CN112007149A - Novel composite immunologic adjuvant and application thereof - Google Patents
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
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- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
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- 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
- A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
Abstract
A novel composite immunologic adjuvant comprises the following raw materials: 10-100 mug oligonucleotide immune stimulant, 5-100 mug activating antibody and 1-20mg aluminum salt, wherein the amino acid sequence of a heavy chain variable region of the activating antibody is as follows: 1, SEQ.1; the amino acid sequence of the light chain variable region is SEQ.2. After antigen immunization, the novel composite immunologic adjuvant can generate higher antibody serum titer compared with the traditional Freund adjuvant or the existing water-soluble adjuvant on the market.
Description
Technical Field
The invention relates to a novel composite immunologic adjuvant and application thereof in development of animal immune antibodies and preparation of vaccines, belonging to the technical field of bioengineering.
Background
The vaccine can effectively control and prevent the occurrence and spread of serious infectious diseases, and plays an important role in public health. Immunoadjuvants are key components of many vaccines, and can greatly improve the immunogenicity of vaccines, reduce the dose of antigens required to elicit effective immunity, and provide long-term immune protection for humans or animals. However, no single adjuvant can meet the requirements of all vaccines. The use of a single adjuvant has many limitations, including problems of weak immunity induction capability, short duration of immune response, and the like. For example, alum and MF59 adjuvants are commonly approved adjuvants for human vaccines, both of which typically induce a Th 2-type humoral immune response but only a weak cellular immune response, and do not provide effective protection against intracellular invading pathogens such as viruses, bacteria, protozoa, and the like. In contrast, the composite immunologic adjuvant can act synergistically, stimulate and activate various types of immune cells such as dendritic cells, macrophages, lymphocytes and the like through the components. The composite vaccine adjuvant is particularly suitable for subunit vaccines with weak immunogenicity. Meanwhile, for the population with low immunity and easy infection, such as neonates, old people and the like, the compound adjuvant can greatly reduce the dosage of the antigen and realize effective immune protection.
In addition, the development of the existing biological antibody medicines is rapid, and the market value reaches billions of dollars. 80% of antibody drugs on the market are prepared by immunizing mice with target antigens to obtain a lead antibody sequence and then performing later-stage genetic engineering modification. Therefore, whether the mice can be successfully immunized or not is a key technical link for screening the antibodies of each research unit. At present, the traditional mouse immunization adopts Freund adjuvant/incomplete Freund adjuvant to be mixed and emulsified with antigen for mouse immunization. However, this method has three problems: 1. the antigen needs to be mixed with an adjuvant and then emulsified, the operation is complex, the emulsification time and the emulsification force are different, and the immune effect on mice is also different. 2. The conventional ultrasonic emulsification damages antigens and influences the immune effect of the antigens. 3. For human and mouse antigens with high homology or low immunogenicity, it is difficult to immunize mice with Freund's adjuvant/incomplete Freund's adjuvant to generate high titer serum.
Disclosure of Invention
The invention aims to provide a novel composite immunologic adjuvant and application thereof.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a novel composite immunologic adjuvant comprises the following raw materials:
10-100 μ g of oligonucleotide immunostimulant;
5-100 μ g of activating antibody;
1-20mg of aluminum salt;
the heavy chain variable region amino acid sequence of the activating antibody is as follows: 1, SEQ.1; the amino acid sequence of the light chain variable region is SEQ.2.
The preferable technical scheme is as follows: the sequence of the oligonucleotide immunostimulatory substance is: and (C) T, a, C, G, a, C, G, T, and performing a phosphorothioation modification on each base.
The preferable technical scheme is as follows: the aluminum salt is aluminum hydroxide.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: the application of a novel composite immunologic adjuvant stimulates animals to produce high-titer serum antibodies.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. the novel composite immunologic adjuvant does not need emulsification when in use, and the antigen and the adjuvant are mixed and then directly injected for immunization in a water-soluble mode.
2. After antigen immunization, the novel composite immunologic adjuvant can generate higher antibody serum titer compared with the traditional Freund adjuvant or the existing water-soluble adjuvant on the market.
3. The novel composite immunologic adjuvant can break the immune tolerance of mice, and can successfully immunize the mice to obtain antibodies for antigens with high homology or low immunogenicity of human mice.
Drawings
FIG. 1 schematic representation of the PSG1003-VH-CH plasmid.
FIG. 2PSG1003-VL-CL plasmid schematic.
FIG. 3 detection of activating antibody SDS-PAGE.
FIG. 4 serum antibody titers ELISA detection after immunization of mice.
FIG. 5 enzyme-linked immunosorbent assay.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-5. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1: novel composite immunologic adjuvant and application thereof
Preparation of oligonucleotide immune stimulant
The sequence of the oligonucleotide immunostimulatory substance is:
and (C) T, a, C, G, a, C, G, T, and performing a phosphorothioation modification on each base. The synthesis of oligonucleotide immunostimulants was performed by hong Xun Biotech, Suzhou, Inc.
Preparation of activating antibodies
The activating antibody has the heavy chain gene sequence SEQ No.3 and the light chain gene sequence SEQ No.4, the corresponding heavy chain variable region sequence SEQ No.1 and light chain variable region sequence SEQ No.2, and the subtype selected by the antibody is mouse IgG 1. Cloning the heavy chain gene sequence and the light chain gene sequence into a PSG1003 plasmid vector by a molecular cloning enzyme digestion and connection method to obtain PSG1003-VH-CH and a plasmid PSG1003-VL-CL, which are respectively shown in figure 2 and figure 3. The two plasmids were extracted using a plasmid macroextraction kit for subsequent transient expression. Will be provided withThe 293F cells are recovered from liquid nitrogen, and are placed in a carbon dioxide incubator for culture and suspension culture under the culture condition of 37 ℃ and 5% carbon dioxide, and the rotation speed of a shaking bottle is 120 rpm/min. Two 15ml sterile centrifuge tubes were prepared before cell transient transformation, 5ml KPM and 100. mu.g sterile plasmid DNA (PSG1003-VH-CH and PSG1003-VL-CL ratio 1:1, 50. mu.g each) were added to one of them, gently blown and mixed; adding 5ml KPM and 500 μ l TA-293 transfection reagent into the other branch, and gently blowing, beating and mixing; transferring all liquid in the centrifuge tube containing the transfection reagent into the centrifuge tube containing the plasmid, and gently blowing, beating and uniformly mixing; standing for 10 minutes at room temperature to prepare a plasmid-vector compound; taking out the cells from the constant temperature shaking table, adding the prepared plasmid-vector complex while shaking, and returning CO2Culturing in a constant temperature shaking table. After 24 hours of transfection, 600. mu.l of 293 cell protein expression enhancer (KE-293) was added to increase the expression of the product, and the supernatant was collected 6 days after transfection, and all reagents for transient transfer were purchased from happy Ri Biotech Ltd. Then, the antibody was purified from the cell culture supernatant using a Protein A purification column, and the purity of the antibody was determined by SDS-PAGE.
As shown in FIG. 3, the results of SDS-PAGE experiments showed that high purity activating antibody was obtained by transient expression. The antibody subtype can also be replaced by mouse IgG2a, mouse IgG2b or mouse IgG 3.
SEQ No.1：
QVQLKESGPGLVQPSQTLSLTCTVSGFSLTGYNLHWVRQPPGKGLEWMGRMRYDGDTYYNSVLKSRLSISRDTSKNQVFLKMNSLQTDDTAIYYCTRDGRGDSFDYWGQGVMVTVSS。
SEQ No.2：
DIVMTQAALPNPVPSGESASITCRSSQSLVYKDGQTYLNWFLQRPGQSPQLLTYWMSTRASGVSDRFSGSGSGTYFTLKISRVRAEDAGVYYCQQVREYPFTFGSGTKLEIK.
SEQ No.3：
CAGGTCCAGCTGAAGGAAAGCGGCCCCGGCCTGGTCCAGCCCTCTCAGACACTGAGCCTGACCTGCACCGTCTCCGGCTTCTCCCTGACAGGCTACAACCTGCACTGGGTGAGACAGCCCCCCGGCAAAGGACTGGAATGGATGGGCAGAATGAGATACGACGGCGACACCTACTACAACAGCGTGCTGAAGAGCAGACTGAGCATCAGCAGGGACACCTCCAAGAACCAGGTGTTCCTGAAGATGAATAGCCTGCAGACCGACGACACCGCCATCTACTATTGCACAAGGGACGGCAGAGGCGACAGCTTCGACTACTGGGGCCAGGGAGTGATGGTGACCGTGAGCAGTGCTAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCCCACGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCGCAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGAATACAAACGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAATGA。
SEQ No.4：
GACATTGTGATGACCCAGGCCGCCCTGCCCAACCCCGTGCCTTCTGGAGAGAGCGCTAGCATTACCTGCAGATCCTCCCAGAGCCTGGTGTATAAAGACGGCCAGACCTACCTGAACTGGTTCCTGCAGAGACCCGGACAGTCCCCCCAGCTGCTGACCTACTGGATGTCAACCAGAGCCTCCGGCGTGTCCGACAGATTCTCTGGGAGCGGAAGCGGAACCTACTTCACCCTGAAAATCTCCAGAGTGAGAGCCGAAGACGCCGGCGTGTACTACTGCCAGCAGGTCAGAGAATATCCCTTTACCTTCGGAAGCGGAACAAAGCTGGAGATCAAGAGAGCAGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTTGA。
Third, animal immunization
Male Balb/C healthy mice (purchased from Beijing Wittingerihua) of about 6-8 weeks of age were selected, and the animal immunization groups are shown in Table I. Experiment group a: the antigen was mixed with Freund's complete adjuvant and PBS, and after complete emulsification, 100. mu.g of antigen was administered to each individual at multiple sites on the back. The second and third immunizations are immunized with incomplete Freund's adjuvant. In experiment B, antigen is mixed with the compound adjuvant without emulsification, and is injected into abdominal cavity after being mixed for 30 minutes at room temperature. Experiment group C, antigen was mixed with oligonucleotide immunostimulant and aluminum salt, mixed for 30 minutes at room temperature, and then injected intraperitoneally. Four groups were tested, and the antigen was mixed with aluminum salt at room temperature for 30 minutes and then injected intraperitoneally. After each group of animals was immunized three times, the tails were bled for subsequent serum titer detection. The immunization is carried out at intervals of three weeks, and blood is taken for detection after one week of three-immunization. The antigen is HER3 recombinant protein.
TABLE I animal immunization group
Fourth, serum titer detection
The ELISA method is used for detecting the serum titer, and the specific method is as follows:
(1) coating an enzyme label plate with free antigen of 1 mu g/ml, wherein each hole is 100 mu L, and the enzyme label plate is coated overnight at 4 ℃;
(2) throwing away redundant antigen, lightly patting the antigen with paper, blocking the antigen with a solution of PBS + 1% and 0.05% Tween 20and 1% BSA, blocking the antigen at 300 μ l/hole and standing the antigen overnight at 4 ℃;
(3) removing the blocking solution, washing with PBST for 3 times, and gently patting on paper;
(4) diluting serum according to gradient, sucking 100 μ l, adding onto ELISA plate, performing indirect ELISA, and incubating at 37 deg.C for 1 hr;
(5) removing redundant supernatant, washing with PBST for 3 times, gently patting on paper, adding secondary antibody HRP-conj mu gated AffiniP mu re Goat Anti-Mo mu se IgG Fc, incubating at 50 mu l/hole, and incubating at 37 ℃ for 30 min;
(6) removing redundant secondary antibody, washing with PBST for 3 times, gently patting on paper, adding TMB, 50 μ l/hole, and developing at room temperature for 15 min;
(7) 2M HC was added at 50. mu.l/well and the plate was read with a microplate reader at 450 nm.
The experimental results show that:
as shown in FIG. 4, the ordinate represents the absorbance of ELISA test samples, and the abscissa represents the different dilution times of mouse serum, and the larger the value, the larger the dilution times. The four curves A-D are respectively the curves made by the average value of ELISA detection values after four groups of animal serum are diluted.
The experiment group B contains all the components of the invention patent, and the antibody serum titer is obviously better than that of the experiment group A immunized by the traditional Freund adjuvant. Meanwhile, the experiment group C and the experiment group D only contain partial adjuvant components of the invention patent, and the effect of the experiment group B is better than that of the experiment group C and the experiment group D, which shows that each component in the novel composite immunologic adjuvant has an important function and plays a role in synergistically strengthening the immunity.
Fifth, preparation of mouse antibody immunized by compound adjuvant
1) Hybridoma fusion: 7-10 days before fusion, thawing SP2/0 cells, observing cell growth state every day, and making the cells in logarithmic growth phase by using half-exchange liquid, wherein the living cell rate of SP2/0 cells used in fusion is required to be more than 95%. Feeder cells were additionally prepared: the cells exuded from the abdominal cavity of the normal mouse were inoculated on a 96-well culture plate one day before the fusion, at a concentration of 100. mu.l/well. Polyethylene glycol 1500, lml each. 3 days after the last immunization, the mice were bled, soaked in 75% alcohol for 3min, opened, the spleen was removed, washed, splenocytes were separated with a splenocyte separator, and single cell suspension was collected. The SP2/0 cells and the immunized mouse splenocytes were resuspended in 1640 incomplete medium and counted on a hemocytometer according to the ratio of splenocytes: mixing SP2/0 cells at a ratio of 1:3 in a 50ml sterile centrifuge tube, adding 1640 incomplete culture medium to 40ml after fully mixing; centrifuging at 1.500rpm for 5 min; centrifuging, removing supernatant and liquid on tube wall (can be sucked dry with sterilized absorbent paper strips), gently beating mixed precipitate of SP2/0 cells and immune mouse cells to loosen the precipitate, and immersing the bottom of the centrifuge tube in warm water at 37 ℃; taking out 1ml of the incubated fusion agent PEG1450 from the incubator, and uniformly dripping the 1ml of the incubated fusion agent PEG1450 into the cell mixed sediment within 60 seconds (rotating the centrifugal tube while dripping); standing and incubating for 45s, taking out 1640 incomplete culture medium preheated in a 37 ℃ incubator, uniformly dripping 1ml and 60s into the precipitate (rotating a centrifugal tube while dripping), uniformly dripping 1ml and 30s, and then slowly dripping all the residual 45ml of culture medium; after mixing by gentle inversion, centrifuging at 1,500rpm for 5min, and discarding the supernatant; resuspend the cell pellet with 200ml of complete medium solution containing feeder cells and HAT; after cell fusion, the HAT selective medium pair was half-changed every other day. The HT selective medium was used 10-14 days later. The 1640 complete medium was used after another 10-14 days. And (3) observing whether the 96-well clone exists, selecting a well-grown clone well for detection, carrying out amplification culture on the positive clone well, expanding the well-grown clone well from the 96-well plate to a 24-well plate, and finally expanding the well-grown clone well into a culture bottle.
2) Hybridoma detection and cloning:
indirect ELISA: coating carrier with HER3 antigen, standing overnight at 4 deg.C, blocking with irrelevant protein (BSA) at 37 deg.C for 2h, adding sample to be tested, incubating at 37 deg.C for 1h, washing with 0.05% Tween 20-PBS for 5-6 times, adding secondary antibody (such as HRP-GAM), incubating at 37 deg.C for 2h, washing, developing with substrate (OPD), and stopping solution (3M H)2SO4) The reaction was stopped and OD492 was read on a microplate reader. Taking hybridoma cells with positive ELSIA signal value and stable growth, adjusting the counting to 10/ml, inoculating 100 mu l/well of the hybridoma cells on a 96-well plate, growing the hybridoma cells, ensuring that the result of the monoclonality positive rate is 100%, and if the result of the monoclonality positive rate is not reached, subcloning again. And (3) performing expanded culture on the screened positive clones, preserving one part of the positive clones as cell seeds by freezing, culturing the other part of the positive clones, purifying the antibody by using a ProteinA purification column, and performing ELISA detection on the purified antibody to verify whether the antibody is combined with the antigen or not.
The experimental results are as follows:
as shown in FIG. 5, 6 hybridoma cells were obtained by hybridoma fusion and ELSIA screening, and the antibody prepared by fusion was confirmed to bind to the antigen HER3 protein by ELSIA.
The immunoadjuvant developed by the embodiment can effectively immunize a mouse to generate a high-titer antibody with serum titer, a hybridoma cell line can be successfully obtained by utilizing a hybridoma fusion method, the generated antibody can be effectively combined with an antigen, and the newly developed immunoadjuvant can be further proved to be used for development and preparation of the antibody.
Example 2: novel composite immunologic adjuvant
A novel composite immunologic adjuvant comprises the following raw materials:
oligonucleotide immunostimulant 10 μ g;
activating antibody 5 μ g;
the heavy chain variable region amino acid sequence of the activating antibody is as follows: 1, SEQ.1; the amino acid sequence of the light chain variable region is SEQ.2.
The preferable technical scheme is as follows: the sequence of the oligonucleotide immunostimulatory substance is: and (C) T, a, C, G, a, C, G, T, and performing a phosphorothioation modification on each base.
The preferred embodiment is: the aluminum salt is aluminum hydroxide.
Example 3: novel composite immunologic adjuvant
A novel composite immunologic adjuvant comprises the following raw materials:
100 μ g of oligonucleotide immunostimulant;
activating antibody 100 μ g;
20mg of aluminum salt;
the heavy chain variable region amino acid sequence of the activating antibody is as follows: 1, SEQ.1; the amino acid sequence of the light chain variable region is SEQ.2.
The preferred embodiment is: the sequence of the oligonucleotide immunostimulatory substance is: and (C) T, a, C, G, a, C, G, T, and performing a phosphorothioation modification on each base.
The preferred embodiment is: the aluminum salt is aluminum hydroxide.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Claims (4)
1. A novel composite immunologic adjuvant is characterized in that: the composite immunologic adjuvant comprises the following raw materials:
10-100 mug of oligonucleotide immune stimulant;
5-100 mug of activating antibody;
1-20mg of aluminum salt;
the heavy chain variable region amino acid sequence of the activating antibody is as follows: 1, SEQ.1; the amino acid sequence of the light chain variable region is SEQ.2.
2. The novel composite immunoadjuvant of claim 1, characterized in that: the sequence of the oligonucleotide immunostimulatory substance is: and (C) T, a, C, G, a, C, G, T, and performing a phosphorothioation modification on each base.
3. The novel composite immunoadjuvant of claim 1, characterized in that: the aluminum salt is aluminum hydroxide.
4. The use of the novel composite immunoadjuvant according to any one of claims 1 to 3, characterized in that: the animals are stimulated to produce high titer serum antibodies.
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