CN111875706B - Single-domain antibody of anti-human IgE protein and application thereof - Google Patents
Single-domain antibody of anti-human IgE protein and application thereof Download PDFInfo
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Abstract
The invention relates to the field of blood purification, in particular to a single domain antibody of anti-human IgE protein and application thereof. The structural general formula of the amino acid sequence of the single domain antibody is as follows: MDVQLQESGGG-X1‑LSC‑X2‑GRFTIS‑X3-GQGTQVTVSS; wherein X1Is an amino acid sequence of 1-9 amino acids, X2Is an amino acid sequence of 1-43 amino acids, X3Is an amino acid sequence containing 1 to 38 amino acids. The invention screens the soluble single-domain antibody which can be expressed by pronucleus or eukaryon by the phage display technology, and has low production cost. The immunoadsorbent synthesized by coupling the obtained single-domain antibody to a solid phase carrier has high human IgE adsorption amount and good specificity; and the nonspecific adsorption of human IgG is low. The single domain antibody of the anti-human IgE protein provides a new choice for treating allergic diseases.
Description
Technical Field
The invention relates to the field of blood purification, in particular to a single domain antibody of anti-human IgE protein and application thereof.
Background
Hypersensitivity reactions are among the most ill people, affecting more than 25% of the world's total population. Among them, the type I allergy is also called immediate type anaphylaxis or anaphylaxis, and is an immune system disease caused by IgE binding to the high affinity receptor on the surface of mast cells and basophils, such as allergic asthma, allergic urticaria, allergic dermatitis, allergic rhinitis, and anaphylactic shock caused by penicillin, which are common in clinical practice. When an allergic reaction occurs, the concentration of IgE in the patient body reaches 10 times or more of the normal value, and particularly, the concentration of allergen-specific IgE can reach about 1000 times of the normal value (GOULD H J, SUTTON B J, etc. 2003).
At present, the drug therapy is the main method for treating allergic diseases, and mainly adopts immunosuppressant drugs. These pharmacotherapies have good therapeutic effects on accidental anaphylaxis, but have great side effects on chronic and intractable allergic diseases such as allergic asthma and the like which need to be taken for a long time. In recent years, the treatment of IgE-mediated type I allergy by blood adsorption purification has become a trend of choice. Specific IgE adsorption products were developed successively by germany, american, astronand, whirlwind and germany, thieneuss.
Single domain antibodies, which were found in 1993 in the humoral immune system of mammalian camelids (Camelidae), consist of a single variable region, 1 hinge region and 2 constant regions (CH2 and CH3) and have a molecular weight of 15 kD. The heavy chain antibody has the advantages of easy expression, good solubility, strong stability and the like, the binding affinity of the heavy chain antibody is close to that of the traditional antibody and can be as low as 100pmol/L (GOULD H J, SUTTON B J and the like, 2004), and the heavy chain antibody is widely applied to the fields of drug research and development and the like in recent years.
Disclosure of Invention
Therefore, in view of the disadvantages of the prior art, the present invention aims to provide a single domain antibody against human IgE protein and its application, which can specifically adsorb human IgE.
In order to achieve the purpose, the invention adopts the technical scheme that: a single domain antibody of anti-human IgE protein, the amino acid sequence of the single domain antibody has the following structural general formula:
MDVQLQESGGG-X1-LSC-X2-GRFTIS-X3-GQGTQVTVSS；
wherein X1Is an amino acid sequence of 1-9 amino acids, X2Is an amino acid sequence of 1-43 amino acids, X3Is an amino acid sequence containing 1 to 38 amino acids.
The amino acid sequence containing the invention has a structural general formula: MDVQLQESGGG-X1-LSC-X2-GRFTIS-X3the-GQGTQVTVSS single-domain antibodies can specifically adsorb human IgE, thereby achieving the purpose of clearing the IgE.
As a preferred embodiment of the single domain antibody, the amino acid sequence of the single domain antibody is shown in any one of SEQ ID NO. 1-SEQ ID NO. 10.
In addition, the present invention provides nucleotides encoding the single domain antibodies.
The invention also provides an immunoadsorbent, which contains the single-domain antibody.
As a preferred embodiment of the immunoadsorbent of the present invention, the immunoadsorbent is obtained by coupling the single domain antibody to a solid support.
In a preferred embodiment of the immunoadsorbent of the present invention, the solid phase support is at least one of chitosan microspheres, agarose microspheres, sephadex, resin, and cellulose microspheres.
In a preferred embodiment of the immunoadsorbent of the present invention, the solid phase carrier is preferably an agarose carrier.
The agarose carrier has the best biocompatibility due to the surface rich in hydroxyl, and is suitable for application in blood purification.
In a preferred embodiment of the immunoadsorbent of the present invention, the coupling is performed by epoxy activation.
The activated epoxy group is connected with the ethylenediamine and the glutaraldehyde, so that the length of the spacer arm is increased, the coupling amount can be ensured to the maximum extent, and the adsorption performance of the filler is improved.
As a preferred embodiment of the immunoadsorbent of the present invention, the coupling step is as follows:
(1) taking a solid phase carrier filler, adding an alkaline solution and an epichlorohydrin solution, cleaning the filler after reaction, and pumping to dry;
(2) adding a PBS solution and an ethylenediamine solution into the filler dried in the step (1), washing the filler after reaction, and drying;
(3) adding a PBS (phosphate buffer solution) solution and a glutaraldehyde solution into the pumped filler in the step (2), washing the filler by reverse osmosis water after reaction, and performing suction filtration;
(4) and (4) adding the single domain antibody and the carbonate buffer solution into the filter filler obtained in the step (3), washing the filter filler after reaction, and drying the filter filler.
In a preferred embodiment of the immunoadsorbent of the present invention, the alkaline solution is NaOH solution.
As a preferred embodiment of the immunoadsorbent of the present invention, the coupling comprises the following specific steps:
(1) taking a solid phase carrier filler, adding 2M NaOH with 3 times of volume, adding an epoxy chloropropane solution according to the volume ratio of 1: 0.3-1: 0.9, reacting at the temperature of 35-42 ℃ and the rpm of 100-200 for 1-4 h, cleaning the filler, and draining;
(2) adding an equal volume of PBS solution into the filler dried in the step (1), adding an ethylenediamine solution according to a volume ratio of 1: 0.3-1: 0.9, reacting at 35-42 ℃ at 100-200 rpm for 1-5 h, cleaning the filler, and drying;
(3) adding an isometric PBS solution into the filler drained in the step (2), adding a glutaraldehyde solution according to a volume ratio of 1: 1.0-1: 2.0, reacting at 35-42 ℃ at 100-200 rpm for 1-5 h, washing the filler by reverse osmosis water, and filtering;
(4) and (4) adding the single domain antibody protein solution into the filter filler obtained after the suction filtration in the step (3) according to the feeding amount of 10mg/mL, wherein the buffer solution is a carbonate buffer solution with the same volume as the filler, reacting for 1-5 h at the temperature of 25-35 ℃, washing the filler, and drying.
The invention also provides application of the single domain antibody in preparing a medicament for treating and/or preventing allergic diseases caused by IgE.
As a preferred embodiment of the use according to the present invention, the IgE-induced allergic disease comprises asthma, atopic dermatitis, allergic bronchopulmonary aspergillosis, allergic rhinitis, eczema or urticaria, food allergy.
The invention also provides application of the immunoadsorbent in the field of blood purification.
The invention has the beneficial effects that:
(1) the invention uses human IgEc epsilon 2-4 region immune alpaca expressed by eukaryon, collects peripheral blood mononuclear cell PBMC, amplifies variable region sequence by molecular biology method, constructs specific single domain antibody phage library. Then adding helper phage, coating human IgE, using ELISA plate for 3-4 rounds of panning, selecting strain with high specificity and sequencing to obtain gene sequence of single domain antibody. The constructed phage library has the capacity of 1013The method ensures the diversity of genes and lays a good foundation for subsequently screening sequences with better specificity.
(2) The invention uses colibacillus or yeast as host cell, to express single-domain antibody in vitro, to screen out soluble expressed strain. The advantages of Escherichia coli include high production speed and easy production control. Saccharomyces cerevisiae, a recognized safe eukaryotic microorganism which does not produce any toxin, does not secrete too many homologous proteins in the growth process, and can simplify the downstream processing process, can also be selected for expression. The yeast contains secretory organelles of endoplasmic reticulum, Golgi apparatus and other similar mammal cell systems, and can effectively form intramolecular disulfide bonds to produce glycosylated foreign protein.
(3) The invention screens the soluble single-domain antibody which can be expressed by pronucleus or eukaryon by the phage display technology, and has low production cost. The immunoadsorbent synthesized by coupling the obtained single-domain antibody to a solid phase carrier has high human IgE adsorption amount and good specificity; and the non-specific adsorption to human IgG is low, the IgG is the highest content in human immunoglobulin, about 7-16g/L, and the adsorption performance to the IgG is high, so that the site of the antibody combined with human IgE is shielded, and the adsorption performance of the IgE is reduced. The IgE immunoadsorbent of the invention provides a new choice for treating allergic diseases.
Drawings
FIG. 1: SDS-PAGE electrophoresis detection result chart of the single domain antibody prepared in the example 3; wherein the molecular weight of the single domain antibody is 14 KD.
Detailed Description
The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. All the technologies implemented based on the above-mentioned contents of the present invention should fall within the scope of the claims of the present application.
The experimental methods described in the following examples are all conventional methods unless otherwise specified; the kit biomaterials, if not specifically indicated, are commercially available.
Example 1 construction of an antibody library binding to human immunoglobulin E (IgE) alpaca phage
The alpaca is immunized by human IgEc epsilon 2-4 expressed by eukaryotic cells, PBMC of peripheral blood mononuclear cells is collected, RNA is extracted and is reversely transcribed into cDNA, after the cDNA is amplified by PCR, enzyme digestion is carried out by using hindIII and NotI, the cDNA is connected to a phagemid vector pHIAT-1, and the Escherichia coli TG1 is transformed to form an original phage library. Adding helper phage M13KO7 into TG1 strain grown to log phase, culturing overnight, centrifuging to collect supernatant, precipitating phagemid with PEG, re-suspending phage with PBS, filtering with 0.45um filter, and sterilizing to obtain phage VHH antibody library with a capacity of 4.8 × 1013。
EXAMPLE 2 screening of monoclonal phage antibodies
The titer determined by the helper phage M13KO7 was 1X 1012pfu/ml. Invasion with helper phage M13KO7Dyed TG 1.
(1) First screening: coating natural human IgE on an ELISA plate, keeping the temperature at 5 mu g/hole overnight at 4 ℃, and washing the plate for 3 times; dissolving BSA with PBST to a concentration of 3%, 200. mu.L/well, blocking at 37 ℃ for 2h, and washing the plate for 3 times; adding 100 mu L of phage library solution into each hole, incubating for 2h at 37 ℃, and washing the plate for 6 times; mu.L of glycine buffer (gly-HCl) was added to each well and shaken gently at room temperature for 10min, the eluate was aspirated and neutralized with 80. mu.L of Tris-HCl buffer. The 10. mu.L of eluate was titrated, the remainder was added to 5mL of TG1 strain grown to logarithmic phase, infected at 37 ℃ for 30min, added to preheated 2 XYT medium to a total volume of 10mL, at 37 ℃, 250rpm, for 30min, added to ampicillin to a final concentration of 100. mu.g/mL, and cultured for 2 h. Adding kanamycin to a final concentration of 70 μ g/mL, culturing overnight at 4 deg.C and 4000rpm, centrifuging for 15min, collecting supernatant, adding 5mL PEG/NaCl, ice-cooling for 1h, cooling at 4 deg.C and 9000rpm, centrifuging for 20min, and discarding supernatant. The centrifuge tube was placed upside down on absorbent paper and patted dry. Then, the mixture was resuspended in 1ml of PBS, centrifuged at 14000rpm for 10min at 4 ℃ to remove the residual cell debris, and the pellet was the phage antibody particles.
2) And (3) second screening: coating natural human IgE on an ELISA plate, washing the plate for 3 times, wherein the concentration of the natural human IgE is 1 mu g/hole; washing the plate 3 times with 3% skimmed milk powder; adding 100 mu L of phage library solution into each hole, incubating for 2h at 37 ℃, and washing the plate for 10 times; the rest of the operations are the same as the first round of screening.
3) And (3) third screening: coating natural human IgE on an ELISA plate, washing the plate for 3 times, wherein the concentration of the natural human IgE is 0.1 mu g/hole; blocking with 3% BSA, and washing the plate 3 times; adding 100 mu L of phage library solution into each hole, incubating for 2h at 37 ℃, and washing the plate for 15 times; the rest of the operations are the same as the first round of screening.
After three rounds of screening, the recovery rate of the anti-IgE phage single-chain antibody is 2.0X 10 of the first round-6Lifted to 7.5X 10 of the 3 rd wheel-3In total, 3750 times of enrichment is achieved.
4) Infecting TG1 bacteria with phage, spreading on a culture dish, randomly picking 44 monoclonals from the cultured plate, using the original library as negative control, ELISA detecting, and selecting OD450Those with a value 2.1 times greater than the original library were primary positive clones. A total of 41 positive clones were selected and sequenced. 41 positive clones were sent for testingAnd (4) sequencing. According to the sequencing result, 10 sequences are screened as alternative sequences after the repetitive sequences are removed, and the amino acid sequences of the 10 sequences are respectively shown as SEQ ID NO. 1-SEQ ID NO. 10.
Example 3 in vitro recombinant expression and antibody purification
The gene sequence with the amino acid sequence shown as SEQ ID NO.1 is transferred into a PET28 plasmid through enzyme cutting sites NcoI and XhoI, and is expressed in Escherichia coli BL21(DE 3). After the scale-up culture with LB medium containing 70. mu.g/mL kanamycin, the cells were collected, sonicated (5 s on, 10s off, 20min on), centrifuged at 10000rpm for 10min, and the supernatant was collected. Purifying by using a his label and a nickel ion chelating filler, and collecting an elution peak to obtain the alpaca single-domain antibody SD 1. The expression level of the single-domain antibody SD1 was determined to be 30 mg/L.
Example 4 in vitro recombinant expression and antibody purification
The gene sequence with the amino acid sequence shown as SEQ ID NO.2 is transferred into YCPIac33 plasmid through enzyme cutting sites BamH1 and EcoRI and expressed in Saccharomyces cerevisiae BJ 5457. The resulting culture was expanded in YPDA medium containing 50. mu.g/mL of ampicillin, and the supernatant was collected. Purifying by using a his label and a nickel ion chelating filler, and collecting an elution peak to obtain the alpaca single-domain antibody. The expression level of the single domain antibody was determined to be 20 mg/L.
Example 5 Synthesis of immunoadsorbent
(1) Taking 10mL of agarose (GE sepharose 4FF) filler, adding 30mL of 2M NaOH solution and 6mL of epichlorohydrin solution, reacting for 2.5h at 40 ℃ and 180rpm, pouring the filler into a sand core funnel, washing by 200mL of reverse osmosis water until no epoxy group remains in a cleaning solution, and vacuumizing for 3 min.
(2) 10mL of PBS solution and 8mL of ethylenediamine solution were added to the above filler, and the mixture was reacted at 37 ℃ and 180rpm for 4 hours. And (5) cleaning the filler by using reverse osmosis water, and performing suction filtration.
(3) 10mL of PBS solution and 12mL of glutaraldehyde were added to the above filler, and the mixture was reacted at 37 ℃ and 180rpm for 4 hours. And (5) cleaning the filler by using reverse osmosis water, and performing suction filtration.
(4) 10mL of a carbonate buffer (1.59 g of sodium carbonate, 2.94g of sodium bicarbonate, volume of 100mL, pH 8.3) containing 10mg/mL of a single domain antibody (prepared in example 3 and having the amino acid sequence shown in SEQ ID NO. 1) was added thereto, and the reaction was carried out at 25 ℃ and 180rpm for 4 hours. Cleaning the filler with reverse osmosis water, draining, adding 20% ethanol, and storing.
Example 6 detection of adsorption Performance of adsorbent to human immunoglobulin
The proteins SD3 and SD4 are obtained by expression and purification according to the method of example 3 (amino acid sequences are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4), immunoadsorbents are respectively prepared according to the method of example 5, the adsorption performance of the immunoadsorbent obtained by static adsorption detection is adopted, 1ml of the immunoadsorbent is mixed with 10ml of healthy human plasma (added with 1500IU/ml of eukaryotic IgE protein) and reacted for 40min by normal temperature oscillation, and an IgE kit (abcam) is used for measuring the IgE difference in the plasma before and after adsorption. The content of human IgG in the plasma before and after adsorption was measured by a biochemical analyzer (Meyer) to determine the amount of nonspecific adsorption.
TABLE 1 detection of adsorption Performance of adsorbents on human immunoglobulin
As can be seen from Table 1, the immunoadsorbent synthesized by coupling the obtained single-domain antibody to a solid phase carrier has high human IgE adsorption capacity and good specificity; and the non-specific adsorption to human IgG is low, the IgG is the highest content in human immunoglobulin, about 7-16g/L, and the adsorption performance to the IgG is high, so that the site of the antibody combined with human IgE is shielded, and the adsorption performance of the IgE is reduced.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
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<213> Artificial Synthesis
<400> 8
Met Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Ala Leu Asp Tyr
20 25 30
Tyr Thr Ile Asn Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
35 40 45
Val Ala Cys Ile Asn Ser Lys Tyr Gly Ala Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Met Arg Leu Val Thr Asn His Gln Ala Thr Cys Leu Asn Pro
100 105 110
Asp Arg Tyr Asp Leu Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 9
<211> 127
<212> PRT
<213> Artificial Synthesis
<400> 9
Met Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Thr Leu Ser Cys Thr Ser Ser Gly Thr Arg Leu Asp Tyr
20 25 30
Tyr Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
35 40 45
Val Ser Ala Ile Gly Arg Thr Asp Asp Arg Ile His Tyr Val Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Glu Asn Thr Val
65 70 75 80
Tyr Leu His Met Asn Asn Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Asp Arg Gly Tyr Ser Ser Phe Thr Pro Leu Ala Leu Gly
100 105 110
Arg Tyr Thr Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 10
<211> 128
<212> PRT
<213> Artificial Synthesis
<400> 10
Met Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Phe His Ser Asp Tyr
20 25 30
Trp Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly
35 40 45
Val Ser Cys Ile Thr Thr Lys Tyr Gly Ala Ser Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asp Asn Leu Lys Pro Glu Asp Thr Ala Leu Tyr Ser Cys
85 90 95
Ala Ala Met Arg Leu Ile Thr Asn His Gln Ala Thr Cys Leu Asn Pro
100 105 110
Ser Arg Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Claims (10)
1. A single domain antibody of anti-human IgE protein is characterized in that the amino acid sequence of the single domain antibody is shown as any one of SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO. 4.
2. Nucleotides encoding the single domain antibody of claim 1.
3. An immunoadsorbent comprising the single domain antibody of claim 1.
4. The immunoadsorbent of claim 3, wherein said immunoadsorbent is obtained by coupling said single domain antibody to a solid support.
5. The immunoadsorbent of claim 4, wherein said solid support is at least one of chitosan microspheres, agarose microspheres, sephadex, resin, cellulose microspheres.
6. The immunoadsorbent of claim 4, wherein said coupling is by epoxy activation.
7. The immunoadsorbent of claim 4, wherein the coupling step is as follows:
(1) taking a solid phase carrier filler, adding an alkaline solution and an epichlorohydrin solution, cleaning the filler after reaction, and pumping to dry;
(2) adding a PBS solution and an ethylenediamine solution into the filler dried in the step (1), washing the filler after reaction, and drying;
(3) adding a PBS (phosphate buffer solution) solution and a glutaraldehyde solution into the pumped filler in the step (2), washing the filler by reverse osmosis water after reaction, and performing suction filtration;
(4) and (4) adding the single domain antibody and the carbonate buffer solution into the filter filler obtained in the step (3), washing the filter filler after reaction, and drying the filter filler.
8. Use of a single domain antibody according to claim 1 for the manufacture of a medicament for the treatment and/or prevention of IgE-induced allergic disease.
9. The use according to claim 8, wherein the IgE-induced allergic disease comprises asthma, atopic dermatitis, allergic bronchopulmonary aspergillosis, allergic rhinitis, eczema or urticaria, food allergy.
10. Use of the immunoadsorbent of any one of claims 3 to 7 for the preparation of a product related to the field of blood purification.
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| CN112940090B (en) * | 2021-03-18 | 2023-02-24 | 广州康盛生物科技股份有限公司 | Protein A immunoadsorption medium capable of resisting irradiation sterilization and high-efficiency sterilization |
| CN113461823B (en) * | 2021-07-08 | 2022-12-20 | 广州康盛生物科技股份有限公司 | Single-domain antibody targeting human IgE, humanized single-domain antibody and application thereof |
| WO2024003376A1 (en) | 2022-07-01 | 2024-01-04 | Alk-Abelló A/S | Displacers of ige-fceri |
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