CN108359645A - A kind of preparation and application of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus - Google Patents
A kind of preparation and application of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus Download PDFInfo
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- CN108359645A CN108359645A CN201810135244.9A CN201810135244A CN108359645A CN 108359645 A CN108359645 A CN 108359645A CN 201810135244 A CN201810135244 A CN 201810135244A CN 108359645 A CN108359645 A CN 108359645A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14311—Parvovirus, e.g. minute virus of mice
- C12N2750/14322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14311—Parvovirus, e.g. minute virus of mice
- C12N2750/14334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/015—Parvoviridae, e.g. feline panleukopenia virus, human Parvovirus
Abstract
The present invention provides a kind of preparations and application of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus, and the preparation method comprises the following steps：A, the VP60 genes of RHDV2 are expanded；B, the VP60 gene clonings for obtaining step A obtain plasmid in baculovirus transfer vector；C, the plasmid transformed competence colibacillus cell for obtaining step B, obtains recombinant plasmid；D, the recombinant plasmid for obtaining step C in liposome-mediated lower transfection insect cell to get II type capsid protein baculoviral of the expression rabbit hemorrhagic disease virus.The present invention successfully constructs the baculoviral of expression II type capsid protein of rabbit hemorrhagic disease, and makes its successful expression on insect cell, and eventually by protein electrophoresis, immunoblotting, Electronic Speculum observation have carried out the identification of viral expression product.The baculoviral is used for preventing II type of rabbit hemorrhagic disease in the following research that can be used for carrying out novel rabbit hemorrhagic disease subunit vaccine, with this.
Description
Technical field
The present invention relates to biotechnologies, specifically, being related to a kind of II type clothing of expression rabbit hemorrhagic disease virus
The preparation and application of glutelin baculoviral.
Background technology
II type of rabbit hemorrhagic disease virus (RHDV2) is also known as novel rabbit pest, is that one kind is new, has flowed extensively at present
Row is in Europe, the zoonosis of Australia.The virus can cause the rabbit illness that classical rabbit pestilence seedling was immunized, while can also
Infect hare, pathogenicity rate is high, and the virus can infect that classical rabbit hemorrhagic disease virus cannot infect be less than 40
The young age rabbit of age in days, caused main clinic symptoms include acute hepatic necrosis, tracheorrhagia, Pulmonary hemorrhage, liver bleeding
And necrosis etc., while mostly with spleen kidney enlargement in clinic.The virus is in the substituted classical rabbit of Australia at present
The trend of pest, and rabbit keeping development has also been seriously affected in the European virus, seriously endanger the safety of wild animal.It is quick-fried
The country for sending out rabbit pest novel, such as Holland, France, Italy, Poland etc. are still prevented newly using the inactivated vaccine method of classics
Type rabbit pest, but traditional inactivated vaccine its be possible to damage or change effective antigenic determinant in inactivation process, generate
Immune effect and hold time relatively short, and need multiple injection inactivated vaccine, the demand to amount of antigen is bigger,
Cost is in contrast also relatively high；Toxicity may also be generated or there is immune response potentially unfavorable to body, at the same time
There is also the potential dangers for disseminating strong poison to a certain extent for inactivated vaccine, it is most important that in the preparation process of inactivated vaccine
It is raw material that lethal rabbit viscera tissue must be infected using RHDV2, and now there has been no about novel Rabbit pest virus at home
Report, it is whether also unknown caused by the not perfect property due to detection means.The capsid protein of RHDV2 is the master of RHDV2
Want structural proteins, by the VP60 gene codes of the virus, the albumen and can self assembly become virus-like particle, virus-like
Particle is free of virulent nucleic acid, can simulate cause of disease stimulation body and generate immunoprotective effec, and utilize baculoviral table
Up to the capsid protein of system expression, have the function of posttranslational modification so that the albumen of recombination structurally and functionally with natural egg
It is white closer.
The vaccine prepared currently with baculovirus expression system production is widely used the prevention in various diseases, such as
Hepatitis B and human papilloma virus etc..Huge economic loss may be brought to China's rabbit keeping in view of RHDV2, preparing should
The subunit vaccine of virus and the detection method of studying the virus in the future have great importance for China's rabbit keeping, also for
We will continue to study how RHDV develops and provide certain Research foundation as RHDV2 in future.And there has been no using rod-shaped at present
The relevant report of expressing viral RHDV2 capsid proteins.
Invention content
The technical problem to be solved by the present invention is to provide a kind of II type capsid protein bar of expression rabbit hemorrhagic disease virus
The preparation and application of shape virus.
The present invention encodes base with reference to the capsid protein (VP60) of the NCBI II type RHDV2 of rabbit hemorrhagic disease virus announced
Because of open reading frame, a pair of primer containing specific cleavage site is devised, the capsid protein encoding gene of RHDV2 is expanded,
The gene cloning of amplification is carried in baculovirus expression in PFAST-BAC-HTA.
The present invention relates to the preparation researches of II type capsid protein gene recombination baculovirus of rabbit hemorrhagic disease virus, lead to
The capsid protein VP60 full length fragments for crossing amplification II type of rabbit hemorrhagic disease virus, are built rod-shaped by Protocols in Molecular Biology
Viral recombinant vector transfects Sf9 cells, obtains the baculoviral that can express capsid protein, then produces the expression of the virus
Object is identified that the present invention provides one kind can be with successful expression rabbit hemorrhagic disease virus II by related art method
The capsid protein of the method for type capsid protein, expression can be packaged into virus-like particle (virus like with self
Particles), in the following research that can be used for carrying out novel rabbit hemorrhagic disease subunit vaccine, it is used for preventing with this
II type of rabbit hemorrhagic disease.
The purpose of the present invention is what is be achieved through the following technical solutions：
The present invention provides it is a kind of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus preparation method,
Include the following steps：
A, the VP60 genes of RHDV2 are expanded；
B, the VP60 gene clonings for obtaining step A obtain plasmid in baculovirus transfer vector；
C, the plasmid transformed competence colibacillus cell for obtaining step B, obtains recombinant plasmid；
D, the recombinant plasmid for obtaining step C is malicious to get the expression rabbit in liposome-mediated lower transfection insect cell
Property II type capsid protein baculoviral of haemorrhagic virus.
Preferably, in step A, primer sequence such as SEQ ID No.1 and the SEQ ID that the VP60 genes of RHDV2 use is expanded
Shown in No.2.
Preferably, the restriction enzyme site of the primer is respectively GAATTC and TCTAGA.
Preferably, the baculovirus transfer vector is PFASTBAC-HTA；The competent cell is DH10Bac
Competent cell；The insect cell is Sf9 insect cells；The liposome is Cellfectin liposomes.The present invention uses
Baculovirus transfer vector PFASTBAC-HTA contain His labels, be conducive to the later stage purifying；The Sf9 insects of use are thin
Born of the same parents are free serum cultures, are conducive to the later stage extensive culture that suspends；Higher transfection can get using Cellfectin liposomes
Efficiency, according to other liposomes, then transformation efficiency is not high, is unfavorable for obtaining recombinant baculovirus.
The present invention also provides a kind of II type capsid eggs of expression rabbit hemorrhagic disease virus prepared according to preceding method
White baculoviral.
The present invention also provides a kind of expression productions of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus
Object identification method, includes the following steps：By II type capsid protein baculovirus infection of the expression rabbit hemorrhagic disease virus
It is cultivated after insect cell, takes the vial supernatant of culture, by Western-blot and IFA methods to expressing viral
Characteristic protein is identified.
Preferably, the ratio of expression rabbit hemorrhagic disease virus II the type capsid protein baculoviral and insect cell
It is 1:10~1:100.
Preferably, the condition of culture is：28 DEG C of culture 96h~120h.
The present invention also provides a kind of application of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus, institutes
It states to apply and includes：Rabbit viral bleeding is prepared using II type capsid protein baculoviral of the expression rabbit hemorrhagic disease virus
II type subunit vaccine of disease, or prepare the application in RHDV2 Enzyme-linked Immunosorbent Assay reagents.
There are immunological responses for the monoclonal antibody of the product of baculovirus expression of the present invention and anti-His albumen.
Simultaneously as the baculovirus vector expression utilized is high, and contain His labels, the purification step in later stage can be facilitated,
It can be prepared into II type subunit vaccine of rabbit hemorrhagic disease, can also be used as and establish the examination of RHDV2 Enzyme-linked Immunosorbent Assays in the future
Agent prepares the coating antigen in research and development.
Compared with prior art, the present invention has following advantageous effect：
When the advantages of being better than prokaryotic expression system the present invention is based on insect baculovirus expression system-expression foreign protein
With complete post translational processing Modifying Capability, expression of the RHDV capsid proteins on insect cell is carried out.In expression RHDV2
During albumen, it is biologically active to show that experiment obtains by the methods of immunoblotting, indirect immunofluorescence
RHDV2-VP60 albumen, and accurately identify expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus.And lead to
It crosses Electronic Speculum observation and finds that the virus protein of expression can be assembled into virus-like particle (virus like particles).Mesh
Before, the vaccine based on baculovirus expression system production is widely used, because baculoviral can only infect non-spinal animal, and
Its host insect cell infected can not extraneous parasitic, therefore, using the VP60 albumen of expression system expression have compared with
High production practices meaning.
Description of the drawings
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon：
The PCR amplification figure of Fig. 1 RHDV2-VP60；Wherein：M is DNA standards Trans2KplusII；1 is RHDV2-VP60F/
R amplified fragments；2 be negative control；
The digestion qualification figure of Fig. 2 recombinant plasmids PFAST-RHDV2-VP60；Wherein：M is DNA standards marker
Trans2KplusII；1-3 is parallel tests of the PFastHTA-RHDV2-VP60 through EcoRI and XbaI double digestions；
The PCR amplification figure of Fig. 3 recombinant plasmids Bacmid-RHDV2-VP60；Wherein M：DNA standards marker
Trans2KplusII；1-12：The parallel test of Bacmid-RHDV2-VP60F/R amplified fragments；
The PCR of Fig. 4 recombinant plasmids Bacmid-RHDV2-VP60 is identified；Wherein M：DNA standards marker
Trans2KplusII；1-5：The parallel test of the Bacmid-RHDV2-VP60 PCR products of primer M13F and M13R；
Fig. 5 recombinant plasmids express figure on Sf9 insect cells, and wherein Fig. 5 A are the Sf9 for transfecting Bacmid-RHDV2-VP60
Insect cell；Fig. 5 B are normal Sf9 insect cells；
The expression identification figure of Fig. 6 recombinant proteins；Wherein：M is albumen pre-dyed Marker；1 is Sf9 insect cell supernatants；2 are
Sf9 insect cell pellets；
Fig. 7 recombinant baculovirus mBacmid-RHDV2-VP60 infection Sf9 cellmediated immunities fluoroscopic examinations (IFA)；Its
In：Fig. 7 A：10 times of fluorescence microscope downward views of infection cell；Fig. 7 B：10 times of fluorescence microscope downward views of normal cell；
The Electronic Speculum detection figure of the capsid protein of Fig. 8 baculovirals mBacmid-RHDV2-VP60 expression.
Specific implementation mode
With reference to specific embodiment, the present invention is described in detail.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
For personnel, without departing from the inventive concept of the premise, several changes and improvements can also be made.These belong to the present invention
Protection domain.
Embodiment 1
1.1 strains, plasmid, cell
PJET1.2-VP60 plasmids are given by Wageningen University's virology laboratory, PFASTBAC-HTA plasmids,
DH10bac competent cells are purchased from Invitrogen companies, and it is limited that Trans1-T1 competent cells are purchased from the full formula gold biology in Beijing
Company, Sf9 insect cells are preserved by this laboratory.
1.2 main agents
Small amount gel reclaims kit is purchased from Shanghai life work biology Co., Ltd；Mini-scale plasmid extracts kit is purchased from
Axygen companies；Restriction enzyme EcoRI and XbaI, T4DNA ligase are purchased from NEB companies；DNA standards marker
Trans2KplusII, PEASY-Blunt-Zero cloning vector are purchased from the complete biological Co., Ltd of formula gold in Beijing；Phusion DNA-
Polymerase, SFM900II Insect culture medium, Cellfectin II transfection reagent boxes are purchased from Thermo-fisher companies；Mouse
The sheep anti-mouse igg of source His labels primary antibody, HRP labels and FITC labels is purchased from CST companies；The chemical reagent such as ethyl alcohol, isopropanol are equal
It is purchased from Shanghai life work biology Co., Ltd.
The design and synthesis of 1.3 primers
Amplification and identification of the pair of primers for VP60 genes are devised according to RHDV2 sequence accession number base sequences.
RHDV2F(SEQ ID No.1)：5’-CGGAATTCATGGAGGGCAAAGCCCGC—3’；
RHDV2R(SEQ ID No.2)：5’-GCTCTAGATCAGACATAAGAAAAGCCATTGG-3’；
Underscore is respectively EcoRI and XbaI restriction enzyme enzyme sites, and the target gene fragment of amplification is 1740bp.
Reference《Bac-to-Bac baculovirus expression systems》Service manual is synthesized for identifying that the general of recombinant virus draws
Object M13Forward (SEQ ID No.3):5’-GTTTTCCCAGTCACGAC-3’；M13Reverse(SEQ ID No.4):5’-
CAGGAAACAGCTATGAC-3 ', primer transfer to Shanghai to give birth to work biosynthesis.
The amplification of 1.4 target gene
Using pJET1.2-VP60 plasmids as template, RHDV2F/RHDV2R is primer, utilizes Phusion DNA
Polymerase carries out the high-fidelity specific amplification of segment, obtains target fragment RHDV2-VP60.As shown in Figure 1, in 1740bp
There is single band in place, it was demonstrated that obtains required target gene fragment.
The structure of 1.5 cloning vector PEASY-RHDV2-VP60 and identification
After room temperature connect half an hour, connection is produced with PEASY-Vector for the target fragment that step 1.4 amplification is obtained
Coated plate after object is converted to Trans1-T1 competent cells is inverted tablet and is incubated overnight in 37 DEG C of constant incubator, picking single bacterium
Row PCR identifications are dropped into, it is to have obtained cloning vector PEASY-RHDV2-VP60 that PCR, which is positive, is that positive bacterium solution carries out by PCR
It preserves.Recombinant plasmid carries out PCR Preliminary Identifications, amplified production 1740bp, with theoretical value phase with primer RHDV2F/RHDV2R
Symbol.
The purifying of 1.6 recombinant plasmid PEASY-RHDV2-VP60
Using the positive bacterium solution of the small upgrading grain extraction step 1.5 of AxyGEN companies, the positive plasmid of purifying is obtained
PEASY-RHDV2-VP60, in -20 DEG C of preservations.
The structure of 1.7 expression vector PFAST-RHDV2-VP60 and identification
The PEASY-RHDV2-VP60 and baculovirus transfer vector PFASTBAC-HTA limits that step 1.6 is obtained
Property restriction endonuclease EcoRI and XbaI processed carries out double digestion in 37 DEG C, is separately recovered after digestion, digestion products T4DNA ligases
16 DEG C of water-baths connect overnight, and connection product is converted to Trans1-T1 competent cells, are inverted tablet in 37 DEG C of constant incubators
It is incubated overnight.Choose bacterium carry out bacterium colony PCR identifications, while recombinate positive plasmid extracting plasmid after through EcoRI and XbaI double digestions
There is the target gene band of 1740bp, size to be consistent with expected results (as shown in Figure 2) afterwards, and serve the raw work biological order-checking in sea into
One step sequencing result, which also confirms that, has been successively inserted into foreign gene, and reading frame is correct, it was demonstrated that successfully constructs recombinant plasmid
PFAST-RHDV2-VP60。
The structure of 1.8 recombinant plasmid Bacmid-RHDV2-VP60 and identification
Step 1.7 is identified that correct recombinant expression carrier PFAST-RHDV2-VP60 converts DH10bac competent cells,
Be applied to containing kanamycins, tetracycline, three kinds of antibiotic of gentamicin tablet on, on tablet simultaneously be coated with X-gal and IPTG,
37 degree of culture 36-48h have been coated on kanamycins, tetracycline, gentamicin three again after cultivating visible white colony dilution
On the tablet of kind antibiotic, it is coated with X-gal and IPTG simultaneously on tablet, again 37 DEG C of culture 36-48h.Picking single bacterium colony, with
RHDV2F/RHDV2R and M13F/M13R carries out bacterium colony PCR identifications, obtains and identifies correct positive bacteria.
The extraction of 1.9 recombinant plasmids
With reference to《Molecular cloning handbook》The positive bacteria obtained using isopropanol-sodium acetate method (alkaline extraction) extraction step 1.8
Recombinant plasmid, reuse RHDV2F/RHDV2R and M13F/M13R and carry out PCR identifications, as a result as shown in Figure 3 and Figure 4,
Amplified production is 4120bp, which proves that swivel base success, recombinant plasmid Bacmid-RHDV2-VP60 are built into target gene
Work(.
Expression of 1.10 recombinant plasmids in insect cell
Sf9 insect cells are cultivated in 28 DEG C of incubators using SFM900II Insect culture mediums, with reference to Invitrogen
Bac-to-bac Baculovirus Expression system are in the liposome-mediated lower recombinations that will be obtained of Cellfectin
Plasmid Bacmid-RHDV2-VP60 carries out transfection Sf 9 insect cell, using non-transfected cells as negative control.Every observing one for 24 hours
The secondary cell growth status in 28 DEG C of incubators collects cell culture fluid in 96h-120h or so, and 3000rpm centrifuges 10min,
Supernatant is recombinant baculovirus liquid, is stored in 4 DEG C, is named as mBacmid-RHDV-VP60.Third generation weight is obtained through passage
Group is viral, carries out Western-blot and analyzes its expression product, when result is positive, is dispensed and frozen in -80 DEG C as kind
Poison.
The identification of 1.11 expression products
1) Western-blot is identified
By the third generation virus mBacmid-RHDV-VP60 passed in step 1.10 according to 1:10~1:100 ratio
Example infection Sf9 insect cells after 28 DEG C are cultivated 72-96 hours, take the supernatant precipitation of the Sf9 insect cells of virus infection to carry out
Sample is carried out SDS-PAGE electrophoresis using Bio-Rad electrophoresis apparatuses by identification, after will be on gel with the half-dried film instrument of walking around of Bio-Rad
Each band be transferred on nitrocellulose filter (NC), using the monoclonal antibody of the anti-His labels in mouse source as primary antibody (1:1000 times of dilutions),
The sheep anti-mouse igg (1 of HRP labels；10000 times are diluted to secondary antibody) carry out Western blot analyze and identify.As shown in figure 5,28
DEG C culture 48h-60h can observe cytopathy under inverted microscope, transfectional cell, which becomes larger, compared with normal cell is rounded, carefully
Karyon increases, and occurs polyhedral body in nucleus, and infection phenomenons occur in about 90% cells of transfection 96h or so.As shown in fig. 6, albumen
There is a characteristic band at 63KDa in matter molecular weight.
2) indirect immunofluorescene assay (IFA)
In 6 orifice plates, the 5th generation recombinant virus is inoculated with to the Sf9 insect cells of exponential phase, culture to generation lesion
48h detects the expression of recombinant protein with indirect immunofluorescence after about infecting, and the monoclonal antibody using mouse source His labels is primary antibody (1:
200 times of dilutions), the sheep anti-mouse igg (1 of FITC labels；5000 times are diluted to secondary antibody) it is identified.The results are shown in Figure 7, Fig. 7 A
There is fluorescence with the cell of the visible virus infection successful expression recombinant protein of comparison of Fig. 7 B, and blank control unstressed configuration.
Western blot and IFA prove that the recombinant virus being capable of successful expression recombinant protein.
3) the Electronic Speculum detection of recombinant protein
Step 1.10 is collected to the Sf9Sf Insect cellcultures supernatant (egg containing recombination after recombinate shape virus infection 72h
In vain), with sonicator smudge cells two minutes (effect 5s, pause 5s) afterwards differential centrifugation after purification, with 1M phosphotungstic acids into
After row negative staining film-making, transmission electron microscope observing (Fig. 8) can find that size is about 30nm, the virus-like particle of polyhedral symmetrical, and accord with
Close the form of typical Caliciviridae virus-like particle.
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make a variety of changes or change within the scope of the claims, this not shadow
Ring the substantive content of the present invention.In the absence of conflict, the feature in embodiments herein and embodiment can arbitrary phase
Mutually combination.
Sequence table
<110>China Agriculture Academe Shanghai Veterinary Institute（China Animal Health and Epidemiology Center Shanghai branch center）
<120>A kind of preparation and application of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus
<130> DAG35715
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
cggaattcat ggagggcaaa gcccgc 26
<210> 2
<211> 31
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
gctctagatc agacataaga aaagccattg g 31
<210> 3
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gttttcccag tcacgac 17
<210> 4
<211> 17
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
caggaaacag ctatgac 17
Claims (8)
1. a kind of preparation method of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus, which is characterized in that packet
Include following steps：
A, the VP60 genes of RHDV2 are expanded；
B, the VP60 gene clonings for obtaining step A obtain plasmid in baculovirus transfer vector；
C, the plasmid transformed competence colibacillus cell for obtaining step B, obtains recombinant plasmid；
D, the recombinant plasmid that step C is obtained is gone out in liposome-mediated lower transfection insect cell to get the expression rabbit viral
Mass formed by blood stasis virus type II capsid protein baculoviral.
2. the preparation side of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus according to claim 1
Method, which is characterized in that in step A, expand primer sequence such as SEQ ID No.1 and the SEQ ID that the VP60 genes of RHDV2 use
Shown in No.2.
3. the preparation side of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus according to claim 2
Method, which is characterized in that the restriction enzyme site of the primer is respectively GAATTC and TCTAGA.
4. the II rod-shaped disease of type capsid protein of expression rabbit hemorrhagic disease virus prepared by a kind of method according to claim 1
Poison.
5. a kind of expression product identification method of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus, feature
It is, includes the following steps：II type capsid protein baculovirus infection insect of the expression rabbit hemorrhagic disease virus is thin
It is cultivated after born of the same parents, takes the vial supernatant of culture, by Western-blot and IFA methods to the feature egg of expressing viral
It is identified in vain.
6. the expression product of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus according to claim 5
Identification method, which is characterized in that the II type capsid protein baculoviral of expression rabbit hemorrhagic disease virus and insect cell
Ratio be 1:10~1:100.
7. the expression product of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus according to claim 5
Identification method, which is characterized in that the condition of culture is：28 DEG C of culture 96h~120h.
8. a kind of application of expression II type capsid protein baculoviral of rabbit hemorrhagic disease virus, which is characterized in that described to answer
With including：Rabbit hemorrhagic disease II is prepared using II type capsid protein baculoviral of the expression rabbit hemorrhagic disease virus
Type subunit vaccine, or prepare the application in II type Enzyme-linked Immunosorbent Assay reagents of RHDV.
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