WO2020155208A1

WO2020155208A1 - Nano-antibody, adsorbent using same as ligand and use thereof

Info

Publication number: WO2020155208A1
Application number: PCT/CN2019/075439
Authority: WO
Inventors: 王玉凤
Original assignee: 康元医疗科技(大连)有限公司
Priority date: 2019-01-29
Filing date: 2019-02-19
Publication date: 2020-08-06
Also published as: CN109627334B; CN109627334A

Abstract

Disclosed are a nano-antibody, an adsorbent using same as a ligand and the use thereof. The amino acid sequence of the nano-antibody includes complementary determining regions and framework regions, wherein in the complementary determining regions, the amino acid sequence of complementary determining region 1 (CDR1) is SEQ ID NO:1; the amino acid sequence of complementary determining region 2 (CDR2) is SEQ ID NO:2; and the amino acid sequence of complementary determining region 3 (CDR3) is SEQ ID NO:3. The above-mentioned nano-antibody and the adsorbent using same as a ligand specifically recognize antigens, thereby effectively removing undesirable inflammatory mediators in blood and achieving a good blood purification effect.

Description

Nanobody, adsorbent using the nanobody as ligand and use thereof

Technical field

The invention belongs to the field of biotechnology, and relates to a nanobody-based immunoaffinity material, in particular to a nanobody, an adsorbent using the nanobody as a ligand, and uses thereof. The adsorbent includes a carrier matrix and a nanobody as a ligand. The material uses a nanobody that specifically recognizes, for example, TNF-α as a ligand, and can reduce the excessive TNF-α level in the patient's body through blood purification, thereby alleviating and/ Or treat diseases such as sepsis.

Background technique

Tumor necrosis factor (TNF) is a cytokine that can induce necrosis of human tumor cells. It is a trimer structure composed of three identical polypeptide chains, which can be combined with TNF receptor (TNF-R) to mediate a variety of biological processes. Among them, TNF-R exists on the surface of a variety of normal and tumor cells. The mechanism of downstream signal transmission after binding to TNF is unclear. It may be related to the activation of protein kinase C (PKC), which catalyzes the phosphorylation of receptor proteins.

Due to the different cells produced, TNF can be divided into two types: TNF-α and TNF-β. Among them, TNF-α is mainly derived from monocytes and macrophages, and the molecular weight of each subunit of its homotrimer is 17kDa.

As an inflammatory mediator, TNF-α has a dual effect on the human body: under normal circumstances, TNF-α presents a protective response to the body, and can participate in the process of resisting infection by bacteria, viruses and parasites, promote tissue repair, and cause Tumor cell apoptosis, through the inflammatory response to clear the body's pathogenic factors, plays an important regulatory role in maintaining the stability of the internal environment and the renewal of tissues. However, during the development of certain diseases (such as sepsis and rheumatoid arthritis), the excessively high blood concentration can lead to an intensified inflammatory response and cause irreversible damage to the patient's body tissues. Researchers found that the maximum concentration of TNF-α in uremia patients can reach 408ng/L, which is about 40 times the level of TNF-α in normal people. In addition, TNF-α enhances the activation and differentiation of macrophages during the onset of sepsis in animals and humans, and releases other pro-inflammatory factors (such as IL-6, IL-8 and MIF), lipid molecules, and responsiveness. Oxygen free radicals and nitrogen free radicals amplify the inflammatory cascade and induce organ dysfunction.

In patients with rheumatoid arthritis (RA), TNF-α can increase the body's secretion and synthesis of chemokines and the expression of cell adhesion molecules, thereby increasing the recruitment rate of lymphocytes and monocytes. TNF-α can also stimulate the expression of antigen-expressing myosin heavy chain (MHC) type II receptors, and induce synovial fibroblasts and chondrocytes to produce prostaglandin E2 (PGE2) and matrix metalloproteinase (MMP) (collagenase) And matrix protease), leading to inflammation and hyperplasia of synovium, and destruction of cartilage tissue (see Non-Patent Document 1).

In addition to the above diseases, TNF-α has also been found to be closely related to the pathogenesis of a variety of malignant tumors, inflammatory bowel disease (IBD), type 2 diabetes (T2D), Crohn’s disease, immunosuppressive disease and multiple sclerosis disease . Therefore, reducing the excess TNF-α in the blood circulation of patients or neutralizing its activity through different ways is of great significance for the prevention, alleviation, treatment and reduction of complications of related diseases.

There are two main ways to treat TNF-α: one is to inhibit the activity of TNF-α or TNF-α receptors in the body through antibody drugs to block TNF-α from working; the other is to use blood purification The method directly adsorbs and removes TNF-α in the blood of patients.

At present, important progress has been made in the development of related antibody drugs, and a variety of monoclonal antibody drugs have been successfully approved for marketing, such as golimumab, infliximab, and adalimumab. For example, Ablynx has developed a series of single domain antibodies against TNF-α using nanobody technology (see Patent Document 1 and Patent Document 2) as therapeutic drugs for autoimmune diseases. These antibody molecules have been shown to effectively interfere with TNF-α-related diseases, such as inflammation, rheumatoid arthritis, Crohn's disease, ulcerative colitis, and inflammatory disease. Intestinal syndrome (inflammatory bowel syndrome), multiple sclerosis (multiple sclerosis), Addison's disease (Addison's disease), autoimmune hepatitis (autoimmune hepatitis), etc. Experimental results prove that drug treatment can significantly reduce the level of TNF-α in the patient's body and effectively alleviate the condition, which further proves that controlling the level of TNF-α is an effective measure to treat related diseases.

However, treatment methods based on monoclonal antibody drugs often have the following problems in clinical use: (1) Although the blood level of TNF-α in the patient is very low, in order to achieve the necessary blood concentration to obtain the drug effect, a greater need For example, the single dose of tocilizumab is 8mg/kg, and each treatment requires an injection of about 400mg, which makes it difficult to avoid high treatment costs. For example, AbbVie’s adalimumab sells as high as 195 Yuan/mg, the average annual treatment cost is 202,000 yuan. (2) High-dose antibody drug infusion also has a higher safety risk. In clinical trials of anti-TNF-α monoclonal antibodies, patients are prone to a variety of adverse reactions, which can cause severe bacterial infections, affect nerve function, and increase the risk of cancer in the lymphatic system and immune diseases (see Non-Patent Document 2).

Blood purification is another effective means to interfere with blood components, which can be used to remove toxic or pathogenic substances from the blood. Commonly used clinical blood purification techniques include hemoperfusion, hemofiltration, hemodialysis, etc., and have been successfully applied to the treatment of renal failure, liver failure, drug poisoning and autoimmune diseases in clinical practice. In recent years, blood purification treatment methods and medical materials for inflammatory factors have also been rapidly developed, and have been used in the treatment of sepsis, rheumatoid arthritis, multiple organ failure, severe pancreatitis and other diseases.

American CytoSorbTM adsorbent (CytoSorbents Corporation, Monmouth Junction, NJ) is a typical resin-based hydrophobic adsorbent, which has been reported to be used for the adsorption and removal of inflammatory factors. The adsorption medium uses polystyrene-divinylbenzene copolymer particles. When applied to hemoperfusion, the total clearance rate of most cytokines in the blood can reach more than 80% within 120 minutes. However, whether CytoSorbTM also filters the critical blood components, nutrients and drugs in the patient's blood during the treatment, and the safety risks caused by this still need to be further evaluated. Zhuhai Jianfan Biotechnology Co., Ltd. invented an adsorbent for hemoperfusion, using a hydrophilic gel medium, such as cellulose, agarose, or polyvinyl alcohol balls, as a carrier, and a hydrophobic para-alkane Aniline and its derivatives are ligands, and it is reported that it can also effectively remove IL-1α, IL-6, IL-8, TNF-α and other cytokines in patients, and the clearance rate of IL-6 can reach 90%. The above (see Patent Document 3). Nankai University invented a nano-composite microsphere adsorbent that can remove inflammatory factors in patients. It uses nano-calcium carbonate-styrene-divinylbenzene as the basic skeleton and a mixture of toluene, gasoline and polycarbon alcohols as porogens. Using benzoyl peroxide as an initiator, this adsorbent has a developed mesoporous structure and a high specific surface area (see Patent Document 4). Although these two adsorbents have achieved good in vitro adsorption effects, non-specific adsorption of TNF-α through hydrophobic interaction still has the disadvantages of low selectivity and high safety risk. Because the adsorption capacity of binding to other blood components far exceeds the adsorption capacity of TNF-α, and the types of other plasma components that can be bound and the ratio of adsorption and removal are not clear, the safety risk during use is difficult to be in the material design link Effective evasion.

The technical principle of immunoadsorption (IA) therapy is based on the specific recognition between specific molecules. Generally, highly specific antigens, antibodies, or molecules with specific physical and chemical affinity are used as adsorption functional groups (ligands), which are coupled to a carrier medium with good blood compatibility through covalent bonds, so as to obtain Highly selective adsorption material. In 1979, Terman et al. used immunosorbents to treat lupus nephritis, which opened up a new application field for immunosorbent technology. Based on the principle of specific molecular recognition, IA has higher selectivity, safety and specificity for eliminating endogenous or exogenous pathogenic factors in the blood.

Long Haibo et al. coupled TNF-α monoclonal antibody to gel microspheres to control the TNF-α-mediated inflammatory response in animal endotoxemia. The experimental results showed that after 2 hours of treatment, the immune adsorption group TNF-α activity is significantly reduced (see Non-Patent Document 3). Wang Qin et al. designed a TNF-α immunoadsorbent. The carrier is polystyrene microspheres coated with polylysine. The anti-TNF-α monoclonal antibody (Z20) is firmly bound by the sieffer base reaction. On the carrier, and made into a specific TNF-α immunoadsorbent column, in vitro experiments have also obtained ideal results (see Non-Patent Document 4).

The existing research and development of immunosorbents against TNF-α all use conventional IgG-type monoclonal antibodies as affinity ligands. Because they need to be prepared from animal cells, the production cost is high. In addition, IgG-type antibodies are structurally unstable, The problem of easy aggregation and degeneration. The above-mentioned problems are the key to the industrialization of immunoaffinity technology and its successful clinical application. Therefore, although in vitro experiments are generally effective, no immunoadsorbent for TNF-α has been used in clinical practice. The effective way to solve this problem is to develop a new type of affinity ligand with stable structure and relatively low cost.

In 1993, Hamers and others accidentally discovered that there is an antibody with only a heavy chain in camels, which is called heavy-chain antibodies (HCAbs). The antigen binding site of this type of antibody is formed only by the variable domain of the heavy chain of HCAbs (VHH) single domain, which is the smallest antibody molecule fragment with complete functions currently available, with a molecular weight of 15kD , Which is only 1/10 of conventional antibodies, is called single domain antibody (sdAb), also called nanobody. Compared with traditional antibodies, Nanobodies have the advantages of good water solubility, acid and alkali resistance, high temperature resistance, high stability, easy expression, weak immunogenicity, high sensitivity, and good tissue penetration. In addition, various test results prove , Various modifications to the Nanobody will not damage its structural integrity, can be compatible with a high-throughput screening platform, and have the advantages of low cost and convenient preparation.

In recent years, nanobody technology has been used in the preparation of immunoadsorbent materials, mainly in the fields of biological separation and analysis. Patent Literature 5 discloses an aflatoxin M1 nanobody 2014AFM-G2, and successfully uses silica gel microspheres or agarose gel as a solid phase carrier to prepare immunosorbents and immunoaffinity adsorption columns for agricultural products and The purification stage of aflatoxin samples in food. Patent Document 6 discloses a highly specific Nanobody against c-Myc, which can purify recombinant proteins containing c-Myc tags. The Capture Select series of chromatographic separation media under Thermo Fisher uses nanobodies as ligands to purify antibodies, antibody fragments and proteins through affinity solutions. For the first time, Dalian University of Technology applied nanobody-based immunoadsorbent materials to the field of blood purification, and developed a specific blood purification adsorbent for β2 microglobulin in patients with renal failure (see Patent Document 7).

Therefore, the highly selective TNF-α immunoadsorbent based on Nanobody of the present invention will further expand the application of Nanobody in the field of blood purification treatment.

Patent Document 1: International Publication WO2004/041862

Patent Document 2: International Publication WO2006/122786

Patent Document 3: CN103585977B

Patent Document 4: CN106334541A

Patent Document 5: CN103869065A

Patent Document 6: CN106890622A

Patent Document 7: CN104098694B

Non-Patent Document 1: Kalden JR. Emerging role of anti-tumor necrosis factor therapy in rheumatic diseases. Arthritis Res, 2002, 4 (Suppl 2): S34-S40.

Non-Patent Literature 2: Hu Xue, Liu Yang. Research progress and adverse reactions of tumor necrosis factor-α inhibitors. Hebei Medicine, 2012, 11, 34: 3477-3480.

Non-Patent Document 3: Long Haibo, Zhang Xun, Hou Fanfan, Experimental study on the treatment of endotoxin shock with immunoadsorbent anti-medium. Chinese Journal of Trauma, 1998, 14(2): 92-95

Non-Patent Document 4: Wang Qin, Wu Xiongfei, Wang Junxia, etc., the development of specific tumor necrosis factor-α immunoadsorbent column and the experimental study of its adsorption rate. Journal of the Third Military Medical University, 2003, 25(24): 2220-2222

Summary of the invention

The present invention is based on solving the existing technical problems, and its purpose is to provide a Nanobody, an adsorbent using the Nanobody as a ligand, and the aforementioned Nanobody or the aforementioned adsorbent for preparing reagents for removing TNF-α And/or use in medical devices, and these uses in immunoassay, enrichment and/or purification.

The present invention is realized through the following technical solutions:

The first aspect of the present invention:

1. Provide a Nanobody. The amino acid sequence of the aforementioned Nanobody includes a complementarity determining region and a framework region. The aforementioned complementarity determining region includes complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 ( CDR3),

The amino acid sequence of the aforementioned complementarity determining region 1 (CDR1) is SEQ ID NO:1,

The amino acid sequence of the aforementioned complementarity determining region 2 (CDR2) is SEQ ID NO: 2,

The amino acid sequence of the aforementioned complementarity determining region 3 (CDR3) is SEQ ID NO: 3.

2. The Nanobody described in 1 above, wherein the aforementioned framework region includes framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4),

The amino acid sequence of the aforementioned framework region 1 (FR1) is SEQ ID NO: 4,

The amino acid sequence of the aforementioned framework region 2 (FR2) is SEQ ID NO: 5,

The amino acid sequence of the aforementioned framework region 3 (FR3) is SEQ ID NO: 6,

The amino acid sequence of the aforementioned framework region 4 (FR4) is SEQ ID NO: 7.

3. The Nanobody according to 1 or 2, wherein the Nanobody further includes a hinge region, and the amino acid sequence of the hinge region is SEQ ID NO: 8.

4. The Nanobody according to 1 or 2, wherein the Nanobody is produced by introducing a gene into Escherichia coli or yeast and using Escherichia coli or yeast expression.

The second aspect of the present invention:

5. An adsorbent is provided, the adsorbent includes a carrier matrix and the Nanobody described in 1 to 4 above.

6. The adsorbent according to 5 above, wherein the carrier matrix is a porous material, and the porous material is selected from one of agarose gel microspheres, cellulose beads, magnetic beads, silica gel microspheres, activated carbon, and resin microspheres .

7. The adsorbent according to 5 or 6, wherein the solid load of the adsorbent is 18 mg/mL or more.

8. The adsorbent according to 5 or 6, wherein the adsorbent is used to specifically recognize TNF-α.

The third aspect of the present invention:

9. To provide a use of the Nanobody described in 1 to 4 or the adsorbent described in 5 to 8 in the preparation of reagents for removing TNF-α and/or medical devices.

10. To provide a use of the Nanobody described in 1 to 4 or the adsorbent described in 5 to 8 in immunoassay, enrichment and/or purification.

Beneficial effect

Because the adsorbent of the present invention has a nanobody with a special structure and a carrier matrix, the nanobody can specifically recognize and bind to TNF-α, thereby effectively removing bad inflammatory mediators from the blood, and because the nanobody itself has low immunogenicity and safety It has the advantages of high sex, so the adsorbent is used in the field of blood purification, which is very safe and effective, and helps to relieve and treat kidney failure, sepsis, rheumatoid arthritis and other diseases. In addition, the adsorbent of the present invention has a large adsorption capacity, and the nanobody itself also has the properties of acid and alkali resistance, high salt resistance, high temperature resistance, etc., so that the immunoaffinity adsorbent can be applied to the enrichment and purification of TNF-α .

Description of the drawings

Figure 1 is the electrophoresis diagram of TNF-α Nanobody expression in whole bacteria;

Figure 2 is an electrophoresis diagram of TNF-α Nanobody after purification.

Description of reference signs:

M: Protein Marker;

1: Electrophoresis of whole bacteria before induction;

2. 3: Electrophoresis of whole bacteria after induction;

4: Sample after purification

detailed description

Hereinafter, examples are given to illustrate the specific implementation of the present invention, but the implementation of the present invention is not limited by the following examples, and any choice and change can be made within the scope that does not affect the technical effect of the present invention. .

In order to make the present invention easier to understand, the terms used are used for the following definitions.

The term "ligand" means that in affinity chromatography, biomolecules that are coupled to a carrier and used to separate specific binding substances are often called ligands.

The term "solid loading" refers to the total amount of ligands coupled per unit volume of the affinity medium (adsorbent).

The specific implementation manners of the present invention are given below in conjunction with the drawings to further illustrate the present invention.

In the Nanobody of the present invention, the amino acid sequence of the Nanobody comprises a complementarity determining region and a framework region. The aforementioned complementarity determining region includes complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3 (CDR3), the aforementioned The amino acid sequence of the complementarity determining region 1 (CDR1) is SEQ ID NO:1, the amino acid sequence of the aforementioned complementarity determining region 2 (CDR2) is SEQ ID NO: 2, and the amino acid sequence of the aforementioned complementarity determining region 3 (CDR3) is SEQ ID NO :3.

For Nanobodies, the complementarity determining regions of different Nanobodies are quite different. As long as the complementarity determining regions in the Nanobodies satisfy the above-mentioned amino acid sequence, the effects of the present invention can be achieved.

For the framework region, it is relatively more conservative, and those skilled in the art will rationally screen the specific sequence structure of the framework region according to the actual use and function of the Nanobody. An amino acid sequence with a sequence homology of 50% or more is preferable, an amino acid sequence with a sequence homology of 70% or more is more preferable, and an amino acid sequence with a sequence homology of 95% or more is most preferable. Further preferably, the framework region of the aforementioned Nanobody includes framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4), and the amino acid sequence of the aforementioned framework region 1 (FR1) is SEQ ID NO: 4, the amino acid sequence of the aforementioned framework region 2 (FR2) is SEQ ID NO: 5, the amino acid sequence of the aforementioned framework region 3 (FR3) is SEQ ID NO: 6, the amino acid sequence of the aforementioned framework region 4 (FR4) It is SEQ ID NO: 7, but the amino acid sequence of these framework regions of the present invention is not limited to this, and can be appropriately selected according to the matching with the complementarity determining region.

Furthermore, the Nanobody further includes a hinge region, and the amino acid sequence of the hinge region is preferably SEQ ID NO: 8, but the amino acid sequence is not limited thereto. By including the hinge region, the flexibility of the Nanobody is increased.

Furthermore, Nanobodies are produced by introducing genes into Escherichia coli or yeast and using Escherichia coli or yeast expression.

The Nanobody of the present invention, as the ligand of the carrier matrix, can be prepared by methods known in the art. As long as the Nanobody of the present invention can be obtained, it can be prepared, for example, by the following method: First, construct the nanobody Non-immune library, and obtain Nanobody gene through screening technology. Secondly, introduce Nanobody gene into Escherichia coli or yeast by means of genetic engineering recombination to obtain genetically engineered bacteria for expressing Nanobody, and then use this genetic engineering The bacteria induce the expression of the nanobody, and finally it is purified by metal chelation chromatography to obtain the pure nanobody that meets the requirements.

The carrier matrix of the present invention is preferably a porous material, and the porous material is preferably selected from one or more of agarose gel microspheres, cellulose beads, magnetic beads, silica gel microspheres, activated carbon, and resin microspheres.

Carriers for the aforementioned adsorbents can be purchased commercially, as specific examples of products, such as Sepharose CL-6B (GE Healthcare, US), Nanomicro series of resin microspheres (Suzhou Nanomicro Technology Co., Ltd.), but It is not limited to these products.

When using the above-mentioned carrier, it is preferable to activate the carrier in advance. The activation can be, for example, but not limited to the following methods: firstly, epoxy activation, secondly diaminopropylimine (DADPA) activation, and finally iodoacetic acid activation, etc.

The aforementioned adsorbent is obtained by coupling the Nanobody to an activated carrier. The specific method is not particularly limited. For example, the purified Nanobody can be reduced to obtain a Nanobody solution, and then the Nanobody solution can be combined with the carrier. Mix, centrifuge, and finally clean and filter the gel to obtain the final adsorbent.

The higher the solid loading amount of the aforementioned Nanobody in the aforementioned adsorbent, the better, which is 18 mg/mL or more, preferably 19 mg/mL or more, and more preferably 20 mg/mL or more. In this range, more Nanobodies can be immobilized on the carrier, so that, for example, TNF-α in blood can be more specifically recognized.

The adsorbent of the present invention can preferably be used to specifically recognize TNF-α.

The nanobody or adsorbent of the present invention can be used to prepare reagents for removing TNF-α, and can also be used to prepare medical devices for removing TNF-α. In addition, the Nanobody or adsorbent of the present invention can also be used in immunodetection, enrichment and/or purification applications.

Example

Example 1

Construction of Anti-TNF-α Nanobody Library

The phage display library used in the present invention is a non-immune library with T7 phage as a carrier. The establishment steps are as follows:

(1) Take the jugular vein blood of a 2-year-old male alpaca, isolate peripheral blood lymphocytes, and extract total RNA (PuerLinkTM RNA Mini Kit, Life Technologies: 12183018A);

(2) Reverse transcription of total RNA into cDNA, and use two rounds of nested PCR to amplify the VHH gene. The first round of nested PCR uses cDNA as the template, and the upstream primer 1 and downstream primer 1 are respectively the upstream and downstream primers. After amplification, a band with a size of 650-750bp is recovered and used as the template for the second round of PCR , The upstream and downstream primers are the upstream primer 2 and the downstream primer 2, respectively. PCR products of 450～500bp are recovered;

Upstream primer 1: 5’-GGTACGTGCT GTTGAACTGT TCC-3’

Downstream primer 1: 5’-CTTGGTGGTCCTGGCTGCTCT-3’

Upstream primer 2: 5’-AAGCTTTTGT GGTTTTGGTG TCTTGGGTTC-3’

Downstream primer 2: 5’-AAGCTTGGGG TCTTCGCTGT GGTGCG-3’

(3) Digest the PCR product with EcoRI and HindIII double enzymes, and perform agarose electrophoresis to recover the 350-500bp gene band, which is the VHH gene fragment;

(4) Connect the T7 vector with T4 ligase (

10-3Cloning Kit, Merck Millipore

70550-3) and VHH gene fragments;

(5) Mix the ligation product with the packaging protein to form a complete T7 phage, and amplify the mixture to obtain the original phage library;

(6) The titer of the original library is 1.5×1010 pfu/mL, and the diversity is 2.0×107.

Example 2

Screening of Nanobodies

First, the antigen was diluted with TBS to 10μg/mL, 100μL was added to a 96-well plate, and incubated at 4°C for 12h. Aspirate the antigen diluent in the well, wash the plate 3 times with TBS, pat dry, add 1% protein-free blocking solution (purchased from Shenggong Bioengineering Co., Ltd.), 300μL/well, incubate at room temperature for 2h (1% protein-free during screening Blocking fluid and 1% BSA are used alternately). Aspirate the blocking agent from the well, wash the plate 6 times with TBST, pat dry, add the amplified phage, 100 μL/well, and incubate at room temperature for 30 min. Wash the plate 10 times with TBST, add T7 elution buffer (1% SDS) to elute the phage, incubate at room temperature for 30 min, and amplify the eluate for the next round of screening.

Example 3

Construction of genetically engineered bacteria

(1) After four rounds of screening, the screening eluate is subjected to solid amplification, plaques are picked, and the plaque amplification fluid is used as a template, and UPprimer3 and DOWNprimer3 are used as the upstream and downstream primers for PCR amplification. increase;

Upstream primer 3: 5’-GGAGCTGTCGTATTCCAGTC-3’

Downstream primer 3: 5'-AACCCCTCAAGACCCGTTTA-3';

(2) Part of the PCR products are outsourced to sequence to obtain the Nanobody sequence information;

(3) Digest another part of the PCR product with NdeI and XhoI, and recover the digested product. At the same time, the pET23a vector was digested and recovered with the same method, and the digested product and vector were ligated with T4 ligase, and the ligated product was transferred to E. coli to obtain a genetically engineered bacteria expressing TNF-α specific nanobody. Take 1 mL of bacterial solution, Entrusted Sangong Bioengineering (Shanghai) Co., Ltd. to sequence. The sequencing results showed that the amino acid sequence of CDR1 is SEQ ID NO: 1, the amino acid sequence of CDR2 is SEQ ID NO: 2, and the amino acid sequence of CDR3 is SEQ ID NO: 3, FR1 The amino acid sequence of FR2 is SEQ ID NO: 4, the amino acid sequence of FR2 is SEQ ID NO: 5, the amino acid sequence of FR3 is SEQ ID NO: 6, and the amino acid sequence of FR4 is SEQ ID NO: 7.

Example 4

Preparation of TNF-α Nanobody

(1) The strain was inoculated into a shake flask at an inoculum of 1%, and cultured overnight at 37°C with 170 r/min shaking. The activated seeds were inoculated into a large culture flask according to the inoculum of 1% and continued to be cultured. When the bacteria grew to the middle and late stages of the logarithmic phase, IPTG with a final concentration of 0.2 mM was added for overnight induction. After the induction, centrifuge at 3900r/min for 20 minutes to obtain wet bacteria containing TNF-α Nanobody. SDS-PAGE was performed on the whole bacteria, and the electrophoresis results are shown in Figure 1, where the loading volume of 2 was 20 μL, and the loading volume of 3 was 30 μL.

(2) Add lysis buffer (10mM imidazole, 500mM NaCl, 0.02M PB, pH7.4) to the obtained wet bacteria at a ratio of 1:10, fully suspend the bacteria, and then use a high-pressure homogenizer to disrupt the cells. The crushing pressure is 700 bar.

(3) Centrifuge the crushing liquid at 4°C and 10000 r/min for 20 minutes, and take the supernatant.

(4) Filter the supernatant through a 0.45μm filter, and then pass the metal chelation chromatography (GE Healthcare, US) to separate and purify the TNF-α nanoantibody. The chromatography packing is Ni Sepharose High Perfomance.

(5) The chromatographic eluate is subjected to SDS-PAGE to determine the purity. The electrophoresis result is shown in Figure 2. The purified protein has a purity of more than 90%, which can be used for subsequent adsorbent synthesis and adsorbent evaluation.

(6) Use the BCA method to determine the protein concentration. After testing, the output of Nanobody is about 252mg/L.

Example 5

Activation of agarose gel

(1) Epoxy activation

①Add 2mL 2mol/L NaOH solution, 1mL epichlorohydrin, and 6mL DMSO to 2mL (1.6g) Sepharose CL-6B agarose gel, and react the reaction system at 40°C and 175r/min. 15 minutes;

②After the reaction is over, use acetone solutions of different concentrations (30%, 70%, 100%, 70%, 30%) to clean the gel, and then use a large amount of water to clean the gel three times in each step;

③After the gel is suction filtered into a wet cake, the next step is to be tested immediately.

(2) DADPA activation

① Mix the epoxy-activated agarose gel with an equal volume of Na ₂ CO ₃ -NaHCO ₃ buffer (0.1mol/L, pH=11);

②Add DADPA to the mixed system and react for 4 hours at 30°C;

③After the reaction, the agarose gel is thoroughly washed with 1mol/L NaCl solution and water in sequence, and it can be stored in 20% ethanol at 4°C.

(3) Iodoacetic acid activation

① Wash the amino sepharose prepared after the reaction in step (2) with water and PBS buffer, and then filter with suction;

②Add PBS buffer (pH=7.2) to iodoacetic acid and prepare a solution with a concentration of 5mg/mL, and mix it evenly with agarose gel in a volume ratio of 1:1;

③Add 2mmoL EDC to 5mmoL NHS according to the ratio of 1mL agarose gel, and react at room temperature for 2.5 hours;

④ After the reaction, wash with water, 1mol/L NaCl solution, and a large amount of water successively, and filter with suction.

Example 6

Coupling of TNF-α Nanobody

(1) Put the purified TNF-α nanobody solution above into a buffer system with a concentration of 7.9 mg/mL by ultrafiltration (0.02mol/L PB with pH 7.4 and 1mmol/L ethylenediaminetetraacetic acid) , And add an appropriate amount of tris (2-carboxyethyl) phosphine for reduction;

(2) Add a certain quality of activated agarose gel to the Erlenmeyer flask, and add 15 times the gel volume of the Nanobody solution, and react in a shaker at 37°C for 4 hours;

(3) After the reaction is over, separate the heart at 10,000 rpm for 5 minutes, and take the supernatant to determine the change in protein concentration before and after the reaction;

(4) Add a small amount of mercaptoglycerol to the adsorbent preliminarily immobilized with TNF-α Nanobody, and react overnight in a shaker at 37°C to seal the remaining sites of the adsorbent;

(5) Wash the gel with water several times and filter with suction to obtain the final product, and store it in a 20% ethanol solution containing 0.02% sodium azide at 4°C.

Example 7

Evaluation of hemolysis performance of adsorbent

The experimental method refers to the 2015 edition of "The Pharmacopoeia of the People's Republic of China" and the "Biological Evaluation of Medical Devices" Chapter 11 "Selection of Interaction with Blood"

(1) Collect 10 mL of blood from the heart of healthy rabbits, add 1 mL of potassium oxalate solution with a mass concentration of 2% (20 g/L), and mix gently to prepare fresh anticoagulated rabbit blood. Take 8 mL of fresh anticoagulated rabbit blood, add 10 mL of sodium chloride injection with a mass concentration of 0.9% (9g/L) for dilution, and prepare freshly diluted anticoagulated rabbit blood.

(2) Weigh 5g of the adsorbent, add it to the test tube, and add 10 mL of normal saline to completely immerse the adsorbent. The negative control is 10 mL of normal saline, and the positive control is 10 mL of distilled water, both of which are 10 mL. Prepare 3 tubes for each sample, negative control and positive control. Put the above test tube in a constant temperature water bath, 37±1℃ constant temperature water bath for 30 minutes, add 0.2 mL diluted anticoagulant rabbit blood (A solution) to each test tube, and continue the water bath for 60 minutes.

(3) Pour out the sample in the above tube, put it in a centrifuge, and centrifuge at 800g for 5 minutes. Aspirate the supernatant and transfer it into a cuvette, and measure the absorbance at 545nm with a spectrophotometer. The absorbance of the test product group and the control group are the average of three tubes. The absorbance of the negative control should not be greater than 0.03, and the absorbance of the positive control tube should be 0.8±0.3, otherwise the test should be repeated. Adjust zero with saline.

(4) The formula for calculating the hemolysis rate of the test product: n=(A1-A2)*100%/(A3-A2), where n is the hemolysis rate (%), A1 is the absorbance of the test product group, and A2 is the negative control group Absorbance, A3 is the absorbance of the positive control group.

Experimental verification shows that the hemolysis rate of the adsorbent measured by the prescribed static contact hemolysis method is almost 0, less than 5% (the criterion for qualified products should generally be less than 5%), which meets the requirements.

Example 8

Evaluation of adsorption capacity of adsorbent in non-blood system

(1) Add a known concentration of TNF-α protein solution to the TNF-α adsorbent prepared in Example 6, the buffer is a pH 8.0 PBS solution, and the glue ratio is 1:15;

(2) At room temperature, use the inversion mixer for static adsorption for 2 hours;

(3) Measure the protein concentration before and after the reaction by BCA method to determine the adsorption of TNF-α.

Table 1 Adsorption performance results of TNF-α adsorbent in non-blood system

As shown in Table 1, when the initial concentration of TNF-α is 1.603 mg/mL, the adsorbent with a solid load of 18.0 mg/mL has a TNF-α adsorption capacity of 19.43 mg/mL, and the corresponding adsorption molar ratio is 0.98, close to 1, the adsorption rate at this time is 75.8%.

Example 9

Determination of adsorption effect of adsorbent on TNF-α in blood

(1) Add a certain amount of TNF-α to the serum, and determine the final concentration to be 1.219ng/mL, which is close to the TNF-α concentration in the blood of liver cancer patients (Zeng Yingling, Ye Xiaoguang. The serum TNF content and the degree of liver damage in hepatitis B patients and the virus The relationship of replication. Journal of Guangzhou Medical College, 2002(30) 3:40-41).

(2) Add the serum solution containing TNF-α to the adsorbent according to the glue ratio of 1:10, and calculate the concentration of TNF-α in the mixed system to be 1.108ng/ml.

(3) At room temperature, use an inversion mixer for adsorption for 2 hours.

(4) Use the TNF-α kit (Solarbio, SEKH-0047) to measure the concentration before and after adsorption to determine the adsorption in the blood.

Table 2 Adsorption performance results of TNF-α adsorbent in blood system

As shown in Table 2, the removal rate of TNF-α in the serum of the adsorbent can reach 55.2%, which can reduce the blood TNF-α level of simulated liver cancer patients to the level of healthy people (the blood TNF-α level of healthy people is 0.39±0.16ng/mL, refer to "Plasma Tumor Necrosis Factor-Content Changes in Patients with Acute Cerebral Infarction"), it can be seen that the adsorbent of the present invention is of great significance in the field of blood purification treatment.

Industrial availability

The nanobody of the present invention, as well as the adsorbent with a special structure of the nanobody and a carrier matrix, can specifically recognize and bind to TNF-α, thereby effectively removing bad inflammatory mediators from the blood, and because the nanobody itself has low immunogenicity and safety It has the advantages of high sex, so the adsorbent is used in the field of blood purification, which is very safe and effective, and helps to relieve and treat kidney failure, sepsis, rheumatoid arthritis and other diseases. In addition, the adsorbent of the present invention has a large adsorption capacity, and the nanobody itself has the properties of acid and alkali resistance, high salt resistance, high temperature resistance, etc., so that the immunoaffinity adsorbent can be applied to the enrichment and purification of TNF-α .

Claims

A Nanobody, characterized in that the amino acid sequence of the Nanobody comprises a complementarity determining region and a framework region, and the complementarity determining region includes complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2) and complementarity determining region 3. (CDR3),

The amino acid sequence of the complementarity determining region 1 (CDR1) is SEQ ID NO:1,

The amino acid sequence of the complementarity determining region 2 (CDR2) is SEQ ID NO: 2,

The amino acid sequence of the complementarity determining region 3 (CDR3) is SEQ ID NO: 3.
The Nanobody of claim 1, wherein the framework region comprises framework region 1 (FR1), framework region 2 (FR2), framework region 3 (FR3) and framework region 4 (FR4),

The amino acid sequence of the framework region 1 (FR1) is SEQ ID NO: 4,

The amino acid sequence of the framework region 2 (FR2) is SEQ ID NO: 5,

The amino acid sequence of the framework region 3 (FR3) is SEQ ID NO: 6,

The amino acid sequence of the framework region 4 (FR4) is SEQ ID NO: 7.
The Nanobody of claim 1 or 2, wherein the Nanobody further comprises a hinge region, and the amino acid sequence of the hinge region is SEQ ID NO: 8.
The Nanobody according to claim 1 or 2, wherein the Nanobody is produced by introducing genes into Escherichia coli or yeast and using Escherichia coli or yeast expression.
An adsorbent, characterized in that the adsorbent comprises a carrier matrix and the nanobody according to claims 1 to 4.
The adsorbent according to claim 5, wherein the carrier matrix is a porous material selected from the group consisting of agarose gel microspheres, cellulose beads, magnetic beads, silica gel microspheres, activated carbon, and resin microspheres. One of the balls.
The adsorbent according to claim 5 or 6, wherein the solid load of the adsorbent is 18 mg/mL or more.
The adsorbent according to claim 5 or 6, wherein the adsorbent is used to specifically recognize TNF-α.
A use of the Nanobody of claims 1 to 4 or the adsorbent of claims 5 to 8 in the preparation of reagents for removing TNF-α or medical devices.
A use of the Nanobody of claims 1 to 4 or the adsorbent of claims 5 to 8 in immunoassay, enrichment or purification.