A recombinant rabies vaccine and its preparation method and application

Abstract

The present invention provides a recombinant rabies vaccine and a preparation method and application thereof, belonging to the technical field of virus vaccine preparations. The recombinant rabies vaccine comprises: a recombinant rabies virus g protein, a pharmaceutical excipient and a vaccine adjuvant; wherein the base sequence of the recombinant rabies virus g protein is as shown in SEQ ID NO.1; the present invention adopts the recombinant rabies virus g protein, a pharmaceutical excipient and a vaccine adjuvant with a sequence of SEQ ID NO.1, and conducts an immune experiment study on BALB / c mice by intramuscular injection, proving that the homemade recombinant rabies vaccine has good immunogenicity.

Description

Technical Field

[0001] The invention belongs to the technical field of virus vaccine preparations, and in particular relates to a recombinant rabies vaccine and a preparation method and application thereof. Background Art

[0002] Rabies virus (RABV) belongs to the genus Lyssavirus, a rhabdovirus that consists of genetically related viruses with an outer shell and contains single-stranded non-segmented negative-strand RNA. Globally, 60,000 people die from rabies each year, with 95% of cases occurring in Asia and Africa. There is currently no effective treatment, and pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) are the main measures for preventing rabies.

[0003] Rabies vaccines can be used to prevent rabies before and after exposure. They are an effective means of preventing and controlling rabies, and have greatly reduced the incidence of rabies in the past few decades. However, all rabies vaccines currently on the market for human use are inactivated vaccines, which require high concentrations of antigens to achieve immune effects, resulting in expensive rabies vaccines. In addition, 4-5 injections are required for post-exposure prevention, and 2 injections are required when exposed again more than 3 months later. Compliance is poor and immune deficiency is easily caused. In addition, if post-exposure prevention is delayed or incomplete, it will still lead to death. Therefore, there is an urgent need to develop a safe, low-cost, short-course, and long-acting vaccine to enhance compliance and effectively prevent rabies.

[0004] The rabies virus G protein is a trimer and the main antigen that induces the production of virus neutralizing antibodies. It can produce immunity against fatal rabies virus infection and is the main component of the vaccine. Adjuvants can enhance the body's immune response to vaccines and induce cellular immunity or humoral immunity with low doses of antigens to achieve protection requirements. Therefore, compared with inactivated vaccines, recombinant rabies vaccines containing adjuvants can enhance the stimulation of the glycoprotein in RABV to the body, improve the immunogenicity of the vaccine, accelerate the immune response, reduce the required dose and number of vaccinations, and reduce the cost of the vaccine.

[0005] In the application of adjuvants in recombinant vaccine preparations, aluminum adjuvants are commonly used. However, rabies vaccines containing aluminum adjuvants have the defect of delaying the release of antigens, thereby causing low antibody levels in the early stage of immunization, which is not conducive to the early prevention and treatment of rabies after exposure, so they are gradually replaced by the current freeze-dried inactivated vaccines.

[0006] In summary, although the existing rabies vaccine can prevent rabies to a certain extent, it has the following defects: first, the high concentration of antigens in the inactivated vaccine makes the vaccine expensive; second, the vaccine has a large dose and frequency of inoculation, poor compliance and easily leads to immune deficiency; in addition, the traditional aluminum adjuvant will delay the release of antigens, affecting the effectiveness of the vaccine in early prevention and treatment. Therefore, it is necessary to develop a new rabies vaccine containing a new adjuvant that can quickly produce antibodies and produce high levels of antibodies to overcome the defects of the existing vaccine. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a recombinant rabies vaccine, comprising:

[0008] Recombinant rabies virus G protein, pharmaceutical excipients and vaccine adjuvants;

[0009] Wherein, the base sequence of the recombinant rabies virus g protein is shown in SEQ ID NO.1;

[0010] Preferably, the amino acid sequence of the recombinant rabies virus g protein is as shown in SEQ ID NO.2.

[0011] Preferably, each milliliter of the recombinant rabies vaccine contains 50 μg-300 μg of recombinant rabies virus G protein.

[0012] Preferably, the pharmaceutical excipients include the following components per milliliter of the recombinant rabies vaccine:

[0013] Polysorbate 80, 100 μg-500 μg;

[0014] Sucrose, 20mg-100mg;

[0015] Sodium chloride, 1mg-9mg.

[0016] Preferably, the vaccine adjuvant is the first adjuvant composition or the second adjuvant composition;

[0017] The first adjuvant composition and the second adjuvant composition both include: DOPC, cholesterol and QS-21.

[0018] Preferably, the first adjuvant composition comprises the following components per milliliter of the recombinant rabies vaccine:

[0019] DOPC, 700 μg-3000 μg;

[0020] Cholesterol, 175 μg-750 μg;

[0021] QS-21, 35μg-120μg.

[0022] Preferably, the second adjuvant composition comprises DOTAP and Poly I:C in addition to DOPC, cholesterol and QS-21.

[0023] Preferably, the second adjuvant composition comprises the following components per milliliter of the recombinant rabies vaccine:

[0024] DOPC, 500 μg-2000 μg;

[0025] Cholesterol, 175 μg-600 μg;

[0026] QS-21, 50 μg-120 μg;

[0027] DOTAP, 70 μg-240 μg;

[0028] Poly I:C, 400μg-900μg.

[0029] In addition, to solve the above problems, the present invention also provides a method for preparing a recombinant rabies vaccine, comprising:

[0030] The recombinant expression plasmid is sequentially subjected to expression treatment and purification treatment to obtain the recombinant rabies virus G protein;

[0031] Take the vaccine adjuvant, add the stock solution of the recombinant rabies virus G protein, adjust the pH value to 6.0-7.0, use injection water to make up the volume and then package, so as to obtain the recombinant rabies vaccine.

[0032] Preferably, the expression processing comprises:

[0033] The recombinant expression plasmid is transformed into DH5α chemical competent cells, and a single clone is selected for sequencing to obtain a genetically engineered monoclonal strain;

[0034] After the monoclonal strain is amplified and cultured, plasmid purification is performed to obtain a transfection-grade plasmid;

[0035] The transfection-grade plasmid is transfected into CHO cells by PEI packaging to express the recombinant protein, and the expression supernatant is collected;

[0036] The purification treatment comprises: ultrafiltration and concentration of the expression supernatant to obtain a concentrated expression supernatant; affinity chromatography of the concentrated expression supernatant to collect the eluate to obtain a stock solution of the recombinant rabies virus G protein.

[0037] Preferably, the preparation method of the recombinant rabies vaccine also includes: preparing the vaccine adjuvant; the vaccine adjuvant is any one of the first adjuvant composition and the second adjuvant composition; wherein, when the vaccine adjuvant is the first adjuvant composition, the preparation of the vaccine adjuvant includes: preparing neutral liposomes using DOPC and cholesterol; mixing phosphate buffer and neutral liposomes, and then adding QS-21 and pharmaceutical excipients to mix to obtain the first adjuvant composition; when the vaccine adjuvant is the second adjuvant composition, the preparation of the vaccine adjuvant includes: preparing neutral liposomes using DOPC and cholesterol; and preparing cationic liposomes using DOTAP, DOPC and cholesterol; mixing QS-21, Poly I:C, cationic liposomes, neutral liposomes and pharmaceutical excipients to obtain the second adjuvant composition.

[0038] In addition, to solve the above problems, the present invention also provides a use of the recombinant rabies vaccine as described above in the preparation of products for treating and / or preventing diseases caused by rabies virus, wherein the products include vaccine products, drugs, diagnostic reagents and detection kits.

[0039] The present invention provides a recombinant rabies vaccine and a preparation method and application thereof, wherein the recombinant rabies vaccine comprises: a recombinant rabies virus g protein, a pharmaceutical excipient and a vaccine adjuvant; wherein the base sequence of the recombinant rabies virus g protein is shown in SEQ ID NO. 1. The present invention adopts the recombinant rabies virus g protein with a sequence of SEQ ID NO. 1, a pharmaceutical excipient and a vaccine adjuvant, and conducts an immune experiment study on BALB / c mice by intramuscular injection, proving that the homemade recombinant rabies vaccine has good immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the chromatogram of the eluted protein in Example 2 of the present invention;

[0041] Figure 2 This is the SDS-PAGE gel electrophoresis diagram after purification in Example 2 of the present invention;

[0042] Figure 3 Graph showing the hemolytic activity test results of different samples in Example 4 of the present invention;

[0043] Figure 4 This is a graph showing the results of DOPC and cholesterol content at 2°C-8°C and 37°C for one week in Example 4 of the present invention;

[0044] Figure 5 This is a graph showing the results of QS-21 content in Example 4 of the present invention after being placed at 2°C-8°C and at 37°C for one week;

[0045] Figure 6 It is a line graph of serum IgG antibody titers of each group in Example 5 of the present invention;

[0046] Figure 7 It is a line graph of serum IgG antibody titers in each group in Example 6 of the present invention.

[0047] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0048] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] The present invention provides a recombinant rabies vaccine, comprising:

[0050] Recombinant rabies virus g protein, pharmaceutical excipients and vaccine adjuvants; the vaccine adjuvant includes QS-21;

[0051] Wherein, the base sequence of the recombinant rabies virus g protein is shown in SEQ ID NO.1;

[0052] Furthermore, the amino acid sequence of the recombinant rabies virus g protein is shown in SEQ ID NO.2.

[0053] The present invention uses a 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 conducts an immune experiment on BALB / c mice by intramuscular injection, which proves that the homemade recombinant rabies vaccine has good immunogenicity.

[0054] Furthermore, each milliliter of the recombinant rabies vaccine contains 50 μg-300 μg of recombinant rabies virus G protein, for example, 50 μg, 80 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, etc.

[0055] Furthermore, the pharmaceutical excipients include the following components in each milliliter of the recombinant rabies vaccine:

[0056] (1) Polysorbate 80, 100 μg-500 μg; for example, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, etc.

[0057] (2) Sucrose, 20 mg to 100 mg; for example, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 800 mg, 100 mg, etc.

[0058] (3) Sodium chloride, 1 mg to 9 mg. For example, 1 mg, 3 mg, 5 mg, 7 mg, 8 mg, 9 mg, etc.

[0059] As mentioned above, Polysorbate 80 is a nonionic surfactant, also known as Tween80. It is a mixture of sorbitol esterified with different amounts of acidified palm oil fatty acids. Polysorbate 80 has good emulsifying, dispersing, stabilizing and surface active properties. In the pharmaceutical field, polysorbate 80 can be used as an adjuvant to improve the solubility and stability of drugs and promote drug absorption. It can be used as an emulsifier and dispersant in drug delivery systems to help drugs mix with body fluids and increase their bioavailability.

[0060] As mentioned above, sucrose can be replaced by an equal amount of trehalose, or a mixture of sucrose and trehalose.

[0061] Further, the vaccine adjuvant is the first adjuvant composition or the second adjuvant composition;

[0062] The first adjuvant composition and the second adjuvant composition both include:

[0063] DOPC, cholesterol, and QS-21.

[0064] As mentioned above, DOPC is the abbreviation of dioleoylphosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine). It is a phospholipid substance used in the biomedical field and drug research. DOPC is a phospholipid whose main component comes from natural sources and has important physiological functions in cell membranes. It is one of the main components in the phospholipid bilayer and can regulate the stability, fluidity and permeability of the cell membrane. As a phospholipid substance, DOPC is composed of two oleic acid groups and one phosphate group with choline. The choline part is hydrophilic and can interact with water molecules, while the oleic acid group is hydrophobic. This allows DOPC molecules to self-assemble in the form of a bilayer phospholipid structure in an aqueous environment to form the basic structural unit of the cell membrane.

[0065] As mentioned above, QS-21 is a saponin compound with multiple biological activities and pharmacological effects. It is mainly used as an immune adjuvant, which can enhance the immunogenicity of vaccines and promote immune response. In the present invention, QS-21 is used together with antigens to improve the immunogenicity of antigens, activate the immune system, and increase the immune response to vaccines in vivo.

[0066] The present invention uses a recombinant rabies virus g protein with a sequence of SEQ ID NO.1 and a vaccine adjuvant. Compared with saponin, QS-21 used in the vaccine adjuvant system has a simpler composition and a defined structure, and can produce high-level specific antibodies and cellular immunity as an adjuvant; in addition, neutral liposomes (DOPC+cholesterol) are added to the adjuvant, and after QS-21 combines with cholesterol in the neutral liposome, it prevents it from combining with the cell membrane, which can further reduce its hemolytic activity and improve the safety of the vaccine.

[0067] Furthermore, the first adjuvant composition comprises the following components per milliliter of the recombinant rabies vaccine:

[0068] (1) DOPC, 700 μg-3000 μg; for example, 700 μg, 800 μg, 1000 μg, 1500 μg, 2000 μg, 2500 μg, 3000 μg, etc.;

[0069] (2) Cholesterol, 175 μg-750 μg; for example, 175 μg, 200 μg, 250 μg, 300 μg, 350 μg, 450 μg, 550 μg, 650 μg, 750 μg, etc.;

[0070] (3) QS-21, 35 μg-120 μg. For example, it can be 35 μg, 45 μg, 50 μg, 60 μg, 70 μg, 100 μg, 110 μg, 120 μg, etc.

[0071] Furthermore, the second adjuvant composition includes DOTAP and Poly I:C in addition to DOPC, cholesterol and QS-21.

[0072] Furthermore, the second adjuvant composition comprises the following components per milliliter of the recombinant rabies vaccine:

[0073] (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.

[0074] (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.;

[0075] (3) QS-21, 50 μg-120 μg; for example, 50 μg, 60 μg, 70 μg, 100 μg, 110 μg, 120 μg, etc.

[0076] (4) DOTAP, 70 μg-240 μg; for example, 70 μg, 90 μg, 100 μg, 120 μg, 150 μg, 200 μg, 220 μg, 240 μg, etc.;

[0077] (5) Poly I:C, 400 μg-900 μg. For example, it can be 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, etc.

[0078] As mentioned above, the full name of DOTAP is 1,2-dioleoyl-3-trimethylammonium-propane. It is a synthetic cationic surfactant.

[0079] As mentioned above, poly I:C is a synthetic double-stranded RNA and an interferon inducer with antiviral and immunomodulatory functions. It is used for chronic hepatitis B, hemorrhagic fever, epidemic encephalitis B, viral keratitis, herpes zoster, various warts and respiratory tract infections.

[0080] In addition, the present invention also provides a method for preparing a recombinant rabies vaccine, comprising:

[0081] S1, sequentially subjecting the recombinant expression plasmid to expression treatment and purification treatment to obtain the recombinant rabies virus G protein;

[0082] 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 diluting the volume with injection water and then packaging to obtain the recombinant rabies vaccine.

[0083] Furthermore, the expression processing includes:

[0084] S11, taking the recombinant expression plasmid and transforming it into DH5α chemical competent cells, and selecting a single clone for sequencing to obtain a genetically engineered transformed single clone strain;

[0085] S12, amplifying and culturing the monoclonal strain, and then purifying the plasmid to obtain a transfection-grade plasmid;

[0086] S13, using PEI to encapsulate and transfect the transfection-grade plasmid into CHO cells to express the recombinant protein, and collecting the expression supernatant.

[0087] In the above step S11, the expression plasmid containing the recombinant DNA is first transformed into the competent cells of E. coli DH5α by chemical methods to allow recombination to occur in the cells. Then, the total DNA is extracted from the single clone and sequenced to ensure successful insertion of the target gene.

[0088] Among them, DH5α is a chemically competent cell whose plasmid can be transformed with exogenous DNA and used in genetic engineering and molecular biology experiments.

[0089] In step S12, the obtained monoclonal strain is cultured and amplified, and then the original strain is lysed using a plasmid extraction kit or other methods to separate the plasmid, remove impurities, and purify the target transfection-grade plasmid.

[0090] In step S13, the purified target transfection-grade plasmid is encapsulated with chemical reagents such as polyethyleneimine (PEI), and then co-cultured with CHO cells. PEI helps the plasmid bind to the cell membrane and be taken up by the cell, thereby achieving the purpose of transfection. Then, through cell culture and induction methods, the target protein is expressed and secreted into the culture supernatant. Finally, the supernatant is collected to obtain the expressed recombinant protein.

[0091] Among them, CHO cells are a type of mammalian cell used for recombinant protein expression, with an efficient translation and glycosylation system that can ensure the production of modifications and activities similar to natural proteins. PEI is a high molecular cationic polymer with good DNA or RNA encapsulation ability and can be used for intracellular plasmid transfection.

[0092] The purification process comprises:

[0093] S14, ultrafiltration and concentration of the expression supernatant to obtain a concentrated expression supernatant;

[0094] S15, performing affinity chromatography on the concentrated expression supernatant, collecting the eluate, and obtaining the stock solution of the recombinant rabies virus G protein.

[0095] As mentioned above, ultrafiltration concentration is a method for separating and concentrating macromolecular substances in a solution. In step S14, the expression supernatant is passed through a membrane with a smaller pore size using ultrafiltration membrane technology, so that the water and small molecules therein pass through the membrane and are lost, while the larger molecular recombinant protein is retained on the membrane, and a concentrated supernatant is obtained. In this way, excess water and impurities can be removed and the concentration of the target protein can be increased.

[0096] As mentioned above, affinity chromatography is a separation and purification technology based on affinity interaction. In step S15, a ligand with specific affinity (such as affinity resin) is used to bind to the target protein, and then non-specifically bound impurities are eluted to finally obtain the target protein. In this way, the recombinant rabies virus g protein can be purified.

[0097] Ultrafiltration concentration and affinity chromatography are processing steps in protein expression and purification technology. Ultrafiltration concentration can increase the concentration of the target protein, while affinity chromatography can achieve efficient purification of the target protein. Reasons for choosing these steps and processing methods include improving the purity of the target protein, removing other impurities, and obtaining sufficient amounts of the desired protein for subsequent experiments or applications.

[0098] Furthermore, the method for preparing the recombinant rabies vaccine also includes:

[0099] The vaccine adjuvant is prepared; the vaccine adjuvant is any one of the first adjuvant composition and the second adjuvant composition;

[0100] Wherein, when the vaccine adjuvant is the first adjuvant composition, the preparation of the vaccine adjuvant comprises:

[0101] (1) Neutral liposomes were prepared using DOPC and cholesterol;

[0102] (2) After mixing the phosphate buffer and the neutral liposome, QS-21 and the pharmaceutical excipients are added and mixed to obtain the first adjuvant composition;

[0103] When the vaccine adjuvant is the second adjuvant composition, the preparation of the vaccine adjuvant comprises:

[0104] (1) preparing neutral liposomes using DOPC and cholesterol; and preparing cationic liposomes using DOTAP, DOPC and cholesterol;

[0105] (2) QS-21, Poly I:C, cationic liposomes, neutral liposomes and pharmaceutical excipients are mixed to obtain the second adjuvant composition.

[0106] The preparation method of the neutral liposome may include: preparing dioleoylphosphatidylcholine (DOPC) and cholesterol in a mass ratio of 4:1, dispersing the lipid components uniformly, and preparing the liposome using an ethanol injection method, a thin film dispersion method, an ultrasonic dispersion method, or a reverse evaporation method;

[0107] Preferably, the ethanol injection method is used for preparation, and the preparation method is: according to the production of colostrum, extrusion molding is carried out using extrusion membranes with pore sizes of 200nm and 100nm in sequence, followed by ultrafiltration and filtration sterilization.

[0108] More preferably, the preparation steps of the neutral liposome are as follows:

[0109] (1) dissolving the two lipid components (DOPC and cholesterol) in the first adjuvant composition in ethanol and shearing them online with the aqueous phase for 1 to 3 times;

[0110] (2) 200 nm liposome extrusion, using a filter membrane with a pore size of 200 nm for filtration, and multiple cycles of extrusion to control the uniformity of particle size;

[0111] (3) 100 nm liposome extrusion, using a filter membrane with a pore size of 100 nm for filtration, and multiple cycles of extrusion to control the uniformity of particle size;

[0112] (4) Use phosphate buffer ultrafiltration replacement, concentration multiples 2-6 times, diafiltration multiples 6-8 times;

[0113] (5) Sterilize by filtering using a sterile filter.

[0114] The physicochemical parameters of the prepared neutral liposomes are:

[0115] (1) Average particle size (D50): 80 nm-140 nm; for example, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, etc.;

[0116] (2) Solution pH: 5.5-7.0; for example, it can be 5.5, 6.0, 6.5, 7.0, etc.; preferably 6.4-6.6; for example, it can be 6.4, 6.5, 6.6, etc.

[0117] The preparation method of the cationic liposome is as follows:

[0118] 1,2-dioleoyl-3-trimethylammonium chloride propane (DOTAP), dioleoylphosphatidylcholine (DOPC) and cholesterol were prepared in a mass ratio of 2:2:1; after the three lipid components were evenly dispersed, liposomes were prepared using ethanol injection method, thin film dispersion method, ultrasonic dispersion method and reverse evaporation method.

[0119] Preferably, the ethanol injection method is used for preparation, and the preparation method is: after ultrafiltration, extrusion molding is performed using extrusion membranes with pore sizes of 200 nm and 100 nm in sequence, and finally filtration and sterilization are performed through a sterile filter.

[0120] More preferably, the preparation steps of the cationic liposomes are as follows:

[0121] (1) dissolving the three lipid components (DOTAP, DOPC and cholesterol) in the second adjuvant composition in ethanol and shearing them online with the aqueous phase for 1 to 3 times;

[0122] (2) Use sucrose acetate buffer for ultrafiltration replacement, with a concentration multiple of 2 to 4 times and a diafiltration multiple of 6 to 8 times;

[0123] (3) 200 nm liposome extrusion, using a filter membrane with a pore size of 200 nm for extrusion, and performing multiple cycles of extrusion to control the uniformity of particle size;

[0124] (4) 100 nm liposome extrusion, using a filter membrane with a pore size of 100 nm for extrusion, and performing multiple cycles of extrusion to control the uniformity of particle size;

[0125] (5) Sterilize by filtering using a sterile filter;

[0126] The physicochemical parameters of the prepared cationic liposomes are:

[0127] (1) Average particle size (D50): 40 nm-100 nm; for example, it can be 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, etc.;

[0128] (2) Solution pH: 4.5-6.5; for example, it can be 4.5, 5.0, 5.5, 6.0, 6.5, etc.;

[0129] (3) Zeta potential: 50 mV -90 mV (pH 6.5). For example, it can be 50 mV, 60 mV, 70 mV, 80 mV, 90 mV, etc.

[0130] The present invention transfects CHO cells with a recombinant plasmid containing SEQ ID NO.1 for expression and then purifies the recombinant rabies virus G protein stock solution, and then adds a vaccine adjuvant to prepare a recombinant rabies vaccine. Immunization experiments conducted by intramuscular injection of BALB / c mice have proven that the homemade recombinant rabies vaccine has good immunogenicity.

[0131] In addition, the present invention also provides a use of the recombinant rabies vaccine as described above in the preparation of products for treating and / or preventing diseases caused by rabies virus, wherein the products include vaccine products, drugs, diagnostic reagents and detection kits.

[0132] The present invention is further described below by means of specific examples, but it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0133] Example 1: Expression processing in the preparation of recombinant rabies virus g protein.

[0134] In the preparation of the recombinant rabies virus g protein in this example, expression treatment for the recombinant expression plasmid was performed.

[0135] (1) Transform the correctly sequenced recombinant expression plasmid into DH5α chemically competent cells. Spread the genetically engineered strain evenly on the pre-prepared LB plate containing 50 μg / mL Kan with a spreading rod, place it upside down in a 37°C incubator, and culture it overnight for 12 to 16 hours. Pick a single colony and inoculate it in a flask containing 10 mL LB culture medium containing 50 μg / mL Kan, and shake it at 37°C 220 rpm overnight;

[0136] (2) The next day, inoculate 0.1% of the culture medium into 1000 mL LB culture medium containing 50 μg / mL Kan and shake at 37°C 220 rpm overnight. Remove the culture, centrifuge at 10,000 g for 15 minutes, and discard the supernatant. Follow the instructions of the EndoFree® Plasmid Giga Kit to purify the plasmid and obtain a transfection-grade plasmid.

[0137] (3) One day before transfection, adjust the density of CHO cells that have been revived and stably passaged to 3.5E6 / mL, with a volume of 650 mL / bottle and culture at 37°C in a shaking incubator overnight.

[0138] (4) During transfection, use 55μg plasmid and 250μg PEI per 25mL transfection volume. Use Opti-MEM as the diluent to prepare an equal volume of PEI solution and DNA solution for mixing. The final volume of the transfection reagent accounts for 1% of the total transfection volume. The equal volume of PEI solution should be slowly added to the plasmid DNA solution. Do not reverse the operation. Do not shake violently to produce bubbles. After mixing, incubate for 15 minutes to fully react. Add the prepared transfection reagent dropwise to the cell culture medium while shaking. Incubate at 5% carbon dioxide, 34°C, and 80rpm / min.

[0139] (5) On the first and third days after transfection, add 8% of the culture volume. After 5-6 days of culture, the cells can be harvested and the supernatant is collected by centrifugation. The cell fermentation liquid containing the recombinant rabies virus G protein is obtained.

[0140] Example 2: Purification of the preparation of recombinant rabies virus g protein.

[0141] The recombinant rabies virus g protein of this example was prepared by protein purification.

[0142] (1) Using 30kD 0.1m 2Ultrafiltration membrane package was used to concentrate 2200 mL of the supernatant of the recombinant rabies virus g protein CHO cell expression to 450 mL.

[0143] (2) Using the SCG protein purification system, 70 mL of concentrated recombinant rabies virus g protein CHO cell expression supernatant was loaded onto the equilibrated affinity chromatography column (Strep Trap XT) at a flow rate of 1 mL / min;

[0144] (3) The column was flushed with equilibrium solution (100 mM Tris-Cl + 150 mM NaCl + 1 mM EDTA-Na2, pH 8.0) at a flow rate of 1 mL / min until the OD280 value of the effluent reached the baseline. Then 70 mL of the concentrated recombinant rabies virus g protein CHO cell expression supernatant was loaded onto the affinity chromatography column at a flow rate of 1 mL / min.

[0145] (4) Flush the column with the equilibrium solution at a flow rate of 1 mL / min until the OD280 value of the effluent reaches the baseline.

[0146] (5) Elute the target protein with elution buffer (100 mM Tris-Cl + 150 mM NaCl + 1 mM EDTA-Na2 + 60 mM Biotin, pH 8.0) at a flow rate of 1 mL / min, collect the effluent, and perform 4-20% SDS-PAGE analysis.

[0147] refer to Figure 1 and Figure 2 After purification by affinity chromatography column (Strep Trap XT), the recombinant rabies virus G protein with high purity was obtained.

[0148] Example 3: Preparation of recombinant rabies vaccine.

[0149] In this example, based on Example 1 and Example 2, a recombinant rabies vaccine was prepared.

[0150] (1) Prepare neutral liposomes.

[0151] 1) The two lipid components (DOPC and cholesterol) were dissolved in ethanol and sheared online with the aqueous phase 1-3 times;

[0152] 2) 200 nm liposome extrusion, using a filter membrane with a pore size of 200 nm for filtration, and multiple cycles of extrusion to control the uniformity of particle size;

[0153] 3) 100 nm liposome extrusion, using a filter membrane with a pore size of 100 nm for filtration, and multiple cycles of extrusion to control the uniformity of particle size;

[0154] 4) Use phosphate buffer ultrafiltration replacement, concentration times 2-6 times, diafiltration times 6-8 times;

[0155] 5) Sterilize by filtering using a sterile filter.

[0156] The physicochemical parameters of the prepared neutral liposomes are:

[0157] 1) Average particle size (D50): 80nm-140nm;

[0158] 2) Solution pH: 6.4-6.6.

[0159] (2) Add phosphate buffer (prepared from sodium dihydrogen phosphate monohydrate and sodium dihydrogen phosphate), neutral liposomes, QS-21 solution and recombinant rabies virus g protein stock solution in sequence, adjust the pH to 6.0-7.0, then use water for injection to make up the volume, filter and sterilize, and then package to obtain the recombinant rabies vaccine. The specific content of each component is shown in the following table:

[0160] Table 1. Dosage of each component of the recombinant rabies vaccine used in Example 3

[0161]

[0162] Example 4: Investigation of dosage of adjuvants and auxiliary materials.

[0163] (1) Changes in physical and chemical properties before and after the addition of QS-21: According to the preparation process of the first adjuvant composition, three batches of neutral liposomes were used to add QS-21 respectively. The changes in particle size distribution and Zeta potential before and after the addition of QS-21 were detected. The results are shown in the following table.

[0164] According to the first adjuvant composition preparation process, three batches of neutral liposomes were used to add QS-21 respectively, and the changes in particle size distribution and Zeta potential before and after the addition of QS-21 were detected. The results are shown in the following table.

[0165] Table 2. Particle size distribution and Zeta potential before and after adding QS-21 in the preparation process of the first adjuvant composition

[0166]

[0167] As can be seen from the table above, after adding QS-21, the average particle size and Zeta potential of the first adjuvant composition did not change significantly, indicating that the addition of QS-21 had no significant effect on the particle size distribution and Zeta potential of the system.

[0168] (2) Investigation of changes in the hemolytic activity of QS-21: In this example, neutral liposomes (cholesterol content of 250 μg) were used to adsorb 50 μg, 100 μg, 200 μg, 300 μg and 400 μg of QS-21, respectively, and 50 μg of QS-21 was used as a control. The above samples were diluted 2-fold in sequence, and chicken red blood cells were added and incubated at room temperature for 30 minutes. The OD value of the supernatant was measured at 570 nm using a UV spectrophotometer. The higher the OD value, the stronger the hemolytic activity.

[0169] Experimental results: as shown in the data in Table 3 below.

[0170] Table 3. Hemolytic activity test table of different samples

[0171]

[0172] Experimental results analysis: Reference Figure 3 When the QS-21 content was not higher than 200 μg, the OD value of the "QS-21+neutral liposome" group had no significant difference from the blank control. This indicated that the neutral liposome containing 250 μg of cholesterol could adsorb at least 200 μg of QS-21, so that it did not produce obvious hemolysis.

[0173] (3) pH range of adjuvant system: Samples with different pH values ​​were placed at 2℃-8℃ and 37℃ for 1 week, and the content of each adjuvant component was measured. The results are shown in the following table.

[0174] Table 4. Content of components in adjuvants at different pH conditions at 2℃-8℃ and 37℃ for 1 week

[0175]

[0176] From the above table and Figure 4 and Figure 5 It can be seen that within the pH range of 6.0-7.5, DOPC and cholesterol have no obvious changes when placed at 2℃-8℃. After being placed at 37℃ for 1 week, their contents decrease to a certain extent, and there is no significant difference in the degree of decrease among samples with different pH values. Considering that the QS-21 content in the sample with pH 7.5 decreases most significantly, and QS-21 is easily hydrolyzed in an alkaline environment, the pH value of the product should not be higher than 7.0.

[0177] Example 5: Screening experiment of recombinant rabies vaccine adjuvant.

[0178] (1) Immunization of BALB / c mice: Experimental groups: SPF BALB / c mice were randomly divided into 11 groups. Except for the freeze-dried human rabies vaccine group, antigen control group and negative control group, which had 8 mice in each group, the remaining 8 groups had 12 mice in each group. The total number of animals was 120. The detailed grouping is shown in the following table.

[0179] Table 5. Mouse grouping and immunization scheme

[0180]

[0181] Immunization method: The test sample was gently shaken before use. Using a 300μL disposable sterile syringe and needle, 50μL of the test sample solution was extracted for each test sample (100μL of the test sample solution was extracted for the freeze-dried human rabies vaccine group) and injected intramuscularly into the outer thigh of the mouse.

[0182] Immunization time: administration on D0 and D7, 2 times in total.

[0183] Serum collection: Blood was collected from experimental animals in each test group on D1, D3, D6, and D10, and serum was separated and stored at -70 ℃ for later use.

[0184] (2) ELISA detection of specific antibodies in antiserum: dilute the antigen to 2 μg / mL with coating solution, coat the ELISA plate with 100 μL / well, and incubate at 4°C overnight. After washing 3 times with washing solution, add 200 μL of blocking solution to each well and block at 25°C for 1 hour. After blocking, pour off the blocking solution, dilute the mouse antiserum and mouse negative serum in multiple ratios and add them to the ELISA plate at 100 μL / well, and incubate at 25°C for 2 hours. After washing 3 times with washing solution, add HRP-labeled goat anti-mouse IgG (1:10000) at 100 μL / well, and incubate at 25°C for 1 hour. After washing 5 times with washing solution, add TMB substrate colorimetric solution, react at 25°C in the dark for 15 minutes, and add 100 μL of stop solution to terminate the reaction. Measure the OD450 value of each well with an enzyme-labeled instrument. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is determined as the titer of the sample.

[0185] (3) Experimental results: The binding IgG antibody titer was detected by ELISA. The antibody curve of the test results is shown in Figure 6 .

[0186] 1) Six days after the first immunization (D6), the serum IgG antibody titers of experimental animals in each group increased.

[0187] The IgG antibody titers induced by the "antigen + first adjuvant composition" and "antigen + second adjuvant composition" experimental groups were similar, and both were higher than the remaining experimental groups.

[0188] 2) Three days after the second immunization (D10), the serum IgG antibody titers of the experimental animals in each group were significantly higher than those of the first immunization.

[0189] The IgG antibody titers induced by the "antigen + first adjuvant composition" and "antigen + second adjuvant composition" experimental groups were similar, and both were higher than the remaining experimental groups.

[0190] In summary, the vaccine preparation prepared by the method of the present invention and adjuvanted with the addition of the first adjuvant composition and the second adjuvant composition can rapidly induce a good specific immune response in BALB / c mice.

[0191] Example 6: Investigation of the immunogenicity of the recombinant rabies vaccine.

[0192] (1) Immunization of BALB / c mice: Experimental grouping: SPF BALB / c mice were randomly divided into three groups, namely, a homemade vaccine immunization group (using the vaccine preparation prepared in Example 3), a commercial vaccine immunization group [commercially available freeze-dried human rabies vaccine (Vero cells)], and a saline immunization group. There were 10 BALB / c mice in each group, for a total of 30 mice, and the detailed grouping is shown below. Among them, the commercial vaccine immunization group used a freeze-dried human rabies vaccine (Vero cells) based on traditional inactivated technology.

[0193] Table 6. Mouse grouping and immunization scheme

[0194]

[0195] Immunization method: The test sample was gently shaken before use, and 100 μL of freeze-dried human rabies vaccine (Vero cells) (commercially available vaccine) was extracted using a 300 μL disposable sterile syringe and needle, and 50 μL of the remaining test sample was extracted for intramuscular injection into the outer thigh of the mouse.

[0196] Immunization time: The first vaccination (D0) is followed by a bonus vaccination 7 days apart, for a total of two vaccinations.

[0197] Serum collection: Blood was collected from the experimental animals in each test group before immunization (D-1) and on D3, D6, D10, D15 and D20 after immunization, and the serum was separated and stored at -70 ℃ for later use.

[0198] (2) ELISA detection of specific antibodies in antiserum: dilute the antigen to 2 μg / mL with coating solution, coat the ELISA plate with 100 μL / well, and incubate at 4°C overnight. After washing 3 times with washing solution, add 200 μL of blocking solution to each well and block at 25°C for 1 hour. After blocking, pour off the blocking solution, dilute the mouse antiserum and mouse negative serum in multiple ratios and add them to the ELISA plate at 100 μL / well, and incubate at 25°C for 2 hours. After washing 3 times with washing solution, add HRP-labeled goat anti-mouse IgG (1:10000) at 100 μL / well, and incubate at 25°C for 1 hour. After washing 5 times with washing solution, add TMB substrate colorimetric solution, react at 25°C in the dark for 15 minutes, and add 100 μL of stop solution to terminate the reaction. Measure the OD450 value of each well with an enzyme-labeled instrument. The dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is determined as the titer of the sample.

[0199] (3) Results: The binding IgG antibody titer was detected by ELISA. The antibody curve of the test results is shown in Figure 7 . After the first immunization, the IgG antibody titer in the serum of animals in the homemade vaccine immunization group was higher than that in the commercial vaccine immunization group, and both were higher than the negative control. After the second immunization, the IgG antibody titer in the serum of each group of experimental animals was significantly higher than that in the first immunization. On D15, the IgG antibody titer in the serum of each group of experimental animals reached a peak. On D20, the induced IgG antibody titer in the serum of animals in the homemade vaccine immunization group remained stable, and the IgG antibody titer in the serum of animals in the other groups decreased.

[0200] In summary, both homemade and commercially available vaccines can produce good specific immune responses in BALB / c mice, and the immunogenicity induced by homemade vaccines is better than that of commercially available vaccines.

[0201] The above are preferred embodiments and corresponding examples of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of the proportion, process, dosage and reaction vessel, such as the use of a continuous flow reactor, which all fall within the scope of protection of the present invention. The above are preferred embodiments and corresponding examples of the present invention. It should be noted that, for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can be made, including but not limited to the adjustment of the proportion, process, dosage, which all fall within the scope of protection of the present invention.

Claims

1. A recombinant rabies vaccine, characterized in that: include: Recombinant rabies virus G protein, pharmaceutical excipients and vaccine adjuvants; the pharmaceutical excipients in each milliliter of the recombinant rabies vaccine are composed of the following components: polysorbate 80, 100 μg-500 μg; sucrose, 20 mg-100 mg; sodium chloride, 1 mg-9 mg; The base sequence of the recombinant rabies virus g protein is shown in SEQ ID NO.1; The amino acid sequence of the recombinant rabies virus g protein is shown in SEQ ID NO.2; The vaccine adjuvant is a first adjuvant composition; The first adjuvant composition consists of the following components per milliliter of the recombinant rabies vaccine: DOPC, 700 μg-3000 μg; Cholesterol, 175 μg-750 μg; QS-21, 35μg-120μg.

2. The recombinant rabies vaccine according to claim 1, characterized in that Each milliliter of the recombinant rabies vaccine contains 50 μg-300 μg of recombinant rabies virus G protein.

3. Use of the recombinant rabies vaccine according to claim 1 or 2 in the preparation of a product for preventing diseases caused by rabies virus, characterized in that: The products include pharmaceuticals.

4. Use of the recombinant rabies vaccine according to claim 1 or 2 in the preparation of a product for preventing diseases caused by rabies virus, characterized in that: The products include vaccine products.

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