrate that the present invention has good immune efficacy in actual applications; having important use value aspect the rabbit hemorrhagic disease prevention and control, possesses good biological safety simultaneously.
Description of drawings
Fig. 1 baculovirus expression system RHDV VP60 gene RT-PCR result
1.RT-PCR product; 2.DNA molecular weight standard (DL-2000)
The enzyme of Fig. 2 baculovirus expression system recombinant transfer vector is cut evaluation
1. digestion with restriction enzyme recombinant transfer vector (Xho I/Sal I); 2.DNA molecular weight standard (DL-2000); 3.DNA molecular weight standard (DL-15000)
The PCR of Fig. 3 baculovirus expression system reorganization Bacmid identifies
1.DNA molecular weight standard (DL-15000); 2.DNA molecular weight standard (DL-2000); 3,4. reorganization Bacmid PCR identifies; 5.Bacmid PCR identifies
Infect the Sf9 cellmediated immunity fluorescence result of Bacmid-VP60 in Fig. 4 baculovirus expression system
Embodiment
One, the amplification of rabbit viral haemorrhagic virus capsid protein VP60 gene and T/A clone
1, the extraction of the total RNA of rabbit hemorrhagic disease virus (RHDV)
Added in the rabbit liver tissue that rabbit hemorrhagic disease takes place in 1: 10 by volume with DEPC (diethypyrocarbonate diethylpyrocarbonate) treated water, grind to form suspension, in the 1.5mL EP pipe of handling with DEPC of packing into, in-20 ℃ of multigelations 3 times, on high speed freezing centrifuge, with 7200r/min, centrifugal 20min gets supernatant 200 μ L, add Trizol (TaKaRa company) 800 μ L, behind the vibration mixing, room temperature is placed 10min, adds chloroform 210 μ L, behind the thermal agitation, room temperature is placed 1min, 4 ℃, the centrifugal 15min of 10000r/min, get upper strata water 750 μ L, add the 0.5mL Virahol, mixing is placed 15min for-20 ℃, 4 ℃ of centrifugal 10min of 10000r/min, remove supernatant, slowly add 75% ethanol 1mL washing, 4 ℃ of centrifugal 5min of 8000r/min, remove supernatant, drying at room temperature 20min adds DEPC water 10 μ L, makes abundant dissolving.
2, design synthetic primer
According to the RHDV genome sequence VP60 sequence (AY523410) in the GenBank database, utilize Primer 5.0 softwares to design voluntarily, by the precious biotechnology company limited (TaKaRa) in Dalian synthetic primer:
P1：5’-ATA
GTCGACATGGAGGGCAAAG-3’ (Sal I)
P2：5’-GC
CTCGAGTCAGACATAAGAAAAGCCA-3’ (Xho I)
Underscore partly is a restriction enzyme site
3, amplification capsid protein VP60 gene:
The reverse transcription system is: 10 * Buffer, 2 μ l, 10mM dNTPs 2 μ l, downstream primer 1 μ l, RNA enzyme inhibitors 0.5 μ l, RNA template 12 μ l, AMV ThermoScript II 0.5 μ l, H
2O (DEPC processing) 2 μ l, cumulative volume is 20 μ l, moment centrifugal mixing, 65 ℃ the reaction 15min, hatch 1h for 42 ℃, 95 ℃ of 5min ,-20 ℃ of preservations are standby.
The PCR reaction system is: 10 * Reaction buffer, 2.5 μ l, 25mM MgCl
21.5 μ l, 2.5mM dNTPS0.5 μ l, 50mM P1 0.5 μ l, 50mM P2 0.5 μ l, template ribonucleic acid 4.0 μ l, ddH
2O 15 μ l, EX TaqTMpolymerase 0.5 μ l, cumulative volume 25 μ l, moment centrifugal mixing, adopt the heat lid to carry out pcr amplification reaction: 94 ℃ of sex change 3min, 94 ℃ of sex change 1min, 57 ℃ of annealing 1min, 72 ℃ are extended 2min, after 30 circulations, 72 ℃ are extended 10min, finish reaction.The PCR product is carried out agarose gel electrophoresis identify that the capsid protein VP60 gene amplification segment size of acquisition is 1740bp;
4, T/A clone and evaluation
Behind the electrophoresis purpose fragment is downcut, adopt the nucleic acid gel to reclaim test kit Gel Extraction Kit (TaKaRa company) and carry out purified pcr product, then according to the explanation of pMD-19T-Vector test kit, the purpose fragment cloning is gone into pMD-19T-Vector (TaKaRa company), transformed into escherichia coli (Escherichia coli) DH5 α competent cell.Select single bacterium colony and cultivate and extract plasmid, cut and the electrophoresis evaluation through the respective limits enzyme, obtain recombinant plasmid pMD-19T-VP60, positive colony is delivered to TaKaRa (Daliang City) and is checked order.
5, the sequence comparison and analysis of Kuo Zeng VP60 gene utilizes DNAstar 5.0 sequence analysis softwares that the VP60 gene of delivering on the sequencing result of VP60 and the GenBank is carried out the homology of nucleotide sequence analysis.
Two, the structure of recombinant baculovirus plasmid Bacmid-VP60:
Digest donor plasmid pFastBac respectively with restriction enzyme Sal I and Xho I
TM1 (available from Invitrogen company) and pMD-19T-VP60 reclaims the purpose fragment, behind 4 ℃ of connections of T4DNA Ligase 12h, and transformed into escherichia coli DH5 α competent cell.Screening positive clone obtains recombinant transfer vector pFastBac
TM1-VP60 is with pFastBac
TMThe 1-VP60 plasmid transform the E.coli DH10Bac competent cell (available from Invitrogen company) contain shuttle vectors Bacmid and, after 37 ℃ of LB liquid nutrient mediums are cultivated 4h, getting 100 μ l converted products is applied to and contains 50 μ g/ml kantlex, 7 μ g/ml gentamicins, the LB flat board of 10 μ g/ml tsiklomitsins and 100 μ g/ml X-gal and 20 μ g/mL IPTG, behind 37 ℃ of cultivation 48h, select white colony, continuing inoculation on three kinds of antibiotic LB flat boards in same containing goes down to posterity once, obtain pure culture, inoculation contains in three kinds of antibiotic LB substratum, 24h is cultivated in 37 ℃ of joltings (180r/min), then with the special solution I that extracts high-molecular weight recombinant shuttle plasmid DNA, II, III follows these steps to extract the Bacmid plasmid and (sees the baculovirus expression system handbook, the C version, the appendix chapter, extract reorganization BacmidDNA joint, the 51-52 page or leaf, 2002, tech_service@invitrogen.com).With reorganization Bacmid DNA is template, carries out PCR with general pUCM13 upstream primer and specificity VP60 downstream primer and identifies.Set up the DHl0Bacmid plasmid control group that does not insert exogenous genetic fragment simultaneously, carry out PCR with pUCM13 upstream and downstream primer and identify.PCR reaction system and pcr amplification reaction condition are seen the baculovirus expression system handbook.0.8% agarose gel electrophoresis is accredited as male recombinant plasmid called after Bacmid-VP60.
Three, transfectional cell obtains recombinant virus
1 transfectional cell and purifying
With Lipofectamin
TM2000 is cotransfection reagent, according to explanation, with Bacmid-VP60 transfection Sf9 monolayer cell.Every 12h observes once after the transfection, and when cytopathy was obvious, collecting cell and supernatant liquor were as recombinant baculovirus stoste-20 ℃ preservation.The recombinant baculovirus called after Bac-VP60 that will contain goal gene VP60.Gone down to posterity with 1% volume ratio inoculation Sf9 cell (available from Invitrogen company) in twice of f1 disease venom freeze thawing back, obtain the 2nd generation recombinant virus.With the 2nd generation recombinant virus be that seed culture of viruses carries out plaque purification.
2 identify
(1) RT-PCR identifies
Collect the Sf9 cell of the 2nd generation infection recombinant virus, extract cell RNA according to preamble rabbit hemorrhagic disease virus (RHDV) method for extracting total RNA, carry out RT-PCR with goal gene upstream and downstream primer, 1% agarose gel electrophoresis is identified, the result shows that electrophoretic band is 1740bp, consistent with the goal gene size, the illustration purpose gene is gone in the Sf9 cell with the baculovirus genome conformity.
(2) evaluation of expression product
Indirect immunofluorescence detects (IFA)
On 24 porocyte culture plates, the recombinant virus of purifying is inoculated the Sf9 cell of logarithmic phase, be cultured to when pathology takes place and detect Recombinant Protein Expression with indirect immunofluorescence (IFA), the result shows, the Sf9 cell that Bacmid-VP60 infects has very strong specificity fluorescent, and Bacmid infection Sf9 control cells does not have special fluorescence, illustrate that VP60 albumen obtains expressing, and expression product is present in the insect cell.
Hemagglutination test (HA) and blood clotting suppress (HI) test
Get in the lysis of infecting recombinant virus cleer and peaceful cells and supernatant 50 μ l respectively after round bottom macropore blood-coagulation-board is made 2 * doubling dilution with physiological saline, add equal-volume 1% people " O " type red cell suspension, after the vibration evenly, place 4 ℃ of effects 45 minutes, observations (HA), simultaneously with other recombinant baculovirus as negative control, with 1: 5 homogenate supernatant of the sick rabbit liver of RHD as positive control.In addition, add 50 μ L RHDV serum in round bottom macropore blood-coagulation-board, after making 2 * doubling dilution with physiological saline then, the expressing protein that adds 4 HAUs is in every hole, blood-coagulation-board is put 37 ℃ of effects 30 minutes, and every hole adds 1% people " O " type red cell suspension, 50 μ l, shakes up immediately, put 4 ℃ of effects 45 minutes, observations (HI).The result shows, the equal energy of cleer and peaceful cells and supernatant aggegation 1% people " O " type red cell suspension in the recombinant virus-infected cell cracking, and other recombinant baculovirus can not aggegation 1% people " O " type red cell suspension, positive control is set up, and shows that the VP60 albumen of expression has the blood clotting characteristic the same with RHDV.120h receives poison behind the inoculation recombinant virus, finds that cleer and peaceful cells and supernatant hemagglutinative titer all can reach 2 in the cells infected cracking
12More than.The proteic blood clotting characteristic of the VP60 of Biao Daing can be suppressed by the RHDV hyper-immune serum simultaneously.
The test of four immunoprotections
The preparation of 1 immunizing antigen
Recombinant baculovirus is inoculated in the Sf9 cell, receives poison behind the 120h, the HA that surveys expressing protein tires, and is diluted to HA with aseptic sterile saline and tires 2
8
2 experimental animals divide into groups, inoculate and attack poison
HA is surveyed in the blood sampling before immunity of 15 non-immunizing rabbits to tire, and be divided into 3 groups, every group 5, first group of above-mentioned antigen of injection 1mL, second group of above-mentioned antigen of injection 2mL, organize in contrast for the 3rd group, injecting normal saline was adopted a blood and is surveyed HI and tire every 7 days between duration of immunity, tiring with HA in the 22nd day after immunity, (hepatic tissue: sterile saline=1: 9) 1mL attacks greater than 10 the strong malicious liver suspension of RHD, observed 14 days, and the record result.
3 test-results
15 non-immunizing rabbit blood samplings before immunity are surveyed HI and are tired 2
0～2
3, first group of all strong living in attacking back 14 days of poison, protection ratio is 100%, the second group of all strong living in attacking back 14 days of poison, and protection ratio is 100%, and these two groups of rabbit serum HI before attacking poison tires 2
5～2
7Between, attack all death in malicious back three days for the 3rd group, symptom is a typical rabbit hemorrhagic disease (table 1).
After the immunity of table 1 various dose to the protectiveness of RHDV
| Test group | First group | Second group | The 3rd group |
| Size of |
5 | 5 | 5 |
| Attack the dead quantity in poison back | 0 | 0 | 5 |
| |
100％ | 100％ | 0％ |
Five vaccine production: cultivate the Sf9 cell to logarithmic phase, inoculate corresponding recombinant virus, there are after the pathology 5 days and receive poison in cell, surveying blood clotting HA tires, adjust antigenic solutions with sterilization phosphate buffer soln PBS PH 7.2, after 2 ‰ formaldehyde solution deactivations, by antigen: the aluminium glue adjuvant is 9: 1 ratio adding adjuvants, mixing is the rabbit hemorrhagic disease recombinant vaccine of acquisition.
The animal immune test-results shows; the non-immunizing rabbit made from the reorganization rabbit viral haemorrhagic virus capsid protein of vaccine immunity; the immunity back is with the RHD strong virus attack; immunizing rabbit obtains 100% provide protection; illustrate that the present invention has good immune efficacy in actual applications; having important use value aspect the rabbit hemorrhagic disease prevention and control, possesses good biological safety simultaneously.
Sequence table
＜110〉Jiangsu Province Agriculture Science Institute
＜120〉rabbit viral haemorrhagic virus capsid protein gene recombination baculovirus and vaccine
＜130〉specification sheets
<140>00
<141>2008-01-12
<160>2
<170>PatentIn version 3.1
<210>1
<211>22
<212>DNA
＜213〉synthetic
<220>
＜221〉primer
<222>(1)..(22)
<223>
<400>1
atagtcgaca tggagggcaa ag 22
<210>2
<211>27
<212>DNA
＜213〉synthetic
<220>
＜221〉primer
<222>(1)..(27)
<223>
<400>2
gcctcgagtc agacataaga aaagcca 27
Claims (4)
1. rabbit viral haemorrhagic virus capsid protein gene recombination baculovirus is characterized in that, is made up by following steps to form:
1) extraction of the total RNA of rabbit hemorrhagic disease virus added in the rabbit liver tissue that rabbit hemorrhagic disease takes place in 1: 10 by volume with diethylpyrocarbonate DEPC water, grind to form suspension, in the 1.5mL EP pipe of handling with DEPC of packing into, in-20 ℃ of multigelations 3 times, on high speed freezing centrifuge, with 7200r/min, centrifugal 20min gets supernatant 200 μ L, add Trizol 800 μ L, behind the vibration mixing, room temperature is placed 10min, adds chloroform 210 μ L, behind the thermal agitation, room temperature is placed 1min, 4 ℃, the centrifugal 15min of 10000r/min, get upper strata water 750 μ L, add the 0.5mL Virahol, mixing is placed 15min for-20 ℃, 4 ℃ of centrifugal 10min of 10000r/min, remove supernatant, slowly add 75% ethanol 1mL washing, 4 ℃ of centrifugal 5min of 8000r/min, remove supernatant, drying at room temperature 20min adds DEPC water 10 μ L, makes abundant dissolving;
2) the synthetic following primer of design:
P1：5’-ATA
GTCGACATGGAGGGCAAAG-3’ Sal I
P2：5’-GC
CTCGAGTCAGACATAAGAAAAGCCA-3’ Xho I
3) amplification capsid protein VP60 gene:
The reverse transcription system is: 10 * Buffer, 2 μ l, 10mM dNTPs 2 μ l, downstream primer 1 μ l, RNA enzyme inhibitors 0.5 μ l, RNA template 12 μ l, AMV ThermoScript II 0.5 μ l, DEPC water 2 μ l, cumulative volume is 20 μ l, moment centrifugal mixing, 65 ℃ the reaction 15min, hatch 1h for 42 ℃, 95 ℃ of 5min ,-20 ℃ of preservations are standby;
The PCR reaction system is: 10 * Reaction buffer, 2.5 μ l, 25mM MgCl
21.5 μ l, 2.5mM dNTPS0.5 μ l, 50mM P1 0.5 μ l, 50mM P2 0.5 μ l, template ribonucleic acid 4.0 μ l, ddH
2O 15 μ l, EX TaqTMpolymerase 0.5 μ l, cumulative volume 25 μ l, moment centrifugal mixing, adopt the heat lid to carry out pcr amplification reaction: 94 ℃ of sex change 3min, 94 ℃ of sex change 1min, 57 ℃ of annealing 1min, 72 ℃ are extended 2min, after 30 circulations, 72 ℃ are extended 10min, finish reaction; The PCR product is carried out agarose gel electrophoresis identify that the capsid protein VP60 gene amplification segment size of acquisition is 1740bp;
4) structure of recombinant baculovirus plasmid Bacmid-VP60:
The VP60 gene of amplification is cloned into transfer vector pFastBac earlier
TMIn 1, obtain recombinant transfer vector pFastBac
TM1-VP60 is then with pFastBac
TMThe 1-VP60 plasmid transforms the E.coliDH10Bac competent cell that contains shuttle vectors Bacmid, converted product is applied to the LB flat board that contains three kinds of microbiotic, X-gal and IPTG, behind 37 ℃ of cultivation 48h, select white colony and cultivate and extract plasmid, as template, carry out PCR with general pUCM13 upstream primer and specificity VP60 downstream primer and identify male recombinant plasmid called after Bacmid-VP60;
5) transfection: with recombinant baculovirus plasmid Bacmid-VP60 transfection Sf9 cell, obtain recombinant virus through purification Identification, and frozen in 4 ℃ ,-20 ℃ and liquid nitrogen.
2. the vaccine of the described rabbit viral haemorrhagic virus capsid protein gene recombination baculovirus of claim 1 preparation.
3. the preparation method of the described vaccine of claim 2, comprise: cultivate the Sf9 cell to logarithmic phase, inoculate corresponding recombinant virus, there were after the pathology 5 days to receive poison in cell, survey blood clotting HA to tire, adjust antigenic solution with sterilization phosphate buffer soln PBS PH7.2, after 2 ‰ formaldehyde solution deactivations, in antigen: the aluminium glue adjuvant is that 9: 1 ratios add adjuvant, and mixing is the rabbit hemorrhagic disease recombinant vaccine of acquisition.
4. the application of the viral hemorrhagic disease viral capsid proteins of the described heavy rabbit viral haemorrhagic virus capsid protein gene recombination baculovirus eukaryotic expression of claim 1 aspect preparation rabbit hemorrhagic disease diagnostic antigen.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102304529A (en) * | 2011-08-31 | 2012-01-04 | 中国农业科学院生物技术研究所 | Preparation method of rabbit hemorrhagic fever virus empty capsid antigen |
| CN102604898A (en) * | 2012-04-17 | 2012-07-25 | 江苏省农业科学院 | VP60 protein recombinant baculovirus carrying type O foot and mouth disease virus B cell epitope |
| CN103033622A (en) * | 2011-12-26 | 2013-04-10 | 武汉中博生物股份有限公司 | PCV2 (porcine circovirus 2) ELISA (enzyme-linked immuno sorbent assay) antigen detection kit as well as preparation method and applications thereof |
| CN104436187A (en) * | 2014-11-10 | 2015-03-25 | 张文波 | Vaccine for expressing rabbit hemorrhagic disease virus VP60 protein |
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| CN104436187A (en) * | 2014-11-10 | 2015-03-25 | 张文波 | Vaccine for expressing rabbit hemorrhagic disease virus VP60 protein |
| CN108359645A (en) * | 2018-02-09 | 2018-08-03 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | A kind of preparation and application of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus |
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| CN111575315A (en) * | 2020-05-29 | 2020-08-25 | 青岛易邦生物工程有限公司 | Rabbit viral hemorrhagic disease virus type II VLP vaccine |
| CN111705083A (en) * | 2020-06-29 | 2020-09-25 | 江苏省农业科学院 | Rabbit hemorrhagic disease virus type 2 capsid protein gene recombinant baculovirus, vaccine, preparation method and application thereof |
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