CN107164252B - Subunit vaccine of RHDV - Google Patents
Subunit vaccine of RHDV Download PDFInfo
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- CN107164252B CN107164252B CN201610131290.2A CN201610131290A CN107164252B CN 107164252 B CN107164252 B CN 107164252B CN 201610131290 A CN201610131290 A CN 201610131290A CN 107164252 B CN107164252 B CN 107164252B
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Abstract
The invention provides a subunit vaccine of RHDV, and particularly the inventor obtains a method for preparing rabbit hemorrhagic disease virus VLP particles through extensive and intensive research. The invention provides a genetic engineering cell, which is a eukaryotic cell, and an expression cassette for expressing rabbit hemorrhagic disease virus VP60 protein is integrated in the genome of the cell; or the cell contains an expression vector which contains an expression cassette for expressing the rabbit hemorrhagic disease virus VP60 protein; and said genetically engineered cell expresses said VP60 protein intracellularly, and said VP60 protein forms a virus-like particle (VLP) inside said genetically engineered cell. The invention also provides a virus-like particle (VLP) expressed by the genetically engineered cell. Experimental results show that the gene engineering cell and the method can efficiently prepare the rabbit hemorrhagic disease virus VLP particles.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a subunit vaccine of RHDV.
Background
Rabbit Hemorrhagic Disease (RHD) is an acute, septic, highly contagious infectious Disease of rabbits caused by Rabbit Hemorrhagic Disease Virus (RHDV), and is characterized mainly by lethality and bleeding of systemic parenchymal organs. In the rabbit breeding industry, the harm is huge.
Virus Like Particles (VLPs) are hollow Particles containing one or more structural proteins of the Virus, are free of viral nucleic acids (DNA/RNA), cannot replicate autonomously, are identical or similar in morphology to the true virions, and can be presented to immune cells in the same way as viral infection, effectively inducing the immune system of the body to produce an immunoprotective response. Since VLPs are not infectious, they are presented to immune cells as immunogens in the same way as viral infections, effectively inducing the immune system of the body to produce an immunoprotective response, and host, viral and vaccine factors together determine the level of protection produced by VLPs.
The existing live virus vaccine has the defect that once the virus activity is recovered, the safety can not be ensured. Meanwhile, inactivated virus vaccines can cause safety problems due to incomplete inactivation processes. But the VLPs vaccine has no infectivity, good stability, difficult inactivation and wide development prospect.
Disclosure of Invention
The invention aims to provide an RHDV subunit vaccine and application thereof.
In the first aspect of the invention, a genetically engineered cell is provided, wherein the genetically engineered cell is a eukaryotic cell and integrates an expression cassette for expressing rabbit hemorrhagic disease virus VP60 protein into the genome of the cell; or the cell contains an expression vector which contains an expression cassette for expressing the rabbit hemorrhagic disease virus VP60 protein;
and said genetically engineered cell expresses said VP60 protein intracellularly, and said VP60 protein forms a virus-like particle (VLP) inside said genetically engineered cell.
In another preferred embodiment, the eukaryotic cell is a yeast cell, preferably a pichia pastoris cell.
In another preferred embodiment, the expression cassette comprises the following elements operably linked 5 'to 3': a promoter, a start codon, an ORF sequence of the VP60 protein, and a stop codon.
In another preferred embodiment, said initiation codon is directly linked to the ORF sequence of said VP60 protein.
In another preferred embodiment, the expression cassette does not contain a secretory expression element.
In another preferred embodiment, the expression cassette does not contain a secretion peptide, a leader peptide, or a signal peptide.
In another preferred example, the gene sequence of the VP60 protein is shown in SEQ ID No. 1.
In another preferred embodiment, the expression vector takes pichia pastoris pPICX vector as a framework.
In a second aspect of the invention, there is provided a Virus Like Particle (VLP) expressed by the genetically engineered cell of the first aspect of the invention.
In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the virus-like particle of the first aspect of the present invention, and a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition comprises a vaccine composition.
In a fourth aspect of the invention, there is provided a method of making a VLP comprising the steps of:
culturing a cell according to the first aspect of the invention under conditions suitable for expression, thereby expressing a virus-like particle (VLP) according to the second aspect of the invention; and
isolating the Virus Like Particle (VLP).
In a fifth aspect of the invention, there is provided an isolated codon-optimized polynucleotide encoding a polypeptide as set forth in SEQ ID No. 2; and the polynucleotide is selected from the group consisting of:
(a) a polynucleotide having a sequence as shown in SEQ ID No. 1;
(b) polynucleotide having a nucleotide sequence homology of 95% or more (preferably 98% or more) with the sequence shown in SEQ ID No. 1;
(c) 1, or a polynucleotide in which 1 to 60 (preferably 1 to 30, more preferably 1 to 10) nucleotides are truncated or added at the 5 'end and/or the 3' end of the polynucleotide shown in SEQ ID No.: 1;
(e) a polynucleotide complementary to any one of the polynucleotides of (a) - (c).
In a sixth aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the fifth aspect of the invention.
In a seventh aspect of the invention, there is provided a host cell comprising an expression vector according to the sixth aspect of the invention or having integrated into its genome a polynucleotide according to the fifth aspect of the invention.
In an eighth aspect of the invention, there is provided a pharmaceutical composition comprising a virus-like particle (VLP) according to the second aspect of the invention, a polynucleotide according to the fifth aspect of the invention or an expression vector according to the sixth aspect of the invention or a host cell according to the seventh aspect of the invention, and a pharmaceutically acceptable carrier and/or adjuvant.
In another preferred embodiment, the pharmaceutical composition comprises a vaccine composition.
In another preferred embodiment, the vaccine composition further comprises an adjuvant.
In another preferred embodiment, the adjuvant comprises alumina, saponin, quil A, muramyl dipeptide, mineral or vegetable oil, vesicle-based adjuvant, nonionic block copolymer or DEAE dextran, cytokines (including IL-1, IL-2, IFN-r, GM-CSF, IL-6, IL-12, and CpG).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the restriction enzyme identification of pPIC3.5K-VP60 expression vector.
FIG. 2 shows the expression of RHDV VP60 protein in Pichia pastoris detected by SDS-PAGE and Western blot.
FIG. 3 shows the experimental observation of RHDV virus particles by electron microscopy negative staining.
FIG. 4 shows the results of the test rabbit disease profile.
Detailed Description
The inventor obtains a method for preparing rabbit hemorrhagic disease virus VLP particles through extensive and intensive research, and experimental results show that the rabbit hemorrhagic disease virus VLP particles can be efficiently prepared according to the method.
Before the present invention is described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methodologies and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now exemplified.
Rabbit Hemorrhagic Disease Virus (Rabbit Hemorrhagic Disease Virus, RHDV)
Rabbit Hemorrhagic Disease (RHD) is RHDV which is a member of Caliciviridae and comprises a main structural protein VP60, VP60 can induce the generation of neutralizing antibodies in animals, is directly related to immune response and is a virus immunoprotective antigen.
The natural gene sequence of RHDV VP60 is as follows:
>gi|358409917|gb|JN165233.1|Rabbit hemorrhagic disease virus isolateTC/China/2007VP60gene,partial cds
ATGGAGGGCAAAGCCCGCACAGCGCCGCAAGGCGAAGCAGCAGGCACTGCTACCACAGCATCAGTTCCCGGAACCACGACCGACGGCATGGATCCTGGAGTAGTGGCCGCGACTAGTGTGGTCACTGCAGAAAATTCATCCGCATCGGTTGCAACGGCGGGGATTGGCGGCCCACCCCAACAGGTGGACCAACAAGAAACATGGAGGACAAACTTTTACTACAATGATGTTTTCACTTGGTCCGTCGCGGATGCGCCCGGCAGCATTCTCTACACTGTCCAACACTCTCCACAGAACAACCCATTCACAGCCGTACTGAGCCAGATGTACGCTGGCTGGGCTGGTGGCATGCAGTTCCGCTTCATAGTTGCTGGATCAGGTGTGTTTGGTGGGCGACTGGTCGCGGCTGTGATACCACCAGGCATCGAGATTGGACCAGGGTTGGAGGTCAGGCAATTTCCTCATGTCGTCATCGACGCCCGTTCACTCGAACCTGTTACCATCACCATGCCAGACTTGCGTCCCAACATGTACCATCCAACTGGTGACCCTGGCCTTGTCCCCACACTAGTCCTTAGTGTTTACAACAACCTCATCAACCCGTTTGGTGGGTCCACCAACGCAATCCAGGTAACAGTGGAAACGAGGCCGAGTGATGACTTTGAGTTCGTGATGATTAGAGCCCCCTCCAGCAAAACTGTTGACTCAATCTCACCCGCAGGCCTCCTCACGACCCCAGTCCTCACTGGTGTTGGCAATGACAACAGGTGGAACGGCCAAATAGTGGGACTGCAACCAGTACCTGGGGGGTTTTCCACGTGCAACAGGCATTGGAACCTGAACGGCAGCACGTATGGCTGGTCAAGCCCTCGGTTTGCCGACATTGACCATCGAAGAGGCAGTGCAAGTTATTCTGGGAACAACTCCACCAACGTGCTTCAGTTTTGGTACGCTAATGCTGGGTCTGCAATTGACAACCCTATCTCCCAGGTTGCACCGGACGGCTTCCCTGACATGTCATTCGTGCCCTTTAACAGCCCCAACATTCCGACCGCGGGGTGGGTCGGGTTTGGTGGTATTTGGAACAGTAACAACGGTGCCCCCGCTGCTACAACTGTGCAGGCCTATGAGTTAGGTTTTGCCACTGGGGCACCAAATAACCTCCAGCCCACCACCAACACTTCAGGTGCACAGACTGTCGCTAAGTCCATTTATGCCGTGGTAACCGGCACAAACCAAAATCCAACCGGACTGTTTGTGATGGCCTCGGGTGTTATCTCCACCCCAAACGCCAGCGCCGTCACATACACGCCCCAACCAGACAGAATTGTGACTACACCCGGCACTCCTGCCGCCGCACCTGTGGGTAAGAACACACCCATCATGTTCGCGTCTGTTGTCAGGCGCACCGGTGACGTCAACGCCGCAGCTGGGTCAACCAACGGGACCCAGTACGGCACGGGCTCCCAACCACTGCCAGTAACAATTGGACTTTCGCTCAACAACTACTCGTCAGCACTCATGCCTGGGCAGTTTTTCGTTTGGCAGTTAACCTTTGCATCTGGTTTCATGGAGATTGGCTTAAGTGTGGACGGGTACTTTTATGCAGGAACAGGAGCCTCAACCACGCTCATTGACTTGACTGAACTCATTGACGTACGCCCCGTGGGACCCCGGCCGTCCAAGAGCACACTCGTGTTCAACCTGGGGGGCACAACCAATGGCTTTTCTTATGTCTGA(SEQ ID NO.3)
based on the natural gene sequence of the RHDV VP60 protein, the present inventors obtained an optimized gene sequence encoding VP60 protein polypeptide suitable for expression in yeast through extensive screening.
The optimized gene sequence of RHDV VP60 is as follows:
>ATGGAAGGAAAAGCCAGAACCGCTCCACAAGGTGAAGCTGCCGGAACCGCTACTACCGCCTCCGTTCCTGGAACTACTACCGATGGAATGGACCCAGGAGTTGTTGCCGCTACTTCCGTTGTTACTGCTGAGAACTCCTCTGCCTCTGTTGCTACTGCTGGTATCGGTGGTCCTCCACAGCAAGTTGATCAACAAGAAACCTGGCGTACCAACTTCTACTATAACGACGTTTTCACTTGGTCTGTTGCTGACGCCCCAGGTTCTATCTTGTACACTGTTCAACATTCTCCACAAAACAACCCATTCACCGCCGTTTTGTCCCAGATGTACGCTGGTTGGGCTGGTGGTATGCAGTTCAGATTCATTGTTGCCGGTTCCGGTGTCTTCGGTGGTAGATTGGTCGCTGCTGTCATCCCACCTGGTATTGAGATCGGTCCAGGTTTGGAGGTCCGTCAATTCCCACACGTTGTTATCGACGCCCGTTCTTTGGAGCCAGTTACCATCACTATGCCAGATTTGAGACCTAACATGTACCACCCAACTGGTGATCCTGGTCTTGTCCCTACTTTGGTTTTGTCTGTTTACAACAATTTGATCAACCCATTTGGTGGTTCCACCAACGCTATTCAGGTTACCGTCGAGACCAGACCATCCGACGACTTCGAGTTTGTCATGATCCGTGCTCCATCCTCCAAGACTGTTGACTCCATCTCCCCAGCTGGTTTGTTGACTACTCCAGTCTTGACCGGTGTCGGAAACGACAACCGTTGGAACGGTCAGATTGTCGGTTTGCAACCTGTCCCTGGAGGTTTCTCTACCTGCAACCGTCACTGGAACTTGAACGGTTCCACTTACGGTTGGTCCTCTCCAAGATTCGCCGATATCGACCACAGAAGAGGTTCCGCCTCTTACTCCGGAAATAACTCCACCAACGTCTTGCAGTTCTGGTACGCCAACGCCGGTTCTGCCATCGACAACCCAATTTCCCAAGTTGCCCCAGACGGTTTCCCAGATATGTCTTTTGTTCCATTCAACTCTCCTAACATCCCAACTGCCGGTTGGGTTGGTTTCGGTGGTATCTGGAACTCCAACAACGGTGCTCCTGCTGCCACCACTGTCCAGGCTTACGAACTTGGTTTCGCCACCGGTGCTCCTAACAACTTGCAGCCAACTACTAACACTTCCGGTGCTCAGACCGTTGCCAAATCCATCTACGCTGTTGTCACCGGTACCAATCAGAACCCAACCGGTTTGTTTGTTATGGCTTCCGGAGTCATCTCCACTCCTAACGCCTCTGCTGTTACCTACACCCCACAGCCTGACAGAATTGTCACTACCCCAGGAACTCCTGCTGCCGCTCCAGTTGGAAAGAACACTCCTATCATGTTCGCCTCCGTCGTTCGTAGAACCGGTGACGTTAACGCTGCCGCTGGTTCCACTAACGGTACCCAATACGGTACCGGTTCTCAGCCATTGCCAGTCACCATTGGTTTGTCTTTGAATAACTATTCCTCTGCCTTGATGCCAGGTCAGTTCTTTGTCTGGCAGTTGACCTTCGCTTCCGGATTCATGGAGATCGGTTTGTCTGTTGATGGATACTTTTACGCCGGTACCGGTGCCTCTACCACTCTTATCGATCTTACCGAATTGATCGATGTCCGTCCAGTTGGTCCTAGACCTTCCAAGTCCACCTTGGTCTTCAACTTGGGAGGTACTACCAATGGTTTTTCTTACGTTTAA(SEQ ID NO.1)
the amino acid sequence of the natural VP60 protein polypeptide is as follows:
>gi|358409918|gb|AEU09705.1|VP60,partial[Rabbit hemorrhagic diseasevirus]
MEGKARTAPQGEAAGTATTASVPGTTTDGMDPGVVAATSVVTAENSSASVATAGIGGPPQQVDQQETWRTNFYYNDVFTWSVADAPGSILYTVQHSPQNNPFTAVLSQMYAGWAGGMQFRFIVAGSGVFGGRLVAAVIPPGIEIGPGLEVRQFPHVVIDARSLEPVTITMPDLRPNMYHPTGDPGLVPTLVLSVYNNLINPFGGSTNAIQVTVETRPSDDFEFVMIRAPSSKTVDSISPAGLLTTPVLTGVGNDNRWNGQIVGLQPVPGGFSTCNRHWNLNGSTYGWSSPRFADIDHRRGSASYSGNNSTNVLQFWYANAGSAIDNPISQVAPDGFPDMSFVPFNSPNIPTAGWVGFGGIWNSNNGAPAATTVQAYELGFATGAPNNLQPTTNTSGAQTVAKSIYAVVTGTNQNPTGLFVMASGVISTPNASAVTYTPQPDRIVTTPGTPAAAPVGKNTPIMFASVVRRTGDVNAAAGSTNGTQYGTGSQPLPVTIGLSLNNYSSALMPGQFFVWQLTFASGFMEIGLSVDGYFYAGTGASTTLIDLTELIDVRPVGPRPSKSTLVFNLGGTTNGFSYV(SEQ ID NO.2)
genetically engineered cell
The invention provides a genetic engineering cell, which is a eukaryotic cell, and an expression cassette of rabbit hemorrhagic disease virus VP60 protein is integrated in the genome of the cell; or the cell contains an expression vector which contains an expression cassette of the rabbit hemorrhagic disease virus VP60 protein;
and said genetically engineered cell expresses said VP60 protein intracellularly, and said VP60 protein forms a virus-like particle (VLP) inside said genetically engineered cell.
In another preferred example, the cell is a yeast cell, preferably a pichia cell, more preferably a pichia KM71 strain.
In another preferred embodiment, the expression cassette comprises the following elements operably linked 5 'to 3': a promoter, a start codon, an ORF sequence of the VP60 protein, and a stop codon.
In the present invention, the term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.
Compositions and methods of administration
The present invention also provides a composition comprising: (i) a recombinant virus-like particle (VLP) of the invention or a polynucleotide of the invention that can encode a recombinant virus-like particle, and (ii) a pharmaceutically or immunologically acceptable excipient or adjuvant.
In the present invention, the term "comprising" means that various ingredients can be applied or present together in the composition of the present invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.
The compositions of the present invention include pharmaceutical compositions and vaccine compositions.
The compositions of the invention may be monovalent (comprising only one recombinant virus-like particle or polynucleotide) or multivalent (comprising a plurality of recombinant virus-like particles or polynucleotides).
The pharmaceutical or vaccine compositions of the present invention may be prepared in a variety of conventional dosage forms, including (but not limited to): injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays and the like.
(1) Pharmaceutical composition
The pharmaceutical composition of the present invention comprises (or contains) a therapeutically effective amount of the recombinant virus-like particle or polynucleotide of the present invention.
The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic agent that treats, alleviates, or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The effect can be detected, for example, by antigen levels. Therapeutic effects also include reduction of physiological symptoms. The precise effective amount for a subject will depend upon the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is not useful to specify an exact effective amount in advance. However, for a given situation, routine experimentation may be used to determine the effective amount.
For the purposes of the present invention, an effective dose is about 0.001 mg/kg to 1000 mg/kg, preferably about 0.01 mg/kg to 100 mg/kg of body weight of the recombinant virus-like particle administered to a subject.
The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent (e.g., a recombinant virus-like particle of the invention). The term refers to such pharmaceutical carriers: they do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. Suitable carriers may be large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acid (polylactic acid), polyglycolic acid and the like. Such vectors are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (mackpub.co., n.j.1991).
Pharmaceutically acceptable carriers in the compositions may include liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. Generally, the compositions can be prepared as injectables, e.g., as liquid solutions or suspensions; it can also be prepared into solid forms suitable for preparing solutions or suspensions, liquid vehicles before injection. Liposomes are also included in the definition of pharmaceutically acceptable carriers.
(ii) Vaccine composition
The vaccine (composition) of the invention may be prophylactic (i.e. to prevent disease) or therapeutic (i.e. to treat disease after disease).
These vaccines comprise an immunizing antigen (including the recombinant virus-like particles of the invention), and are typically combined with "pharmaceutically acceptable carriers" including any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and the like. Such vectors are well known to those of ordinary skill in the art. In addition, these carriers may act as immunostimulants ("adjuvants"). Alternatively, the antigen may be conjugated to a bacterial toxoid such as a toxoid from a pathogen such as diphtheria, tetanus, cholera, helicobacter pylori, and the like.
Preferred adjuvants to enhance the effect of the immunological composition include, but are not limited to: (1) aluminum salts (alum) such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations such as (a) MF59 (see WO90/14837), (b) SAF, and (c) Ribi Adjuvant System (RAS) (Ribi Immunochem, Hamilton, MT), (3) saponin adjuvant; (4) freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g., interferon), macrophage colony stimulating factor (M-CFS), Tumor Necrosis Factor (TNF), etc.; (6) detoxified variants of bacterial ADP-ribosylating toxins (e.g., e.coli heat labile toxin LT); and (7) other substances that act as immunostimulants to enhance the effectiveness of the composition.
Vaccine compositions, including immunogenic compositions (e.g., which may include an antigen, a pharmaceutically acceptable carrier, and an adjuvant), typically contain diluents such as water, saline, glycerol, ethanol, and the like. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like may be present in such vehicles.
More particularly, vaccines, including immunogenic compositions, comprise an immunologically effective amount of an immunogenic polypeptide, as well as the other desired components described above. An "immunologically effective amount" refers to an amount that is therapeutically or prophylactically effective for administration to an individual as part of a single dose or a continuous dose. The amount will depend upon the health and physiological condition of the individual being treated, the class of individual being treated (e.g., human), the ability of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the assessment of the medical condition by the treating physician, and other relevant factors. It is expected that the amount will be within a relatively wide range and can be determined by routine experimentation.
Typically, the vaccine composition or immunogenic composition can be prepared as an injectable formulation, such as a liquid solution or suspension; it can also be made into solid form suitable for preparing solution or suspension, or liquid excipient before injection. The formulation may also be emulsified or encapsulated in liposomes to enhance the adjuvant effect.
Furthermore, the vaccine composition of the present invention may be a monovalent or multivalent vaccine.
(iii) Route of administration and dosage
Once the composition of the invention is formulated, it can be administered directly to the subject. The subject to be treated may be a mammal, especially a human.
When used as a vaccine, the recombinant virus-like particles of the present invention can be administered directly to an individual by known methods. These vaccines are typically administered using the same route of administration as conventional vaccines and/or mimicking the route of infection by pathogens.
Routes of administration of the pharmaceutical or vaccine compositions of the invention include (but are not limited to): intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, nasal, oral, or other parenteral routes of administration. If desired, the routes of administration may be combined, or adjusted according to the disease condition. The vaccine composition may be administered in a single dose or in multiple doses, and may include administration of booster doses to elicit and/or maintain immunity.
The recombinant virus-like particle vaccine should be administered in an "effective amount", i.e., an amount of recombinant virus-like particle sufficient to elicit an immune response in the chosen route of administration effective to promote protection of the host against the associated disease.
Representative diseases include (but are not limited to): rabbit hemorrhagic disease virus infection, etc.
The amount of recombinant virus-like particles selected in each vaccine dose is based on the amount that elicits an immunoprotective response without significant side effects. Generally, after infection of the host cells, each dose of vaccine is sufficient to contain about 1. mu.g-1000 mg, preferably 1. mu.g-100 mg, more preferably 10. mu.g-50 mg of protein. Standard research methods including observing antibody titers and other responses in a subject can be used to determine the optimal amount of a particular vaccine. The need for booster doses can be determined by monitoring the level of immunity provided by the vaccine. After the antibody titer in serum is assessed, booster doses of immunization may be selected. Administration of adjuvants and/or immunostimulants can enhance the immune response to the proteins of the invention.
The preferred method is to administer the immunogenic composition by injection from the parenteral (subcutaneous or intramuscular) route.
In addition, the vaccines of the present invention may be administered in conjunction with other immunomodulators, or with other therapeutic agents.
The main advantages of the invention are:
(1) realizes the expression in the genetically engineered cell to form the rabbit hemorrhagic disease virus-like particle (VLP) for the first time;
(2) the recombinant rabbit hemorrhagic disease virus-like particles can induce animals to generate immune response against rabbit hemorrhagic disease viruses;
(3) the optimized RHDV VP60 gene sequence can be efficiently expressed in yeast cells and can form VLP, so that the invention provides a method for preparing rabbit hemorrhagic disease virus-like particles in large quantity.
(4) The recombinant rabbit hemorrhagic disease virus-like particle can be specifically combined with an anti-rabbit hemorrhagic disease virus antibody, and can be used for detecting the rabbit hemorrhagic disease virus antibody.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1 construction of Pichia pastoris engineering bacteria expressing RHDV VP60 protein VLP
First, experimental material
1. Strains and plasmids
E.coli DH5 α; the Pichia pastoris host strain GS115 and the yeast expression plasmid pPIC3.5K are both purchased from Invitrogen; the RHDV VP60 gene (optimized) was synthesized by jingzhi, japan.
2. Tool enzyme, antibody, kit and main chemical reagent
DNA restriction enzymes BamHI and Not I enzymes were purchased from NEB, Taq DNA polymerase, DNA Marker, dNTPs; t4DNA was purchased from TaKaRa. The plasmid extraction kit and the gel recovery kit are purchased from Qiagen company; PVDF membrane (0.45 micron size) is a product of Roche; PMSF was purchased from bi yun corporation; goat anti-mouse IgG labeled with horseradish peroxidase was purchased from Shanghai Kyoho Biotech Co., Ltd. The mouse anti-RHDV VP60 protein monoclonal antibody is purchased from Beijing Jia Mei Nuo Biotech Co. Yeast Nitrogen Base (Without amino acid) is purchased from BD company, and DAB horseradish peroxidase color development kit and D-Biotin are products of Shanghai BioEngineers, Biotech, Inc. G418 is a GIBCO product.
3. Main instrument
The PCR apparatus was purchased from ABI, the protein electrophoresis apparatus and the Western blot transfer apparatus were purchased from Bio-Rad, the electron microscope was a product of Beckman, the constant temperature incubator and the constant temperature shaking table were purchased from Shanghai Zhicheng Analyzer manufacturing Co., Ltd, and the nucleic acid electrophoresis apparatus and the small protein decolorization shaking table were a product of Beijing Heix.
4. Preparation of Main Medium and reagents
Culture medium
LB liquid cultureBase: 10g tryptone, 5g yeast extract, 10g sodium chloride in about 800mL ddH2Dissolving O, diluting to 1000mL, sterilizing with high pressure steam at 121 deg.C for 20min, and storing at 4 deg.C.
LB solid medium: on the basis of preparing a liquid culture medium, adding agar powder into the liquid culture medium of 15g/1000mL, sterilizing with high-pressure steam at 121 ℃ for 20min, cooling to about 55 ℃, adding corresponding antibiotics, paving a plate, and storing at 4 ℃.
Yeast culture medium stock solution formula:
YPD medium: 1% yeast extract, 2% peptone, 2% glucose. Dissolving 10g of yeast extract, 20g of peptone and 20g of glucose in 900mL of deionized water, sterilizing at 121 ℃ for 20min, and adding bacterial culture agar powder to 20g/L before sterilizing to prepare a flat plate.
YPD G418 resistant plates: the preparation method is similar to YPD plate, after sterilization, sterile G418 stock solution is added until the culture solution is cooled to 55 ℃ until the final concentration is 1mg/mL, 2mg/mL and 3mg/mL respectively, and the culture medium without G418 can be stored at room temperature for 3 months. After G418 is added, the mixture should be stored at 4 ℃ in the dark for 1-2 weeks.
10 × GY (10% glycerol): mixing glycerol with ddH2Mixing O at a ratio of 1:9, filtering for sterilization or autoclaving, and storing at 4 deg.C.
10 × M (5% methanol): reacting methanol with ddH2O is mixed in a ratio of 1:19, filtered to sterilize and stored at 4 ℃.
10 xd (20% glucose solution): at 500mL ddH2Dissolving 200g D-glucose in O, adding ddH after fully dissolving2O to a final volume of 1000 mL. Filtering for sterilization or autoclaving, and storing at 4 deg.C for one year.
10 XYNB: at 700mL ddH2Dissolving 134g YNB in O, adding ddH2O to 1000 mL. After filtration sterilization, the mixture was stored at 4 ℃ for one year.
500 xb (0.02% biotin): 20mg of biotin was dissolved in 100mL of deionized water, and sterilized by filtration and used at 4 ℃ for one year.
1M (pH 6.0) phosphate buffer: 132mL of 1M K2HPO，868mL of 1M KH2PO4 solution, adjusting pH to 6.0 with KOH or phosphoric acid, autoclaving, and storing at room temperature.
MD and MM plates:
MD：1.34％YNB，4×10-5% Biotin, 1% glycerol.
MM：1.34％YNB，4×10-5% Biotin, 0.5% methanol.
Sterilized with 800mL of sterile water and 15g of agarose, cooled to 60 ℃ and then added with 100mL of 10 XYNB solution, 2mL of 500 XB, 100mL of 10 XD (MD) or 10 XM (MM) solution, poured out of the plate, coagulated and stored at 4 ℃ for 3 months.
BMGY/BMMY medium:
yeast powder 10g, peptone 20g, using 700ml ddH2Dissolving O, cooling to room temperature, sterilizing with high pressure steam at 121 deg.C for 20min, cooling to room temperature, and adding the following reagents: 100mL of 1M pH6.0 potassium phosphate buffer, 100mL of 10 XYNB, 2mL of 500 XB, 100mL of 10 GY or 10 XM, and left at 4 ℃ for 2 months.
Reagent
Nucleic acid electrophoresis related solution
50 XTAE buffer (pH 8.5): measuring 242g of Tris alkali and Na2EDTA-2H2Placing 37.2g of O into a 1L beaker, adding 800mL of deionized water, fully stirring for dissolving, adding 57.1mL of acetic acid, fully stirring, adding deionized water to 1L, and storing at room temperature.
Protein electrophoresis related solution
5 × Tris-glycine electrophoresis buffer: weighing 15.1g Tris base, 94g glycine (electrophoresis grade) (Ph 8.3), 5.0g SDS, adding about 800mL deionized water, stirring to dissolve, adding ddH2O is subjected to constant volume to 1L and is stored at room temperature;
1.5M Tris buffer (pH 8.8): 18.1g Tris base, dissolved in appropriate amount of ddH2Adjusting the pH value to 8.8 by using HC1, and metering to 100 mL;
1.0M Tris buffer (pH6.8): 12.1g Tris base, dissolved in appropriate amount of ddH2Adjusting the pH value to 6.8 by using HCl, and fixing the volume to 100 mL;
protein loading buffer (5 ×): 250mM Tris-Cl (pH6.8), 5% (V/V) beta-mercaptoethanol (beta-ME), 10% SDS, 0.5% (W/V) bromophenol blue, 50% glycerol.
Preparation method of 5 mL: measuring 1.25mL of 1M Tris-Cl (pH6.8) in a 15mL centrifuge tube, weighing 0.5g SDS and 0.025g bromophenol blue, mixing, adding 2.5mL glycerol, and supplementing ddH2And (4) dissolving O, metering to 5mL, and subpackaging in small parts. Before use, 25 beta-ME is added into every 500 mu L of the buffer solution and mixed evenly to obtain the reductive loading buffer solution.
12% SDS-gel and 5% concentrated gel
Protein Western-blot related reagent
And (3) membrane transfer buffer solution: weighing 2.9g of glycine, 5.8g of Tris base and 0.37g of SDS, weighing 200mL of methanol, adding ddH2O to 1000 mL. After dissolution, the mixture is stored at room temperature.
TBS buffer (100mM Tris-HCl, pH7.5, 150mM NaCl): 10mL of 1mol/L Tris-HCl (pH7.5), 8.8g NaCl was added with ddH2And O is metered to 1000 mL.
TBST buffer: TBS buffer solution containing 0.05% Tween-20, Tween-200.5 mL and TBS 1000mL are taken, and the TBS buffer solution can be used after being uniformly mixed and is prepared as before.
Sealing liquid: TBST buffer containing 5% skimmed milk powder, 5g skimmed milk powder, and TBST 100 mL. After dissolution, the mixture was stored at 4 ℃. When in use, the temperature is recovered to room temperature, and the dosage is covered by the membrane surface, thus the disposable mask is used for one time.
Second, Experimental methods
1. Construction of recombinant expression plasmid pPIC3.5K-VP60
According to the published gene sequence of the RHDV VP60 protein, codon optimization is carried out, a large number of sequence fragments are optimized and designed, and artificial synthesis is respectively carried out.
After artificially synthesizing a VP60 gene sequence, carrying out double enzyme digestion on the pPIC3.5K carrier by using Bam HI and EcoR I; then, a nucleic acid gel recovery kit is used for recovering a VP60 gene and a pPIC3.5K vector corresponding fragment after enzyme digestion, a VP 4 ligase is used for respectively carrying out a ligation reaction on a VP60 gene (Bam HI/EcoR I) and a pPIC9K vector (Bam HI/EcoR I), a ligation product is transformed into escherichia coli DH5 alpha competence, the ligation product is coated on an Amp-resistant LB flat plate, inverted culture is carried out at 37 ℃ for 12-16h, 5 clones are selected from each plate for colony PCR identification, small quality-improved grains are preliminarily obtained after the amplification culture of positive colonies to obtain pPIC9K-VP60, and the plasmid with correct enzyme digestion size is sent to Jinzhi company of Suzhou for gene sequencing after double enzyme digestion identification by Bam HI/EcoRI.
In the process of expressed gene optimization, the inventor finds that different VP60 gene sequences obtained by optimization have large difference in expression level, and many gene sequences cannot form viral particles or the formed viral particles have low immunogenicity. After a large number of screening and verification, a VP60 gene sequence capable of being efficiently expressed in yeast is obtained, and a VP60 protein expressed by the sequence can form correctly folded VLP particles in cells, so that the VLP is very suitable for preparing VLPs with natural VP60 immunogenicity, and the specific sequence is as follows.
Optimized gene sequence of RHDV VP60
>ATGGAAGGAAAAGCCAGAACCGCTCCACAAGGTGAAGCTGCCGGAACCGCTACTACCGCCTCCGTTCCTGGAACTACTACCGATGGAATGGACCCAGGAGTTGTTGCCGCTACTTCCGTTGTTACTGCTGAGAACTCCTCTGCCTCTGTTGCTACTGCTGGTATCGGTGGTCCTCCACAGCAAGTTGATCAACAAGAAACCTGGCGTACCAACTTCTACTATAACGACGTTTTCACTTGGTCTGTTGCTGACGCCCCAGGTTCTATCTTGTACACTGTTCAACATTCTCCACAAAACAACCCATTCACCGCCGTTTTGTCCCAGATGTACGCTGGTTGGGCTGGTGGTATGCAGTTCAGATTCATTGTTGCCGGTTCCGGTGTCTTCGGTGGTAGATTGGTCGCTGCTGTCATCCCACCTGGTATTGAGATCGGTCCAGGTTTGGAGGTCCGTCAATTCCCACACGTTGTTATCGACGCCCGTTCTTTGGAGCCAGTTACCATCACTATGCCAGATTTGAGACCTAACATGTACCACCCAACTGGTGATCCTGGTCTTGTCCCTACTTTGGTTTTGTCTGTTTACAACAATTTGATCAACCCATTTGGTGGTTCCACCAACGCTATTCAGGTTACCGTCGAGACCAGACCATCCGACGACTTCGAGTTTGTCATGATCCGTGCTCCATCCTCCAAGACTGTTGACTCCATCTCCCCAGCTGGTTTGTTGACTACTCCAGTCTTGACCGGTGTCGGAAACGACAACCGTTGGAACGGTCAGATTGTCGGTTTGCAACCTGTCCCTGGAGGTTTCTCTACCTGCAACCGTCACTGGAACTTGAACGGTTCCACTTACGGTTGGTCCTCTCCAAGATTCGCCGATATCGACCACAGAAGAGGTTCCGCCTCTTACTCCGGAAATAACTCCACCAACGTCTTGCAGTTCTGGTACGCCAACGCCGGTTCTGCCATCGACAACCCAATTTCCCAAGTTGCCCCAGACGGTTTCCCAGATATGTCTTTTGTTCCATTCAACTCTCCTAACATCCCAACTGCCGGTTGGGTTGGTTTCGGTGGTATCTGGAACTCCAACAACGGTGCTCCTGCTGCCACCACTGTCCAGGCTTACGAACTTGGTTTCGCCACCGGTGCTCCTAACAACTTGCAGCCAACTACTAACACTTCCGGTGCTCAGACCGTTGCCAAATCCATCTACGCTGTTGTCACCGGTACCAATCAGAACCCAACCGGTTTGTTTGTTATGGCTTCCGGAGTCATCTCCACTCCTAACGCCTCTGCTGTTACCTACACCCCACAGCCTGACAGAATTGTCACTACCCCAGGAACTCCTGCTGCCGCTCCAGTTGGAAAGAACACTCCTATCATGTTCGCCTCCGTCGTTCGTAGAACCGGTGACGTTAACGCTGCCGCTGGTTCCACTAACGGTACCCAATACGGTACCGGTTCTCAGCCATTGCCAGTCACCATTGGTTTGTCTTTGAATAACTATTCCTCTGCCTTGATGCCAGGTCAGTTCTTTGTCTGGCAGTTGACCTTCGCTTCCGGATTCATGGAGATCGGTTTGTCTGTTGATGGATACTTTTACGCCGGTACCGGTGCCTCTACCACTCTTATCGATCTTACCGAATTGATCGATGTCCGTCCAGTTGGTCCTAGACCTTCCAAGTCCACCTTGGTCTTCAACTTGGGAGGTACTACCAATGGTTTTTCTTACGTTTAA
2. Recombinant expression plasmid transformed pichia pastoris
2.1 Mass preparation and linearization of plasmid DNA
Mass extraction of plasmids: single colonies identified by digestion to contain positive clones were inoculated in 3mL LB medium (containing 100g/mL Amp) and shaken overnight at 37 ℃. When the bacteria grow to late log phase, the concentration of the bacteria is about OD600When the concentration is 0.60, the culture is transferred to a 500mL LB culture bottle (containing 100g/mL Amp), cultured with shaking at 37 ℃ for 12-16 hours, collected, centrifuged at 4000rpm4 ℃ for 10 minutes, and the supernatant is removed, and the plasmid is macro-sized according to the procedure of a plasmid macro-kit of Qiagen.
Linearized digestion of plasmid DNA:
the enzyme digestion system is as follows:
the enzyme was cleaved at 37 ℃ for 3 hours, and an equal volume of phenol was added: chloroform, mixing, centrifuging at 12000rpm for 10 min, sucking the upper aqueous phase, adding equal volume of chloroform: isoamyl alcohol, mixing, centrifuging at 12000rpm for 10 minutes, sucking the upper water phase, adding 1/4 volume of ammonium acetate and 2 times volume of absolute ethyl alcohol, standing at-20 ℃ for 20 minutes, then centrifuging at 12000rpm for 15 minutes, pouring out the supernatant, washing the precipitate with 70% ethanol and absolute ethyl alcohol respectively once, drying at 37 ℃, and then suspending in 15 mu L of TE buffer solution.
2.2 linearized plasmid electrotransformation of Pichia cells
Preparation of competent cells:
(1) a single colony of Pichia pastoris was inoculated into a 50mL Erlenmeyer flask containing 5mL YPD medium and cultured overnight at 220rpm at 28 ℃.
(2) 0.1-0.5mL of the culture was inoculated into 500mL of fresh medium and cultured overnight to an OD600 of about 1.3-1.5.
(3) The cells were collected by centrifugation at 1500 Xg for 5 minutes at 4 ℃ and the pellet was redissolved in 500mL of frozen sterile water.
(4) The cells were harvested by centrifugation at 1500 Xg for 5 minutes at 4 ℃ and the pellet redissolved in 250mL of frozen sterile water.
(5) The cells were harvested by centrifugation at 1500 Xg for 5min at 4 ℃ and the pellet was redissolved in 20mL of frozen 1M sorbitol.
(6) The cells were harvested by centrifugation at 1500 Xg for 5 minutes at 4 ℃ and the pellet redissolved in 1mL of frozen 1M sorbitol.
(7) Subpackaging into prefreezing 1.5mL centrifuge tubes, and subpackaging 80 μ L each centrifuge tube.
And (3) electric conversion:
(1) take 5-20. mu.g of linearized DNA, dissolve in 5-10. mu.L of TE, mix with 80. mu.L of electroporation competent cells, and then transfer to a frozen 0.2cm electroporation cuvette.
(2) Incubate the electric rotor on ice for 5 minutes.
(3) And (3) transformation: the electrotransformation instrument is Bio-Rad GenePulse, the voltage is 1500V, the capacitance is 25 muF, and the resistance is 200 omega.
(4) Add immediately l mL "frozen 1M sorbitol to the transformation cup and transfer the solution from the cup to a 1.5mL sterile centrifuge tube.
(5) 200-.
3. Screening of Pichia pastoris multicopy transformants by G418 resistance
For yeast electrically transformed with pPIC3.5K plasmid, after single colony growth on MD plate, several colonies were randomly selected and dropped into 96-well plate previously added with 100. mu.L YPD liquid medium, cultured at 28 ℃ for 16-20h, and then 10. mu.L each of the bacterial liquid was inoculated into another 96-well plate previously added with 100. mu.L LYPD. After synchronization, 10. mu.L of the inoculum was inoculated onto YPD plates containing 1mg/mL, 2mg/mL and 3mg/mL G418 resistance. Culturing at 28 deg.C for 2-5 days to obtain single colony.
4PCR method for identifying positive recombinants
Selecting part of yeast from single colony, adding 5 μ L of 1% helicase/SCE solution, shaking gently at 37 deg.C for 3 hr, performing enzymolysis to break cell wall, heating at 100 deg.C for 15 min, diluting to 20 μ L, taking 5 μ L as template, and performing PCR reaction with primers El and E2.
Reaction system: h2O75. mu.L, dNTPs (2.5mM/each) 5. mu.L, 10 XTaq enzyme reaction buffer 10. mu.L, template 5. mu.L, primer El 2. mu.L, primer E22. mu.L.
And (3) PCR reaction conditions: adding Taq enzyme 1 μ L/tube (4 μm) at 95 deg.C for 5min, and performing 30 temperature cycles at 72 deg.C for 10 min
Temperature cycling conditions; 95 ℃ for 30 seconds, 60 ℃ for 30 seconds, 72 ℃ for 120 seconds. The product identification method is the same as above.
5 induced expression of fusion gene in Pichia pastoris
1. Single clones on GS115-pPIC3.5K-VP60 yeast plates (2mg/mL) were picked up on YPD plates with grids drawn on the back, one single clone was grown on each grid, and the colonies were cultured in an incubator at 28 ℃.
After 2.60h yeast growth on the plate, 3 clones were picked each into 50mL centrifuge tubes containing 10mL BMGY medium, recorded, covered with four layers of sterile gauze, and incubated at 28 ℃ 250rpm to an OD600 of about 2-6 (log phase growth, about 16-18 h).
3. Centrifuging at 1500g for 5min at room temperature, collecting cells, removing supernatant, re-suspending the cells with BMMY until OD600 is 1.0, remaining 10mL of culture solution in the tube, discarding the excess, covering the culture with four layers of sterilized gauze, and placing in a shaking table for continuous induction expression.
After 4.24 hours, methanol was supplemented to a final concentration of 0.5% (about 1mL of 5% methanol, the amount of medium was checked to ensure correct addition of methanol, since evaporation would reduce the volume of the medium) to continue induction.
After 5.24h, the cells were harvested by centrifugation at 1500 Xg for 5 minutes.
6. Collecting cells, centrifuging at room temperature at maximum speed for 2-3min, collecting 80mg wet yeast (adding equal weight of beads, 150 μ L of lysate) for crushing, and detecting protein expression by SDS-PAGE and ELISA.
6 preparation of Yeast cell protein extract
Cells collected after induction of expression were taken and 80mg of yeast wet-weight was added to each 1.5mL eppendorf tube. Wash 2 times with ice cold deionized water, add 160. mu.L of ice cold lysis buffer (containing 100mM PMSF) and add an equal volume of acid wash beads (0.5 mM). The mixture was vigorously shaken for 30 seconds, then ice-cooled for 30 seconds, and this shaking and ice-cooling operation was repeated 12 times. Centrifuging at 12000rpm for 15 min, and collecting supernatant to obtain protein extract.
7SDS-PAGE and Western blot
1) Taking 20 microliter of yeast cell extract, adding 5 microliter of 5 xSDS-PAGE loading buffer, mixing uniformly, boiling for 5min at 100 ℃;
2) and (3) mounting the protein gel on an electrophoresis frame, putting the electrophoresis frame into an electrophoresis tank, pouring electrophoresis liquid, removing a comb, adding 25 mu L of the processed protein sample into a sample adding hole, and performing SDS-PAGE electrophoresis: after electrophoresis is carried out for 20min under the voltage of 100V, the voltage is adjusted to be 150V after the protein strips start to move to the separation gel, and electrophoresis is carried out for 1 h;
3) transferring the protein to a PVDF membrane by a wet transfer method after electrophoresis is finished, and converting the membrane current by 280mA for 1 h;
4) after the film is transferred, putting the film into a small plastic box, adding 5% of skimmed milk powder, shaking and sealing for 1h at room temperature in a shaking table to eliminate non-specific background;
5) after sealing, washing the membrane for 3 times (5 min each time) by TBST;
6) adding Anti-RHDV VP60 monoclonal antibody diluted with 1% skimmed milk powder (TBST configuration) as primary antibody (1:1000 dilution), and shaking and incubating for 2h (or incubating overnight at 4 deg.C) at room temperature;
7) recovering primary antibody after incubation, washing the membrane for 3 times and 5 min/time by using TBST;
8) adding a HRP-labeled Goat-Anti-mouse secondary antibody, and incubating for 1h on a shaking table at room temperature;
9) washing with TBST for 5min for 3 times;
10) the membrane was transferred to freshly prepared DAB solution for color development, carefully observed, and once the protein hybridization bands were visualized, the reaction was stopped by rinsing with water and transferred to PBST solution.
8 electronic microscope observation and identification of VLP formed by VP60 protein expressed in Pichia pastoris
And selecting a yeast cell protein extract sample for electron microscope observation. The method comprises the steps of adopting a conventional negative dyeing technology, mixing an equal amount of sample and 2% phosphotungstic acid dye solution, dripping the mixture onto a carrier net with a film by using a capillary tube, sucking excessive liquid, drying the carrier net at 37 ℃, observing by using an electron microscope, and estimating the actual size of particles according to the diameter and the magnification of the particles on a picture.
Third, experimental results
1 construction of recombinant expression plasmid pPIC3.5K-VP60
The recombinant plasmid pPIC3.5K-VP60 was cut into two fragments of about 7000bp and 5500bp by BsrGI enzyme (FIG. 1); the plasmid is initially confirmed to be a positive recombinant plasmid, the plasmid is sent to Jinzhi company for sequencing, the sequencing result is aligned by ClustalW2 in EBI, and the sequence is completely consistent with the optimized VP60 cDNA sequence.
FIG. 1 shows the restriction enzyme identification of pPIC3.5K-VP60 expression vector, Lane 1: undigested pPIC3.5K-VP 60; lane 2 digested with BsrG I; lane 3 molecular weight marker.
The pPIC3.5K-VP60 and the BsrG I enzyme-digested pPIC3.5K-VP60 are synchronously subjected to agarose gel electrophoresis, and the detection result shows that the pPIC3.5K-VP60 recombinant vector is about 12000bp, and the BsrG I enzyme-digested recombinant vector pPIC3.5K-VP60 has two fragments of about 7000bp and 5500bp
2, transforming the pichia pastoris cell by the recombinant plasmid pPIC3.5K-VP60, screening pichia pastoris multicopy transformants by G418 resistance, and identifying a positive recon by a PCR method.
3Western blot to identify expression of VP60 protein in Pichia pastoris cells
3 KM71 single colonies are selected and inoculated into BMGY liquid culture medium, the temperature is 28 ℃, the bacteria is shaken at 250rpm and cultured until the bacterial liquid is 0D 600-6, after centrifugation, BMMY heavy suspension bacteria is added to ensure that the bacterial liquid is 0D 600-1, then the bacterial liquid is inoculated into the BMMY culture medium for induced expression, and as a negative control, the KM71 strain which is transformed into pPIC3.5k empty vector is synchronously induced and expressed. Adding 100 Xmethanol into the culture medium every 24h to make the final concentration of the protein reach 0.5%, performing induced culture for 48h, centrifuging to collect thallus precipitate, crushing, extracting protein, and performing Western blot detection on the protein extract. The results showed that the protein bands were present at around 60kDa in the pPIC3.5k-VP60/KM71 protein extract sample, whereas no target protein band was detected in the pPIC3.5K/KM71 protein extract sample.
The result shows that the VP60 protein is successfully expressed in Pichia pastoris, and the expression quantity of three clones selected after screening has no significant difference.
FIG. 2 shows the expression of RHDV VP60 protein in Pichia pastoris detected by SDS-PAGE and Western blot.
4-electron microscopy of VLP particles formed by VP60 protein
The results of the electron microscope negative staining experiment on the yeast cell protein extract sample after methanol induction are shown in FIG. 3: after negative staining, obvious virus particles can be observed under an electron microscope, and the result shows that the RHDV VP60 protein expressed in pichia pastoris can be assembled into virus particles.
FIG. 3 shows the experimental observation of RHDV virus particles by electron microscopy negative staining.
Example 2 RHDV Virus-like particle vaccine Activity assay
The virus-like particle vaccine of rabbit viral hemorrhagic disease virus (RHDV) is prepared by producing VP60 structural protein of RHDV by using a yeast expression system, then assembling in vitro to form virus-like particles, and adding a proper amount of adjuvant. The tests of bacteria, mould, mycoplasma and exogenous virus are carried out according to the existing method of Chinese animal pharmacopoeia, and all the tests meet the specified standards. The safety and immunopotency tests for the vaccines produced by the present invention are now summarized below.
1 seed of Vibrio species
1.1 sources of virus seeds for testing
The virus strain used in the effect detection is aseptically used for collecting the liver tissue of a rabbit infected with RHDV and died of illness, then the tissue grinding is carried out (3 mL of PBS containing antibiotics is added into 1g of liver tissue, and the antibiotics are penicillin and streptomycin which are 1000IU/mL respectively), the supernatant is obtained after being centrifuged at 4000r/min for 10 minutes after being ground, and the supernatant is stored, identified and supplied by the engineering technology center of Shanghai Haili biological Limited company.
1.2 test reagents
Inactivated vaccines for rabbit viral hemorrhagic disease virus for efficacy test are purchased from Nanjing Tianbang Biotechnology GmbH; the rabbit viral hemorrhagic disease virus-like particle antigen is prepared by the engineering center and is mixed by adding a proper amount of adjuvant, and the antigen content is 1mg/mL and 2mg/mL respectively; other chemical reagents are all domestic superior pure.
1.3 test materials
Surgical trays and dissecting instruments, tissue grinders, syringes, blood collectors, emulsifiers, sony cameras, and the like.
1.4 test animals
Healthy SPF rabbits weighing 1.5kg or more at the age of 45-60 days, purchased from Qingdaokang Biotech, Inc., were used for this test.
2 Immunopotentiality test of vaccine
2.1 grouping of Experimental animals
16 SPF rabbits were randomly divided into 4 groups of 4 animals each: the first group was a PBS control group, 1mL PBS was injected subcutaneously; the second group is a commercial vaccine immunization group, and 1mL of commercial vaccine is injected subcutaneously; the third group was a low dose antigen immunization group, injected subcutaneously with 1mL of antigen (1 mg/mL); the fourth group was a high dose antigen immunization group, which was injected subcutaneously with 1mL of antigen (2 mg/mL).
2.2 comparison of immune Effect
After 21 days of immunization, 1ml of RHDV virus solution is injected subcutaneously into each group of rabbits. And (4) clinically observing for 7 days after challenge, recording clinical reaction conditions, counting the death number of each group of rabbits, dissecting the infected dead rabbits in time, recording the eye pathological changes dissected by the diseased dead rabbits, photographing, and evaluating the immune effect of the vaccine.
3 results
3.1 clinical manifestations of symptoms
The PBS group test shows that the infected rabbits show typical symptoms of rabbit fever after 4-28h of infection, the body temperature rises to peak value (40-41.5 ℃) after 16-34h, wherein the body temperature of 3 diseased rabbits lasts for 10-18h respectively, and the infected rabbits die 7-12.5 h after the body temperature begins to drop. After the temperature is obviously raised, the spirit is depressed, and the appetite is reduced and the drinking desire is enhanced; further, the respiratory is rapid, the mucous membrane is cyanotic, oliguria, proteinuria and somnolence can be seen; abdominal distension and constipation appear, short-term excitation appears before death, struggle, the cage is rushed, then two forelimbs lie on the ground, two hind limbs stand up, the whole body shakes and twitches, finally the limbs lie on the ground, the limbs slide continuously, finally the limbs twist to one side, the angle bow is in a reversed posture, blood coagulation is poor and is dark red, conjunctiva congestion occurs, blood pollution exists around nostrils, bright red foam blood sometimes flows out, gingival bleeding occurs, and the limbs jump suddenly and are tragic; the bloody stream of the vulva of the individual diseased rabbits. The eye lesions during the autopsy are consistent with acute rabbit plague. The rest of the immunized rabbits showed a transient anorexia and a depressed spirit within 3 days after the challenge, but then recovered to be normal.
3.2 mortality statistics
Two rabbits (2/4) die after about 60h, one rabbit die (1/4) after about 84h and the rest rabbits die after 7d after toxin attack; no rabbit in the immune group died after being attacked, which shows that the vaccine has good immune effect.
3.3 pathological anatomy Observation
The pathological anatomy results of the rabbits after virus attack are shown in fig. 4, the viscera changes of the rabbits after the virus attack of the PBS group accord with the rabbit plague, and the viscera of the rabbits of the immunization group are not abnormal.
4 summary of laboratory trial products
The research takes RHDV virus-like particles produced by a yeast expression system as antigens, produces and manufactures vaccines under the condition of a laboratory, and carries out efficacy test on the vaccines, and the result shows that the produced vaccines meet the standard specified by the vaccine specification. The result of the immunity challenge test shows that the vaccination test rabbit is safe and reliable to animals, has no toxic and side effect, and the vaccine has good immunity effect.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (6)
1. A genetically engineered cell, wherein the genetically engineered cell is a eukaryotic cell, and an expression cassette for expressing rabbit hemorrhagic disease virus VP60 protein is integrated in the genome of the cell; or the cell contains an expression vector which contains an expression cassette for expressing the rabbit hemorrhagic disease virus VP60 protein;
and said genetically engineered cell expresses said VP60 protein intracellularly and said VP60 protein forms a virus-like particle inside said genetically engineered cell;
in the expression cassette of the rabbit hemorrhagic disease virus VP60 protein, the gene sequence of the VP60 protein is shown as SEQ ID NO. 1;
and the eukaryotic cell is a pichia pastoris cell.
2. A method of preparing a virus-like particle comprising the steps of:
culturing the cell of claim 1 under conditions suitable for expression, thereby expressing the virus-like particle; and
isolating the virus-like particle.
3. An isolated codon-optimized polynucleotide encoding a polypeptide as set forth in SEQ ID No. 2; and the sequence of the polynucleotide is shown in SEQ ID NO. 1.
4. An expression vector comprising the polynucleotide of claim 3.
5. A host cell comprising the expression vector of claim 4 or having the polynucleotide of claim 3 integrated into its genome.
6. A pharmaceutical composition comprising the polynucleotide of claim 3 or the expression vector of claim 4 or the host cell of claim 5, and a pharmaceutically acceptable carrier and/or adjuvant.
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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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