CN114836416B - CpG ODN adjuvant and its application in antibody production - Google Patents
CpG ODN adjuvant and its application in antibody production Download PDFInfo
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Abstract
The invention relates to a CpG ODN adjuvant and application thereof in antibody production, belonging to the technical field of polyclonal antibody production. The application of the CpG ODN adjuvant in antibody production is that one or more of CpGODN-1, cpG ODN-2, cpG ODN-3, cpG ODN-4, cpG ODN-5, cpG ODN-6, cpGODN-7, cpG ODN-8, cpG ODN-9 and CpG ODN-10 sequences are synthesized and then mixed, and then mixed with cationic liposome reagent in equal volume, and the mixture is used as a CpG ODN mixed adjuvant for antibody production. The CpG ODN mixed adjuvant provided by the invention can shorten the conventional animal immune cycle from 10 weeks to 4 weeks. The titer of the produced polyclonal antibody product is obviously higher than that of the control group, and the antibody yield is also higher than that of the control group.
Description
Technical Field
The invention belongs to the technical field of polyclonal antibody production, and particularly relates to a CpG ODN adjuvant and application thereof in antibody production.
Background
Polyclonal antibodies refer to animals immunized with an antigen comprising multiple epitopes that stimulate the production of different antibodies to multiple epitopes by multiple B cell clones of the body, and the resulting immune serum is essentially a mixture containing multiple antibodies. In vitro diagnostic reagents, immunoturbidimetry reagents can use monoclonal antibodies or polyclonal antibodies, which are costly because only limited epitopes can be recognized and complete recognition of target proteins in the sample cannot be guaranteed. Therefore, polyclonal antibodies are mostly used as the turbidimetric immunoassay reagent in the market at present. The polyclonal antibody has low cost and can be produced in batch, but has high requirements on production cycle, antibody titer, batch-to-batch difference and the like. The general preparation flow of polyclonal antibodies comprises antigen preparation, animal immunization, titer detection, serum collection and treatment, and antibody purification. The animal immunization mainly uses proper adjuvant to match antigen to inoculate animal, and makes repeated inoculation according to a certain immunization scheme so as to make it produce specific antibody as quickly, high-effectively and largely as possible.
CpG ODN adjuvant is a deoxynucleotide sequence containing unmethylated cytosine guanine, and can enhance antigen processing presentation and induce humoral immunity. CpG ODNs, by activating TLR9 signaling, can synergistically promote activation of naive B cells, induced B cells, and memory B cells with B cell antigen receptor signaling, and further differentiate into plasma cells that secrete antibody molecules. TLR9 is expressed primarily by plasma cells and B cells, B lymphocytes exposed to TLR9 agonists are more readily activated by antigen to reflect their utility as immunopotentiators. In general, since naive B cells do not have the ability to express TLR9, they do not respond to the activation of CpG ODN, and thus the naive B cells must be stimulated with antigen in advance to ensure differentiation into plasma cells after activation by CpG ODN. The CpG ODN can be artificially synthesized, can be applied to various antigens, is preserved in a freeze-dried powder state, is stable in state and convenient to transport, and provides a powerful foundation support for industrialization of the CpG ODN serving as an immunoadjuvant.
The prior art discloses specific sequences, preparation and application methods of CpG ODN adjuvant in various human vaccines and animal vaccines, which can obviously enhance the effect of the vaccine. In the production process of the polyclonal antibody, a proper CpG ODN adjuvant is selected, a reasonable scheme is designed, and the traditional immunization technology is matched, so that the function of an immune enhancer is exerted, the production period of the polyclonal antibody is shorter, and the antibody titer is higher.
Disclosure of Invention
The invention aims to provide a CpG ODN adjuvant and application thereof in antibody production so as to solve the problems in the background technology.
The aim of the invention can be achieved by the following technical scheme:
CpG ODN adjuvant with specific sequence shown in Table 1.
TABLE 1
| CpG ODN designation | Sequence(s) |
| CpG ODN-1 | TGACTGTGAACGTTCGAGAAGA |
| CpG ODN-2 | ATTCCTGCACGTTCGAGTCCATT |
| CpG ODN-3 | CCTTCATTCCGTTCGGGTACATT |
| CpG ODN-4 | TTTCACCTCGTTCGTTACCTGAC |
| CpG ODN-5 | AACTGTAACCGTTCGTCCACTAC |
| CpG ODN-6 | TTACAATCACGAACGTTCATCG |
| CpG ODN-7 | AACTTCACGTTCGACTTTCTAGA |
| CpG ODN-8 | ATCCATTCGTTCGAATCATACCTA |
| CpG ODN-9 | AATTCTTCGTTCGATCGTTCGTAA |
| CpG ODN-10 | TACTCCCTCGTTCGATCGCCTCAGT |
One or more sequences of the 10 sequences are synthesized, mixed and then mixed with a cationic liposome reagent to be used as CpG ODN mixed adjuvant for antibody production.
Further, the cationic liposome reagent is a reagent well known to those skilled in the art, such as Lipofectamine cationic liposome reagent, PPL Fectin cationic liposome reagent, etc.
Preferably, the application of the CpG ODN adjuvant in antibody production specifically comprises the following steps:
step one, antigen preparation;
step two, preparing a CpG ODN mixed adjuvant: synthesizing the sequences in Table 1, mixing with the cationic liposome reagent in equal volume, standing for 30min, and standing for use;
step three, immune test and antibody extraction:
i. basic immunization is carried out by using Freund's complete adjuvant, and CpG ODN mixed adjuvant is injected into animals after 3 days;
performing first boosting by using Freund's incomplete adjuvant after 7 days, and simultaneously injecting CpG ODN mixed adjuvant into the animal;
a second boost using the procedure in step ii after 14 days;
a third boost using the procedure in step ii after 21 days;
serum final collection was performed 28 days later and titers were detected and antibodies purified.
The invention has the beneficial effects that:
the use of CpG ODN mixed adjuvants for polyclonal antibody production according to the present invention can reduce the conventional animal immune cycle from 10 weeks to 4 weeks. The titers of the polyclonal antibody products produced were significantly higher than those of the control (control 1-2), and the antibody yields were also higher than those of the control (control 1-2).
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, 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.
Example 1
Application test of CpG ODN adjuvant in antibody production
Step one, antigen preparation:
using recombinant human Procalcitonin (PCT) protein recombinantly expressed by escherichia coli as an immunogen, wherein the antigen concentration is 1mg/mL; the basic immune antigen was 500ul recombinant PCT protein, mixed with 500ul freund's complete adjuvant (Sigma, F5881), and sonicated for 1 min, repeated 3 times; the booster antigen was 500ul recombinant PCT protein, mixed with 500ul incomplete freund's adjuvant (Sigma, F5506), sonicated for 1 min, and repeated 3 times;
step two, preparing a CpG ODN mixed adjuvant:
the synthetic CpG ODN-1, the CpG ODN-2, the CpG ODN-3, the CpG ODN-4, the CpG ODN-5, the CpG ODN-6, the CpG ODN-7, the CpG ODN-8, the CpG ODN-9 and the CpG ODN-10. Dissolving with ddH2O to obtain mother solution of 5mg/mL, mixing 0.1mL each, and mixing by vortex; before use, the CpG ODN and Lipofectamine 2000 (Thermo, 11668019) are mixed according to the volume ratio of 1:1, and are used after standing for 30 minutes;
step three, immune test and antibody extraction:
3.1 immunization protocol: conventional cycle group: boosting is carried out every two weeks after basic immunization, and blood collection is carried out two weeks after five days of immunization. Basic immunization was inoculated with Freund's complete adjuvant emulsified with antigen, 3 days later with CpG ODN adjuvant alone. Boost was vaccinated with Freund's incomplete adjuvant emulsified with antigen, with CpG ODN mixed adjuvant injected alone.
3.2 immunization Process
3.2.1 animal preparation: 8 healthy New Zealand white rabbits of 8 weeks in size were selected, 4 in each of the conventional cycle group and the optimized cycle group, and 2 in each of the experimental group and the control group.
3.2.2 Pre-sampling blood (for negative reference)
The rabbits were carefully placed in a fixed rack, and the small cotton balls were smeared with alcohol at the vascular site to dilate the blood vessels. About 10mL of blood was withdrawn from the ear vein with a syringe, the wound was pressed appropriately to avoid bleeding, and then the wound was sterilized with an alcohol cotton ball. The collected blood was inactivated at 37℃for 30min, and finally allowed to coagulate overnight at 4℃to release serum. The coagulated blood was centrifuged at 10000R/min for 10min, and the supernatant was collected.
3.2.3 animal Vaccination
The antigen of two rabbits was injected at 1mL, the rabbits were carefully removed from the cages, each rabbit was immunized at 4 sites, both back and thigh root, 250ul each site, needle was inserted subcutaneously at 45 degree angle at 1-2cm, and the residence time was several seconds after injection was completed to prevent outflow of antigen. CpG ODN mixed adjuvant was injected alone, 4 sites were immunized on each rabbit, 125ul each site, and the antigen injection sites were staggered. The vaccination was performed according to the cycle in the group immunization protocol.
3.2.4 serum treatment and antibody purification
3.2.4.1 serum treatment: blood collection is carried out after immunization is completed, the collected blood is inactivated at 37 ℃ for 30min, and finally the collected blood is subjected to overnight at 4 ℃ to coagulate and release serum. The coagulated blood was centrifuged at 10000R/min for 10mi, and the supernatant was collected.
3.2.4.2 preparation of antigen affinity column: an appropriate amount of NHS-Activated Beads 4FF was taken, washed three times with 1mM HCl solution and once with coupling solution. The dissolved sample was added to washed NHS-Activated Beads 4FF, NHS-Activated Beads 4FF: sample solution volume comparison 1:1, a step of; the reaction was allowed to react at 28℃overnight with shaking for 3h or 4 ℃. After the reaction was completed, a coupling sample was collected to examine the coupling efficiency. Cleaning the filler by deionized water, adding a sealing liquid with the volume of 2 times of that of the column, and carrying out oscillation reaction for 1h at 28 ℃; taking out the reaction system, draining the sealing liquid in the reaction system, cleaning the resin with deionized water with the volume of 3 times of the column volume, repeatedly flushing the cleaning liquid 1, the deionized water, the cleaning liquid 2 and the deionized water for 2 times, then storing in a protection liquid with the same volume, and storing at the temperature of 2-8 ℃.
3.2.4.3 antigen affinity purification: the serum of each batch to be treated was taken out and filtered with a sterile 0.22um membrane. Taking out the coupled antigen affinity chromatographic column, sucking the filler in the column into serum, and placing the serum in a spin mixer for incubation at 4 ℃ overnight or mixing at room temperature for 2 hours; the incubated serum is subjected to column chromatography, the flow rate is controlled, effluent liquid is collected to be FT, then the chromatographic column is washed by 1 XPBS pH 7.4, the effluent liquid is collected to be W0, finally the chromatographic column is washed by 0.1M glycine buffer solution, the eluent liquid is collected to be E, and balance liquid is evenly added into the E to enable the pH value of the eluent liquid to be recovered to be neutral; and determining whether to pass through the column again according to the amount of protein until no protein exists.
3.2.4.4 sample treatment: the purified eluate was dialyzed against 20mL of 1-2L of 1 XPBS; ultrafiltering the dialyzed sample with 50KD concentration tube, sampling the concentrated sample to determine antibody concentration, subpackaging, placing into frost crack preventing tube, marking, and storing at-20deg.C.
3.2.5 potency assay
1 XCBS was prepared, the antigen was diluted to 1ug/mL with CBS, added to the ELISA plate, and left overnight at 4℃per well at 100 ul. The samples in the ELISA plate were discarded, the plate was washed 2 times, blocking solution was added and incubated at 37℃for 2h per well 150 ul. Discarding sealing liquid in the ELISA plate, putting into a plate beating machine, centrifuging, and standing at 4 ℃ for standby. Diluting serum with antibody diluent according to a ratio of 1:1000, adding 210ul of diluted serum into a first hole by a dilution plate, adding 140ul of antibody diluent into each hole, sucking 70ul of the diluted serum from the first hole to a second hole, uniformly mixing, sucking 70ul of the diluted serum from the second hole to a third hole, uniformly mixing, performing row-by-row dilution until reaching a seventh hole, reserving an eighth hole as negative control of the antibody diluent, transferring the diluted antibody to a coating plate, and placing the coating plate into a constant temperature box at 37 ℃ for incubation for 40min. Discarding samples in the ELISA plate, placing into a plate washer, washing the plate 5 times with 0.05% TWEN20 washing solution, and placing into a plate beating machine for centrifugal drying. After 1g G (H+L) of HRP-labeled goat anti-mouse was diluted 1:10000 with antibody dilution, 100ul of the antibody was added to each well, and the ELISA plate was placed in a 37℃incubator for 40min. Discarding samples in the ELISA plate, placing into a plate washer, washing the plate 5 times with 0.05% TWEN20 washing solution, and placing into a plate beating machine for centrifugal drying. TMB working solution (mixing TMB A and TMB in equal volumes, and mixing in situ) was prepared, 100ul of each well was added, incubated in a 37℃incubator for 10min, and 50ul of stop solution was added to each well. The wavelength of 450nm is selected, the OD value is measured by an enzyme label instrument, and data are recorded.
Example 2
Application test of CpG ODN adjuvant in antibody production
In comparison with example 1, the immunization protocol in step three was replaced by the following protocol, the other steps being identical:
optimization cycle group: boosting is carried out at intervals of one week after basic immunization, and blood collection is carried out one week after the end of four-day immunization. Basic immunization was inoculated with Freund's complete adjuvant emulsified with antigen, 3 days later with CpG ODN adjuvant alone. Boost was vaccinated with Freund's incomplete adjuvant emulsified with antigen, with CpG ODN mixed adjuvant injected alone.
Comparative example 1 (i.e., control group 1)
Antibody production:
in comparison to example 1, ddH2O was used in equal amounts instead of CpG ODN mixed adjuvants, the remainder being identical.
Comparative example 2 (i.e., control group 2)
Antibody production:
in comparison to example 2, ddH2O was used in equal amounts instead of CpG ODN mixed adjuvants, the remainder being identical.
Example 3
Analysis of results:
1. potency comparison: the titer data of the antibodies obtained in examples 1-2 and comparative examples 1-2 are shown in Table 2.
TABLE 2
From the data in Table 2, it can be seen that the results of the antibody titers obtained in examples 1-2 are superior to those obtained in comparative examples 1-2, and that the results of the antibody titers obtained in example 2 are superior to those obtained in example 1.
2. Yield comparison: the titer data of the antibodies obtained in examples 1-2 and comparative examples 1-2 are shown in Table 3.
TABLE 3 Table 3
From the data in Table 3, it can be seen that the antibody production in example 2 is higher than that in example 1, comparative examples 1-2.
Example 4
Application test of CpG ODN adjuvant in antibody production: in comparison with example 2, the CpG ODN synthetic sequence was replaced, and the rest was the same as in example 2;
the specific synthetic sequences are shown in Table 4. After the completion of antibody production, antibody titer (indicated by OD value) was measured by using a 111ng/mL antigen concentration-coated ELISA plate, and antibody production was measured as shown in Table 4.
TABLE 4 Table 4
As can be seen from Table 4, the sequences obtained in examples 4-1, 4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 4-11, 4-12, 4-13, 4-14, 4-15, 4-16, 4-17, 4-18 were used in antibody production, and the resulting antibody titers and yields were superior to those of the test results for antibody production in comparative example 2.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing is merely illustrative and explanatory of the invention, as various modifications and additions may be made to the particular embodiments described, or in a similar manner, by those skilled in the art, without departing from the scope of the invention or exceeding the scope of the invention as defined in the claims.
Sequence listing
<110> Anhui world Gene technologies Co., ltd
<120> CpGODN adjuvant and its use in antibody production
<140> CN2022105744224
<141> 2022-05-24
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 1
tgactgtgaa cgttcgagaa ga 22
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 2
attcctgcac gttcgagtcc att 23
<210> 3
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 3
ccttcattcc gttcgggtac att 23
<210> 4
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 4
tttcacctcg ttcgttacct gac 23
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 5
aactgtaacc gttcgtccac tac 23
<210> 6
<211> 22
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 6
ttacaatcac gaacgttcat cg 22
<210> 7
<211> 23
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 7
aacttcacgt tcgactttct aga 23
<210> 8
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 8
atccattcgt tcgaatcata ccta 24
<210> 9
<211> 24
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 9
aattcttcgt tcgatcgttc gtaa 24
<210> 10
<211> 25
<212> DNA
<213> Artificial sequence (artificial sequence)
<400> 10
tactccctcg ttcgatcgcc tcagt 25
Claims (4)
1. A CpG ODN adjuvant, characterized by: consists of the following sequences: cpG ODN-1 is TGACTGTGAACGTTCGAGAAGA; cpG ODN-2 is ATTCCTGCACGTTCGAGTCCATT; cpG ODN-3 is CCTTCATTCCGTTCGGGTACATT; cpG ODN-4 is TTTCACCTCGTTCGTTACCTGAC; cpG ODN-5 is AACTGTAACCGTTCGTCCACTAC; cpG ODN-6 is TTACAATCACGAACGTTCATCG; cpG ODN-7 is AACTTCACGTTCGACTTTCTAGA; cpG ODN-8 is ATCCATTCGTTCGAATCATACCTA; cpG ODN-9 is AATTCTTCGTTCGATCGTTCGTAA; cpG ODN-10 was TACTCCCTCGTTCGATCGCCTCAGT.
2. Use of CpG ODN adjuvant in the production of human procalcitonin recombinant protein antibodies, characterized in that: the CpG ODN-1, cpG ODN-2, cpG ODN-3, cpG ODN-4, cpG ODN-5, cpG ODN-6, cpG ODN-7, cpG ODN-8, cpG ODN-9 and CpG ODN-10 sequences in the CpG ODN adjuvant of claim 1 are synthesized, and then mixed with the cationic liposome reagent in equal volume, and used as a CpG ODN mixed adjuvant for antibody production.
3. Use of a CpG ODN adjuvant according to claim 2 for the production of human procalcitonin recombinant protein antibodies, characterized in that: the method comprises the following steps:
step one, antigen preparation;
step two, preparing a CpG ODN mixed adjuvant: synthesizing sequences CpG ODN-1, cpG ODN-2, cpG ODN-3, cpG ODN-4, cpG ODN-5, cpG ODN-6, cpG ODN-7, cpG ODN-8, cpG ODN-9 and CpG ODN-10, mixing with cationic liposome reagent in equal volume, standing for 30min for use;
and step three, immune test and antibody extraction.
4. Use of a CpG ODN adjuvant according to claim 3 for the production of human procalcitonin recombinant protein antibodies, characterized in that: the specific operation of the third step is as follows:
i. basic immunization is carried out by using Freund's complete adjuvant, and CpG ODN mixed adjuvant is injected into animals after 3 days;
performing first boosting by using Freund's incomplete adjuvant after 7 days, and simultaneously injecting CpG ODN mixed adjuvant into the animal;
a second boost using the procedure in step ii after 14 days;
a third boost using the procedure in step ii after 21 days;
serum collection was performed 28 days later and titers were detected and antibodies purified.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105749275A (en) * | 2016-03-30 | 2016-07-13 | 山东农业大学 | Nucleic acid slow release adjuvant and preparation and use methods thereof |
| CN107987159A (en) * | 2017-11-28 | 2018-05-04 | 上海药明生物技术有限公司 | It is a kind of to use heavy dose of DNA immunization technique that the method that animal improves Serum Antibody titre is immunized |
| CN112089833A (en) * | 2020-08-14 | 2020-12-18 | 中山大学 | Universal CpG ODN nano-particle adjuvant and preparation method and application thereof |
| CN112159814A (en) * | 2020-10-29 | 2021-01-01 | 中国农业科学院兰州兽医研究所 | CpG oligodeoxynucleotide, preparation and application thereof |
| CN112626073A (en) * | 2020-10-26 | 2021-04-09 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105749275A (en) * | 2016-03-30 | 2016-07-13 | 山东农业大学 | Nucleic acid slow release adjuvant and preparation and use methods thereof |
| CN107987159A (en) * | 2017-11-28 | 2018-05-04 | 上海药明生物技术有限公司 | It is a kind of to use heavy dose of DNA immunization technique that the method that animal improves Serum Antibody titre is immunized |
| CN112089833A (en) * | 2020-08-14 | 2020-12-18 | 中山大学 | Universal CpG ODN nano-particle adjuvant and preparation method and application thereof |
| CN112626073A (en) * | 2020-10-26 | 2021-04-09 | 中国科学院昆明动物研究所 | CpG-ODN with specific immunostimulation effect on PRRSV and application thereof |
| CN112159814A (en) * | 2020-10-29 | 2021-01-01 | 中国农业科学院兰州兽医研究所 | CpG oligodeoxynucleotide, preparation and application thereof |
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