CN117298263A - Recombinant rabies vaccine and preparation method and application thereof - Google Patents
Recombinant rabies vaccine and preparation method and application thereof Download PDFInfo
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- CN117298263A CN117298263A CN202311265714.0A CN202311265714A CN117298263A CN 117298263 A CN117298263 A CN 117298263A CN 202311265714 A CN202311265714 A CN 202311265714A CN 117298263 A CN117298263 A CN 117298263A
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- vaccine
- recombinant rabies
- recombinant
- adjuvant
- protein
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Classifications
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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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55577—Saponins; Quil A; QS21; ISCOMS
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/20011—Rhabdoviridae
- C12N2760/20111—Lyssavirus, e.g. rabies virus
- C12N2760/20134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
Abstract
The invention provides a recombinant rabies vaccine, a preparation method and application thereof, and belongs to the technical field of virus vaccine preparations. Wherein the recombinant rabies vaccine comprises: recombinant rabies virus g protein, pharmaceutical excipients and vaccine adjuvants; wherein, the base sequence of the recombinant rabies virus g protein is shown as SEQ ID NO. 1; the invention adopts recombinant rabies virus g protein with the sequence of SEQ ID NO.1, pharmaceutical excipients and vaccine adjuvants, and the immune experimental study of the BALB/c mice given by intramuscular injection proves that the self-made recombinant rabies vaccine has good immunogenicity.
Description
Technical Field
The invention belongs to the technical field of virus vaccine preparations, and particularly relates to a recombinant rabies vaccine, a preparation method and application thereof.
Background
Rabies Virus (Rabies Virus, RABV) belongs to the genus rhabdovirus, consisting of genetically related encapsidated viruses, containing single-stranded non-segmented negative-strand RNA. There is currently no effective treatment, pre-exposure prophylaxis (Pre-exposure prophylaxis, prEP) and Post-exposure prophylaxis (Post-exposure prophylaxis, PEP) in humans are the primary measures for the prophylaxis of rabies.
Rabies vaccine, which can be used for pre-exposure and post-exposure prevention of rabies, is an effective means for preventing and controlling rabies, and greatly reduces the occurrence of rabies in the past decades. However, currently, all rabies vaccines for people on the market are inactivated vaccines, and high concentration of antigen is needed to achieve the immune effect, so that the rabies vaccine has high cost; and the medicine is used for preventing and treating after exposure, 4-5 needles are required to be inoculated, 2 needles are required to be reinforced when the medicine is exposed again for more than 3 months, compliance is poor, and immune deficiency is easy to cause; if delayed or incomplete prevention is provided after exposure, death may still result. Therefore, there is an urgent need to develop a vaccine that is safe, low cost, short duration, long lasting to enhance compliance and effectively prevent rabies.
The rabies virus G protein is a trimer, is a main antigen for inducing the production of virus neutralizing antibodies, can generate immunity aiming at the fatal infection of the rabies virus, and is a main component for playing a role of a vaccine. The adjuvant can enhance the immune response of the organism to the vaccine, and the low dose antigen induces cell immunity or humoral immunity to meet the protection requirement. Therefore, compared with the inactivated vaccine, the recombinant rabies vaccine containing the adjuvant can enhance the stimulation of glycoprotein in RABV to organisms, improve the immunogenicity of the vaccine, accelerate the immune response, reduce the required dose and the vaccination times and reduce the vaccine cost.
In the use of recombinant vaccine formulation adjuvants, aluminum adjuvants are routinely employed. However, the rabies vaccine containing the aluminum adjuvant has the defect of delaying the release of antigens, so that the antibody level in the early stage of immunization is lower, and the prevention and the control of the early stage after the rabies exposure are not facilitated, so that the rabies vaccine is gradually replaced by the current freeze-dried inactivated vaccine.
In summary, although the existing rabies vaccine can prevent rabies to a certain extent, the following defects exist: first, the high concentration of antigen in an inactivated vaccine results in the vaccine being expensive; secondly, the vaccination dose and times of the vaccine are more, the compliance is poor and immune deficiency is easy to cause; in addition, conventional aluminum adjuvants can delay the release of antigens, affecting the effectiveness of the vaccine in early control. Therefore, there is a need to develop a novel rabies vaccine comprising a novel adjuvant which can rapidly produce antibodies and which produces high levels of antibodies, to overcome the drawbacks of the existing vaccines.
Disclosure of Invention
In order to solve the above problems, the present invention provides a recombinant rabies vaccine comprising:
recombinant rabies virus g protein, pharmaceutical excipients and vaccine adjuvants;
wherein, the base sequence of the recombinant rabies virus g protein is shown as SEQ ID NO. 1;
preferably, the amino acid sequence of the recombinant rabies virus g protein is shown as SEQ ID NO. 2.
Preferably, 50-300 μg of recombinant rabies virus g protein is included per milliliter of the recombinant rabies vaccine.
Preferably, the pharmaceutical excipients comprise the following components in each milliliter of the recombinant rabies vaccine:
polysorbate 80, 100 μg-500 μg;
sucrose, 20mg-100mg;
sodium chloride, 1mg-9mg.
Preferably, the vaccine adjuvant is a first adjuvant composition or a second adjuvant composition;
both the first adjuvant composition and the second adjuvant composition include: DOPC, cholesterol and QS-21.
Preferably, the first adjuvant composition comprises the following components in amounts per ml of the recombinant rabies vaccine:
DOPC，700μg-3000μg；
cholesterol, 175 μg-750 μg;
QS-21，35μg-120μg。
preferably, the second adjuvant composition comprises DOTAP and Poly I: C in addition to DOPC, cholesterol and QS-21.
Preferably, the second adjuvant composition comprises the following components in amounts per ml of the recombinant rabies vaccine:
DOPC，500μg-2000μg；
cholesterol, 175 μg-600 μg;
QS-21，50μg-120μg；
DOTAP，70μg-240μg；
Poly I:C，400μg-900μg。
in addition, in order to solve the above problems, the present invention also provides a preparation method of a recombinant rabies vaccine, comprising:
the recombinant expression plasmid is subjected to expression treatment and purification treatment in sequence to obtain recombinant rabies virus g protein;
and adding a vaccine adjuvant into the stock solution of the recombinant rabies virus g protein, adjusting the pH value to 6.0-7.0, and subpackaging after using water for injection to fix the volume to obtain the recombinant rabies vaccine.
Preferably, the expression process includes:
transforming the recombinant expression plasmid into DH5 alpha chemically competent cells, and selecting a monoclonal for sequencing to obtain a monoclonal strain transformed by genetic engineering;
amplifying and culturing the monoclonal strain, and then purifying the plasmid to obtain a transfection-grade plasmid;
packaging by PEI, transfecting the transfection-grade plasmid into CHO cells, carrying out recombinant protein expression, and collecting an expression supernatant;
the purification treatment comprises: carrying out ultrafiltration concentration on the expression supernatant to obtain concentrated expression supernatant; and carrying out affinity chromatography on the concentrated expression supernatant, and collecting eluent to obtain the stock solution of the recombinant rabies virus g protein.
Preferably, the preparation method of the recombinant rabies vaccine further comprises the following steps: preparing the vaccine adjuvant; the vaccine adjuvant is any one of a first adjuvant composition and a second adjuvant composition; wherein, when the vaccine adjuvant is the first adjuvant composition, the preparing obtains the vaccine adjuvant, including: neutral liposome is prepared by DOPC and cholesterol; mixing phosphate buffer solution and neutral liposome, and then adding QS-21 and pharmaceutical excipients for mixing to obtain the first adjuvant composition; when the vaccine adjuvant is the second adjuvant composition, the preparing obtains the vaccine adjuvant, including: neutral liposome is prepared by DOPC and cholesterol; and, preparing cationic liposome by utilizing DOTAP, DOPC and cholesterol; and mixing QS-21, poly I, C, cationic liposome, neutral liposome and pharmaceutical excipients to obtain the second adjuvant composition.
In addition, in order to solve the problems, the invention also provides the application of the recombinant rabies vaccine in preparing products for treating and/or preventing diseases caused by rabies viruses, wherein the products comprise vaccine products, medicaments, diagnostic reagents and detection kits.
The invention provides a recombinant rabies vaccine, a preparation method and application thereof, wherein the recombinant rabies vaccine comprises the following components: recombinant rabies virus g protein, pharmaceutical excipients and vaccine adjuvants; wherein, the base sequence of the recombinant rabies virus g protein is shown as SEQ ID NO. 1. The invention adopts recombinant rabies virus g protein with the sequence of SEQ ID NO.1, pharmaceutical excipients and vaccine adjuvants, and the immune experimental study of the BALB/c mice given by intramuscular injection proves that the self-made recombinant rabies vaccine has good immunogenicity.
Drawings
FIG. 1 is a chromatogram of eluted proteins in example 2 of the present invention;
FIG. 2 is a diagram of SDS-PAGE gel after purification in example 2 of the present invention;
FIG. 3 is a graph showing the results of the hemolytic activity test of different samples in example 4 of the present invention;
FIG. 4 is a graph showing DOPC and cholesterol levels obtained by standing at 2℃to 8℃and 37℃for 1 week in example 4 of the present invention;
FIG. 5 is a graph showing QS-21 content results for a period of 1 week between 2℃and 8℃and 37℃in example 4 of the present invention;
FIG. 6 is a plot of serum IgG antibody titers for each group of example 5 of the present invention;
FIG. 7 is a plot of serum IgG antibody titers for each group of example 6 of the present invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention provides a recombinant rabies vaccine, comprising:
recombinant rabies virus g protein, pharmaceutical excipients and vaccine adjuvants; the vaccine adjuvant comprises QS-21;
wherein, the base sequence of the recombinant rabies virus g protein is shown as SEQ ID NO. 1;
furthermore, the amino acid sequence of the recombinant rabies virus g protein is shown as SEQ ID NO. 2.
The invention adopts recombinant rabies virus g protein with a base sequence of SEQ ID NO.1 (and an amino acid sequence of SEQ ID NO. 2), pharmaceutical excipients and vaccine adjuvants, and the immune experimental study of the BALB/c mice given by intramuscular injection proves that the self-made recombinant rabies vaccine has good immunogenicity.
Further, each milliliter of the recombinant rabies vaccine comprises 50-300 mug of recombinant rabies virus g protein. For example, 50 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, etc. may be used.
Further, the pharmaceutical excipients comprise the following components in each milliliter of the recombinant rabies vaccine:
(1) Polysorbate 80, 100 μg-500 μg; for example, it may be 100. Mu.g, 200. Mu.g, 300. Mu.g, 400. Mu.g, 500. Mu.g, etc.
(2) Sucrose, 20mg-100mg; for example, 20mg, 30mg, 40mg, 50mg, 60mg, 70mg, 800mg, 100mg, etc. are possible.
(3) Sodium chloride, 1mg-9mg. For example, 1mg, 3mg, 5mg, 7mg, 8mg, 9mg, etc. may be mentioned.
Polysorbate 80 (Polysorbate 80) is a nonionic surfactant, also known as Tween 80, described above. It is a mixture of sorbitol esterified with varying amounts of acidified palm oil fatty acids. Polysorbate 80 has good emulsifying, dispersing, stabilizing and surface active properties, and in the pharmaceutical field, polysorbate 80 can be used as an adjuvant to increase the solubility and stability of the drug and promote absorption of the drug. It can be used as emulsifier and dispersant in drug delivery system to help drug mix with body fluid and increase bioavailability.
As mentioned above, sucrose may be replaced with an equivalent amount of trehalose, or a mixture of sucrose and trehalose.
Further, the vaccine adjuvant is a first adjuvant composition or a second adjuvant composition;
both the first adjuvant composition and the second adjuvant composition include:
DOPC, cholesterol and QS-21.
DOPC is an abbreviation for dioleoyl phosphatidylcholine (1, 2-dioleoyl-sn-glycero-3-phosphaline). It is a phospholipid substance and is applied to the biomedical field and the pharmaceutical research. DOPC is a phospholipid whose main component is derived from natural sources and has important physiological functions in cell membranes. It belongs to one of the main components in phospholipid bilayer, and can regulate the stability, flowability and permeability of cell membrane. As a phospholipid, DOPC consists of two oleic acid groups and one phosphonic acid group with choline. The choline moiety is hydrophilic and can interact with water molecules, while the oleic acid group is hydrophobic. This enables the DOPC molecules to self-assemble in aqueous environments in the form of bilayer phospholipid structures, forming the basic building blocks of cell membranes.
The QS-21 is a saponin compound and has various biological activities and pharmacological actions. It is mainly used as an immunological adjuvant, and can enhance the immunogenicity of vaccines and promote immune responses. In the present invention, QS-21 is used together with an antigen, which can enhance the immunogenicity of the antigen, activate the immune system, and increase the immune response to the vaccine in vivo.
The invention adopts recombinant rabies virus g protein with the sequence of SEQ ID NO.1 and vaccine adjuvant. Wherein QS-21 employed in the vaccine adjuvant system is more single in component and structurally defined than saponins, and can produce antibodies and cellular immunity with high levels of specificity as an adjuvant; in addition, neutral liposome (DOPC+cholesterol) is added into the adjuvant, and after QS-21 is combined with cholesterol in the neutral liposome, the combination of the neutral liposome and a cell membrane is prevented, so that the hemolytic activity of the neutral liposome can be further reduced, and the safety of the vaccine is improved.
Further, the first adjuvant composition comprises the following components in amounts per milliliter of the recombinant rabies vaccine:
(1) DOPC,700 μg-3000 μg; for example, 700 μg, 800 μg, 1000 μg, 1500 μg, 2000 μg, 2500 μg, 3000 μg, etc. can be used;
(2) Cholesterol, 175 μg-750 μg; for example, it may be 175 μg, 200 μg, 250 μg, 300 μg, 350 μg, 450 μg, 550 μg, 650 μg, 750 μg, etc.;
(3) QS-21, 35 μg-120 μg. For example, 35 μg, 45 μg, 50 μg, 60 μg, 70 μg, 100 μg, 110 μg, 120 μg, etc. may be used.
Further, DOTAP and Poly I: C are included in the second adjuvant composition in addition to DOPC, cholesterol and QS-21.
Further, the second adjuvant composition comprises the following components in amounts per milliliter of the recombinant rabies vaccine:
(1) DOPC,500 μg-2000 μg; for example, 500 μg, 600 μg, 800 μg, 1000 μg, 1200 μg, 1500 μg, 1800 μg, 2000 μg, etc. may be used.
(2) Cholesterol, 175 μg-600 μg; for example, 175 μg, 200 μg, 250 μg, 300 μg, 350 μg, 450 μg, 550 μg, 600 μg, etc. may be used;
(3) QS-21, 50 μg-120 μg; for example, 50 μg, 60 μg, 70 μg, 100 μg, 110 μg, 120 μg, etc. may be used.
(4) DOTAP,70 μg-240 μg; for example, it may be 70 μg, 90 μg, 100 μg, 120 μg, 150 μg, 200 μg, 220 μg, 240 μg, etc.;
(5) Poly I: C,400 μg-900 μg. For example, it may be 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, etc.
The DOTAP is referred to as 1, 2-dioleoyl-3-trimethylammoniopropane (1, 2-dioleoyl-3-trimethylammonium chloride). This is a synthetic cationic surfactant.
The polyinosinic-polyinosinic (PolyI: C) is an artificially synthesized double-stranded ribonucleic acid, is an interferon inducer, has antiviral and immunoregulatory functions, and is used for treating chronic hepatitis B, epidemic hemorrhagic fever, epidemic encephalitis B, viral keratitis, herpes zoster, various warts, respiratory tract infections and the like.
In addition, the invention also provides a preparation method of the recombinant rabies vaccine, which comprises the following steps:
s1, sequentially carrying out expression treatment and purification treatment on the recombinant expression plasmid to obtain recombinant rabies virus g protein;
s2, taking a vaccine adjuvant, adding the stock solution of the recombinant rabies virus g protein, adjusting the pH value to 6.0-7.0, and subpackaging after using water for injection to fix the volume to obtain the recombinant rabies vaccine.
Further, the expression processing includes:
s11, transforming the recombinant expression plasmid into DH5 alpha chemically competent cells, and selecting a monoclonal for sequencing to obtain a monoclonal strain transformed by genetic engineering;
s12, carrying out plasmid purification after amplifying and culturing the monoclonal strain to obtain transfection-grade plasmid;
s13, packaging by PEI, transfecting the transfection-grade plasmid into CHO cells, carrying out recombinant protein expression, and collecting an expression supernatant.
In step S11, the expression plasmid containing the recombinant DNA is first chemically transformed into competent E.coli DH 5. Alpha. Cells to cause intracellular recombination. Then total DNA is extracted from single clone and sequenced to ensure successful insertion of target gene.
Among them, DH 5. Alpha. Is a chemically competent cell whose plasmid can be transformed by exogenous DNA for use in genetic engineering and molecular biology experiments.
In step S12, the obtained monoclonal strain is cultured and amplified. Then, the original strain is cracked by using a plasmid extraction kit and other methods, plasmids are separated, impurities are removed, and the target transfection-grade plasmids are purified from the plasmids.
In step S13, the purified target transfection-grade plasmid is coated with a chemical reagent such as Polyethylenimine (PEI), and then co-cultured with CHO cells. The PEI can assist the combination of plasmid and cell membrane and uptake by cell, thus achieving transfection purpose. Then, the target protein is expressed and secreted into the culture supernatant by cell culture, induction and other methods. The supernatant is finally collected to obtain the expressed recombinant protein.
Among them, CHO cells are mammalian cells used for recombinant protein expression, have efficient translation and glycosylation systems, and can ensure the production of modifications and activities similar to those of natural proteins. PEI is a high molecular cationic polymer with good DNA or RNA wrapping capability, and can be used for transfection of intracellular plasmids.
The purification treatment comprises:
s14, carrying out ultrafiltration concentration on the expression supernatant to obtain concentrated expression supernatant;
s15, carrying out affinity chromatography on the concentrated expression supernatant, and collecting eluent to obtain the stock solution of the recombinant rabies virus g protein.
Ultrafiltration concentration is a method of separating and concentrating macromolecular substances in a solution as described above. In step S14, the expressed supernatant is passed through a membrane with a small pore size by ultrafiltration membrane technology, so that water and small molecular substances therein are lost through the membrane, while the recombinant protein with a large molecule is retained on the membrane, and a concentrated supernatant is obtained. Thus, excessive moisture and impurities can be removed, and the concentration of the target protein can be increased.
The affinity chromatography is a separation and purification technique based on affinity interaction. In step S15, a ligand having specific affinity (e.g., affinity resin) is used to bind the target protein, and then non-specifically bound impurity substances are eluted, to finally obtain the target protein. Thus, purification of recombinant rabies virus g protein can be achieved.
Ultrafiltration concentration and affinity chromatography are the processing steps in protein expression and purification techniques. The concentration of the target protein can be improved by ultrafiltration concentration, and the high-efficiency purification of the target protein can be realized by affinity chromatography. The reasons for selection of these steps and treatments include increasing the purity of the target protein, removing other impurities, and obtaining a sufficient amount of the desired protein for subsequent experimentation or use.
Further, the preparation method of the recombinant rabies vaccine further comprises the following steps:
preparing the vaccine adjuvant; the vaccine adjuvant is any one of a first adjuvant composition and a second adjuvant composition;
wherein, when the vaccine adjuvant is the first adjuvant composition, the preparing obtains the vaccine adjuvant, including:
(1) Preparing neutral liposome by DOPC and cholesterol;
(2) Mixing phosphate buffer solution and neutral liposome, and then adding QS-21 and pharmaceutical excipients for mixing to obtain the first adjuvant composition;
when the vaccine adjuvant is the second adjuvant composition, the preparing obtains the vaccine adjuvant, including:
(1) Neutral liposome is prepared by DOPC and cholesterol; and, preparing cationic liposome by utilizing DOTAP, DOPC and cholesterol;
(2) And mixing QS-21, poly I, C, cationic liposome, neutral liposome and pharmaceutical excipients to obtain the second adjuvant composition.
The preparation method of the neutral liposome can comprise the following steps: preparing dioleoyl phosphatidylcholine (DOPC) and cholesterol according to a mass ratio of 4:1, uniformly dispersing lipid components, and preparing liposome by using an ethanol injection method, a film dispersion method, an ultrasonic dispersion method and a reverse evaporation method;
preferably, the preparation is carried out by using an ethanol injection method, and the preparation method comprises the following steps: and (3) producing colostrum, sequentially using extrusion films with the pore diameters of 200nm and 100nm for extrusion molding, and then performing ultrafiltration, filtration and sterilization.
More preferably, the neutral liposome is prepared by the following steps:
(1) Dissolving two lipid components (DOPC and cholesterol) in the first adjuvant composition in ethanol, and shearing with water phase for 1-3 times;
(2) Extruding 200nm liposome, filtering with 200 nm-pore filter membrane, circularly extruding for multiple times, and controlling particle size uniformity;
(3) Extruding 100nm liposome, filtering with a filter membrane with the aperture of 100nm, circularly extruding for multiple times, and controlling the uniformity of particle size;
(4) Ultrafiltration and replacement are carried out by using phosphate buffer solution, the concentration multiple is 2-6 times, and the washing and filtering multiple is 6-8 times;
(5) The bacteria were filtered off using a sterile filter.
The physical and chemical parameters of the prepared neutral liposome are as follows:
(1) Average particle diameter (D50): 80nm-140nm; for example, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, etc.;
(2) Solution pH: 5.5-7.0; for example, it may be 5.5, 6.0, 6.5, 7.0, etc.; preferably 6.4 to 6.6; for example, 6.4, 6.5, 6.6, etc.
The preparation method of the cationic liposome comprises the following steps:
is prepared from 1,2 dioleoyl 3 trimethyl ammonium propane chloride (DOTAP), dioleoyl phosphatidylcholine (DOPC) and cholesterol according to a mass ratio of 2:2:1; after the three lipid components are uniformly dispersed, preparing the liposome by using an ethanol injection method, a film dispersion method, an ultrasonic dispersion method and a reverse evaporation method.
Preferably, the preparation is carried out by using an ethanol injection method, and the preparation method comprises the following steps: sequentially using extrusion films with the pore diameters of 200nm and 100nm for extrusion molding after ultrafiltration, and finally filtering and sterilizing by a sterile filter.
More preferably, the cationic liposome is prepared by the following steps:
(1) Dissolving the three lipid components (DOTAP, DOPC and cholesterol) in the second adjuvant composition in ethanol, and shearing with the aqueous phase for 1-3 times;
(2) Ultrafiltration replacement is carried out by using sucrose acetate buffer solution, the concentration multiple is 2-4 times, and the washing filtration multiple is 6-8 times;
(3) Extruding 200nm liposome, extruding with 200nm pore size filter membrane, circularly extruding for several times, and controlling particle size uniformity;
(4) Extruding 100nm liposome, extruding with a filter membrane with the aperture of 100nm, circularly extruding for a plurality of times, and controlling the uniformity of the particle size;
(5) Filtering and sterilizing by using a sterile filter;
the physical and chemical parameters of the prepared cationic liposome are as follows:
(1) Average particle diameter (D50): 40nm-100nm; for example, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, etc.;
(2) Solution pH: 4.5-6.5; for example, it may be 4.5, 5.0, 5.5, 6.0, 6.5, etc.;
(3) Zeta potential: 50mV-90mV (pH 6.5). For example, 50mV, 60mV, 70mV, 80mV, 90mV, etc.
The recombinant rabies vaccine is prepared by carrying out purification after transfection of CHO cells with recombinant plasmid containing SEQ ID NO.1 sequence for expression to obtain recombinant rabies virus g protein stock solution, and then adding a vaccine adjuvant. The immunization experiment study of the BALB/c mice by intramuscular injection proves that the self-made recombinant rabies vaccine has good immunogenicity.
In addition, the invention also provides application of the recombinant rabies vaccine in preparing a product for treating and/or preventing diseases caused by rabies viruses, wherein the product comprises a vaccine product, a medicament, a diagnostic reagent and a detection kit.
The invention is further illustrated by the following specific examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
Example 1: expression treatment in the preparation of recombinant rabies virus g protein.
The recombinant rabies virus g protein preparation of this example was subjected to expression treatment for recombinant expression plasmids.
(1) The recombinant expression plasmid with correct sequencing was transformed into DH 5. Alpha. Chemically competent cells. Uniformly coating a genetic engineering strain on a prepared LB plate containing 50 mug/mL Kan by a coating rod, inverting the coating rod in a 37 ℃ incubator, culturing overnight for 12-16 hours, picking a single colony, inoculating the colony into a triangular flask containing 10mL LB culture solution containing 50 mug/mL Kan, and shaking at 220rpm at 37 ℃ overnight;
(2) The next day was inoculated at 0.1% into 1000mL LB medium containing 50. Mu.g/mL Kan, and shaken at 37℃for 220rpm overnight. The culture was removed, centrifuged at 10000g for 15 minutes, and the supernatant was discarded. According toPlasmid Giga Kit instructions were run and plasmids were purified to give transfection-grade plasmids.
(3) The day before transfection, the CHO cells recovered in advance and stably passaged were adjusted to a density of 3.5E6/mL, a volume of 650 mL/bottle, and shaking culture at 37℃overnight.
(4) In the transfection, 55 mug of plasmid 250 mug of PEI is used per 25mL of transfection volume, the PEI solution and the DNA solution are prepared into an equal volume dilution solution by taking Opti-MEM as the dilution solution to prepare a mixed preparation, and the final volume of the transfection reagent accounts for 1% of the total volume of the transfection. An equal volume of PEI solution should be slowly added to the plasmid DNA solution, not reversible, not shaking vigorously to generate bubbles, and incubated for 15 minutes after mixing to allow for sufficient reaction. The prepared transfection reagent was added dropwise to the cell culture solution while shaking, and incubated at 34℃and 80rpm with 5% carbon dioxide.
(5) The first day and the third day after transfection are supplemented with 8% of culture volume, and the culture can be carried out for 5-6 days for harvesting, and supernatant is obtained by centrifugation. Obtaining the cell fermentation liquid containing the recombinant rabies virus g protein.
Example 2: purifying and treating recombinant rabies virus g protein.
In the preparation of the recombinant rabies virus g protein of this example, a protein purification treatment was performed.
(1) By 30kD 0.1m 2 And (3) packaging an ultrafiltration membrane, and concentrating 2200mL of recombinant rabies virus g protein CHO cell expression supernatant by ultrafiltration to 450mL.
(2) Applying 70mL of concentrated recombinant rabies virus g protein CHO cell expression supernatant to an equilibrated affinity chromatography column (Strep Trap XT) at a flow rate of 1 mL/min by using an SCG protein purification system;
(3) With equilibration solution (100 mM Tris-Cl+150mM NaCl+1mM EDTA-Na 2 pH 8.0) the column was washed at a flow rate of 1 mL/min until the effluent OD280 reached baseline. 70mL of the concentrated recombinant rabies virus g protein CHO cell expression supernatant is loaded to an affinity chromatography column at a flow rate of 1 mL/min.
(4) The column was washed with equilibration solution at a flow rate of 1 mL/min until the effluent OD280 reached baseline.
(5) With eluent (100 mM Tris-Cl+150mM NaCl+1mM EDTA-Na 2 +60mM Biotin,pH8.0) eluting the target protein at a flow rate of 1 mL/min, collecting the effluent, and performing 4-20% SDS-PAGE analysis.
Referring to fig. 1 and 2, the recombinant rabies virus g protein with higher purity is obtained after purification by an affinity chromatography column (Strep Trap XT).
Example 3: preparation of recombinant rabies vaccine.
In this example, the preparation of recombinant rabies vaccine was carried out based on examples 1 and 2.
(1) Neutral liposome is prepared.
1) Dissolving two lipid components (DOPC and cholesterol) in ethanol, and shearing with water phase for 1-3 times;
2) Extruding 200nm liposome, filtering with 200 nm-pore filter membrane, circularly extruding for multiple times, and controlling particle size uniformity;
3) Extruding 100nm liposome, filtering with a filter membrane with the aperture of 100nm, circularly extruding for multiple times, and controlling the uniformity of particle size;
4) Ultrafiltration and replacement are carried out by using phosphate buffer solution, the concentration multiple is 2-6 times, and the washing and filtering multiple is 6-8 times;
5) The bacteria were filtered off using a sterile filter.
The physical and chemical parameters of the prepared neutral liposome are as follows:
1) Average particle diameter (D50): 80nm-140nm;
2) Solution pH: 6.4-6.6.
(2) Sequentially adding phosphate buffer solution (prepared from monosodium phosphate monohydrate and disodium phosphate), neutral liposome, QS-21 solution and recombinant rabies virus g protein stock solution, adjusting pH to 6.0-7.0, fixing volume with injectable water, filtering for sterilization, and packaging to obtain recombinant rabies vaccine. The contents of the specific components are shown in the following table:
table 1, amounts of the components of the recombinant rabies vaccine employed in example 3
Example 4: and (5) inspecting the dosage of the adjuvant and auxiliary materials.
(1) Changes in physicochemical properties before and after QS-21 addition: according to the first adjuvant composition formulation procedure, three batches of neutral liposomes were used to separately incorporate QS-21 and the particle size distribution and Zeta potential change before and after QS-21 incorporation was measured and the results are shown in the following table.
According to the first adjuvant composition formulation procedure, three batches of neutral liposomes were used to separately incorporate QS-21 and the particle size distribution and Zeta potential change before and after QS-21 incorporation was measured and the results are shown in the following table.
TABLE 2 particle size distribution and Zeta potential tables before and after QS-21 addition during formulation of the first adjuvant composition
As can be seen from the above table, the average particle size and Zeta potential of the first adjuvant composition did not change significantly after QS-21 addition. It is shown that the addition of QS-21 has no obvious effect on the particle size distribution and Zeta potential of the system.
(2) Variation of hemolytic Activity of QS-21 examination: in this example, 50. Mu.g, 100. Mu.g, 200. Mu.g, 300. Mu.g and 400. Mu.g QS-21 were adsorbed using neutral liposomes (cholesterol content 250. Mu.g) respectively, with 50. Mu.g QS-21 as a control. The samples are respectively and continuously diluted by 2 times, chicken red blood cells are added for incubation for 30 minutes at room temperature, the OD value of the supernatant is measured at 570nm by an ultraviolet spectrophotometer, and the higher the OD value is, the stronger the hemolysis is.
Experimental results: as shown in the data in table 3 below.
TABLE 3 Experimental Table of hemolytic Activity of different samples
Analysis of experimental results: referring to FIG. 3, when the QS-21 content was not higher than 200. Mu.g, the OD value of the "QS-21+ neutral liposomes" group was not significantly different from that of the blank control. It was demonstrated that neutral liposomes containing 250. Mu.g cholesterol adsorbed at least 200. Mu.g QS-21 without causing significant hemolysis.
(3) Adjuvant system pH range: samples of different pH values were placed at 2℃to 8℃and 37℃for 1 week and the contents of the components of the adjuvants were determined, and the results are shown in the following table.
TABLE 4 content of each component of adjuvant fraction at 2-8℃and 37℃under different pH conditions for 1 week
As can be seen from the above table and the graphs of FIG. 4 and FIG. 5, DOPC and cholesterol have no obvious change when placed at 2-8deg.C within the pH range of 6.0-7.5, and the content of DOPC and cholesterol has a certain decrease when placed at 37deg.C for 1 week, and the decrease amplitude of samples with different pH values has no obvious difference; considering that the sample QS-21 content at pH7.5 is most significantly reduced and QS-21 is easily hydrolyzed in an alkaline environment, the pH of the preparation should be not higher than 7.0.
Example 5: recombinant rabies vaccine adjuvant screening experiments.
(1) Immunization of BALB/c mice: experimental grouping: SPF-class BALB/c mice were randomly divided into 11 groups, and except for 8 groups of rabies vaccine, antigen control group and negative control for freeze-dried human, the remaining 8 groups were 12 groups, and the total number of animals was 120, and the detailed group is shown in the following table.
Table 5, mice grouping and immunization protocol
Immunization mode: the test samples were gently shaken and homogenized before use, and 50. Mu.L of the test sample solution was extracted from each test sample (100. Mu.L of the test sample solution was extracted from the rabies vaccine group for freeze-dried human) using a 300. Mu.L disposable sterile syringe and needle, and intramuscular injection was performed outside the thighs of the mice.
Immunization time: d0 D7 was administered 2 times in total.
Serum collection: the experimental animals of each experimental group are collected at D1, D3, D6 and D10, serum is separated, and the serum is frozen at-70 ℃ for standby.
(2) ELISA detection of specific antibodies in antisera: the antigen was diluted to 2. Mu.g/mL with coating solution, 100. Mu.L/well coated ELISA plate, overnight at 4 ℃. After 3 washes with wash solution, 200. Mu.L of blocking solution was added to each well and blocked at 25℃for 1 hour. After blocking, the blocking solution was removed, the mouse antisera was diluted in a double ratio and the mouse negative serum was added to ELISA plates, 100. Mu.L/well, and incubated at 25℃for 2 hours. After 3 washes with wash solution, HRP-labeled goat anti-mouse IgG (1:10000) was added, 100. Mu.L/well, and incubated at 25℃for 1 hour. After washing 5 times with the washing solution, TMB substrate developing solution was added, the reaction was stopped by light-shielding at 25℃for 15 minutes and 100. Mu.L of stop solution was added. The OD450 values of each well were determined with a microplate reader. The dilution corresponding to wells with OD values greater than 2.1 times the OD value of the set negative control was determined as the titer of the sample.
(3) Experimental results: the bound IgG antibody titer was detected by ELISA and the antibody curve of the detection results is shown in fig. 6.
1) After 6 days of immunization (D6), serum IgG antibody titers were increased in each group of experimental animals.
The "antigen+first adjuvant composition" and "antigen+second adjuvant composition" experimental groups induced IgG antibody titers that were both higher than the remaining experimental groups.
2) Serum IgG antibody titers were significantly higher for each group of experimental animals than for the first immunization 3 days (D10) after the second immunization.
The "antigen+first adjuvant composition" and "antigen+second adjuvant composition" experimental groups induced IgG antibody titers that were both higher than the remaining experimental groups.
Taken together, it is clear that the vaccine formulation based on the first and second adjuvant compositions prepared by the method of the present invention, with the addition of adjuvants, can rapidly induce a good specific immune response in BALB/c mice.
Example 6: immunogenicity investigation of recombinant rabies vaccine.
(1) Immunization of BALB/c mice: experimental grouping: SPF-class BALB/c mice were randomly divided into 3 groups, which were a self-made vaccine immunization group (vaccine preparation prepared by using example 3), a commercial vaccine immunization group [ a commercial lyophilized human rabies vaccine (Vero cells) ], and a physiological saline immunization group, respectively. A total of 30 BALB/c mice per group, grouped in detail as follows. Wherein, the vaccine immunization group on the market adopts freeze-dried human rabies vaccine (Vero cells) based on the traditional inactivation technology.
Table 6, mice grouping and immunization protocol
| Experimental group | Quantity of | Injection mode | Number of injections | Injection dosage |
| Homemade vaccine | 10 | Intramuscular injection | 2 | 0.1 dose |
| Commercially available vaccine | 10 | Intramuscular injection | 2 | 0.1 dose |
| Physiological saline | 10 | Intramuscular injection | 2 | 0.1 dose |
Immunization mode: the test sample was gently shaken before use, 100. Mu.L of human rabies vaccine (Vero cells) (commercially available vaccine) was lyophilized using a 300. Mu.L disposable sterile syringe and needle, and 50. Mu.L of the remaining test sample was withdrawn, and intramuscular injection was performed on the outside of the thigh of the mouse.
Immunization time: the first immunization (D0) was added 7 days apart, totaling two immunizations.
Serum collection: the experimental animals of each experimental group were collected for D3, D6, D10, D15 and D20 days before the immunization (D-1) and after the immunization, and serum was isolated and frozen at-70℃for use.
(2) ELISA detection of specific antibodies in antisera: the antigen was diluted to 2. Mu.g/mL with coating solution, 100. Mu.L/well coated ELISA plate, overnight at 4 ℃. After 3 washes with wash solution, 200. Mu.L of blocking solution was added to each well and blocked at 25℃for 1 hour. After blocking, the blocking solution was removed, the mouse antisera was diluted in a double ratio and the mouse negative serum was added to ELISA plates, 100. Mu.L/well, and incubated at 25℃for 2 hours. After 3 washes with wash solution, HRP-labeled goat anti-mouse IgG (1:10000) was added, 100. Mu.L/well, and incubated at 25℃for 1 hour. After washing 5 times with the washing solution, TMB substrate developing solution was added, the reaction was stopped by light-shielding at 25℃for 15 minutes and 100. Mu.L of stop solution was added. The OD450 values of each well were determined with a microplate reader. The dilution corresponding to wells with OD values greater than 2.1 times the OD value of the set negative control was determined as the titer of the sample.
(3) Results: the bound IgG antibody titer was detected by ELISA and the antibody curve of the detection results is shown in fig. 7. After one immunization, the titer of IgG antibodies in the serum of animals immunized by the self-made vaccine is higher than that of animals immunized by the commercial vaccine, and the titer of IgG antibodies in the serum of animals immunized by the self-made vaccine is higher than that of negative control animals. After the second immunization, the titer of IgG antibodies in serum of each group of experimental animals is obviously higher than that of the first immunization. At day D15, igG antibody titers peaked in the serum of each group of experimental animals. At day D20, igG antibody titers were kept stable in sera of animals of the self-made vaccine immunization group, and IgG antibody titers were reduced in sera of animals of the remaining groups.
In summary, self-made and commercial vaccines produced good specific immune responses in BALB/c mice, and the immunogenicity induced by self-made vaccines was superior to that of commercial vaccines.
While the foregoing is directed to the preferred embodiments and examples of the present invention, it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the inventive concepts, including but not limited to, adjustments in the ratio, flow, amount and reaction vessel, such as the use of a continuous flow reactor, which are within the scope of the present invention. While the preferred embodiments and examples of the present invention have been described, it should be noted that those skilled in the art may make various modifications and improvements without departing from the inventive concept, including but not limited to, adjustments of proportions, procedures, and amounts, which fall within the scope of the present invention.
Claims (11)
1. A recombinant rabies vaccine comprising:
recombinant rabies virus g protein, pharmaceutical excipients and vaccine adjuvants;
wherein, the base sequence of the recombinant rabies virus g protein is shown as SEQ ID NO. 1;
preferably, the amino acid sequence of the recombinant rabies virus g protein is shown as SEQ ID NO. 2.
2. The recombinant rabies vaccine according to claim 1, characterized in that it comprises 50 μg-300 μg of recombinant rabies virus g protein per milliliter of said recombinant rabies vaccine.
3. The recombinant rabies vaccine according to claim 1, wherein the pharmaceutical excipients comprise the following components per milliliter of the recombinant rabies vaccine:
polysorbate 80, 100 μg-500 μg;
sucrose, 20mg-100mg;
sodium chloride, 1mg-9mg.
4. The recombinant rabies vaccine of claim 1, wherein the vaccine adjuvant is a first adjuvant composition or a second adjuvant composition;
both the first adjuvant composition and the second adjuvant composition include: DOPC, cholesterol and QS-21.
5. The recombinant rabies vaccine according to claim 4, wherein the first adjuvant composition comprises the following components in amounts per milliliter of the recombinant rabies vaccine:
DOPC，700μg-3000μg；
cholesterol, 175 μg-750 μg;
QS21，35μg-120μg。
6. the recombinant rabies vaccine of claim 4, wherein the second adjuvant composition further comprises DOTAP and Poly I: C in addition to DOPC, cholesterol and QS-21.
7. The recombinant rabies vaccine according to claim 6, wherein the second adjuvant composition comprises the following components in amounts per milliliter of the recombinant rabies vaccine:
DOPC，500μg-2000μg；
cholesterol, 175 μg-600 μg;
QS21，50μg-120μg；
DOTAP，70μg-240μg；
Poly I:C，400μg-900μg。
8. a method of preparing a recombinant rabies vaccine comprising:
the recombinant expression plasmid is subjected to expression treatment and purification treatment in sequence to obtain recombinant rabies virus g protein;
and adding a vaccine adjuvant into the stock solution of the recombinant rabies virus g protein, adjusting the pH value to 6.0-7.0, and subpackaging after using water for injection to fix the volume to obtain the recombinant rabies vaccine.
9. The method of preparing a recombinant rabies vaccine according to claim 8, wherein said expression processing comprises:
transforming the recombinant expression plasmid into DH5 alpha chemically competent cells, and selecting a monoclonal for sequencing to obtain a monoclonal strain transformed by genetic engineering;
amplifying and culturing the monoclonal strain, and then purifying the plasmid to obtain a transfection-grade plasmid;
packaging by PEI, transfecting the transfection-grade plasmid into CHO cells, carrying out recombinant protein expression, and collecting an expression supernatant;
the purification treatment comprises:
carrying out ultrafiltration concentration on the expression supernatant to obtain concentrated expression supernatant;
and carrying out affinity chromatography on the concentrated expression supernatant, and collecting eluent to obtain the stock solution of the recombinant rabies virus g protein.
10. The method of preparing a recombinant rabies vaccine according to claim 8, further comprising:
preparing the vaccine adjuvant; the vaccine adjuvant is any one of a first adjuvant composition and a second adjuvant composition;
wherein, when the vaccine adjuvant is the first adjuvant composition, the preparing obtains the vaccine adjuvant, including:
neutral liposome is prepared by DOPC and cholesterol;
mixing phosphate buffer solution and neutral liposome, and then adding QS-21 and pharmaceutical excipients for mixing to obtain the first adjuvant composition;
when the vaccine adjuvant is the second adjuvant composition, the preparing obtains the vaccine adjuvant, including:
neutral liposome is prepared by DOPC and cholesterol; and, preparing cationic liposome by utilizing DOTAP, DOPC and cholesterol;
and mixing QS-21, poly I, C, cationic liposome, neutral liposome and pharmaceutical excipients to obtain the second adjuvant composition.
11. Use of a recombinant rabies vaccine according to any one of claims 1-7 for the preparation of a product for the treatment and/or prevention of diseases caused by rabies virus, characterized in that said product comprises vaccine products, medicaments, diagnostic reagents and detection kits.
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