CN107164252A - A kind of RHDV subunit vaccine - Google Patents
A kind of RHDV subunit vaccine Download PDFInfo
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- CN107164252A CN107164252A CN201610131290.2A CN201610131290A CN107164252A CN 107164252 A CN107164252 A CN 107164252A CN 201610131290 A CN201610131290 A CN 201610131290A CN 107164252 A CN107164252 A CN 107164252A
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/525—Virus
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- C12N2770/00011—Details
- C12N2770/16011—Caliciviridae
- C12N2770/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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Abstract
The invention provides a kind of RHDV subunit vaccine, specifically the present inventor by extensive in-depth study, obtain a kind of method for preparing rabbit hemorrhagic disease virus VLP particles.The invention provides a kind of genetically engineered cell, the genetically engineered cell expresses the expression cassette of rabbit hemorrhagic disease virus VP 60 albumen to be integrated with eukaryotic, and the genome of the cell；Or containing expression vector in the cell, the expression vector contains the expression cassette for expressing the rabbit hemorrhagic disease virus VP 60 albumen；And the genetically engineered cell is in VP60 albumen described in intracellular expression, and the VP60 albumen is internally formed virus-like particle (VLP) in the genetically engineered cell.Present invention also offers the virus-like particle (VLP) by described genetically engineered cell expression.Test result indicates that, rabbit hemorrhagic disease virus VLP particles can efficiently be prepared according to the genetically engineered cell and method of the present invention.
Description
Technical field
The invention belongs to biological technical field, specifically, the present invention relates to a kind of RHDV subunit vaccine.
Background technology
Rabbit haemorrhagic disease (Rabbit Hemorrhagic Disease, RHD) is by rabbit hemorrhagic disease virus (Rabbit
Hemorrhagic Disease Virus, RHDV) caused by rabbit a kind of acute, septic, highly contagious disease, with
Lethal and whole body organa parenchymatosum bleeding are principal character.In rabbit aquaculture, endanger huge.
Virus-like particle (Virus Like Particles, VLP) is the sky containing viral one or more structural proteins
Heart particle, without viral nucleic acid (DNA/RNA), it is impossible to autonomous replication, its morphologically or phase identical with real virion
Seemingly, immunocyte can be presented to by the approach being infected with virus, effectively induces the immune system of body to produce and be immunized
Protection reaction.Because VLPs is without infectivity, as immunogene, it can be presented to immune by the approach as virus infection
Cell, effectively induces the immune system of body to produce immunoprotection reaction, and host, virus and vaccine factor are together decided on
The body protection level that VLPs is produced.
Recover once the defect of current live-virus vaccine is exactly virus activity, security it cannot be guaranteed that.Meanwhile, inactivation of viruses
Vaccine not exclusively can cause safety issue because of inactivation process.But VLPs vaccines, without infectivity, stability is good, not volatile
It is living, with vast potential for future development.
The content of the invention
It is an object of the invention to provide the subunit vaccine of RHDV a kind of and its application.
In the first aspect of the present invention there is provided a kind of genetically engineered cell, the genetically engineered cell is eukaryotic,
And the expression cassette of expression rabbit hemorrhagic disease virus VP 60 albumen is integrated with the genome of the cell；Or contain in the cell
There is expression vector, the expression vector contains the expression cassette for expressing the rabbit hemorrhagic disease virus VP 60 albumen；
And the genetically engineered cell is in VP60 albumen described in intracellular expression, and the VP60 albumen is in the gene work
Journey cell interior formation virus-like particle (VLP).
In another preference, the eukaryotic is yeast cells, preferably Pichia pastoris.
In another preference, described expression cassette includes the elements below that 5' to 3' is operably connected：Promoter, rise
The ORF sequences and terminator codon of beginning codon, the VP60 albumen.
In another preference, described initiation codon and the ORF sequences of the VP60 albumen are joined directly together.
In another preference, described expression cassette does not contain secreting, expressing element.
In another preference, described expression cassette does not contain secretion peptide, guiding peptide or signal peptide.
In another preference, the gene order such as SEQ ID NO. of the VP60 albumen:Shown in 1.
In another preference, the expression vector is using Pichia pastoris pPICX carriers as skeleton.
The second aspect of the present invention is there is provided a kind of virus-like particle (VLP), and the VLP is by first aspect present invention institute
The genetically engineered cell expression stated.
The third aspect of the present invention includes first party of the present invention there is provided a kind of pharmaceutical composition, described pharmaceutical composition
Virus-like particle described in face, and pharmaceutically acceptable carrier.
In another preference, described pharmaceutical composition includes vaccine combination.
There is provided a kind of method for preparing VLP, including step for the fourth aspect of the present invention：
Under conditions suitable for the expression, the cell described in culture first aspect present invention, so as to give expression to the present invention second
Virus-like particle (VLP) described in aspect；With
Separate the virus-like particle (VLP).
There is provided a kind of polynucleotides through codon optimization of separation, the polynucleotides for the fifth aspect of the present invention
Encode SEQ ID NO.:Polypeptide shown in 2；And the polynucleotides are selected from the group：
(a) sequence such as SEQ ID NO.:Polynucleotides shown in 1；
(b) nucleotide sequence and SEQ ID NO.:The multinuclear of homology >=95% (preferably >=98%) of sequence shown in 1
Thuja acid；
(c) such as SEQ ID NO.:5 ' the ends and/or 3 ' ends of polynucleotides shown in 1 are truncated or addition 1-60 (preferably 1-
30, more preferably 1-10) nucleotides polynucleotides；
(e) polynucleotides complementary with any described polynucleotides of (a)-(c).
The sixth aspect of the present invention is there is provided a kind of expression vector, and the expression vector contains fifth aspect present invention institute
The polynucleotides stated.
The seventh aspect of the present invention contains sixth aspect present invention there is provided a kind of host cell, described host cell
Described expression vector, or it is integrated with genome the polynucleotides described in fifth aspect present invention.
The eighth aspect of the present invention contains second aspect of the present invention there is provided a kind of pharmaceutical composition, described composition
Polynucleotides described in described virus-like particle (VLP), fifth aspect present invention or the table described in sixth aspect present invention
Up to the host cell described in carrier or seventh aspect present invention, and pharmaceutically acceptable carrier and/or auxiliary material.
In another preference, described pharmaceutical composition includes vaccine combination.
In another preference, described vaccine combination also contains adjuvant.
In another preference, described adjuvant includes aluminum oxide, saponin(e, quil A, muramyl dipeptide, mineral oil or plant
Thing oil, the adjuvant based on vesica, non-ionic block copolymer or deae dextran, cell factor (including IL-1, IL-2, IFN-
R, GM-CSF, IL-6, IL-12 and CpG).
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
It can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 shows the digestion identification of pPIC3.5K-VP60 expression vectors.
Fig. 2 shows expression of the SDS-PAGE and Western blot detection RHDV VP60 albumen in Pichia pastoris.
Fig. 3 shows negative staining electron microscope Germicidal efficacy RHDV virions.
Fig. 4 shows experiment rabbit pathological anatomy result.
Embodiment
The present inventor's in-depth study by extensive, obtains a kind of method for preparing rabbit hemorrhagic disease virus VLP particles, real
Test result to show, the method according to the invention can efficiently prepare rabbit hemorrhagic disease virus VLP particles.
Before describing the present invention, it should be understood that the invention is not restricted to described specific method and experiment condition, because this
Class method and condition can change.It should also be understood that its purpose of term used herein is only that description specific embodiment, and
And it is not intended to be restricted, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein are respectively provided with such as art of the present invention with scientific terminology
The identical meanings that are generally understood that of those of ordinary skill.As used herein, in use, term in the numerical value specifically enumerated is mentioned
" about " mean that the value can change from the value enumerated and be not more than 1%.For example, as used herein, " about 100 " include 99 Hes for statement
101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although can be used and heretofore described similar or of equal value any method in the implementation or test of the present invention
And material, herein place enumerate preferred method and material.
Rabbit hemorrhagic disease virus (Rabbit Hemorrhagic Disease Virus, RHDV)
Rabbit haemorrhagic disease (Rabbit Hemorrhagic Disease, RHD) is that RHDV is Caliciviridae member, includes master
Want structural proteins VP60, VP60 to can induce in animal body and produce neutralizing antibody, it is directly related with immune response, it is virus immunity
Protective antigens.
RHDV VP60 native gene sequence is as follows：
>gi|358409917|gb|JN165233.1|Rabbit hemorrhagic disease virus isolate
TC/China/2007VP60gene,partial cds
ATGGAGGGCAAAGCCCGCACAGCGCCGCAAGGCGAAGCAGCAGGCACTGCTACCACAGCATCAGTTCCCGGAACCAC
GACCGACGGCATGGATCCTGGAGTAGTGGCCGCGACTAGTGTGGTCACTGCAGAAAATTCATCCGCATCGGTTGCAA
CGGCGGGGATTGGCGGCCCACCCCAACAGGTGGACCAACAAGAAACATGGAGGACAAACTTTTACTACAATGATGTT
TTCACTTGGTCCGTCGCGGATGCGCCCGGCAGCATTCTCTACACTGTCCAACACTCTCCACAGAACAACCCATTCAC
AGCCGTACTGAGCCAGATGTACGCTGGCTGGGCTGGTGGCATGCAGTTCCGCTTCATAGTTGCTGGATCAGGTGTGT
TTGGTGGGCGACTGGTCGCGGCTGTGATACCACCAGGCATCGAGATTGGACCAGGGTTGGAGGTCAGGCAATTTCCT
CATGTCGTCATCGACGCCCGTTCACTCGAACCTGTTACCATCACCATGCCAGACTTGCGTCCCAACATGTACCATCC
AACTGGTGACCCTGGCCTTGTCCCCACACTAGTCCTTAGTGTTTACAACAACCTCATCAACCCGTTTGGTGGGTCCA
CCAACGCAATCCAGGTAACAGTGGAAACGAGGCCGAGTGATGACTTTGAGTTCGTGATGATTAGAGCCCCCTCCAGC
AAAACTGTTGACTCAATCTCACCCGCAGGCCTCCTCACGACCCCAGTCCTCACTGGTGTTGGCAATGACAACAGGTG
GAACGGCCAAATAGTGGGACTGCAACCAGTACCTGGGGGGTTTTCCACGTGCAACAGGCATTGGAACCTGAACGGCA
GCACGTATGGCTGGTCAAGCCCTCGGTTTGCCGACATTGACCATCGAAGAGGCAGTGCAAGTTATTCTGGGAACAAC
TCCACCAACGTGCTTCAGTTTTGGTACGCTAATGCTGGGTCTGCAATTGACAACCCTATCTCCCAGGTTGCACCGGA
CGGCTTCCCTGACATGTCATTCGTGCCCTTTAACAGCCCCAACATTCCGACCGCGGGGTGGGTCGGGTTTGGTGGTA
TTTGGAACAGTAACAACGGTGCCCCCGCTGCTACAACTGTGCAGGCCTATGAGTTAGGTTTTGCCACTGGGGCACCA
AATAACCTCCAGCCCACCACCAACACTTCAGGTGCACAGACTGTCGCTAAGTCCATTTATGCCGTGGTAACCGGCAC
AAACCAAAATCCAACCGGACTGTTTGTGATGGCCTCGGGTGTTATCTCCACCCCAAACGCCAGCGCCGTCACATACA
CGCCCCAACCAGACAGAATTGTGACTACACCCGGCACTCCTGCCGCCGCACCTGTGGGTAAGAACACACCCATCATG
TTCGCGTCTGTTGTCAGGCGCACCGGTGACGTCAACGCCGCAGCTGGGTCAACCAACGGGACCCAGTACGGCACGGG
CTCCCAACCACTGCCAGTAACAATTGGACTTTCGCTCAACAACTACTCGTCAGCACTCATGCCTGGGCAGTTTTTCG
TTTGGCAGTTAACCTTTGCATCTGGTTTCATGGAGATTGGCTTAAGTGTGGACGGGTACTTTTATGCAGGAACAGGA
GCCTCAACCACGCTCATTGACTTGACTGAACTCATTGACGTACGCCCCGTGGGACCCCGGCCGTCCAAGAGCACACT
CGTGTTCAACCTGGGGGGCACAACCAATGGCTTTTCTTATGTCTGA(SEQ ID NO.3)
According to the native gene sequence of RHDV VP60 albumen, the present inventor obtains by a large amount of screenings to be adapted in yeast
The optimization gene sequence of the coding VP60 polypeptides of middle expression.
RHDV VP60 optimization gene sequence is as follows：
>ATGGAAGGAAAAGCCAGAACCGCTCCACAAGGTGAAGCTGCCGGAACCGCTACTACCGCCTCCGTTCC
TGGAACTACTACCGATGGAATGGACCCAGGAGTTGTTGCCGCTACTTCCGTTGTTACTGCTGAGAACTCCTCTGCCT
CTGTTGCTACTGCTGGTATCGGTGGTCCTCCACAGCAAGTTGATCAACAAGAAACCTGGCGTACCAACTTCTACTAT
AACGACGTTTTCACTTGGTCTGTTGCTGACGCCCCAGGTTCTATCTTGTACACTGTTCAACATTCTCCACAAAACAA
CCCATTCACCGCCGTTTTGTCCCAGATGTACGCTGGTTGGGCTGGTGGTATGCAGTTCAGATTCATTGTTGCCGGTT
CCGGTGTCTTCGGTGGTAGATTGGTCGCTGCTGTCATCCCACCTGGTATTGAGATCGGTCCAGGTTTGGAGGTCCGT
CAATTCCCACACGTTGTTATCGACGCCCGTTCTTTGGAGCCAGTTACCATCACTATGCCAGATTTGAGACCTAACAT
GTACCACCCAACTGGTGATCCTGGTCTTGTCCCTACTTTGGTTTTGTCTGTTTACAACAATTTGATCAACCCATTTG
GTGGTTCCACCAACGCTATTCAGGTTACCGTCGAGACCAGACCATCCGACGACTTCGAGTTTGTCATGATCCGTGCT
CCATCCTCCAAGACTGTTGACTCCATCTCCCCAGCTGGTTTGTTGACTACTCCAGTCTTGACCGGTGTCGGAAACGA
CAACCGTTGGAACGGTCAGATTGTCGGTTTGCAACCTGTCCCTGGAGGTTTCTCTACCTGCAACCGTCACTGGAACT
TGAACGGTTCCACTTACGGTTGGTCCTCTCCAAGATTCGCCGATATCGACCACAGAAGAGGTTCCGCCTCTTACTCC
GGAAATAACTCCACCAACGTCTTGCAGTTCTGGTACGCCAACGCCGGTTCTGCCATCGACAACCCAATTTCCCAAGT
TGCCCCAGACGGTTTCCCAGATATGTCTTTTGTTCCATTCAACTCTCCTAACATCCCAACTGCCGGTTGGGTTGGTT
TCGGTGGTATCTGGAACTCCAACAACGGTGCTCCTGCTGCCACCACTGTCCAGGCTTACGAACTTGGTTTCGCCACC
GGTGCTCCTAACAACTTGCAGCCAACTACTAACACTTCCGGTGCTCAGACCGTTGCCAAATCCATCTACGCTGTTGT
CACCGGTACCAATCAGAACCCAACCGGTTTGTTTGTTATGGCTTCCGGAGTCATCTCCACTCCTAACGCCTCTGCTG
TTACCTACACCCCACAGCCTGACAGAATTGTCACTACCCCAGGAACTCCTGCTGCCGCTCCAGTTGGAAAGAACACT
CCTATCATGTTCGCCTCCGTCGTTCGTAGAACCGGTGACGTTAACGCTGCCGCTGGTTCCACTAACGGTACCCAATA
CGGTACCGGTTCTCAGCCATTGCCAGTCACCATTGGTTTGTCTTTGAATAACTATTCCTCTGCCTTGATGCCAGGTC
AGTTCTTTGTCTGGCAGTTGACCTTCGCTTCCGGATTCATGGAGATCGGTTTGTCTGTTGATGGATACTTTTACGCC
GGTACCGGTGCCTCTACCACTCTTATCGATCTTACCGAATTGATCGATGTCCGTCCAGTTGGTCCTAGACCTTCCAA
GTCCACCTTGGTCTTCAACTTGGGAGGTACTACCAATGGTTTTTCTTACGTTTAA(SEQ ID NO.1)
The amino acid sequence of natural VP60 polypeptides is as follows：
>gi|358409918|gb|AEU09705.1|VP60,partial[Rabbit hemorrhagic disease
virus]
MEGKARTAPQGEAAGTATTASVPGTTTDGMDPGVVAATSVVTAENSSASVATAGIGGPPQQVDQQETWRTNFYYNDV
FTWSVADAPGSILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFP
HVVIDARSLEPVTITMPDLRPNMYHPTGDPGLVPTLVLSVYNNLINPFGGSTNAIQVTVETRPSDDFEFVMIRAPSS
KTVDSISPAGLLTTPVLTGVGNDNRWNGQIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFADIDHRRGSASYSGNN
STNVLQFWYANAGSAIDNPISQVAPDGFPDMSFVPFNSPNIPTAGWVGFGGIWNSNNGAPAATTVQAYELGFATGAP
NNLQPTTNTSGAQTVAKSIYAVVTGTNQNPTGLFVMASGVISTPNASAVTYTPQPDRIVTTPGTPAAAPVGKNTPIM
FASVVRRTGDVNAAAGSTNGTQYGTGSQPLPVTIGLSLNNYSSALMPGQFFVWQLTFASGFMEIGLSVDGYFYAGTG
ASTTLIDLTELIDVRPVGPRPSKSTLVFNLGGTTNGFSYV(SEQ ID NO.2)
Genetically engineered cell
The invention provides a kind of genetically engineered cell, the genetically engineered cell is eukaryotic, and the cell
Genome in be integrated with the expression cassette of rabbit hemorrhagic disease virus VP 60 albumen；Or contain expression vector in the cell, it is described
Expression vector contains the expression cassette of the rabbit hemorrhagic disease virus VP 60 albumen；
And the genetically engineered cell is in VP60 albumen described in intracellular expression, and the VP60 albumen is in the gene work
Journey cell interior formation virus-like particle (VLP).
In another preference, the cell is yeast cells, preferably Pichia pastoris, more preferably Pichia pastoris
KM71 bacterial strains.
In another preference, described expression cassette includes the elements below that 5' to 3' is operably connected：Promoter, rise
The ORF sequences and terminator codon of beginning codon, the VP60 albumen.
In the present invention, term " being operably connected " means such configuration, wherein regulating and controlling sequence is placed in relative to many
The appropriate location of the coded sequence of nucleotides so that regulating and controlling sequence instructs the expression of coded sequence.
Composition and application process
Present invention also offers a kind of composition, it contains：(i) recombinant virus sample particle (VLP) or this hair of the invention
The polynucleotides of bright codified recombinant virus sample particle, and (ii) acceptable excipient or assistant pharmaceutically or in immunology
Agent.
In the present invention, term " containing " represents that various composition can together be applied to or be present in the composition of the present invention.
Therefore, term " mainly by ... constitute " and " consist of " are included in term " containing ".
The composition of the present invention includes pharmaceutical composition and vaccine combination.
The composition of the present invention can be monovalent (only containing a kind of recombinant virus sample particle or polynucleotides), can also
It is multivalence (containing a variety of recombinant virus sample particles or polynucleotides).
The pharmaceutical composition or vaccine combination of the present invention can be prepared into various regular dosage forms, including (but do not limit
In)：Injection, granula, tablet, pill, suppository, capsule, suspension, spray etc..
(1) pharmaceutical composition
The pharmaceutical composition of the present invention includes the recombinant virus sample particle of the present invention or multinuclear of (or containing) therapeutically effective amount
Thuja acid.
Term " therapeutically effective amount " used herein refers to therapeutic agent treatment, alleviates or prevent the amount of target disease or situation,
Or show the amount of detectable treatment or prevention effect.The effect can be detected for example, by antigen levels.Therapeutic effect
Also the reduction of physical symptoms is included.For a certain object accurate effective dose depend on the object build and health status,
The combination of therapeutic agent and/or therapeutic agent that the nature and extent of illness and selection are given.Therefore, preassigning accurately has
Effect amount is useless.However, for certain given situation, the effective dose can be determined with normal experiment.
For the purposes of the present invention, effective dosage is to give individual about 0.001 mg/kg to 1000 mg/kgs,
It is preferably about the recombinant virus sample particle of 0.01 mg/kg to 100 mg/kg body weight.
Pharmaceutical composition can also contain pharmaceutically acceptable carrier.Term " pharmaceutically acceptable carrier " refers to for controlling
Treat the carrier of agent (recombinant virus sample particle of the invention) administration.The term refers to some such medicament carriers：Themselves
The antibody for producing and being harmful to the individual for receiving said composition is not induced, and does not have undue toxicity after administration.Suitable carrier can
To be big, be metabolized slow macromolecular, such as protein, polysaccharide, PLA (polylactic acid), polyglycolic acid.This
A little carriers are well known to those of ordinary skill in the art.In Remington ' s Pharmaceutical Sciences (Mack
Pub.Co., N.J.1991) in can find discussing fully on pharmaceutically acceptable carrier or excipient.
The upper acceptable carrier of combination of traditional Chinese medicine may include liquid, such as water, salt solution, glycerine and ethanol.In addition, these are carried
Complementary material, such as wetting agent or emulsifying agent, pH buffer substance are there is likely to be in body.Generally, composition can be made
Injectable agent, such as liquid solution or suspension；It may also be fabricated which and be adapted to supplying solution or suspension, liquid excipient before the injection
Solid form.Liposome is also included within the definition of pharmaceutically acceptable carrier.
(ii) vaccine combination
The vaccine (composition) of the present invention can be preventative (i.e. prevention disease) or curative (be controlled after illness
Treat disease).
These vaccines include immunising antigen (including recombinant virus sample particle of the present invention), and generally with " pharmaceutically may be used
The carrier of receiving " is combined, and these carriers include itself not inducing times for producing the antibody for being harmful to the individual for receiving said composition
What carrier.Suitable carrier is typically big, is metabolized slow macromolecular, such as protein, polysaccharide, PLA, polyglycolic acid, ammonia
Base acid polymer, amino acid copolymer, lipid aggregates (such as oil droplet or liposome).These carriers are ordinary skills
Known to personnel.In addition, these carriers can play immunostimulant (" adjuvant ").In addition, antigen can also be with bacterium class
Toxin (such as diphtheria, lockjaw, cholera, the toxoid of helicobacter pylori pathogen) is coupled.
The preferred adjuvant of enhancing immune composition effect includes but is not limited to：(1) aluminium salt (alum), such as aluminium hydroxide, phosphorus
Sour aluminium, aluminum sulfate etc.；(2) oil-in-water emulsion formula, for example, (a) MF59 (referring to WO90/14837), (b) SAF, and (c)
Ribi adjuvant systems (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvant；(4) Freund Freund's complete adjuvants
And Freund Freund's incomplete adjuvants (IFA) (CFA)；(5) cell factor, such as interleukin (such as IL-1, IL-2, IL-4, IL-5, IL-6,
IL-7, IL-12 etc.), interferon (such as interferon), macrophage colony stimulatory factor (M-CFS), TNF (TNF)
Deng；(6) the detoxification variant of bacterial ADPribosylating toxin (such as E.coli LT LT)；And (7) conduct
Immunostimulant strengthens the other materials of composition effect.
Vaccine combination including immunogenic composition is (for example, it may include antigen, pharmaceutically acceptable carrier
And adjuvant), usually contain diluent, such as water, salt solution, glycerine, ethanol etc..In addition, auxiliary substances, such as wetting agent or emulsification
Agent, pH buffer substance etc. may be present in this kind of carrier.
More specifically, the vaccine including immunogenic composition, the immunogenic polypeptide comprising immunological effective amount,
And above-mentioned other required components." immunological effective amount " refers to gives the amount of individual to treatment with single dose or a continuous agent part
Or prevention is effective.The consumption can be according to treating the health status and physiological status of individual, treat individual classification (such as
People), the ability of individual immunity system synthesis antibody, required degree of protection, the preparation of vaccine, treating physician is to medical conditions
Depending on assessment and other correlative factors.It is expected that the consumption is by relatively wide scope, can be by normal experiment come really
It is fixed.
Generally, injectable agent, such as liquid solution or suspension can be made in vaccine combination or immunogenic composition；Also
It can be made into and be adapted to supplying solution or suspension, the solid form of liquid excipient before the injection.Said preparation is also emulsifiable or is encapsulated in
In liposome, to strengthen adjuvant effect.
In addition, the vaccine combination of the present invention can be unit price or polyvaccine.
(iii) method of administration and dosage
Once being made into the composition of the present invention, object can be directly given by it.Object to be treated can be mammal,
Especially people.
When as vaccine, can with known method by the present invention recombinant virus sample particle be directly applied to individual.It is logical
These vaccines are applied frequently with conventional vaccine identical route of administration and/or simulation pathogenic infection path.
Giving the approach of pharmaceutical composition of the present invention or vaccine combination includes (but being not limited to)：Intramuscular, subcutaneous, skin
Interior, intrapulmonary, intravenous, intranasal, by oral administration or other parenteral route of administration.It is possible if desired to combination medicine-feeding approach, or root
It is adjusted according to disease event.Vaccine combination can be given with single dose or multiple dose, and can include give booster with
Trigger and/or maintain immunity.
Recombinant virus sample particle vaccines should be given with " effective dose ", i.e., the amount of recombinant virus sample particle is in selected administration
Immune response is adequate to bring about in path, can effectively promote the disease for protecting host's resistance related.
Representational disease includes (but being not limited to)：Rabbit hemorrhagic disease virus infection etc..
The amount of selected recombinant virus sample particle in each vaccine dose part, is by can trigger protective immune response and nothing
Depending on the amount of obvious side effect.Generally, after host cells infected, each dose of vaccine is enough containing about 1 μ g-1000mg, compared with
It is 1 μ g-100mg, more preferably 10 μ g-50mg protein goodly.Can with include IgG titers in the object of observation and it is other instead
The standard research techniques answered determine the optimum amount of specific vaccine.It can be determined by monitoring the immunity level of vaccine offer
Whether need to strengthen dosage.After the IgG titers in have evaluated serum, it may be necessary to from enhancing dose immunizations.Apply
The immune response of the protein to the present invention can be improved with adjuvant and/or immunostimulant.
Method for optimizing is to give immunogenic composition from parenteral (subcutaneously or intramuscularly) approach by injection.
In addition, the vaccine of the present invention can together be given with reference to other immunomodulators, or together with other therapeutic agents
Give.
Main advantages of the present invention are：
(1) realize first in genetically engineered cell internal representations formation rabbit hemorrhagic disease virus virus-like particle (VLP)；
(2) restructuring rabbit hemorrhagic disease virus virus-like particle of the invention can induce animal body generation anti-rabbit hemorrhagic disease virus
Immune response；
(3) optimized RHDV VP60 gene orders of the invention can be expressed efficiently in yeast cells, and can
VLP is formed, therefore the invention provides a kind of method that can largely prepare rabbit hemorrhagic disease virus virus-like particle.
(4) restructuring rabbit hemorrhagic disease virus virus-like particle of the invention can specifically with anti-rabbit hemorrhagic disease virus antibody
With reference to can be used for the detection of antibodies against rabbit hemorrhagic disease virus.
With reference to specific embodiment, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental method of unreceipted detailed conditions in the following example, generally according to conventional strip
Part such as U.S. Sambrook.J etc. writes《Molecular Cloning: A Laboratory room guide》(Huang Peitang etc. is translated, Beijing：Science Press, 2002)
Described in condition, or according to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage and number be by weight
Calculate.Experiment material and reagent used can be obtained from commercially available channel unless otherwise instructed in following examples.
The structure of the expression RHDV VP60 albumen of embodiment 1 VLP Pichia yeast engineering
First, experiment material
1. bacterial strain and plasmid
Bacillus coli DH 5 alpha；Pichia pastoris Host Strains GS115 and expression plasmid of yeast pPIC3.5K are purchased from
Invitrogen companies；RHDV VP60 genes (after optimization) are synthesized by this Suzhou Jin Weizhi companies.
2. toolenzyme, antibody, kit and Primary Chemical
DNA restriction enzyme BamHI and Not I enzymes purchased from NEB companies, Taq archaeal dna polymerases, DNA Marker,
dNTPs；T4DNA is purchased from TaKaRa companies.Plasmid extraction kit, gel reclaims kit are purchased from Qiagen companies；
Pvdf membrane (0.45 micron of specification) is Roche Products；PMSF is purchased from green skies company；The mountain of horseradish peroxidase-labeled
Sheep anti-mouse igg is purchased from Shanghai base peak bio tech ltd.The anti-RHDV VP60 protein monoclonal antibodies of mouse are purchased from the good U.S. in Beijing
Promise bio tech ltd.Yeast Nitrogen Base (Without amino acid) are purchased from BD companies, DAB horseradishes
Peroxidase colour reagent box and D-Biotin are Shanghai Sheng Gong bioengineering limited company product.G418 is GIBCO
Products.
3. key instrument
PCR instrument is purchased from ABI companies, and protein electrophoresis instrument and Western blot transfer devices are purchased from Bio-Rad companies, electronics
Microscope is Beckman Products, and constant incubator and constant-temperature shaking incubator manufacture limited purchased from Shanghai intelligence city analytical instrument
Company, nucleic acid electrophoresis apparatus and small-sized protein decolouring shaking table are the Products of Beijing 61.
4. the preparation of main medium and reagent
Culture medium
LB fluid nutrient mediums：By 10g tryptones, 5g yeast extracts, 10g sodium chloride about 800mL ddH2O dissolves
After be settled to 1000mL, in after 121 DEG C of high pressure steam sterilization 20min, 4 DEG C of preservations.
LB solid mediums：On the basis of fluid nutrient medium is prepared, agar is added with 15g/1000mL fluid nutrient mediums
Powder, 121 DEG C of high pressure steam sterilization 20min treat that temperature is down to 55 DEG C or so, add corresponding antibiotic and paved plate, 4 DEG C of guarantors
Deposit.
Yeast culture medium liquid storage is formulated:
YPD culture mediums：1% yeast extract, 2% peptone, 2% glucose.10g is dissolved in 900mL deionized waters
Yeast extract and 20g peptones, 20g glucose, 121 DEG C of sterilizing 20min prepare flat board and then used in the preceding plus Bacteria Culture that sterilizes
Agar powder is to 20g/L.
YPD G418 resistant panels：Compound method is similar to YPD flat boards, adds nothing when waiting nutrient solution to be cooled to 55 DEG C after sterilizing
Bacterium G418 storing liquids to final concentration is respectively 1mg/mL, 2mg/mL, 3mg/mL, does not add G418 culture medium to be stored in room temperature, 3
Used in individual month.Plus after G418,4 DEG C must be put and be kept in dark place, used in 1-2 weeks.
10 × GY (10% glycerine)：By glycerine and ddH2O presses 1:9 ratio mixing, filtration sterilization or autoclaving, storage
In 4 DEG C.
10 × M (5% methanol)：By methanol and ddH2O presses 1:19 ratio mixing, filtration sterilization is stored in 4 DEG C.
10 × D (20% glucose solution)：In 500mL ddH2200g D-Glucoses are dissolved in O, are fully added after dissolving
ddH2O to final volume 1000mL.Filtration sterilization or autoclaving, are stored in 4 DEG C, 1 year and use.
10×YNB：In 700mL ddH2134g YNB are dissolved in O, ddH is added2O constant volumes are to 1000mL.After filtration sterilization,
It is stored in 4 DEG C, 1 year and uses.
500 × B (0.02% biotin)：20mg biotin are dissolved in 100mL deionized waters, filtration sterilization is stored in 4
DEG C, used in 1 year.
1M (pH 6.0) phosphate buffer：By 132mL 1M K2HPO, 868mL 1M KH2PO4 solution is mixed, and is used
KOH or phosphorus acid for adjusting pH value are stored in room temperature to 6.0 after autoclaving.
MD and MM flat boards：
MD：1.34%YNB, 4 × 10-5%Biotin, 1% glycerine.
MM：1.34%YNB, 4 × 10-5%Biotin, 0.5% methanol.
Add 15g agaroses to sterilize in 800mL sterilized waters, be cooled to after 60 DEG C plus 100mL 10 × YNB solution, 2mL 500
× B, 100mL10 × D (MD) or 10 × M (MM) solution, are down flat after plate, solidification to be stored in 4 DEG C, 3 months and use.
BMGY/BMMY culture mediums：
Dusty yeast 10g, peptone 20g, uses 700mLddH2O dissolves, and is cooled to room temperature, 121 DEG C of high pressure steam sterilization 20min,
Treat that temperature is down to room temperature, add following reagent：100mL 1M pH6.0 kaliumphosphate buffers, 100mL 10 × YNB, 2mL 500
× B, 100mL 10 × GY or 10 × M, are positioned over 4 DEG C, can put 2 months.
Reagent
Nucleic acid electrophoresis related solution
50 × TAE buffer solutions (pH8.5)：Measure Tris alkali 242g, Na2EDTA-2H2O 37.2g are placed into 1L beaker
In, 800mL deionized water is added, is sufficiently stirred for dissolving, adds deionized water to arrive after adding 57.1mL acetic acid, stirring fully
1L, room temperature preservation.
Protein electrophoresis related solution
5 × Tris- glycine running buffers：Weigh 15.1g Tris alkali, 94g glycine (electrophoresis level) (Ph 8.3),
SDS 5.0g, add about 800mL deionized water, stirring and dissolving, plus ddH2O is settled to 1L, room temperature preservation；
1.5M Tris buffer solutions (pH 8.8)：18.1g Tris alkali, is dissolved in appropriate ddH2O, pH is adjusted to 8.8 with HC1, fixed
Hold to 100mL；
1.0M Tris buffer solutions (pH6.8)：12.1g Tris alkali, is dissolved in appropriate ddH2O, pH to 6.8, constant volume are adjusted with HCl
To 100mL；
Albumen loading loading buffer (5 ×)：250mMTris-Cl (pH6.8), 5% (V/V) beta -mercaptoethanol (β-
ME), 10%SDS, 0.5% (W/V) bromophenol blue, 50% glycerine.
5mL collocation method：1.25mL 1M Tris-Cl (pH6.8) are measured in 15mL centrifuge tubes, 0.5g is weighed
Mix and hook after SDS, 0.025g bromophenol blue, add 2.5mL glycerine, add ddH2O dissolving after constant volume arrive 5mL, aliquot dispense.Use
β-the ME that 25 are added in preceding every 500 μ L are mixed, then are reproducibility sample-loading buffer.
12%SDS separation gels and 5% concentration glue
Protein Western-blot related reagents
Transferring film buffer solution：Glycine 2.9g, Tris alkali 5.8g, SDS 0.37g is weighed, methanol 200mL, plus ddH is measured2O
To 1000mL.Room temperature preservation after dissolving.
TBS buffer solutions (100mM Tris-HCl, pH7.5,150mM NaCl)：Take 1mol/L Tris-HCl (pH7.5)
10mL, NaCl 8.8g, use ddH2O is settled to 1000mL.
TBST buffer solutions：TBS buffer solutions containing 0.05%Tween-20, take Tween-20 0.5mL, TBS 1000mL, mix
It can be used after even, matching while using.
Confining liquid：TBST buffer solutions containing 5% skimmed milk power, skimmed milk power 5g, TBST 100mL.4 DEG C of preservations after dissolving.
In use, recovering room temperature, consumption is disposable to cover film surface.
2nd, experimental method
1. recombinant expression plasmid pPIC3.5K-VP60 structure
According to the gene order of the RHDV VP60 albumen of announcement, carry out codon optimization, the substantial amounts of sequence of optimization design
Fragment, and carry out respectively artificial synthesized.
With pPIC3.5K carriers Bam HI and EcoR I double digestions after artificial synthesized VP60 gene orders；Nucleic acid is used afterwards
Gel reclaims kit reclaims VP60 genes and pPIC3.5K carrier respective segments after digestion, with T4 ligases respectively by VP60
Gene (Bam HI/EcoR I) and pPIC9K carriers (Bam HI/EcoR I), are attached reaction, connection product conversion large intestine
Bacillus DH5 α competence, connection product is applied on the LB flat boards of Amp resistances, and 37 DEG C are inverted culture 12-16h, every 5 grams of plate picking
Grand progress bacterium colony PCR identifications, positive bacterium colony expands small upgrading grain after culture and tentatively obtains pPIC9K-VP60, through Bam HI/EcoR
I double digestions identify that the correct plasmid of digestion size send Suzhou Jin Weizhi companies to carry out gene sequencing.
In expressing gene optimization process, the inventors discovered that, optimize the different VP60 gene orders obtained, in expression
Difference is huge in level, and many gene orders can not form viral particles, or the viral particles immunogene formed
Property is very low.By a large amount of screenings, checking, obtain can in yeast high efficient expression VP60 gene orders, and the sequence
The VP60 albumen of expression can form the VLP particles correctly folded in cell, be especially suitable for natural VP60 immunogenicities
VLP preparation, its particular sequence is as follows.
RHDV VP60 optimization gene sequence
>ATGGAAGGAAAAGCCAGAACCGCTCCACAAGGTGAAGCTGCCGGAACCGCTACTACCGCCTCCGTTCC
TGGAACTACTACCGATGGAATGGACCCAGGAGTTGTTGCCGCTACTTCCGTTGTTACTGCTGAGAACTCCTCTGCCT
CTGTTGCTACTGCTGGTATCGGTGGTCCTCCACAGCAAGTTGATCAACAAGAAACCTGGCGTACCAACTTCTACTAT
AACGACGTTTTCACTTGGTCTGTTGCTGACGCCCCAGGTTCTATCTTGTACACTGTTCAACATTCTCCACAAAACAA
CCCATTCACCGCCGTTTTGTCCCAGATGTACGCTGGTTGGGCTGGTGGTATGCAGTTCAGATTCATTGTTGCCGGTT
CCGGTGTCTTCGGTGGTAGATTGGTCGCTGCTGTCATCCCACCTGGTATTGAGATCGGTCCAGGTTTGGAGGTCCGT
CAATTCCCACACGTTGTTATCGACGCCCGTTCTTTGGAGCCAGTTACCATCACTATGCCAGATTTGAGACCTAACAT
GTACCACCCAACTGGTGATCCTGGTCTTGTCCCTACTTTGGTTTTGTCTGTTTACAACAATTTGATCAACCCATTTG
GTGGTTCCACCAACGCTATTCAGGTTACCGTCGAGACCAGACCATCCGACGACTTCGAGTTTGTCATGATCCGTGCT
CCATCCTCCAAGACTGTTGACTCCATCTCCCCAGCTGGTTTGTTGACTACTCCAGTCTTGACCGGTGTCGGAAACGA
CAACCGTTGGAACGGTCAGATTGTCGGTTTGCAACCTGTCCCTGGAGGTTTCTCTACCTGCAACCGTCACTGGAACT
TGAACGGTTCCACTTACGGTTGGTCCTCTCCAAGATTCGCCGATATCGACCACAGAAGAGGTTCCGCCTCTTACTCC
GGAAATAACTCCACCAACGTCTTGCAGTTCTGGTACGCCAACGCCGGTTCTGCCATCGACAACCCAATTTCCCAAGT
TGCCCCAGACGGTTTCCCAGATATGTCTTTTGTTCCATTCAACTCTCCTAACATCCCAACTGCCGGTTGGGTTGGTT
TCGGTGGTATCTGGAACTCCAACAACGGTGCTCCTGCTGCCACCACTGTCCAGGCTTACGAACTTGGTTTCGCCACC
GGTGCTCCTAACAACTTGCAGCCAACTACTAACACTTCCGGTGCTCAGACCGTTGCCAAATCCATCTACGCTGTTGT
CACCGGTACCAATCAGAACCCAACCGGTTTGTTTGTTATGGCTTCCGGAGTCATCTCCACTCCTAACGCCTCTGCTG
TTACCTACACCCCACAGCCTGACAGAATTGTCACTACCCCAGGAACTCCTGCTGCCGCTCCAGTTGGAAAGAACACT
CCTATCATGTTCGCCTCCGTCGTTCGTAGAACCGGTGACGTTAACGCTGCCGCTGGTTCCACTAACGGTACCCAATA
CGGTACCGGTTCTCAGCCATTGCCAGTCACCATTGGTTTGTCTTTGAATAACTATTCCTCTGCCTTGATGCCAGGTC
AGTTCTTTGTCTGGCAGTTGACCTTCGCTTCCGGATTCATGGAGATCGGTTTGTCTGTTGATGGATACTTTTACGCC
GGTACCGGTGCCTCTACCACTCTTATCGATCTTACCGAATTGATCGATGTCCGTCCAGTTGGTCCTAGACCTTCCAA
GTCCACCTTGGTCTTCAACTTGGGAGGTACTACCAATGGTTTTTCTTACGTTTAA
2. recombinant expression plasmid converts Pichia pastoris
The a large amount of preparations and linearisation of 2.1 DNAs
A large amount of extractions of plasmid：(contain 3mL LB culture mediums are inoculated in through single bacterium colony of the digestion identification containing positive colony
100g/mL Amp) in, 37 DEG C of shaken overnights.When bacterial growth to late log phase, the dense about OD of bacterium600When=0.60, this is cultivated
Thing is gone in 500mL (Amp containing 100g/mL) LB blake bottles, 37 DEG C of shaken cultivations 12~16 hours, collects bacterium solution, 4000rpm
4 DEG C centrifuge 10 minutes, remove supernatant, according to the big upgrading grain of the big extraction reagent kit specification step of the plasmid of Qiagen companies.
The linearized enzyme digestion of DNA：
Digestion system is as follows：
37 DEG C of digestions 3 hours, plus isometric phenol：Chloroform, is mixed, and 12000rpm is centrifuged 10 minutes, draws upper strata aqueous phase, then
Plus isometric chloroform：Isoamyl alcohol, is mixed, and 12000rpm is centrifuged 10 minutes, draws upper strata aqueous phase, plus the ammonium acetate of 1/4 volume and 2
The absolute ethyl alcohol of times volume, places 20 minutes in -20 DEG C, and then 12000rpm is centrifuged 15 minutes, is removed supernatant, is used 70% ethanol
Precipitation is washed respectively once with absolute ethyl alcohol, puts and 15 μ L TE buffer solutions are resuspended in after 37 DEG C of dryings.
2.2 linearization plasmids electricity conversion Pichia pastoris
The preparation of competent cell：
(1) inoculation Pichia pastoris single bacterium is fallen within the 50mL triangular flasks equipped with 5mL YPD culture mediums, 28 DEG C of 220rpm cultures
Overnight.
(2) 0.1-0.5mL cultures are taken to be inoculated in 500mL fresh cultures, incubated overnight is about 1.3- to OD600
1.5。
(3) 4 DEG C, cell is collected by centrifugation for 5 minutes in 1500 × g, and precipitation is dissolved in the sterilized water of 500mL frosts again.
(4) 4 DEG C, cell is collected by centrifugation for 5 minutes in 1500 × g, and precipitation is dissolved in the sterilized water of 250mL frosts again.
(5) 4 DEG C, cell is collected by centrifugation for 5 minutes in 1500 × g, and precipitation is dissolved in the 1M sorbierites of 20mL frosts again.
(6) 4 DEG C, cell is collected by centrifugation for 5 minutes in 1500 × g, and precipitation is dissolved in the 1M sorbierites of 1mL frosts again.
(7) packing is into the 1.5mL centrifuge tubes of pre-freeze, and every centrifuge tube dispenses 80 μ L.
Electricity conversion：
(1) DNA for taking 5-20 μ g to linearize, is dissolved in 5-10 μ L TE, turning competent cell with 80 μ L electricity mixes, then
It is transferred in the 0.2cm of frost electric revolving cup.
(2) electric revolving cup is incubated on ice 5 minutes.
(3) convert：Electroporation is Bio-Rad GenePulse, voltage 1500V, electric capacity 25 μ F, the Ω of resistance 200.
(4) add immediately l mL " the 1M sorbierites of frost in conversion cup, the solution in electric revolving cup go to 1.5mL without
In bacterium centrifuge tube.
(5) 200-600 μ L solution is taken to be applied to MD flat boards, His+/Muts phenotypic clonings are selected in 28 DEG C of cultures.
3. utilize G418 resistance screening Pichia pastoris multicopy transformants
Saccharomycete for carrying out electric conversion using pPIC3.5K plasmids, after growing monoclonal bacterium colony on MD plates, with
Machine selects some bacterium colonies into 96 orifice plates that with the addition of 100 μ L YPD fluid nutrient mediums in advance, 28 DEG C of cultures 16-20h, Ran Houzai
10 μ L bacterium solutions are respectively taken to connect bacterium into other one piece 96 orifice plates that with the addition of 100 μ LYPD in advance.After synchronization, then take 10 μ
L bacterium solutions connect bacterium to the YPD flat boards containing 1mg/mL, 2mg/mL and 3mg/mL G418 resistances.28 DEG C of culture 2-5d single bacteriums to be grown
Fall.
4PCR methods identify positive recombinant
Picking partial yeast bacterium is in 5 1% glusulases of μ L/SCE solution in single bacterium colony, and 37 DEG C of jog 3h digest broken wall,
100 DEG C heating 15 minutes after be diluted to 20 μ L, take 5 μ L to make template, with primer El and E2 enter performing PCR reaction.
Reaction system：H2O 75 μ L, dNTPs (2.5mM/each) 5 μ L, 10 × Taq enzyme reaction buffer 10 μ L, the μ of template 5
The μ L of L, primer El 2 μ L, primer E2 2.
PCR reaction conditions：95 DEG C 5 minutes, plus the μ L/ of Taq enzyme 1 pipe (4 Μ), 30 temperature cycles, 72 DEG C 10 minutes
Thermal cycling conditions；95 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 120 seconds.Product authentication method is ibid.
Induced expression of 5 fusions in Pichia pastoris
1. monoclonal on picking GS115-pPIC3.5K-VP60 yeast flat board (2mg/mL) is to the YPD of back side frame
Flat board, each grid, which is grown in a monoclonal, 28 DEG C of incubators, to be cultivated.
Yeast growth gets up on 2.60h rear plates, and picking 3 is cloned into the 50mL containing 10mL BMGY culture mediums respectively
In centrifuge tube, make a record, be capped four layers of sterile gauze, 28 DEG C of 250rpm cultivate to OD600 about 2-6 (logarithmic phase grow, greatly
About 16-18h).
3. room temperature 1500g centrifugation 5min, collect cell, remove supernatant, cell is resuspended to OD600=1.0 with BMMY, pipe is interior
Remaining 10mL nutrient solutions, unnecessary to discard, culture is capped four layers of sterile gauze, is put into shaking table and continues induced expression.
After 4.24 hours, adding methanol, (about 1mL 5% methanol checks the amount of culture medium, really to final concentration of 0.5%
Protect it is correct add methanol because evaporation can reduce the volume of culture medium) to continue induction.
1500 × g after 5.24h, is collected by centrifugation cell in 5 minutes.
6. cell is collected, room temperature maximum (top) speed centrifugation 2-3min, often pipe takes the saccharomycete of 80mg weight in wet bases (to add same after centrifugation
Etc. the beads of weight, 150 μ L lysate) crushed, SDS-PAGE and ELISA method detection protein expression situation.
The preparation of 6 yeast cells protein extracts
The cell collected after induced expression is taken to add the saccharomycete of 80mg weight in wet bases in every 1.5mL eppendorf pipe.
Washed 2 times with ice-cold deionization, add the ice-cold μ L of lysis buffer (PMSF containing 100mM) 160 and add isometric pickling
beads(0.5mm).Intense oscillations 30 seconds, then ice bath 30 seconds, repeat this vibration bath operation on the rocks 12 times.12000rpm is centrifuged
15 minutes, supernatant is taken to produce protein extract.
7SDS-PAGE and Western blot
1) the μ L of yeast cell extract 20 are taken, 5 × SDS-PAGE loading buffer, 5 microlitres of mixings, 100 DEG C are added
Boil 5min；
2) protein adhesive is arranged on electrophoresis frame, is put into electrophoresis tank, pours into electrophoresis liquid, take out comb, in well
The protein sample that 25 μ L are handled well is added, SDS-PAGE electrophoresis is carried out：Under 100V voltages after electrophoresis 20min, treat that protein band is opened
It is 150V, electrophoresis 1h that beginning, which is moved to after separation gel and adjusts voltage,；
3) using wet transfer method by protein delivery to pvdf membrane after electrophoresis is finished, transferring film electric current 280mA turns 1h；
4) transferring film terminates film being put into plastic caddy, adds 5% skimmed milk power and sways closing 1h to disappear in room temperature shaker
Except non-specific background；
5) closing is finished, and TBST washes film 3 times, each 5min；
6) the Anti-RHDV VP60 monoclonal antibodies for adding 1% skimmed milk power (TBST configurations) dilution are used as primary antibody (1:
1000 dilutions), sway incubation 2h (or 4 DEG C of overnight incubations) in room temperature shaker；
7) it is incubated and terminates to reclaim primary antibody, film is washed with TBST 3 times, 5min/ times；
8) plus HRP marks Goat-Anti-mouse secondary antibodies, in being incubated at room temperature 1h on shaking table；
9) washed with TBST 3 times, each 5min；
10) film is gone in Fresh DAB solution and developed the color, meticulous inspection, once western hybridization band shows,
Terminating reaction is rinsed with water immediately, is gone in PBST solution.
The VP60 albumen formation VLP expressed in 8 electron microscopic observations identification Pichia pastoris
Choosing yeast cells protein extract sample is used for electron microscopic observation.Using conventional negative group velocity, the sample of equivalent
Dropped to after product and the mixing of 2% phosphotungstic acid dye liquor with capillary in the contained network of film, suck excessive liquid and contained network is put in 37
Electron microscopic observation is carried out after DEG C drying, and the actual size of particle is estimated according to the diameter and multiplication factor of particle on photo.
3rd, experimental result
1 recombinant expression plasmid pPIC3.5K-VP60 structure
Recombinant plasmid pPIC3.5K-VP60 is about 7000bp into size through BsrGI digestions and 5500bp two bar segments (figure
1)；Positive recombinant plasmid tentatively is confirmed as, this plasmid Song Jinwei will company is sequenced, sequencing result is through the ClustalW2 in EBI
Compare, and the VP60 cDNA sequences optimized are completely the same.
Fig. 1 shows the digestion identification of pPIC3.5K-VP60 expression vectors, Lane 1:Indigested pPIC3.5K-
VP60；Lane 2:Digested with BsrG I；Lane 3:Molecular weight marker.
PPIC3.5K-VP60 and the pPIC3.5K-VP60 after BsrG I digestions synchronously enter row agarose gel electrophoresis, detect
As a result pPIC3.5K-VP60 recombinant vectors about 12000bp is shown, and the recombinant vector pPIC3.5K- after BsrG I digestions
Two bar segments that it is about 7000bp and 5500bp that VP60, which occurs,
After 2 recombinant plasmid pPIC3.5K-VP60 conversion Pichia pastoris, with G418 resistance screening Pichia pastoris multicopies
Transformant and PCR methods identification positive recombinant.
3Western blot identification VP60 albumen is expressed in Pichia pastoris
3 KM71 single bacterium colonies of picking are seeded in BMGY fluid nutrient mediums, and 28 DEG C, 250rpm shakes bacterium and cultivated to 0D600=
6, BMMY resuspension thalline are added after centrifugation makes bacterium solution 0D600=1, is then seeded in BMMY culture mediums and carries out induced expression, makees
For negative control, the KM71 bacterial strains for converting pPIC3.5k empty carriers synchronously carry out induced expression.Added every 24h into culture medium
100 × methanol makes its whole agriculture degree reach after 0.5%, Fiber differentiation 48h, and bacterial sediment is collected by centrifugation, and carries out extracting egg after crushing
In vain, Protein Extraction liquid carries out Western blot detections.As a result pPIC3.5k-VP60/KM71 protein extract samples are shown in
There is protein band at 60kDa or so places, but purpose egg is not detected in pPIC3.5K/KM71 protein extract samples
Informal voucher band.
This result shows that the expression quantity for three clones that the success of VP60 albumen is expressed in Pichia pastoris, is selected after screening does not have
There were significant differences.
Fig. 2 shows expression of the SDS-PAGE and Western blot detection RHDV VP60 albumen in Pichia pastoris.
The VLP particles of 4 electron microscopic observation VP60 albumen formation
Yeast cells protein extract sample after methanol induction is done into negative staining electron microscope experiment, as a result such as Fig. 3：After negative staining
It is observed that obvious virion, as a result shows that the RHDV VP60 albumen expressed in Pichia pastoris being capable of group under Electronic Speculum
Dress up virion.
Fig. 3 shows negative staining electron microscope Germicidal efficacy RHDV virions.
The RHDV virus sample particle vaccines Activity determinations of embodiment 2
Rabbit hemorrhagic disease virus (RHDV) virus sample particle vaccines of the present invention are to utilize yeast expression system production
RHDV VP60 structural proteins, then assembling forms virus-like particle in vitro, and adds the vaccine that appropriate adjuvant is made.By existing
OK《Chinese veterinary pharmacopoeia》Method has carried out the inspection of bacterium and mould, mycoplasma and exogenous virus, meets defined standard.It is existing
The security and immuning effect test of vaccine produced by the invention are summarized as follows.
1 seed culture of viruses
1.1 inspections are originated with seed culture of viruses
The sterile rabbit liver tissue for taking infection RHDV to die of illness of effect inspection seed culture of viruses system used, then carries out tissue grinder (1g livers
Dirty tissue adds the PBS 3mL containing antibiotic, and antibiotic is penicillin, each 1000IU/mL of streptomysin), 4000r/min after grinding
Centrifugation takes supernatant in 10 minutes, by the Industry Technology Center keeping of Shanghai Hai Li Biological Co., Ltd., identification and supply.
1.2 test reagent
Effect inspection rabbit hemorrhagic disease virus inactivated vaccine used is purchased from Nanjing Tianbang Bio-industry Co., Ltd.；Rabbit poison
Property haemorrhagic virus sample particulate antigen, which is prepared by this engineering center and adds appropriate adjuvant, to be mixed, and antigenic content is respectively
1mg/mL and 2mg/mL；Other chemical reagent are domestic top pure grade.
1.3 test material
Operation tray and disscting instrument, tissue grinder, syringe, hemostix, emulsifier, Sony's camera etc..
1.4 experimental animal
More than the body weight 1.5kg of 45-60 ages in days healthy SPF rabbit, purchased from Qingdao health mcroorganism Science and Technology Ltd., use
In this experiment.
The immune efficacy of 2 vaccines is examined
2.1 experimental animals are grouped
16 SPF rabbits are randomly divided into 4 groups, every group 4：First group is PBS control group, and 1mL PBS are subcutaneously injected；Second
Group is commercially available vaccine immune group, and 1mL commercially available vaccines are subcutaneously injected；3rd group is low dosage antigen immune group, and 1mL is subcutaneously injected and resists
Former (1mg/mL)；4th group is high dose antigen immune group, and 1mL antigens (2mg/mL) are subcutaneously injected.
2.2 immune effects compare
RHDV virus liquids 1ml is subcutaneously injected in each group rabbit immunization after 21 days.Clinical observation 7 days after poison are attacked, record is clinical
Response situation simultaneously counts the dead number of each group rabbit, and the dead rabbit of infection sequela is dissected in time, rabbit dissection of dying of illness is recorded
Observe pathological change and take pictures, evaluate vaccine immune effect.
3 results
3.1 clinical symptoms are showed
PBS group experimental infection rabbits are raised after the classical symptom for rabbit pest occur, 16-34h respectively at 4-28h after infection, body temperature
Body temperature rises to peak value (40-41.5 DEG C), wherein 3 sick rabbit high temperature continue 10-18h respectively, it is small that body temperature begins to decline rear 7-12.5
When it is dead.Spirit is showed after heating substantially depressed, anorexia drink is intended to strengthen；And then be short of breath, Visual mucous membrane cyanosis is few
Urine, albuminuria, somnolence；Present and bolted in abdominal distension, constipation, the on one's deathbed short-term excitement of preceding performance, struggle, cage, then the sleeping ground of two forelimbs,
Two hind legs are propped, general tremor, are twitched, and are finally lain on one's side on the ground, four limbs are constantly slided, last twisting side, opisthotonos posture,
Bad blood clotting is in kermesinus, and conjunctival congestion, nares are polluted with blood, and cerise foam-like blood is flowed out sometimes
Liquid, gingiva bleeding gingival hemorrhage, horrible cry of takeofing suddenly；Indivedual disease rabbit vaginal orifices are bled.Lesion is observed during cut open inspection and meets acute rabbit pest.Remaining is immunized
There is transient anorexia after poison is attacked in group rabbit in 3d, spirit is depressed, but recovers therewith normal.
3.2 death conditions are counted
PBS group rabbit 60h or so after poison is attacked dead two (2/4), 84h or so dead one (1/4), a remaining sight
Examine 7d not dead；Immune group rabbit attacks not dead after poison, and this explanation immune effect of vaccine is good.
3.3 pathological anatomy are observed
Rabbit is attacked after poison, and pathological anatomy result meets rabbit pest as shown in figure 4, PBS group rabbit attack internal organs change after poison, and exempts from
Epidemic disease group rabbit internal organs no abnormality seen.
4 laboratory trial products are summarized
The RHDV virus-like particles that this research is produced using yeast expression system are manufactured in laboratory conditions as antigen
Vaccine, and efficacy test has been carried out to vaccine, as a result show, the vaccine of production meets standard as defined in this vaccine code.Exempt from
Epidemic disease challenge test result shows that vaccination tests rabbit is reliable to animal safety, has no toxic side effect, and vaccine has well
Immune effect.
All documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after the above-mentioned instruction content of the present invention has been read, those skilled in the art can
To be made various changes or modifications to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Claims (10)
1. a kind of genetically engineered cell, it is characterised in that the genetically engineered cell is eukaryotic, and the base of the cell
Because being integrated with the expression cassette of expression rabbit hemorrhagic disease virus VP 60 albumen in group；Or contain expression vector in the cell, it is described
Expression vector contains the expression cassette for expressing the rabbit hemorrhagic disease virus VP 60 albumen；
And the genetically engineered cell is in VP60 albumen described in intracellular expression, and the VP60 albumen is thin in the genetic engineering
Born of the same parents are internally formed virus-like particle (VLP)；
Preferably, the eukaryotic is yeast cells, more preferably Pichia pastoris.
2. genetically engineered cell as claimed in claim 1, it is characterised in that the expression of the rabbit hemorrhagic disease virus VP 60 albumen
In box, the gene order such as SEQ ID NO. of the VP60 albumen:Shown in 1.
3. a kind of virus-like particle (VLP), it is characterised in that the VLP is as the genetically engineered cell table described in claim 1
Reach.
4. a kind of pharmaceutical composition, it is characterised in that described pharmaceutical composition includes the virus-like particle described in claim 3,
Pharmaceutically acceptable carrier.
5. pharmaceutical composition as claimed in claim 4, it is characterised in that described pharmaceutical composition includes vaccine combination.
6. a kind of method for preparing VLP, it is characterised in that including step：
Under conditions suitable for the expression, the cell described in culture claim 1, so as to give expression to the virus described in claim 3
Sample particle (VLP)；With
Separate the virus-like particle (VLP).
7. a kind of polynucleotides through codon optimization of separation, it is characterised in that the polynucleotide encoding SEQ ID NO.:
Polypeptide shown in 2；And the polynucleotides are selected from the group：
(a) sequence such as SEQ ID NO.:Polynucleotides shown in 1；
(b) nucleotide sequence and SEQ ID NO.:Many nucleosides of homology >=95% (preferably >=98%) of sequence shown in 1
Acid；
(c) such as SEQ ID NO.:5 ' the ends and/or 3 ' ends of polynucleotides shown in 1 are truncated or addition 1-60 (preferably 1-30,
More preferably 1-10) nucleotides polynucleotides；
(e) polynucleotides complementary with any described polynucleotides of (a)-(c).
8. a kind of expression vector, the expression vector contains the polynucleotides described in claim 7.
9. a kind of host cell, described host cell contains the expression vector described in claim 8, or in genome it is whole
Close the polynucleotides having the right described in requirement 7.
10. a kind of pharmaceutical composition, described composition contains virus-like particle (VLP) described in claim 3, claim
The expression vector described in polynucleotides or claim 8 described in 7 or the host cell described in claim 9, and medicine
Acceptable carrier and/or auxiliary material on.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110317794A (en) * | 2019-06-12 | 2019-10-11 | 吉林特研生物技术有限责任公司 | A kind of recombinant baculovirus, Raccoon dog parvovirus recombinant subunit vaccine and preparation method thereof |
| 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 |
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| WO2002018426A2 (en) * | 2000-09-01 | 2002-03-07 | Consejo Superior De Investigaciones Cientificas | Rabbit hemorrhagic disease vaccine and antigens |
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| EP0704528A2 (en) * | 1994-07-08 | 1996-04-03 | Cyanamid Iberica, Sa | Rabbit hemorrhagic disease virus (RHDV) recombinant capsids and proteins, diagnostic kits and vaccines containing them |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110317794A (en) * | 2019-06-12 | 2019-10-11 | 吉林特研生物技术有限责任公司 | A kind of recombinant baculovirus, Raccoon dog parvovirus recombinant subunit vaccine and preparation method thereof |
| 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 |
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