Anti-GLP-1R nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to an anti-GLP-1R nano antibody as well as a preparation method and application thereof. The invention provides an anti-GLP-1R nano antibody, and also provides a construction method of the anti-GLP-1R nano antibody. The method comprises the following steps: constructing GLP-1R overexpression plasmids and overexpression cell strains, immunizing alpaca by using the plasmids and the cells to obtain PBMC cells, extracting total RNA, carrying out reverse transcription to obtain cDNA, amplifying antibody gene segments through PCR, connecting the segments to phage expression vectors, and electrically transforming the constructed vectors into SS320 competent cells to finally obtain the GLP-1R immune antibody library. The method comprises the following steps: carrying out cell panning for one round, carrying out Cell ELISA detection to obtain an antibody combined with GLP-1R, carrying out sequencing to obtain an antibody sequence, obtaining antibody protein through mammalian cells, and carrying out FACS detection by using CHOK1-GLP-1R cells to finally obtain the antibody combined with GLP-1R. The method is simple and convenient to operate, and the activity of the antibody obtained by using a mammalian cell expression system is closer to that of a natural antibody.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and specifically relates to an anti-GLP-1R nanobody, a preparation method thereof and an application thereof. Background Art

[0002] Diabetes is considered the fastest growing health problem in the world, affecting approximately 10% of adults worldwide. Approximately 90% of diabetes cases are type 2 diabetes mellitus (T2DM). T2DM is caused by pancreatic β-cell dysfunction and the resulting lack of insulin.

[0003] Most current drugs for T2DM do not target β cells, so the therapeutic effect gradually weakens during the course of diabetes treatment. Taking metformin is a common method for treating type 2 diabetes. When a single drug is not enough, sulfonylureas, α-glucosidase inhibitors, thiazolidinediones, and insulin injections can be added for treatment. However, these drugs may cause side effects such as hypoglycemia, weight gain, and cardiovascular risks.

[0004] Studies have found that the natural peptide secreted in the intestine, insulinoceptin, can promote insulin secretion, which provides a new direction for the treatment of T2DM. Insulinoceptin has become the preferred injection therapy for the treatment of T2DM because it targets β cells and has a strong hypoglycemic effect and significant safety. Insulinoceptin includes glucagon-like 1 (GLP-1) and glucose-dependent insulin secretagogue polypeptide (GIP). Although GLP-1 and GIP can promote insulin secretion, the method of using GIP alone to treat T2DM is controversial due to its therapeutic effect; GLP-1, as an endogenous agonist of GLP-1R, has the effects of suppressing appetite, promoting insulin secretion and weight loss, and is a promising natural anti-diabetic product. However, in the body, GLP-1 is cleaved at the second amino acid (alanine) at its N-terminus by dipeptidyl peptidase 4 (DPP-4) immediately after secretion, which will lead to rapid degradation of GLP-1 in the patient's body, limiting its application in treatment; although DPP-4 inhibitors and GLP-1 analogs have been used in the treatment of T2DM and have shown good effects, GLP-1R agonists are more popular because they can better control body weight and cardiovascular disease compared with inhibitors and analogs; and compared with insulin and sulfonylureas, they reduce the risk of hypoglycemia.

[0005] Therefore, developing an anti-GLP-1R antibody drug as a T2DM treatment drug has broad application prospects. Anti-GLP-1R antibody drugs can also be used to treat obesity and other diseases. Summary of the invention

[0006] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an anti-GLP-1R nanobody, a preparation method thereof and an application thereof.

[0007] The technical solution of the present invention to solve the technical problem is as follows:

[0008] In the first aspect of the present invention, an anti-GLP-1R nanobody is provided, wherein the anti-GLP-1R nanobody comprises a heavy chain variable region, wherein the heavy chain variable region comprises a framework region and a complementarity determining region; the complementarity determining region comprises CDR1, CDR2 and CDR3, and the amino acid sequence of the complementarity determining region is selected from any one of the following two groups:

[0009] 1) The amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3.

[0010] 2) The amino acid sequences of CDR1-3 are at least 90% identical to the amino acid sequences of SEQ ID NO. 1-3, respectively.

[0011] Preferably, the heavy chain variable region of the anti-GLP-1R Nanobody has an amino acid sequence as shown in SEQ ID NO:4.

[0012] In the second aspect of the present invention, a nucleic acid encoding the anti-GLP-1R nanobody as described in the first aspect is provided. Preferably, the nucleic acid sequence of the heavy chain variable region of the anti-GLP-1R nanobody is shown in SEQ ID NO.5.

[0013] In the third aspect of the present invention, a vector is provided, which contains the nucleic acid as described in the second aspect. Preferably, the vector is -pComb3XSS.

[0014] In the fourth aspect of the present invention, a host cell is provided, which contains the vector as described in the third aspect. Preferably, the host cell is TG1 or SS320; more preferably, the host cell is SS320 host cell.

[0015] In the fifth aspect of the present invention, a method for preparing an anti-GLP-1R nanobody as described in the first aspect is provided, comprising the following steps: constructing a GLP-1R overexpression plasmid and an overexpression cell line, immunizing alpacas with the plasmid and cells to obtain PBMC cells, extracting total RNA, reverse transcribing into cDNA, amplifying antibody gene fragments by PCR and connecting the fragments to a phage expression vector, and electroporating the constructed vector into SS320 competent cells to finally obtain a GLP-1R immune antibody library; obtaining antibodies binding to GLP-1R through one round of cell panning and Cell ELISA detection, sequencing the antibody sequence, obtaining antibody proteins through mammalian cells, and performing FACS detection using CHOK1-GLP-1R cells to finally obtain antibodies binding to GLP-1R.

[0016] In the present invention, the alpaca immunization, the primers used in the step of amplifying the antibody gene fragment, the panning method, and the construction of the GLP-1R high-expressing cell line are obtained after optimization in conventional operations.

[0017] In the sixth aspect of the present invention, a detection reagent or kit is provided, which contains the nanobody as described in the first aspect or the biomaterial as described in the second, third or fourth aspect.

[0018] In the seventh aspect of the present invention, provided is the use of the anti-GLP-1R nanobody as described in the first aspect in the preparation of a medicament for the treatment of diabetes and obesity, or in the preparation of an agent or medicament for the diagnosis, prevention or treatment of a GLP-1R-related disease or a hypoglycemia-related disease.

[0019] In the eighth aspect of the present invention, there is provided the use of the anti-GLP-1R nanobody as described in the first aspect in the preparation of a GLP-1R detection reagent.

[0020] In the ninth aspect of the present invention, there is provided the use of the anti-GLP-1R nanobody as described in the first aspect in the preparation of a product binding to GLP-1R.

[0021] The present invention has the following technical effects:

[0022] 1) The antibody of the present invention has the characteristics of small molecular weight, strong binding ability with the antigen GLP-1R, high specificity, simple structure and easy engineering.

[0023] 2) The antibodies obtained in the present invention are efficiently expressed by a mammalian protein expression system, and their three-dimensional structure and chemical modification are closer to their state in mammals, and their activity is closer to that of natural antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 These are the results of 7 immune serum titer tests.

[0025] Figure 2 This is the result of the first round of PCR electrophoresis.

[0026] Figure 3 This is the result of the second round of PCR electrophoresis.

[0027] Figure 4 This is the test result of antibody binding to CHOK1-GLP-1R cells. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0029] The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased through regular channels.

[0030] Example 1

[0031] In this embodiment, the method for preparing GLP-1R nanobody comprises the following steps:

[0032] 1) Based on the gene sequence and protein sequence information of GLP-1R (NCBI Reference Sequence: NM_002062.5→NP_002053.3), a GLP-1R overexpression plasmid and CHOK1-GLP-1R cells were constructed; the overexpression plasmid and overexpression cell line were constructed according to routine operations in the art.

[0033] 2) The alpaca is immunized 7 times with the plasmid or cell obtained in step 1), wherein the first two times are plasmid immunizations and the last five times are cell immunizations, to obtain alpaca PBMC cells. The specific immunization method is as follows: the alpaca is immunized for the first and second time with the GLP-1R overexpression plasmid, and immunized with CHOK1-GLP-1R cells and GERBU adjuvant on days 28, 42, 56, 70 and 84. After each immunization for 1 week, blood is collected to detect the Anti-GLP-1R serum titer. The Anti-GLP-1R serum titer is detected by FACS. When the MFI value of the sample to be tested is more than 3 times that of the negative control, the antiserum titer is judged to be positive. The results are as follows: Figure 1 As shown, the serum titer MFI value after the seventh immunization was more than 3 times that of the negative control; one week after the seventh immunization, 50 mL of blood was collected for screening and library construction.

[0034] 3) Dilute the 50 ml blood collected in step 2) twice with PBS, separate the PBMC cells with lymphocyte separation solution (Ficoll reagent), and then add Trizol to dissolve the cells to obtain a cell concentration of 10 7 / ml PBMC cells, stored at -80 degrees.

[0035] 4) RNA from PBMC was extracted using Trizol method, and RNA was reverse transcribed into cDNA using oligo(dT) (the reverse transcription kit was TaKaRa-SMARTcribe Reverse Transcript), and PCR was performed twice using nested PCR to obtain nanobody gene fragments. Figure 2 This is the result of the first round of PCR. The target fragment is about 750 bp. Figure 3 The second round of PCR results show that the target band is about 400 bp. The obtained nano antibody gene fragment is inserted into the phage expression vector, the phage vector with the antibody fragment is obtained and electroporated into the SS320 electroporation competent state, the bacteria are collected, and the GLP-1R immune phage antibody library is obtained.

[0036] 5) Panning of the GLP-1R phage antibody library, packaging the obtained library bacteria into phages, and measuring their titers, which is the Input phage. Take an appropriate amount of Input phage and incubate it with negative screening cells, namely CHOK1 cells, and then incubate it with GLP-1R protein after the incubation. Finally, the phage is eluted with trypsin, infects SS320, and then coated on a plate, single clones are selected for culture, and clone ELISA is performed.

[0037] 6) Clone ELISA: Inoculate CHOK1-GLP-1R cells into a 96-well ELISA plate at 37°C overnight. Add 1% BSA to block at room temperature for 1 hour the next day, wash three times with 0.05% PBST, centrifuge the cultured monoclonal bacterial solution and incubate at room temperature for 1 hour, wash three times with 0.05% PBST, add diluted secondary antibody anti-M13 (1:10000), incubate at room temperature for 40 minutes, wash six times with 0.05% PBST, add TMB colorimetric solution, color for 5-10 minutes, add stop solution to stop the reaction, and detect the OD450 value with an ELISA instrument. Those with a value more than 3 times that of the negative control can be regarded as positive clones, which can be expressed in mammalian cells after sequencing.

[0038] 7) Cell binding detection: cell binding (FACS). The cultured CHOK1-GLP-1R cells were prepared with MACS buffer to a cell number of 2E+05 cells / ml, and 50ul / well of the cell suspension was added to a 96-well plate. The prepared antibody (this antibody is the antibody obtained by sequencing in step 6 and expressed and purified by mammalian cells) was diluted 4 times with MACS buffer, and 50ul of the antibody dilution was added to the designated wells of the 96-well plate and mixed evenly. At the same time, positive control and negative control (positive control: PC-Anti-hGLP-1R antibody, negative control: NC-Anti-HEL-VHH-IgG1 Isotype and NC-Anti-HEL hIgG1-isotype) were added, incubated at 4°C in the dark for 60min, washed 3 times with MACS buffer, and fluorescent secondary antibody was added to resuspend the cells. Incubated at 4°C in the dark for 40min, washed 3 times with MACS buffer, 180ul of MACS buffer was added to resuspend the cells, and the flow cytometer was used for measurement and data analysis. The results are shown in the table. Figure 4 The results showed that NC-Anti-HEL-VHH-IgG1 Isotype and NC-Anti-HEL hIgG1-isotype had no obvious binding to CHOK1-GLP-1R cells, while PC-Anti-hGLP-1Rantibody and antibody numbered ANb1336-7M-21G9 had obvious binding to CHOK1-GLP-1R cells.

[0039] The antibody is numbered ANb1336-7M-21G9, and the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.4, the complementary determining region CDR1 sequence in the heavy chain variable region is shown in SEQ ID NO.1, the CDR2 sequence is shown in SEQ ID NO.2, the CDR3 sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.5.

[0040] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. An anti-GLP-1R nanobody, comprising a heavy chain variable region, wherein the heavy chain variable region comprises a framework region and a complementary determining region; the complementary determining region comprises CDR1, CDR2 and CDR3, wherein: The amino acid sequence of the complementary determining region is selected from any one of the following two groups: 1) The amino acid sequence of CDR1 is shown in SEQ ID NO.1, the amino acid sequence of CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of CDR3 is shown in SEQ ID NO.3; 2) The amino acid sequences of CDR1-3 are at least 90% identical to the amino acid sequences of SEQ ID NO. 1-3, respectively.

2. The anti-GLP-1R nanobody according to claim 1, characterized in that The heavy chain variable region of the anti-GLP-1R nanobody has an amino acid sequence as shown in SEQ ID NO:

4.

3. A nucleic acid encoding the anti-GLP-1R Nanobody according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The heavy chain variable region nucleic acid sequence of the anti-GLP-1R nanobody is shown in SEQ ID NO.

5.

5. A carrier, characterized in that Containing the nucleic acid as claimed in claim 3.

6. A host cell, characterized in that Contains the carrier as claimed in claim 5.

7. The method for preparing the anti-GLP-1R nanobody according to claim 2, characterized in that: The method comprises the following steps: constructing a GLP-1R overexpression plasmid and an overexpression cell line, immunizing alpacas with the plasmid and cells to obtain PBMC cells, extracting total RNA, reversely transcribing into cDNA, amplifying antibody gene fragments by PCR and connecting the fragments to a phage expression vector, and electrotransforming the constructed vector into SS320 competent cells to finally obtain a GLP-1R immune antibody library; obtaining antibodies binding to GLP-1R through one round of cell panning and Cell ELISA detection, sequencing to obtain antibody sequences, obtaining antibody proteins through mammalian cells, performing FACS detection with CHOK1-GLP-1R cells, and finally obtaining antibodies binding to GLP-1R.

8. A detection reagent or kit, characterized in that: Containing the Nanobody as claimed in claim 1 or 2 or the biomaterial as claimed in any one of claims 4 to 6.

9. Use of the anti-GLP-1R nanobody according to claim 1 or 2 in the preparation of a drug for treating diabetes and obesity, or in the preparation of an agent or drug for diagnosing, preventing or treating a GLP-1R-related disease or a hypoglycemia-related disease.

10. Use of the anti-GLP-1R nanobody according to claim 1 or 2 in the preparation of a GLP-1R detection reagent.

11. Use of the anti-GLP-1R nanobody according to claim 1 or 2 in the preparation of a product binding to GLP-1R.

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