Recombinant single-chain antibodies against 25-oh vitamin d3 and preparation and use thereof

Abstract

The application provides a kind of anti 25-OH vitamin D3 Recombinant single-chain antibody and its preparation, application.The preparation method includes: S1, synthesis 25-OH-VD3-scFv single-chain antibody sequence, then insert into PTT5 carrier, and transfect single-chain antibody sequence containing PTT5 carrier to HEK293 cell in culture and collect cell culture supernatant;S2, the cell culture supernatant is used Ni Sepharose Excel filler to enrich the target protein, obtain elution component;S3, the elution component is separated and purified using Nuvia HP Q anion exchange filler, and obtains penetration component;S4, the pH of the penetration component is adjusted to 6.0±0.5, and then the conductivity of the penetration component is adjusted to 12.0±1.0 mS / cm using buffer and sodium chloride solution;S5, linear gradient elution is carried out using Capto SP ImpRes cation exchange filler for 40 column bed volumes, and then different elution peaks in the gradient are classified and collected to obtain anti 25-OH vitamin D3 Recombinant single-chain antibody.

Description

Technical Field

[0001] This invention relates to a recombinant single-chain antibody against 25-OH vitamin D3, its preparation, and its application. Background Technology

[0002] Single-chain antibody fragments (scFvs) are antibodies composed of variable regions of the heavy and light chains linked by a short peptide (linker) of 15-20 amino acids. scFvs retain their affinity for antigens well and possess characteristics such as small molecular weight, strong penetrability, and weak antigenicity. Therefore, their advantages include the ability to remove non-specific competitive surface proteins, resulting in clearer tumor imaging backgrounds; increased drug concentration through penetration into tumor tissue; low immunogenicity, eliminating rejection reactions in human anti-mouse antibodies; short half-life in vivo, facilitating detoxification and excretion; and easy binding to toxins or enzyme genes, enabling the direct acquisition of immunotoxins or enzyme-labeled antibodies. They have broad application prospects in antiviral therapy, tumor treatment, autoimmune disease treatment, and targeted drug therapy.

[0003] Single-chain antibodies are mainly synthesized artificially using genetic engineering techniques, and can be completed in both prokaryotic and eukaryotic expression systems.

[0004] However, due to the complex and diverse charge isoforms of the expressed proteins caused by intracellular processing and modification, they exhibit low purity and poor uniformity, which directly leads to poor protein stability, easy degradation, insufficient affinity, and is not conducive to later preservation and use. Summary of the Invention

[0005] This invention provides a recombinant single-chain antibody against 25-OH vitamin D3, its preparation and application, which can effectively solve the above-mentioned problems.

[0006] This invention is implemented as follows: This invention provides a method for preparing a recombinant single-chain antibody against 25-OH vitamin D3, comprising the following steps: S1, synthesize the 25-OH-VD3-scFv single-chain antibody sequence, insert it into the PTT5 vector, and transfect the PTT5 vector containing the single-chain antibody sequence into HEK293 cells, culture and collect the cell culture supernatant; S2, The cell culture supernatant is enriched with the target protein using Ni Sepharose Excel packing material to obtain the elution fraction; S3, the eluted components are separated and purified using Nuvia HP Q anion exchange packing material to obtain the breakthrough components; S4, adjust the pH of the permeation component to 6.0±0.5, and then adjust the conductivity of the permeation component to 12.0±1.0 mS / cm using buffer solution and sodium chloride solution; S5, using Capto SP ImpRes cation exchange packing material, linear gradient elution was performed for 40 column volumes. Then, different peaks within the gradient were classified and collected to obtain recombinant single-chain antibody against 25-OH vitamin D3.

[0007] The present invention further provides a recombinant single-chain antibody against 25-OH vitamin D3, wherein the recombinant single-chain antibody against 25-OH vitamin D3 is obtained according to the method described above.

[0008] The present invention further provides a recombinant single-chain antibody against 25-OH vitamin D3, the amino acid sequence of which is shown in SEQ ID No. 1.

[0009] The present invention further provides an application of the above-mentioned recombinant single-chain antibody against 25-OH vitamin D3 in the detection of 25-OH vitamin D3.

[0010] The beneficial effects of this invention are as follows: This invention uses nickel ion affinity chromatography and anion exchange chromatography to enrich and initially purify single-chain antibodies, thereby preparing single-chain antibodies with a purity of not less than 95%. Subsequently, strong cation exchange chromatography is used to further purify the charge isomers, removing unstable and low-affinity charge isomer components, thus finally preparing a highly stable and high-affinity recombinant single-chain antibody against 25-OH vitamin D3. The single-chain antibody sequence provided by this invention can efficiently and specifically bind to 25-OH VD3 and can be used for the development of detection methodologies for 25-OH VD3. Attached Figure Description

[0011] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0012] Figure 1 This invention provides a method for preparing a recombinant single-chain antibody against 25-OH vitamin D3.

[0013] Figure 2 This is a cation exchange chromatography pattern of a recombinant single-chain antibody against 25-OH vitamin D3 provided in an embodiment of the present invention.

[0014] Figure 3These are the HPLC detection results of single-chain antibody samples that failed the cation exchange chromatography of this invention.

[0015] Figure 4 From Figure 2 The HPLC results of the single-chain antibody sample with elution peak 1 collected in the sample.

[0016] Figure 5 From Figure 2 The HPLC detection results of the single-chain antibody sample with elution peak 2 collected in the sample.

[0017] Figure 6 From Figure 2 The HPLC detection results of the single-chain antibody sample with elution peak 3 collected in the sample.

[0018] Figure 7 This invention is from Figure 2 The purity SDS-PAGE images of the three elution peaks collected.

[0019] Figure 8 The images are SDS-PAGE graphs showing the stability of single-chain antibody samples that did not pass the cation exchange chromatography of this invention at 0, 1, 3, and 7 days at 37°C.

[0020] Figure 9 Corresponding to Figure 2 SDS-PAGE images of the stability results of single-chain antibody samples with three peak components at 37℃ for 0, 1, 3, and 7 days.

[0021] Figure 10 Corresponding to Figure 3 Medium single-chain antibodies and Figure 2 The image shows the ELISA results of the three peak components of the single-chain antibody sample.

[0022] Figure 11 Corresponding to Figure 2 The image shows the ELISA results of the stability of the three peak components of the single-chain antibody sample at 37℃. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Reference Figure 1 As shown, this embodiment of the invention provides a method for preparing a recombinant single-chain antibody against 25-OH vitamin D3, comprising the following steps: S1, synthesize the 25-OH-VD3-scFv single-chain antibody sequence, insert it into the PTT5 vector, and transfect the PTT5 vector containing the single-chain antibody sequence into HEK293 cells, culture and collect the cell culture supernatant; S2, The cell culture supernatant is enriched with the target protein using Ni Sepharose Excel packing material to obtain the elution fraction; S3, the eluted components are separated and purified using Nuvia HP Q anion exchange packing to obtain the breakthrough components; S4, adjust the pH of the permeation component to 6.0±0.5, and then adjust the conductivity of the permeation component to 12.0±1.0 mS / cm using buffer solution and sodium chloride solution; S5, using Capto SP ImpRes cation exchange packing material, linear gradient elution was performed for 40 column volumes. Then, different peaks within the gradient were classified and collected to obtain recombinant single-chain antibody against 25-OH vitamin D3.

[0025] As a further improvement, in step S1, the steps of synthesizing the 25-OH-VD3-scFv single-chain antibody sequence, inserting it into the PTT5 vector, and transfecting the PTT5 vector containing the single-chain antibody sequence into HEK293 cells specifically include: The 25-OH-VD3-scFv single-chain antibody sequence was synthesized and then inserted into the PTT5 vector according to FreeStyle. TMThe 293 Expression System User Manual describes the transfection of the PTT5 vector containing the single-chain antibody sequence into HEK293 cells. In other embodiments, the 25-OH-VD-scFv single-chain antibody sequence can be obtained using other plasmid construction, cell transfection, culture methods, or synthesis methods known in the art, which will not be elaborated here.

[0026] The sequence of the 25-OH-VD3-scFv single-chain antibody is SEQ ID No. 1: QVRIEESHPTIVRPSQHLGITCTVSFFSPDNYGVGSVHQAPSKAIEELWGIDRTGWRWYNRAIKSRILITRDTSKSMVTIILSFVRIENTAVFYCARVWYSPNFFKFGYFDTWGPMILVKVASGGGGSGGGGSGG GGSQATITQWTSTSKAIGRSVSITCSGSYSNIGYDNYVSQFPVVPSKAPKIIIYDTITRASPVPKRFSGSRSGDTKTITLNSIQTEKSADSYCWSFDSTDFGIFGAGTRITWIGINDIFEAQKIEWHETGHHHHHHH As a further improvement, in step S2, the specific steps of enriching the target protein in the cell culture supernatant using Ni SepharoseExcel packing material to obtain the elution components include: The cell culture supernatant was used to enrich the target protein and obtain the elution fraction by controlling the linear flow rate of 300-600 cm / h with Ni Sepharose Excel packing material, wherein the particle size of the Ni Sepharose Excel packing material is 90±5 micrometers.

[0027] As a further improvement, in step S3, the step of separating and purifying the eluted components using Bio Rad Nuvia HP Q anion exchange packing to obtain the breakthrough components includes: The eluted fraction is separated and purified using Nuvia HP Q anion exchange chromatography at a controlled linear flow rate of 300-600 cm / h to obtain the permeate fraction. The Nuvia HP Q anion exchange chromatography packing has a particle size of ≤90 μm, preferably 40 ± 5 μm for Bio Rad Nuvia HP Q packing. In steps S2 and S3, nickel affinity chromatography and anion exchange chromatography are used to enrich and initially purify the single-chain antibody, thereby preparing a single-chain antibody with a purity of not less than 95% in the permeate fraction.

[0028] As a further improvement, in step S4, the step of adjusting the pH of the permeation component from 3.5 to 6.0 ± 0.5, and then adjusting the conductivity of the permeation component to 12.0 ± 1.0 mS / cm using buffer solution and sodium chloride solution includes: The pH of the permeation component was adjusted to 6.0 ± 0.5 using 20 mM acetate buffer, and the conductivity of the permeation component was adjusted to 12.0 ± 1.0 mS / cm using 50 mM disodium hydrogen phosphate-citric acid buffer and 1.0 M sodium chloride solution.

[0029] As a further improvement, in step S5, the step of linear gradient elution of 40 column volumes using Capto SP ImpRes cation exchange packing includes: Capto SP ImpRes cation exchange media were used for linear gradient elution of 40 column volumes at a linear flow rate of 150-300 cm / h. The Capto SP ImpRes cation exchange media had a particle size of 40±5 μm and a column height of 200±20 mm. The gradient elution buffer was a 50 mM disodium hydrogen phosphate-citrate buffer with a pH of 6.0. The NaCl gradient range in the gradient elution was between 100 mM and 500 mM.

[0030] As a further improvement, in step S5, the step of classifying and collecting different peaks within the gradient includes: classifying and collecting different peaks within the gradient according to A280, thereby obtaining single-chain antibodies with a purity of not less than 98%.

[0031] It is understood that in step S5 of this invention, the charge isomers are further purified by strong cation exchange chromatography to remove the unstable and low-affinity charge isomer components, thereby preparing a highly stable and high-affinity anti-25-OH VD3 single-chain antibody. Through multiple experiments, it has been verified that when the pH, conductivity, particle size, and column height of the cation exchange packing exceed the set values, the separation effect decreases, and the unstable and low-affinity charge isomer components cannot be removed.

[0032] The present invention further provides a recombinant single-chain antibody against 25-OH vitamin D3, wherein the recombinant single-chain antibody against 25-OH vitamin D3 is obtained according to the method described above.

[0033] The present invention further provides a recombinant single-chain antibody against 25-OH vitamin D3, the amino acid sequence of which is shown in SEQ ID No. 1.

[0034] This invention further provides an application of the above-mentioned recombinant single-chain antibody against 25-OH vitamin D3 in the detection of 25-OH vitamin D3. The single-chain antibody sequence provided by this invention can bind to 25-OHVD3 efficiently and specifically, and can be used for the development of methodologies for the detection of 25-OHVD3.

[0035] Example 1 The above-mentioned single-chain antibody sequence was synthesized by Shanghai Sangon Biotech (Shanghai) Co., Ltd., and inserted into the PTT5 vector according to Thermo Fisher Scientific's FreeStyle protocol. TM Following the instructions in the 293 Expression System User Manual, transfect the vector containing the single-chain antibody sequence into 293-F cells, culture them, and collect the cell culture supernatant. Take the cell supernatant from HEK293 cells and enrich the target protein using Ni Sepharose Excel (Cytiva, particle size: 90 μm, linear flow rate 300-600 cm / h), finally collecting the elution fraction. Take the elution fraction from Ni Sepharose Excel chromatography and purify it using Nuvia HP Q (Bio Rad, particle size: 40 μm, linear flow rate 300-600 cm / h), collecting the breakthrough fraction; the final purity can reach over 90%. Take the breakthrough fraction from Nuvia HP Q chromatography, adjust the pH to 6.0 with 20 mM acetate buffer (pH 3.5), then adjust the conductivity to 12.0 mS / cm using 50 mM disodium hydrogen phosphate-citrate buffer (1.0 M NaCl, pH 6.0), and then use Capto SP... ImpRes (Cytiva, particle size: 40 μm, column height: 200 ± 20 mm, linear flow rate 150-300 cm / h) was used for linear gradient elution of 40 column volumes. The different peaks within the gradient were determined based on A280 (see [link to relevant documentation]). Figure 2 (As shown) Collect them by category.

[0036] from Figure 2 As can be seen, there are three elution peaks: elution peak 1, elution peak 2, and elution peak 3. Figure 2 The three elution peaks in the figure represent the results of separating the charge isomers in the expressed single-chain antibody using the method of this invention. The three peaks shown correspond to the three charge isomers.

[0037] Test example: The HPLC detection results for the three charge isomers corresponding to the three separated peaks are as follows: Figure 4-6 As shown. Figure 4-6 The HPLC detection results correspond to elution peak 1, elution peak 2, and elution peak 3, respectively. Figure 3These are the HPLC detection results for single-chain antibody samples that failed the cation exchange chromatography of this invention. As can be seen from 3-6, Figure 3 The HPLC chromatograms of single-chain antibody samples that did not utilize this invention were messy and heterogeneous. In contrast, the samples obtained by this invention appeared homogeneous, and different retention times were observed for different peak components, indicating that the charge isomers were well separated and collected.

[0038] from Figure 7 As can be seen, the purity analysis of the purified components using SDS-PAGE indicates that the final sample purity can reach over 98%. From... Figure 8 It can be seen that single-chain antibody samples that failed the cationic chromatography of this invention exhibited significant degradation at 37°C, ultimately showing two bands. From... Figure 9 It can be seen that no degradation was found in the single-chain antibody samples obtained by this invention, and the results are clearly comparable.

[0039] Please see Figure 10-11 The purification results were analyzed by ELISA to compare the affinity. The results showed that the single-chain antibody samples obtained by this invention all had high affinity, and there were also significant differences between different elution components, which helps to screen the optimal sample. In addition, the stability at 37℃ did not decrease significantly, indicating good stability and no degradation.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a recombinant single-chain antibody against 25-OH vitamin D3, characterized in that, Includes the following steps: S1. Prepare the 25-OH-VD3-scFv single-chain antibody sequence, insert it into the PTT5 vector, and transfect the PTT5 vector containing the single-chain antibody sequence into HEK293 cells. Culture and collect the cell culture supernatant. The amino acid sequence of the 25-OH-VD3-scFv single-chain antibody is shown in SEQ ID No.

1. S2, The cell culture supernatant is enriched with the target protein using Ni Sepharose Excel packing material to obtain the elution fraction; S3, the eluted components are separated and purified using Nuvia HP Q anion exchange packing to obtain the breakthrough components; S4, adjust the pH of the permeation component to 6.0±0.5, and then adjust the conductivity of the permeation component to 12.0±1.0 mS / cm using buffer solution and sodium chloride solution; S5, using Capto SP ImpRes cation exchange packing material, linear gradient elution was performed for 40 column volumes. Then, different elution peaks within the gradient were classified and collected to obtain recombinant single-chain antibody against 25-OH vitamin D3.

2. The method for preparing the recombinant single-chain antibody against 25-OH vitamin D3 as described in claim 1, characterized in that, In step S1, the steps of preparing the 25-OH-VD3-scFv single-chain antibody sequence, inserting it into the PTT5 vector, and transfecting the PTT5 vector containing the single-chain antibody sequence into HEK293 cells include: The 25-OH-VD3-scFv single-chain antibody sequence was synthesized and then inserted into the PTT5 vector according to FreeStyle. TM 293 Expression System User Manual: Transfect the PTT5 vector containing the single-chain antibody sequence into HEK293 cells.

3. The method for preparing the recombinant single-chain antibody against 25-OH vitamin D3 as described in claim 1, characterized in that, In step S2, the step of enriching the target protein in the cell culture supernatant using Ni Sepharose Excel packing material to obtain the elution fraction includes: The cell culture supernatant was used to enrich the target protein and obtain the elution fraction by controlling the linear flow rate of 300-600 cm / h with Ni Sepharose Excel packing material, wherein the particle size of the Ni Sepharose Excel packing material is 90±5 micrometers.

4. The method for preparing the recombinant single-chain antibody against 25-OH vitamin D3 as described in claim 1, characterized in that, In step S3, the step of separating and purifying the eluted fraction using Nuvia HP Q anion exchange packing to obtain the breakthrough fraction includes: The eluted components were separated and purified using Nuvia HP Q anion exchange packing material at a controlled linear flow rate of 300-600 cm / h to obtain the breakthrough components, wherein the particle size of the Nuvia HP Q anion exchange packing material was less than or equal to 90 micrometers.

5. The method for preparing the recombinant single-chain antibody against 25-OH vitamin D3 as described in claim 1, characterized in that, In step S4, the steps of adjusting the pH of the permeation component from 3.5 to 6.0±0.5, and then adjusting the conductivity of the permeation component to 12.0±1.0 mS / cm using buffer solution and sodium chloride solution include: The pH of the permeation component was adjusted to 6.0 ± 0.5 using 20 mM acetate buffer, and the conductivity of the permeation component was adjusted to 12.0 ± 1.0 mS / cm using 50 mM disodium hydrogen phosphate-citric acid buffer and 1.0 M sodium chloride solution.

6. The method for preparing the recombinant single-chain antibody against 25-OH vitamin D3 as described in claim 1, characterized in that, In step S5, the step of performing linear gradient elution of 40 column volumes using Capto SP ImpRes cation exchange packing includes: Forty column volumes of linear gradient elution were performed using Capto SP ImpRes cation exchange packing material at a linear flow rate of 150-300 cm / h. The Capto SP ImpRes cation exchange packing material had a particle size of 40±5 μm and a column height of 200±20 mm. The gradient elution buffer was a 50 mM disodium hydrogen phosphate-citrate buffer with a pH of 6.

0. The NaCl gradient range in the gradient elution was between 100 mM and 500 mM.

7. The method for preparing the recombinant single-chain antibody against 25-OH vitamin D3 as described in claim 1, characterized in that, In step S5, the step of classifying and collecting different elution peaks within the gradient includes: Different peaks within the gradient are classified and collected based on A280.

8. A recombinant single-chain antibody against 25-OH vitamin D3, characterized in that, The amino acid sequence of the recombinant single-chain antibody against 25-OH vitamin D3 is shown in SEQ ID No.

1.

9. The application of the recombinant single-chain antibody against 25-OH vitamin D3 as described in claim 8 in the detection of 25-OH vitamin D3, wherein the detection is not for diagnostic purposes.

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