CN113527502B - Nano antibody recombinant protein for treating rheumatoid arthritis - Google Patents

Abstract

The invention discloses a nano antibody recombinant protein for treating rheumatoid arthritis, which belongs to the field of genetic engineering, and is characterized in that the nano antibody recombinant protein is formed by fusing sequences of camel source anti-TNF-alpha and anti-human serum protein nano antibodies together through flexible connecting peptide, and the amino acid sequence of the nano antibody recombinant protein is shown as SEQ ID No.1 or comprises an amino acid sequence which has at least 80%, 85%, 90%, 95%, 98% or 99% of identity with the SEQ ID No.1 and has the same function; the invention provides an anti-TNF-alpha nano antibody recombinant protein which can be used for preventing or treating rheumatoid arthritis and has the advantages of safety, reliability and strong specificity.

Description

A nanobody recombinant protein for the treatment of rheumatoid arthritis

technical field

The invention relates to the technical field of genetic engineering, in particular to a nanobody recombinant protein for treating rheumatoid arthritis.

Background technique

Rheumatoid Arthritis (RA) is a chronic inflammatory and destructive joint disease that affects 0.5-1% of the population in the industrialized world and often results in significant disability that reduces the patient's quality of life.

Angiogenesis in the synovium of patients with RA is considered an important early step in pathogenesis and disease persistence (Taylor, 2002). As in neoplastic disease, angiogenesis promotes dilation of the synovium (Walsh et al., 1998). Vascular growth likely contributes to the proliferation of inflammatory synovial pannus and to the influx of inflammatory leukocytes into the synovial tissue. The synovium of patients with RA contains increased amounts of fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) (Koch, 2003). Serum VEGF concentrations correlate with disease activity and decrease when synovitis can be successfully suppressed by therapy (Taylor, 2002).

Tumor necrosis factor α (tumor necrosis factor alpha, TNFα) is a multifunctional cytokine that participates in important physiological processes such as cell apoptosis and survival, inflammatory response, and immune response. It plays an important role in the occurrence and development of diseases such as Crohn's disease and psoriatic disease. Therefore, TNFα is considered to be a very important drug development target for the treatment of the above-mentioned related diseases. Infliximab (infliximab), adalimumab (adalimumab), golimumab (golimumab) are therapeutic anti-TNFα antibodies, which have shown significant efficacy and safety in the treatment of RA and other diseases. It has become one of the best-selling blockbuster drugs in the world, and the research and development of other therapeutic anti-TNFα antibodies are still hot.

In the early 1990s, the Hamers research group found that camelids can simultaneously process two different types of immunoglobulins, one is a classic antibody consisting of a light chain with two domains and a heavy chain with four domains, and the other One is an antibody that naturally lacks the light chain. This antibody only contains a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not like an artificially engineered single-chain antibody fragment (scFv) It is easy to stick to each other and even gather into blocks. More importantly, the independently cloned and expressed VHH structure has comparable structural stability and antigen-binding activity to the original heavy chain antibody, and is currently the smallest unit known to bind the target antigen. VHH crystals are 2.5nm in length, 4nm in length, and have a molecular weight of only 15KDa, so they are also called nanobodies (Nanobody, Nb).

Contents of the invention

The object of the present invention is to provide a kind of nanobody recombinant protein that is used for the treatment of rheumatoid arthritis, to solve the problem that the above-mentioned prior art exists, the anti-TNF-alpha and anti-human serum albumin (HSA) that derive from camel The sequences of the nanobodies are fused together via a flexible linker peptide to form an anti-TNF-α nanobody (TNF50) with stronger biological activity.

To achieve the above object, the present invention provides the following scheme:

The present invention provides a nanobody recombinant protein, in which the sequences of camel-derived anti-TNF-α and anti-human serum protein nanobodies are fused together through a flexible linker peptide to form a fusion antibody, the amino acid sequence of which is shown in SEQ ID No.1 Or comprise an amino acid sequence having at least 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID No. 1 and having the same function.

The present invention also provides a nucleotide encoding the above-mentioned Nanobody recombinant protein.

Further, its nucleotide sequence is shown in SEQ ID No.2.

The present invention also provides an expression vector, comprising the above-mentioned nucleotides or having at least 80%, 85%, 90%, 95%, 98% or 99% identity with SEQ ID No.2 and encoding the same protein Nucleotide sequence.

The present invention also provides a gene engineering expression bacterium, including the above-mentioned vector.

Further, the genetically engineered bacterium is Escherichia coli Rosetta (DE3).

The present invention also provides a method for constructing the above-mentioned genetic engineering expression bacteria, in which the above-mentioned nucleotides are connected to pET-22b or pET-28a Escherichia coli expression plasmids, the above-mentioned expression vectors are constructed, and transformed into the Escherichia coli In the process, the genetically engineered expression bacteria expressing the nanobody recombinant protein were screened.

The present invention also provides a pharmaceutical composition, comprising the above-mentioned Nanobody recombinant protein and at least one pharmaceutically acceptable excipient.

Further, at least one pharmaceutically acceptable adjuvant is also included.

Pharmaceutical compositions can contain any number of excipients. Excipients that can be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersion or suspension aids, stabilizers, colorants, flavoring agents, coatings, disintegrants, lubricants , sweeteners, preservatives, isotonic agents, and combinations thereof.

The primary vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline or artificial cerebrospinal fluid, which may be supplemented with other materials commonly used in injections. For example, the vehicle or carrier can be neutral buffered saline or saline mixed with serum albumin. Other exemplary pharmaceutical compositions comprise Tris buffer, or acetate buffer, which may also contain sorbitol or a suitable substitute thereof. In one embodiment of the invention, the composition can be prepared for storage by mixing selected components with the desired purity and any formulations, either in lyophilized or aqueous solution. Additionally, therapeutic compositions can be formulated as lyophilizates using suitable excipients such as sucrose.

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (eg, by injection or bolus injection). Depending on the route of administration, the active molecule can be encapsulated in a material that protects it from acids and other natural conditions that could inactivate it.

The present invention also provides an application of the above-mentioned nanobody recombinant protein, the above-mentioned nucleotide, the above-mentioned expression vector, the above-mentioned genetically engineered expression bacteria or the above-mentioned pharmaceutical composition in the preparation of a drug for treating rheumatoid arthritis.

The invention discloses the following technical effects:

The invention provides an anti-TNF-α nanometer antibody recombinant protein, which can be used for the prevention or treatment of rheumatoid arthritis, and has the advantages of safety, reliability and strong specificity.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

Fig. 1 is two kinds of expression plasmids of pET22-TNF50 and pET28-TNF50 constructed by the present invention;

Fig. 2 is the expression verification of TNF50 of the present invention;

Fig. 3 is the verification of TNF50 protein sequence of the present invention;

Fig. 4 is the optimization of culture medium of the present invention;

Fig. 5 is the detection of TNF50 activity in the present invention.

detailed description

Various exemplary embodiments of the present invention will now be described in detail. The detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features and embodiments of the present invention.

It should be understood that the terminology described in the present invention is only used to describe specific embodiments, and is not used to limit the present invention. In addition, regarding the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents are described. In case of conflict with any incorporated document, the contents of this specification control.

It will be apparent to those skilled in the art that various modifications and changes can be made in the specific embodiments of the present invention described herein without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the present invention. The description and examples of the invention are illustrative only.

As used herein, "comprising", "comprising", "having", "comprising" and so on are all open terms, meaning including but not limited to.

Example 1

One, design and construct anti-TNF-α nanobody and its genetic engineering expression bacteria, the steps are as follows:

1. Obtaining TNF50 Nucleic Acid Coding Sequence

(1) The sequences of the camel-derived anti-TNF-α and anti-human serum albumin (HSA) nanobodies were fused together through a flexible linker peptide to obtain an anti-TNF-α nanobody with stronger biological activity (TNF50 )sequence;

(2) Translate the obtained sequence into a nucleotide sequence according to the standard amino acid codon table;

(3) Synthesizing the coding nucleotide sequence of TNF50.

2. Synthesis of Primers

A total of 4 primers were designed and synthesized, as follows:

EC01: ATCGAAGGTCGTGAAGTTCAACTTGTTGAATCAGG;

EC02: GCGAGGAGCTCATTATGATGAAACAGTAACAAGAG;

EC03: AACTTCACGACCTTCGATGCCGCTGCTGTGATGATG;

EC04: AGCTACATCACTCAGACTTCAGCAACCGCACCTGTG.

3. Construction of TNF50 prokaryotic expression vector

In order to construct the prokaryotic expression plasmids pET22-TNF50 and pET28-TNF50 of TNF50, we first synthesized the required primers and named them EC01, EC02, EC03 and EC04 respectively, wherein EC01 and EC04 contain the homologous sequence Xa.

(1) Using the plasmid pET28a as a template, the fragment PT7-Xa was amplified by primers EC03 and EC04;

(2) Using the synthesized TNF50 nucleotide sequence as a template, the fragment Xa-TNF50 is amplified by primers ECO1 and ECO2;

(3) Using the fragments PT7-Xa and Xa-TNF50 as common templates, the fragment PT7-TNF50 is amplified by primers EC02 and EC04;

(PT7-TNF50 contains 6 important elements—important promoter PT7, NcoI restriction enzyme recognition site, 6xHistag tag sequence, protease Xa recognition site, target gene sequence TNF50, SacI restriction enzyme recognition site)

(4) Treat fragment PT7-TNF50, plasmids pET22b and pET28a with restriction enzymes NcoI and SacI, respectively, for 1 hour;

(5) Purify the treated fragments and linearized plasmids through agarose gel, and quantify them with a thermo ultraviolet nucleic acid protein quantifier;

(6) According to the molar ratio of fragments:plasmid=1:1 and 3:1, the ligation reaction mixture was mixed, and the mixture was placed in the 16-type reaction overnight;

(7) Transform the ligated mixture into competent cells of E. coli DH5α;

(8) Pick dozens of single clones from the transformation plate with white colonies and carry out colony PCR;

(9) Part of the positive clones obtained in step (8) were selected and inoculated into a test tube containing 5 mL of LB medium, and 5 μL of ampicillin sodium salt solution (pET22b) or kanamycin sulfate solution (pET28a) was added, placed in Cultivate in a shaker, 37°C, 220rpm, overnight;

(10) extract plasmid;

(11) Treat each tube of plasmids extracted with restriction enzymes EcoRI and EcoRV and perform nucleic acid electrophoresis;

(12) Select the partial positive clones obtained in step (11) and send them to Beijing Jinweizhi Biotechnology Co., Ltd. for sequencing; transfer the correctly sequenced plasmids into Escherichia coli Rosetta (DE3);

(The primers used in the sequencing reaction are universal primers-T7promoter and T7terminator)

(13) Select one positive clone from each of the two expression plasmids to inoculate bacteria according to step (9). When the OD600 is 0.6, add an inducer (IPTG) to a final concentration of 1 mM, and then place it on a shaker to continue culturing for 7 hours, 30 ℃, 220rpm;

(14) Collect 1 mL of each of the two bacterial cells containing expression plasmids into 1.5 mL EP tubes, centrifuge at 12000 rpm for 2 min at room temperature;

(15) Discard the supernatant, add 1mL PBS, gently blow suspension, and repeat the centrifugation in step (14);

(16) repeat the rinsing operation of step (15);

(17) Carefully suck up the supernatant, add 45 μL PBS and 5 μL 5X protein loading buffer, and boil in boiling water for 10 minutes;

(18) Load 20 μL of the sample in step (17) into a pre-prepared 15% polyacrylamide gel, and start electrophoresis; electrophoresis conditions: run stacking gel at 70V, and run separating gel at 140V;

(19) Stop the electrophoresis, cut the separating gel, and do Coomassie brilliant blue color development on one piece of gel; the other piece is subjected to western-blot detection and analysis according to the following steps;

4. Purification and quantification of TNF50.

5. Interaction between TNF50 and human TNF-α. The interaction between TNF50 and its target—human TNF-α can be detected by immunoblotting.

6. TNF50 250mL shake flask culture optimization

7. Detection of biological activity of TNF50.

Carrageenan is a mucopolysaccharide in the cell wall of red algae. It is an anionic linear polymer molecule composed of 1,3α-1,4β galactose. It is divided into κ(1) according to the sulfur contained in each monomer , ι(2) and λ(3) three configurations, wherein the first two will gather into a helical structure to form hard or soft jelly, respectively, and the carrageenan of the λ configuration will not gather into a helical structure, in solution It is a non-condensed state. Symptoms of inflammation such as edema, pain, and erythema appeared immediately after subcutaneous injection of carrageenan in animals. Its molecular mechanism is that carrageenan induces cells to produce stress molecules such as histamine, bradykinin, tachykinin, reactive oxygen species and nitric oxide, which can recruit neutrophils to the injection site, and then the cells can secrete Pro-inflammatory factors, including TNF-α, induce inflammation. In the present invention, the λ carrageenan is used to induce mouse ankle swelling, and the biological activity of the prepared TNF50 is evaluated by measuring the swelling degree of the ankle. The specific method is:

(1) 40 SPF grade KM mice, about 30 grams each, with half male and half female in each group, were reared separately;

(2) After one week of adaptation to the environment, the mice were evenly divided into 5 groups by weight, each group being half male and half male;

(3) Inject 200 μL of PBS intraperitoneally to the 1st and 2nd mice, inject 50 μL of 120 mg/mL aspirin solution into the 3rd group, inject 200 μL of TNF50 PBS solution containing 5 mg/mL into the 4th mouse, and inject the 5th mouse 200 μL of TNF50 PBS solution containing 10 mg/mL;

(3) Half an hour later, inject 25 μL of PBS solution into the ankle of the right hind leg of the first group of mice, and inject 25 μL of PBS solution containing 1% lambda carrageenan into the ankle of the right hind leg of each of the remaining mice;

(4) Measure the diameter of the mouse's hind leg ankle with a vernier caliper every 1 hour after the injection, and each measurement is repeated twice;

(5) Calculate the mouse toe swelling degree for each measurement, and draw the changes of each group into the same "swelling degree-time" table. After the experiment, the experimental site was taken for slice analysis.

result:

Fig. 1 shows two expression plasmids pET22-TNF50 and pET28-TNF50 constructed in the present invention. 10 transformants in the pET22-TNF50 transformation plate and 30 transformants in the pET28-TNF50 transformation plate were randomly selected for PCR verification, and the blank agar near the transformants was used as a negative control template, and 30 transformants were obtained All fragments with the same size as the target band were amplified, but no fragment was changed in the negative (Fig. 1a). Further, 5 transformants of pET22-TNF50 and 5 transformants of pET28-TNF50 verified by PCR were selected for culture and plasmid extraction, and then two restriction enzymes EcoRI and EcoRV were used for restriction map analysis. The results showed that the transformants from 10 transformants All the plasmids excised the target bands belonging to the positive clones. Finally, three of each type of transformants were selected for DNA sequencing, and the results showed that the sequences were consistent with the target sequence (Fig. 1b).

Fig. 2 is the expression verification of TNF50 of the present invention. Since the sequence was not optimized according to the codon preference of Escherichia coli, Escherichia coli Rosetta (DE3) was used as the expression host of TNF50. After the constructed plasmids pET22-TNF50 and pET28-TNF50 were transformed into Rosetta (DE3), they were induced and cultured respectively, and the expression of the target protein was detected by SDS-PAGE and Western-blot. The results showed that both plasmids were successfully expressed in their expression hosts (Fig. 2a, Fig. 2b).

Fig. 3 is the verification of the TNF50 protein sequence of the present invention. In order to further confirm the correctness of the sequence, mass spectrometry identification was carried out. A single band of the target sequence was obtained by solid-phase metal affinity purification and SDS-PAGE. Then, the strip was excised and sent to the Analysis and Testing Center of the International Institute of Biomedicine (Tianjin, China) for protein spectrum analysis. As a result, a total of six peptides matching the target sequence were detected, with a coverage rate of 64%), which basically proved that the detected protein was the target protein.

Purification and quantification of TNF50 protein. Since the N-terminal of TNF50 has a His tag, it was purified by solid-phase Co ²⁺ affinity chromatography. Through the optimization of washing conditions, the optimal concentration of imidazole in the washing solution was finally determined to be 70mM, and the collected elution peaks were buffer exchanged and concentrated with ultrafiltration tubes, and SDS-PAGE electrophoresis was performed, and its purity was detected to be 95 %, the protein concentration after concentration was determined quantitatively by Bradford and BCA kits to be 10 mg/mL.

Fig. 4 is the optimization of the culture medium of the present invention. Nine kinds of Escherichia coli medium were selected to cultivate TNF50 expression host bacteria to select the most suitable medium. Inoculate the same initial bacterial solution in 9 media, and then culture under the same conditions. Then, cells in 1 mL of each culture solution were collected for lysis and SDS-PAGE. The results showed that the presence of TNF50 was hardly detected in M9 (Fig. 4a), so it was discarded in subsequent data processing. During data processing, the absolute expression of TNF50 obtained in the most commonly used Escherichia coli medium (i.e. High-saltLB) and the relative expression of the unit bacterium were used as a reference, and the two results obtained in the remaining medium were divided by Corresponding results obtained in High-salt LB. The results showed that the SB medium was the best in the comparison of the two indicators (Figure 4b, Figure 4c), so the SB medium was used in the optimization of the next culture conditions.

Fig. 5 is the detection of TNF50 activity in the present invention. Firstly, the in vitro binding activity of TNF50 and human TNFα was detected by immunoblotting; then, the antagonistic activity of TNF50 and mouse TNFα in vivo was detected by λ carrageenan-induced mouse ankle swelling.

Human TNF-[alpha] was developed in three lanes by SDS-PAGE in immunoblotting. Next, transfer these three protein bands to NC membranes. Then cut out the three bands on the membrane and treat them separately: Band 1. After blocking, incubate with anti-His tag primary antibody, and then incubate with secondary antibody; Band 2. After blocking, incubate with TNF50, then incubate with anti-His tag Incubate with tag primary antibody, and finally incubate with secondary antibody; band 3. Rinse with guanidine hydrochloride gradient (this can make the protein refold on the membrane, so that the amino acids that are linearly far away from the peptide chain are close to each other in three-dimensional space to prevent antigen expression The position was destroyed during SDS-PAGE electrophoresis), the concentration of guanidine hydrochloride was reduced from 6M to 0M, and the concentration was reduced by half each time until it fell below 0.5, and then washed with PBS, then blocked, then incubated with TNF50, and then the same anti -Incubate with His tag primary antibody, and finally incubate with secondary antibody. Finally, the three processed films were scanned by infrared simultaneously. The results showed that there was no colored band in band 1, but there were colored bands in bands 2 and 3 ( FIG. 5 a ), indicating that TNF50 can bind to human TNFα in vitro.

When conducting animal experiments, we choose two ways of intraperitoneal administration and leg administration. Group 1 (pbs+pbs): Mice were injected intraperitoneally with 200 μL of sterile pbs, and half an hour later, the right leg was injected with 25 μL of sterile pbs; Group 2 (pbs+pbs): mice were injected intraperitoneally with 200 μL of sterile pbs, half an hour later Inject 25 μL of sterile 1% carrageenan solution into the hind right leg; Group 3 (pbs+pbs): intraperitoneally inject 200 μL of sterile low-dose TNF50 (5 mg/mL), and half an hour later inject sterile 1% carrageenan solution into the right leg 25 μL of carrageenan solution; Group 4 (pbs+pbs): 200 μL of sterile high-dose TNF50 (10 mg/mL) was injected intraperitoneally into mice, and 25 μL of sterile 1% carrageenan solution was injected into the right leg half an hour later; Group 5 (pbs+pbs): 50 μL of sterile aspirin solution (ASP, 120 mg/mL) was injected intraperitoneally into the mouse, and 25 μL of sterile 1% carrageenan solution was injected into the right leg half an hour later. The diameter of the swollen part of the leg was measured every 1 hour after administration in each group, and then the experimental results were processed to finally obtain corresponding statistical tables and statistical graphs (Fig. 5b, Fig. 5c). The results indicated that TNF50 could antagonize the activity of mouse TNFα in vivo.

The above-mentioned embodiments are only to describe the preferred mode of the present invention, not to limit the scope of the present invention. Without departing from the design spirit of the present invention, those skilled in the art may make various Variations and improvements should fall within the scope of protection defined by the claims of the present invention.

sequence listing

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Claims (9)

1. An anti-TNF-alpha nano antibody is characterized in that the anti-TNF-alpha nano antibody is formed by fusing sequences of camel source anti-TNF-alpha and anti-human serum protein nano antibodies together through flexible connecting peptide; the amino acid sequence of the anti-TNF-alpha nano antibody is shown in SEQ ID No.1, and the nucleotide sequence of the anti-TNF-alpha nano antibody is shown in SEQ ID No. 2.

2. A nucleic acid encoding the anti-TNF- α nanobody of claim 1.

3. An expression vector comprising the nucleic acid of claim 2.

4. A genetically engineered expression bacterium comprising the expression vector of claim 3.

5. The genetically engineered expression strain of claim 4, wherein the genetically engineered strain is Escherichia coli

Rosetta(DE3)。

6. The method for constructing the genetically engineered expression strain of claim 4 or 5, wherein the nucleic acid of claim 2 is connected to an escherichia coli expression plasmid pET-22b or pET-28a to construct an expression vector of claim 3, the expression vector is transformed into escherichia coli, and the genetically engineered expression strain expressing the nano antibody recombinant protein is obtained through screening.

7. A pharmaceutical composition comprising the anti-TNF- α nanobody of claim 1 and at least

A pharmaceutically acceptable excipient.

8. The pharmaceutical composition of claim 7, further comprising at least one pharmaceutically acceptable adjuvant

And (3) preparing.

9. Use of the anti-TNF- α nanobody of claim 1, the nucleic acid of claim 2, the expression vector of claim 3, the genetically engineered expression bacterium of any one of claims 4 to 5, or the pharmaceutical composition of any one of claims 7 to 8 in the preparation of a medicament for the treatment of rheumatoid arthritis.

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