CN113105546B

CN113105546B - Anti-recombinant human basic fibroblast growth factor nano antibody and application thereof

Info

Publication number: CN113105546B
Application number: CN202010970856.7A
Authority: CN
Inventors: 谢秋玲; 熊盛; 陈纯; 雷云
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-06-06
Filing date: 2018-06-06
Publication date: 2022-02-08
Anticipated expiration: 2038-06-06
Also published as: CN113150141A; CN113150141B; CN113105546A; CN108640993B; CN108640993A

Abstract

The invention discloses an anti-recombinant human basic fibroblast growth factor nanobody and its application. The anti-recombinant human basic fibroblast growth factor nanobody has the advantages of small molecular weight, strong specificity, high affinity, high stability, easy expression, strong cell/vascular penetration, strong antigen binding ability, and can be used in prokaryotic cells. It can effectively neutralize the over-expressed bFGF in patients with tumors and fibrosis, and can effectively inhibit angiogenesis, The growth and metastasis of tumors can also be used to inhibit the formation of fibrosis in the liver, lung or kidney. At the same time, the nanobody can be highly expressed in Escherichia coli, and has a good application prospect in the treatment of tumor antibody drugs or cancer diagnosis kits or reagents.

Description

Anti-recombinant human basic fibroblast growth factor nano antibody and application thereof

The patent application of the invention is a divisional application with the application number of '201810573403.3', the application date of the original application is '06 months 06 days in 2018', the application number is '201810573403.3', and the invention name is 'an anti-recombinant human basic fibroblast growth factor nano antibody and application thereof'.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-recombinant human basic fibroblast growth factor nano antibody and application thereof.

Background

Antibodies (antibodies), also called immunoglobulins (immunoglobulins), are glycoproteins that specifically bind to antigens. Traditional antibodies exist as one or more "Y" shaped monomers, each consisting of 4 polypeptide chains, comprising two identical heavy chains and two identical light chains. The traditional antibody has large molecular weight and complex structure, so that the stability and penetrability are poor, and the development and production cost is high, so that the use cost is high, and the further application of the antibody is limited. Therefore, the development of some novel small molecule antibodies is an important research point, such as: fab antibody, F (ab)₂Antibodies, single chain antibodies (scFv), nanobodies, and the like.

In 1993, Hamers Casterman et al first reported the presence of peculiar antibodies that naturally lack light chains in camelid blood, i.e., Heavy Chain antibodies (HCAb). An Antibody fragment consisting of only Heavy Chain Variable regions, namely a Heavy Chain single Domain Antibody VHH (Variable Domain of Heavy Chain of Heavy-Chain Antibody) can be obtained by cloning the Variable regions through genetic engineering technology. It is also called Nanobody (Nb) because its molecular weight is only one tenth of that of traditional antibodies, and its length is more on the order of a few nanometers. The antibody has the characteristics of small molecular weight, high stability, easy expression, strong cell/blood vessel penetrability, strong antigen binding capacity, capability of expressing in a prokaryotic expression system and the like, and has the advantages of the traditional antibody and micromolecular medicaments. Compared with the traditional macromolecular antibody, the nano antibody almost perfectly overcomes the defects of long development period, low stability, harsh preservation conditions and the like, and small molecular substances are not easy to cause immunogenicity; compared with the same molecules such as small molecule antibodies Fab, scFv and the like, the nano antibody only has one heavy chain, is easier to express, is not easy to stick to each other or even aggregate into blocks like small molecule antibodies such as single chain antibody (scFv) and the like, and therefore becomes a novel antibody molecule in the research field of antibody medicines and reagents, and has good development prospect. At present, the nano antibody has been gradually applied to the fields of disease treatment, disease diagnosis, radioactive imaging and the like, and the application of the nano antibody in the field of disease treatment is a current research hotspot. Nanobodies treat diseases such as: the anti-tumor mechanism is mainly characterized in that the anti-tumor mechanism prevents the combination reaction of antigen and receptor, for example, Coppeters and the like treat arthritis by using the characteristic that the nano antibody of anti-TNF-alpha can antagonize TNF-alpha and the receptor thereof, Rovers and the like treat cancer by using EGF-EGFR nano antibody to prevent EGF from combining with EGFR.

Basic Fibroblast Growth Factor (bFGF), also known as Fibroblast Growth Factor (FGF-2), is a mitogen for fibroblasts purified in 1974 from extracts of bovine pituitary and brain tissue by gospodarodizi et al, consisting of 146 amino acids, and is called basic Fibroblast Growth Factor because its isoelectric point is 9.6. bFGF has very wide biological action and plays an important role in the processes of angiogenesis, wound healing and tissue repair promotion, tissue regeneration promotion and growth and development of nerve tissues.

In some tumor diseases, the abnormal expression of bFGF is closely related to tumorigenesis and development, tumor metastasis, angiogenesis and prognosis, mainly because bFGF not only promotes local angiogenesis through paracrine and autocrine and other pathways, but also directly acts on tumor cells to promote proliferation and accelerate tumor infiltration and metastasis. In-vitro and in-vivo experiments show that bFGF can promote the secretion of various growth factors, has synergistic effect with other angiogenesis promoting factors VEGF, PDGF and the like, and promotes tumorigenesis together. In addition, various tumors can change bFGF/FGFR signal path by regulating bFGF overexpression, so that the aim of promoting the growth of the tumors is achieved. bFGF is involved in the growth of various tumors (such as glioma, rhabdomyoma, leukemia, renal carcinoma, lung cell carcinoma, melanoma and lung cancer), and a considerable part of tumors show high expression of bFGF and bFGF receptors. Wang et al first discovered in human melanoma transplantable tumors that the bFGF/FGFR1 autocrine signaling pathway cycle could promote melanoma progression. Coldren and the like find that bFGF/FGFRs are obviously increased from gene expression data of a series of non-small cell lung cancer cell lines, and suggest that an FGFR-dependent autocrine signaling pathway plays an important role in non-small cell lung cancer. Casanovas et al found that increased bFGF re-elicited angiogenesis and tumor cell growth in a mouse resistance model of pancreatic cancer that inhibited VEGFR, suggesting that bFGF/FGFR signaling may evade VEGF pathway inhibition to promote angiogenesis. Thus, inhibition of the bFGF/FGFR pathway can inhibit tumor vascular growth, inhibit tumor growth, differentiation, infiltration, and metastasis by slowing signaling. Therefore, the bFGF/FGFR system becomes a new target for antitumor therapy. Detection and treatment of this target is therefore of great importance. The current research on bFGF monoclonal antibodies is that of traditional IgG antibodies. In 1989, Reilly et al prepared bFGF monoclonal antibody with neutralizing activity for the first time, and then confirmed the function of bFGF monoclonal antibody in inhibiting tumor and blood vessel growth. The research of domestic bFGF monoclonal antibody starts in 1998, and murine bFGF monoclonal antibody and human bFGF monoclonal antibody which have the function of inhibiting angiogenesis are prepared to thrifty and the like. Although there are research documents and patent reports related to anti-bFGF conventional antibodies and small molecule antibodies such as Fab antibodies, the antibodies prepared in these reports are all from mice or rabbits, and are classic IgG antibodies containing heavy chains and light chains and derivatives thereof, and most of the currently marketed bFGF content detection kits (ELISA method) are conventional IgG antibodies. The traditional IgG antibody production depends on immune animals or cultured mammalian cells, has long production period, high cost and high price, needs low-temperature storage, and has the characteristics of limitation on the application of the IgG antibody in treatment and detection kits. The heavy chain antibody, namely the nano antibody, discovered from the camelid has the characteristics of strong specificity, high affinity, good stability, high temperature resistance, easiness in storage and the like due to the structural specificity, and the production cost is greatly reduced due to the expression of the heavy chain antibody by bacteria or yeast, so that the defects of the traditional IgG antibody can be overcome in application if the heavy chain antibody is successfully developed. The current bFGF antibodies are all classic IgG antibodies containing heavy chains and light chains and derivatives thereof, and no related research reports of novel antibodies (namely nano antibodies) only with heavy chains from camelids exist.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a nano antibody for resisting recombinant human basic fibroblast growth factor, which can specifically neutralize human bFGF (namely FGF-2).

The invention also aims to provide application of the anti-recombinant human basic fibroblast growth factor nano-antibody.

The purpose of the invention is realized by the following technical scheme:

an anti-recombinant human basic fibroblast growth factor nanobody, comprising a variable region domain consisting of framework regions fr (framework regions) and complementary-determining regions CDRs (complementary-determining regions), said variable region domain comprising CDRs 1, CDR2 and CDR3 selected from the group consisting of:

(1) CDR1 shown by any amino acid sequence of SEQ ID No. 1-4;

(2) CDR2 shown by any amino acid sequence of SEQ ID No. 5-8;

(3) CDR3 shown in any amino acid sequence of SEQ ID Nos. 9-12.

The variable region domain comprises a CDR1, a CDR2, and a CDR3 selected from any one of:

(1) CDR1 shown in SEQ ID No.1, CDR2 shown in SEQ ID No.5, and CDR3 shown in SEQ ID No. 9;

(2) CDR1 shown in SEQ ID No.2, CDR2 shown in SEQ ID No.5 or SEQ ID No.6, and CDR3 shown in SEQ ID No.9 or SEQ ID No. 10;

(3) CDR1 shown in SEQ ID No.3, CDR2 shown in SEQ ID No.7, and CDR3 shown in SEQ ID No. 11;

(4) CDR1 shown in SEQ ID No.4, CDR2 shown in SEQ ID No.8, and CDR3 shown in SEQ ID No. 12.

The framework region FR is selected from the group consisting of FR1, FR2, FR3 and FR 4:

(1) FR1 represented by any one of SEQ ID Nos. 13-15;

(2) FR2 represented by any amino acid sequence of SEQ ID Nos. 16-19;

(3) FR3 represented by any amino acid sequence of SEQ ID Nos. 20-24;

(4) FR4 represented by any amino acid sequence of SEQ ID Nos. 25 to 27.

The amino acid sequence of the anti-recombinant human basic fibroblast growth factor nano antibody is selected from any one of the amino acid sequences shown in SEQ ID NO. 28-33; the method comprises the following specific steps:

or

Or

Or

Or

Or

The anti-recombinant human basic fibroblast growth factor nano antibody comprises an amino acid sequence which has at least 90%, preferably at least 95% and more preferably at least 99% of sequence identity with any amino acid sequence shown in SEQ ID No. 28-33.

Any one of the amino acid sequences shown in SEQ ID NO. 28-33 is subjected to substitution, deletion or addition of one or more amino acids.

The substitution preferably comprises one or more amino acid substitutions, preferably conservative amino acid substitutions compared with any one of amino acid sequences shown in SEQ ID NO. 28-33. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions are included.

The nucleotide sequence of the anti-recombinant human basic fibroblast growth factor nano-antibody is preferably as follows:

nucleotide sequences encoding the amino acid sequences shown as SEQ ID NO. 28-33 are respectively and sequentially shown as SEQ ID NO. 34-39.

The nanobody consists of 342 bases, 336 bases or 369 bases, codes 114 amino acids, 112 amino acids or 123 amino acids, and consists of 4 Framework Regions (FRs) and 3 Complementary Determining Regions (CDRs) of the antibody: the CDR1 of the nanobody encodes 10 amino acids, CDR2 encodes 7 amino acids, and CDR3 encodes 11 amino acids; or CDR1 encodes 10 amino acids, CDR2 encodes 7 amino acids, CDR3 encodes 9 amino acids; or CDR1 encodes 10 amino acids, CDR2 encodes 8 amino acids, CDR3 encodes 19 amino acids; 3 CDR regions are specific sequences.

The function of the nano antibody is determined by specific nucleotide sequences in antibody antigenic determinants CDR1, CDR2 and CDR3 (which are functional active regions of the invention), and corresponding amino acid sequences form antibody specific bFGF antigen binding regions.

The application of the anti-recombinant human basic fibroblast growth factor nano antibody comprises the following steps: the functional neutralizing antibody gene specifically combined with bFGF is determined by the gene sequence of CDR specificity in the complementarity determining region of the antibody, and based on the above-mentioned complementarity determining region gene, it can adopt gene engineering method to obtain small molecule gene engineering antibody with several forms by means of construction and expression,such as Fab antibodies, F (ab)₂Antibodies, single chain antibodies (scFv), antibody fusion proteins, and IgG whole antibodies.

A recombinant expression vector contains the nucleotide sequence of the anti-recombinant human basic fibroblast growth factor nano antibody.

A recombinant engineered cell containing the above recombinant expression vector; the recombinant engineering cell can express the anti-recombinant human basic fibroblast growth factor nano antibody.

The nano antibody gene or any gene containing the complementary determining region of the nano antibody gene reconstructed based on the gene can be expressed in prokaryotic cells, yeast cells, insect cells and eukaryotic cells to obtain an antibody product with neutralizing human bFGF, and the gene can be applied to the following applications: (1) used for diagnosing tumor, (2) inhibiting tumor angiogenesis and tumor growth and metastasis, and (3) inhibiting liver, lung and kidney fibrosis.

The anti-recombinant human basic fibroblast growth factor nano antibody is applied to the preparation of a tumor antibody treatment drug or a cancer diagnosis kit or reagent.

The tumor is preferably melanoma.

The cancer diagnosis kit or reagent is preferably a cancer diagnosis kit or reagent for detecting human bFGF.

Compared with the prior art, the invention has the following advantages and beneficial effects:

1. the invention immunizes alpaca with human bFGF (namely FGF-2), then utilizes the alpaca peripheral blood lymphocytes to establish a nano antibody gene library aiming at the human bFGF, couples the human bFGF on an enzyme label plate in the test, utilizes the phage display technology to screen the immune nano antibody gene library (camel heavy chain antibody phage display gene library) by the antigen in the form, thereby obtaining the nano antibody gene aiming at the specificity of the human bFGF, transfers the gene into escherichia coli, and further establishes the nano antibody strain which can be efficiently expressed in the escherichia coli.

2. The anti-human bFGF nano antibody has the characteristics of small molecular weight, strong specificity, high affinity, high stability, easy expression, strong cell/blood vessel penetrability, strong antigen binding capacity, capability of being expressed in a prokaryotic expression system and the like, overcomes the defects of long development period, low stability, harsh storage conditions and the like of the traditional antibody, can effectively neutralize the bFGF excessively expressed in the bodies of patients with tumors and fibrosis, can effectively inhibit the generation of blood vessels and the growth and metastasis of the tumors, and can also be used for inhibiting the formation of fibrosis of the liver, the lung or the kidney and the like.

Drawings

FIG. 1 is a gene electrophoresis diagram of a bFGF nanobody; wherein, Lane 1 is DNA Marker, Lanes 2-7 are PCR amplified nanometer antibody gene segments, and Nb 1-Nb 6 are arranged in sequence.

FIG. 2 is a graph showing the results of 3 rounds of panning of the phage library of bFGF nanobody.

FIG. 3 is a graph of ELISA results for different phage antibody clones; wherein 1 is a positive control, 2 is a negative control, and 3-86 are different phage antibody clones respectively.

FIG. 4 is an electrophoresis diagram of SDS-PAGE after the purification of bFGF nanobody; wherein Lane 1 is a protein Marker, Lanes 2-7 are nanobodies eluted by 300mmol of imidazole eluent, and are Nb 1-Nb 6 in sequence.

FIG. 5 is a graph showing the analysis of the specific binding between bFGF nanobody and bFGF; wherein 1 is a positive control, 2-7 represent different nano antibody clone strains, and Nb 1-Nb 6 are sequentially adopted.

FIG. 6 is an analysis chart of the heat stability experiment result of bFGF nano-antibody; wherein bFGF monoclonal antibody is a positive control, and Nb 1-Nb 6 represent different nano antibody clone strains.

FIG. 7 is a comparison analysis chart of binding sites of bFGF2 nanobody and FGFR 2; wherein Nb 1-Nb 6 represent different nano antibody clone strains.

FIG. 8 is a graph showing the comparative analysis of binding sites of bFGF nanobody and bFGF mab; wherein Nb 1-Nb 6 represent different nano antibody clone strains.

FIG. 9 is a photograph of a melanoma tumor treated with nano-antibody of bFGF; wherein 1 is a negative control group, and 2 is an anti-bFGF nano antibody group.

FIG. 10 is an analysis chart of the results of bFGF nanobody pairing experiments; whereinFIGS. 10A to F show Nb concentrations of 2. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, 0.25. mu.g/mL, 0.125. mu.g/mL, and 0.0625. mu.g/mL in this order_bFGF4(His-Flag tag) and the rest of the bFGF nanobodies.

FIG. 11 is an analysis chart of the results of the detection range of the concentration of bFGF by the established double-sandwich ELISA method.

FIG. 12 is a standard curve diagram of the concentration of bFGF antigen detected by the established double-sandwich ELISA method.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1 construction of anti-recombinant human basic fibroblast growth factor (bFGF) Nanobody

1. Construction of nano antibody library gene

(1) Alpaca (alee Biotechnology & Pharmaceuticals Co., usa) was immunized with recombinant human basic fibroblast growth factor (bFGF, available from Sigma, beijing, buzz) at a concentration of 0.2mg/mL, 0.2mg of bFGF was mixed with freund's adjuvant in equal volume once every two weeks for 4 immunizations, except for complete freund's adjuvant (available from Sigma) for the first time and freund's adjuvant (available from Sigma) for the remaining several times. The immune process stimulates B cells to express antigen-specific single chain antibodies. (2) After 4 immunizations, 100mL of alpaca peripheral blood lymphocytes were extracted, total RNA was extracted using Trizol reagent (purchased from Invitrogen), and cDNA was synthesized by reverse transcription using AMV First Strand cDNA Synthesis kit (for details, see kit product Specification of NEB). (3) Primers were designed to perform PCR amplification of the variable region of heavy chain antibody (see Table 1 for reaction system and Table 2 for PCR conditions). The PCR product was recovered and purified by QIA quick PCR Purification kit (purchased from QIAGEN) to obtain about 330-370 bp of alpaca heavy chain variable region PCR product.

TABLE 1 PCR amplification reaction System for the variable region of the Single-chain antibody of alpaca

TABLE 2 PCR amplification conditions for the alpaca single chain antibody variable region (cover temperature set at 105 ℃ C.)

2. Construction of a Nanobody phage antibody library

(1) Mu.g of pComb3X (from Allle Biotechnology and Pharmaceuticals) phage display vector and 10. mu.g of alpaca heavy chain variable region PCR product were digested with the restriction enzyme Sfi I (from NEB) and the two fragments were ligated using T4 DNA ligase (from NEB). The ligation products were electroporated into electroporation competent cells XL1-Blue (from Allle Biotechnology and Pharmaceuticals) and then 1mL of SOC medium (from NEB) was added five times to the broth in the resuspension cup of the electroporation cuvette, and 5mL of the broth was incubated for 1h at 250rpm in a shaker at 37 ℃. (2) 10mL of SB medium (preparation method: weighing 8g of MOPs, 24g of peptone and 16g of yeast extract, diluting to 800mL, sterilizing at 121 ℃ for 20min, wherein the culture medium contains 10mg/L tetracycline) and 3. mu.L of ampicillin stock solution (100g/L ampicillin) are added into 5mL of the culture solution, 1. mu.L of the culture solution is diluted by 100 times after mixing, and the mixture is uniformly coated on an LB plate (containing 100mg/L ampicillin) to detect the storage capacity. The remaining culture broth was incubated at 37 ℃ and 275rpm for 1 h. To the culture broth, 4.5. mu.L of an ampicillin stock solution (100g/L of ampicillin) was added, and the culture was continued for 1 hour. To the culture broth 2mL of the helper phage VCSM13 (purchased from Allle Biotechnology and Pharmaceuticals; titer 10)¹²～10¹³pfu/mL), SB medium (containing 10mg/L tetracycline) was added to 200mL, and 92.5. mu.L of ampicillin stock (100g/L ampicillin) was added and incubated at 37 ℃ and 275rpm for 2 h. To the culture medium was added 400. mu.L of kanamycin stock solution (35g/L kanamycin) and cultured overnight. (3) The next day, the cells will be cultured overnightThe resulting bacterial solution was centrifuged at 3000g for 15min at 4 ℃. The supernatant was transferred to a 50mL centrifuge tube containing 4% (w/v) PEG-8000 and 3% (w/v) NaCl, vortexed, and incubated at 4 ℃ for 30min for phage precipitation. Centrifuge at 15000 g for 15min at 4 ℃. The supernatant was discarded and the phage pellet resuspended in 2mL TBS. Centrifuging the supernatant at the maximum rotation speed for 5min at the temperature of 4 ℃, and filtering the supernatant by using a 0.22 mu m filter to a sterile EP tube, thus obtaining the phage original library of the anti-bFGF nano antibody.

3. Screening of phage antibody libraries

(1) bFGF antigen (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was diluted to 2. mu.g/well, coated on a 96-well plate, and coated overnight at 4 ℃. The next day, after blocking with 5% skim milk (2.5g of skim milk completely dissolved in 50mL of 0.05% PBST solution) at 37 ℃ for 1 hour, the phage library preparation solution (i.e., the phage stock obtained in step 2) (10)¹⁴pfu/mL) was added to the antigen-coated wells and incubated at 37 ℃ for 2 hours. XL1-blue bacterial suspension (OD 0.8-1) was added thereto at 100. mu.L/well, and the mixture was incubated at room temperature for 15 min. The step is repeated for a plurality of times until 2mL of infected bacterial liquid is obtained. 2mL of the infected inoculum was added to 6mL of SB medium (containing 10mg/L tetracycline) and 1.6. mu.L of ampicillin stock (100g/L ampicillin) was added. Infected bacterial liquid 1. mu.L is diluted 100 times, smeared on LB plate (containing 10mg/L tetracycline), cultured overnight at 37 ℃, and the antibody library titer is determined. After 8mL of the culture was incubated at 37 ℃ and 275rpm for 1 hour, 2.4. mu.L of an ampicillin stock solution (100g/L of ampicillin) was added thereto and the culture was further continued for 1 hour. To the medium was added 1mL of the helper phage VCSM13, SB medium (containing 10mg/L tetracycline) was added to a final volume of 100mL, and 92. mu.L of ampicillin stock (100g/L ampicillin) was added. 100mL of the culture medium was cultured at 37 ℃ and 275rpm for 1.5-2 hours. 200. mu.L of kanamycin stock (35g/L kanamycin) was added and cultured overnight at 37 ℃ and 275 rpm. The next day, the purified phage was used for the next round of panning, and the same panning process was repeated for 3 rounds. The results are shown in fig. 2, in the process of continuous screening, the positive clones are continuously enriched, thereby achieving the purpose of screening the anti-bFGF specific antibody in the antibody library by using the phage display technology.

(2) 84 positive clones were selected from the plates from round 3 panning, inoculated into 1mL SB medium (containing 10mg/L tetracycline, 100mg/L ampicillin) and the helper phage was used as a negative control for this experiment, and the culture was shake-cultured at 37 ℃ for 5 h. Then adding 10 mu L/hole of VCSM13 helper phage, and culturing for 1.5-2 h. mu.L of kanamycin stock (35g/L kanamycin) was added and shake-cultured overnight at 37 ℃. The next day, the culture broth was centrifuged at 3000g for 15min at 4 ℃. mu.L of the supernatant was taken and 200. mu.L of a 5-fold concentrated mixture of 4% PEG-8000 and 3% NaCl was added, and incubated at 4 ℃ for 30 min. The culture was centrifuged at 15000 g for 15min at 4 ℃. Discard the supernatant, add 200. mu.L TBS, and gently shake well. 50 μ L of suspension was added to a 96-well plate coated with 200 ng/well bFGF antigen. The mixed solution of any 3-well sample is used as a positive control, the auxiliary phage is used as a negative control of the experiment, and PBS is used as a blank control of the experiment. The 96-well plate was incubated at 37 ℃ for 1 h. Anti-M13 antibody (purchased from Sigma) was then incubated at 100 ng/well for 1 hour. 100. mu.L/well of TMB color developing solution (from NEB) was added and incubated for 15 mins. And measuring the light absorption value at 450nm, and defining the clone strain with the experimental group OD value more than 3 times of the auxiliary phage OD value as a positive clone strain. The results are shown in FIG. 3, and a total of 69 clones were positive clones, with a positive proportion of 82%. The positive clones were subjected to plasmid extraction and sent to Biotechnology engineering (Shanghai) GmbH for sequencing. DNA sequencing results were analyzed by Bioedit, and clones having the same amino acid sequence were regarded as the same clones, and clones having different amino acid sequences were regarded as different clones, and finally 6 nanobodies having different amino acid sequences were obtained.

The gene sequence of the coding nano antibody is shown as any one of the gene sequences of SEQ ID NO. 34-39; the gene electrophoresis chart of the 6 bFGF nanobody is shown in FIG. 1.

4. Expression and purification of bFGF nanobody

(1) A group of specific primers (shown in Table 3) are used for carrying out PCR amplification on the target gene of the nano antibody on the pComb3x carrier (the reaction system is shown in Table 4, and the PCR conditions are shown in Table 5).

TABLE 3 amplification primers for target genes of Nanobodies

(underlined Hind III and Nde I sites added)

TABLE 4 PCR amplification system for target gene of nano antibody

TABLE 5 PCR amplification reaction time for the target Gene of the Nanobody

The PCR product is recovered and purified by a QIA quick PCR Purification kit (purchased from QIAGEN), and a nano antibody PCR product of about 330-370 bp is obtained. The expression vector pET22b (available from Novagen) and the target gene of the nanobody were digested with restriction enzymes Hind III (available from Thermo) and Nde I (available from Thermo), and the two fragments were ligated with T4 DNA ligase. The ligation product-transformed expression host bacterium BL21 (DE3) (available from Biotechnology, Inc., Shanghai) was plated on plates containing LB solid medium (containing 100mg/L ampicillin) overnight at 37 ℃.

(2) Individual colonies were picked and inoculated into 5mL of LB medium (containing 100mg/L ampicillin) and shake-cultured overnight at 37 ℃. Then, 1mL of overnight strain was inoculated into 400mL of LB medium (containing 100mg/L ampicillin), and shake-cultured at 37 ℃ and when OD was 0.6-1, IPTG (final concentration: 0.5mmol/L) was added, and shake-cultured at 18 ℃ for 20 hours. The next day, the mixture is centrifuged at 6000rpm for 15min at 4 ℃ to collect the bacteria. The mycelia were resuspended in PBS and sonicated for 10 min. Centrifuging at 8000rpm for 30min to obtain supernatant as expression product coarse extractive solution. Purifying the antibody protein by nickel column ion affinity chromatography. In order to obtain the high-purity antibody, an imidazole gradient elution method is adopted, low-concentration imidazole eluent (100 mmol/L) is used for washing impurity bands, high-concentration imidazole eluent (300mmol/L) is used for eluting target protein, and finally the protein with the purity of more than 90% can be prepared. The results are shown in fig. 4, from left to right: lane 1 shows the standard protein molecules, and lanes 2-7 show the protein samples eluted with 300mmol/L imidazole eluate (Nb 1-6 in this order). The result shows that the purity of the nano antibody can reach more than 90 percent after the nano antibody is purified.

Example 2 test of physicochemical Properties and biological Activity of anti-bFGF Nanobody

1. Specificity analysis of anti-bFGF Nanobody

50 ng/well of bFGF, aFGF, KGF, EGF, TNF alpha, VEGF (all from Beijing Yiqian Shenzhou Biotechnology Co., Ltd.), BSA, and milk were added to a 96-well plate and coated overnight at 4 ℃. The next day, after blocking the microplate with 5% skim milk for 2h, positive control bFGF mAb (purchased from Sigma) at a concentration of 100 ng/well, blank control PBS and the anti-bFGF nanobody prepared in example 1 were added, respectively, and incubated at 37 ℃ for 1 h. Then 100 ng/well of anti-Flag tag antibody (purchased from Sigma) was added and incubated for 1 h. Adding 100. mu.L/well of TMB color developing solution, and incubating for 10 min. Finally, the reaction was terminated with 2.29% sulfuric acid, and the absorbance at 450nm was measured. And (3) defining the antibody clone with the absorbance value of the anti-bFGF nano antibody and the corresponding antigen bFGF being more than 3 times that of other control antigens as positive.

The results are shown in fig. 5, in which 6 strains of anti-bFGF nanobody have strong specific binding ability with bFGF, and weak binding ability with 7 other control antigens.

2. Affinity assay for anti-bFGF Nanobodies

And (3) measuring the affinity of the anti-bFGF nano antibody by adopting a surface plasma resonance biosensor. 0.4mol of N-ethyl-N' - (dimethylaminopropyl) carbodiimide and 0.1mol of N-hydroxysuccinimide are mixed in equal volume and then introduced into an instrument at the flow rate of 20 mu L/min to activate the surface of the chip. After diluting bFGF protein to 2mg/mL with 0.2mol of acetic acid buffer solution having a pH of 4.2, the bFGF protein was allowed to flow over the chip surface. After the fixed amount reaches the required amount, PBS buffer is introduced. The remaining ester bonds were inactivated by passing 1mol ethanolamine (pH 8.5) solution for 7 min. Various concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200nM) of the anti-bFGF nanobody were passed into the instrument at a flow rate of 20. mu.L/min. And (3) calculating the affinity of the anti-bFGF nano antibody through instrument software. As shown in Table 6, the anti-bFGF nanobody has high affinity, and the highest affinity reaches 0.69 nM. The affinity constants of the remaining anti-bFGF nanobodies were 11.32nM, 317.60nM, 5.37nM, 12.37 nM and 10.32nM, respectively.

TABLE 6 affinity constants of anti-bFGF nanobody

	Nb1	Nb2	Nb3	Nb4	Nb5	Nb6
							KD(nM)	11.32	317.60	5.37	0.69	12.37	10.32

3. Determination of thermal stability

Placing the screened nano antibodies (namely Nb 1-6) at four different temperatures of 25 ℃, 37 ℃, 60 ℃ and 90 ℃ for 10min, 30min, 60min, 120min and 180min respectively; a bFGF monoclonal antibody (purchased from Sigma, murine antibody) was also used as a positive control. Samples treated at different temperatures and different times are collected, and the binding property of the samples to the bFGF is detected by an ELISA method, namely whether the antibody has the capacity of binding to the bFGF after the samples are treated at different temperatures and different times. The detection result of fig. 6 shows that, compared with the untreated nano antibody, the relative activity of the nano antibody is maintained above 90% after treatment at 25 ℃ and 37 ℃, and the relative activity of the nano antibody is reduced to some extent after treatment at 60 ℃, but still maintained above 80%. But after 90 ℃ treatment, the relative activity of the nano-antibody is remarkably reduced, wherein the relative activity is reduced to 0% after 60min treatment of Nb5, the relative activity is reduced to 0% after 180min treatment of Nb6, and the relative activity of other groups of nano-antibodies is reduced to 0% after 120min treatment. This is a clear advantage over the bFGF monoclonal antibody as a positive control, although the bFGF monoclonal antibody has a relative activity of more than 90% at three different temperatures of 25 ℃, 37 ℃ and 60 ℃, after 90 ℃ treatment, its relative activity has already decreased to 0% within 10 min. Therefore, it was demonstrated that the nanobody has better stability in high-temperature treatment than the conventional murine monoclonal antibody.

4. Competitive binding assays

(1) Detection of Nanobodies (Nb) of the invention by competitive ELISA_bFGF) Competed with FGFR2 (purchased from beijing yinqiao shenzhou biotechnology limited) for the ability to bind bFGF (purchased from beijing yinqiao shenzhou biotechnology limited). In the experiment, a quantitative 100ng of nano antibody and a variable (0-1 mu g) of FGFR2 are added into a flat plate coated with 50ng of bFGF at the same time for incubation, if the content detected by the nano antibody is less and less along with the increase of the concentration of FGFR2, the nano antibody is consistent or similar to the binding epitope of FGFR2 and the bFGF, or the affinity of the nano antibody and the bFGF is different from that of FGFR2 and the bFGF; if the FGFR2 concentration is gradually increased, the content of the nano antibodyAnd if the nano antibody and the FGFR2 are not changed, the binding epitope with the bFGF is not consistent, or the binding tightness between the nano antibody and the bFGF is higher than that between the FGFR2 and the bFGF. As a result, as shown in fig. 7, the Nb2 and Nb4 contents remained stable with the increase of the FGFR2 content, and it can be presumed that their binding epitopes to bFGF are not consistent with those of FGFR2 and bFGF, or that their affinity to bFGF is higher than that of FGFR 2. The binding force of four nano antibodies Nb1, Nb3, Nb5 and Nb6 to bFGF shows a descending trend along with the increase of the concentration of FGFR2, which indicates that the binding epitope of the four nano antibodies to bFGF is consistent with or close to that of FGFR2 to bFGF, or the affinity of the four nano antibodies to bFGF is lower than that of FGFR2 to bFGF.

(2) Detection of Nb by competitive ELISA_bFGFCompeted with the commercial murine monoclonal antibody bFGF mAb (FB-8) (purchased from Sigma) for the ability to bind bFGF. FIG. 8 shows that Nb4 levels off with increasing bFGF mAb levels, presumably by not matching the binding epitope for bFGF to that of bFGF mAb, or that Nb4 has higher affinity for bFGF than bFGF mAb. The binding force of five nano antibodies Nb1, Nb2, Nb3, Nb5 and Nb6 to bFGF shows a descending trend along with the increase of the concentration of bFGF mAb, which indicates that the binding epitopes of the five nano antibodies to bFGF are consistent with or close to the binding epitopes of bFGF mAb and bFGF, or the affinity of the five nano antibodies to bFGF is lower than that of the bFGF mAb to bFGF.

Example 3 use of anti-bFGF Nanobodies for the treatment of melanoma in mice

The inoculation density of the soft skin part of the outer thigh of the right hind limb of 18C 57BL/6J mice (purchased from Guangdong province medical laboratory animal center, SPF grade, 6-7 weeks old, female) is 5 multiplied by 10⁶Mouse melanoma B16 cells (purchased from Shanghai cell Bank of Chinese academy) at 0.1 mL/cell. And when the average longest diameter of the tumor is 1-3 mm, eliminating mice without tumor growth and with the longest diameter larger than 5 mm. The mice were then randomly assigned to negative control groups, anti-bFGF nanobody groups, 6 per group. Mice were administered subcutaneously around the tumor. PBS control group is given 1 XPBS 0.1 mL/L/day, anti-bFGF nanoThe antibody group was administered with bFGF nanobody (Nb6) at 20 mg/kg/day. The administration was carried out 14 times consecutively. Mice were sacrificed 24h after the last dose and tumor masses were dissected and photographed, and the results are shown in fig. 9. As a result, the tumor body weights of the mice in the negative control group and the anti-bFGF nanobody group were found to be (3.45. + -. 1.73) g and (0.96. + -. 0.81) g, respectively, and the tumor inhibition rate of the anti-bFGF nanobody group was 72.24% compared with that in the negative control group. The anti-bFGF nano antibody can obviously inhibit the proliferation of melanoma in a mouse body.

Example 4 use of Nanobody against bFGF as a diagnostic reagent for the detection of bFGF

1. Pairing experiment of anti-bFGF nano antibody

To construct a double sandwich ELISA method capable of detecting the concentration of antigen bFGF, bFGF nanobodies were paired, wrapped with Nb1(His-HA tag), Nb2(His-HA tag), Nb3(His-HA tag), Nb5(His-HA tag), Nb6(His-HA tag) at a concentration of 5. mu.g/mL, and after 2. mu.g/mL of antigen FGF-2 was added, Nb4(His-Flag tag) at a concentration of 2. mu.g/mL, 1. mu.g/mL, and 0.5. mu.g/mL was incubated, followed by Anti-Flag tag-HRP (purchased from Sigma, cat # A8592-2 MG) as a detection antibody, and finally developed with TMB, and absorbance values were measured at a wavelength of 450 nm.

As shown in FIG. 10, the absorbance values of the experiments of pairing Nb4(His-Flag tag) with the remaining bFGF nanobodies at different concentrations were all more than 3 times higher than that of the control group 1 XPBS, and the highest absorbance values were Nb4(His-Flag tag) and Nb4(His-Flag tag)_FGF-26(His-HA tag). Therefore, we determined that Nb6(His-HA tag) was used as a capture antibody in the double sandwich ELISA method for detecting the concentration of the antigen bFGF, and Nb4(His-Flag tag) was used as a detection antibody for the antigen.

2. Determination of concentration range of antigen bFGF (basic fibroblast growth factor) detected by double-sandwich ELISA (enzyme-linked immunosorbent assay) method

After determining two strains of nano antibodies required in the double-sandwich ELISA method for detecting the concentration of the antigen bFGF, adding the antigens with different concentrations, and determining the detection range of the method on the antigen bFGF. The plates were plated with Nb6(His-HA tag) at a concentration of 5. mu.g/mL, plus 2000ng/mL, 1000ng/mL, 500ng/mL, 250ng/mL, 125ng/mL, 62.5ng/mL, 31.25ng/mL, 15.63ng/mL, 7.81ng/mL, 3.91ng/mL, 1.95 ng/mLng/mL, 0.98ng/mL, 0.49ng/mL of bFGF antigen, and incubating with 2. mu.g/mL of Nb_bFGF4(His-Flag tag), followed by Anti-Flag tag-HRP as detection antibody, and finally developed with TMB, and absorbance value was measured at a wavelength of 450 nm.

The results are shown in FIG. 11, which shows that antigen was detected at a concentration of 7.81ng/mL or more by this detection method (FIG. 11A); the results were plotted as a scatter plot with antigen concentration on the abscissa and absorbance values on the ordinate, and it was found that the change in absorbance was significant when the antigen concentration was between 7.81ng/mL and 500ng/mL (FIG. 11B).

Antigen concentration was adjusted to fall between 10ng/mL and 400ng/mL, and then another double sandwich ELISA experiment was performed, in which Nb6(His-HA tag) at a concentration of 5. mu.g/mL was coated, and Nb4(His-Flag tag) at a concentration of 2. mu.g/mL was incubated after antigen bFGF at concentrations of 400ng/mL, 300ng/mL, 200ng/mL, 100ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, and 10ng/mL, and then Anti-Flag tag-HRP was used as a detection antibody, and finally color was developed with TMB, and absorbance was measured at a wavelength of 450 nm.

And (3) drawing a scatter diagram of the result by taking the antigen concentration as an abscissa and the absorbance value as an ordinate, and solving a linear equation and a correlation coefficient of the scatter diagram: the equation is that Y is 0.0045X-0.005 and the correlation coefficient R is²0.98977. The above experimental results show that in the double sandwich ELISA method for detecting an antibody using Nb6(His-HA tag) at a concentration of 5. mu.g/mL as a capture antibody and Nb4(His-Flag tag) at a concentration of 2. mu.g/mL as an antigen, the antigen bFGF can be diluted to eight concentrations, such as 400ng/mL, 300ng/mL, 200ng/mL, 100ng/mL, 80ng/mL, 40ng/mL, 20ng/mL, and 10ng/mL, and a standard curve for sample detection can be plotted using the antigen concentration as abscissa and the absorbance value as ordinate (FIG. 12).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

<120> anti-recombinant human basic fibroblast growth factor nano antibody and application thereof

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<210> 34

<211> 342

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60

gtttctggaa gcaacttcag taacaatgac atgggctggt accgccaggc tccagggaag 120

cagcgcgagg tggtcgcagg tattagtagt gttggacgta caatgtatgg agaccccgtg 180

aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtatct gcaaatgaac 240

agactgaaag ctaaagacac ggccgtctat tactgtcacc tttatggtga ctataggggg 300

actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342

<210> 35

<211> 342

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60

gtttctggaa gcaacttcag tatcaatgac atgggctggt accgccaggc tccggggaag 120

cggcgcgagg tggtcgcagg tattagtagt gttggacgcg caatgtatgc agaccccgtg 180

aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtactt gcaaatgaac 240

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<210> 36

<211> 342

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60

gtttccggaa gcaacttcag tatcaatgac atgggctggt accgccaggc tccagggaag 120

cggcgcgagg tggtcgcagg tattagtagt gttggacgca caatgtatgc agaccccgtg 180

aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtatct gcaaatgaac 240

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<210> 37

<211> 336

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ctgcagcagt ctgggggagg cttggtgcag cctggggggt ctctgagact ctcctgtaaa 60

gcctctagaa acatcttcag tgtcaatcac atgggctatt accgccaggc tccagggaag 120

gagcgcgagc tggtcgcgct tattactccc ggtggtacca gaaactatgc aaactccgtg 180

aagggccgat tcaccatctc caaagacaac gccaagaaca cggtgtatct gcagatgaac 240

agcctgcaac ctgaggacac ggccgtctat tactgtaata cctggccata tgagtctgcc 300

tattcgggcc aggggaccca ggtcaccgtc tccaca 336

<210> 38

<211> 369

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ctgcaggagt ctgggggagg attggtgcag gctgggggct ctctgagact ctcctgtgca 60

gcctctggac gcaccttcag tagcggtgcc atgggctggt tccgccaggc tccagggaag 120

gagcctgagt ttgtggcaac tattacgtgg gatgggggta cgacatacta tgcagactcc 180

gtgaagggcc gatacaccat ctccagagac aacgccaaga atacggtata tctgcaaatg 240

aacgacctga aacctgagga cacggccgtt tattcctgtg cagcgagatc ttatagtgag 300

gcttactact taatcggctc gtccgattat aactactggg gtcaggggac ccaggtcacc 360

gtctcctca 369

<210> 39

<211> 342

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ctgcaggagt ctgggggaga cttggtgcag cctggggggt ctctgagact ctcctgtgaa 60

gtttctggaa gcaacttcag tatcaatgac atgggctggt accgccaggc tccagggaag 120

cgacgcgagg tggtcgcagg tattagtagt gttggacgca caatgtatgg agaccccgtg 180

aagggccgat tcaccatctc cagagacaac gccaagaaca tggtgtatct gcaaatgaac 240

agactgaaac ctgaggacac ggccgtctat tactgtcacc tttatggtga ctataggggg 300

actggtttct ggggcaaggg gacccaggtc accgtctcgt cg 342

<210> 40

<211> 30

<212> DNA

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<400> 40

ataaaacata tgctgcagga gtctggggga 30

<210> 41

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

ataaaacata tgctgcagca gtctggggga 30

<210> 42

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ataaaaaagc ttcttatcgt cgtcatcttt ata 33

Claims

1. an anti-recombinant human basic fibroblast growth factor nanobody, comprising the variable region domain that is made up of framework region FR and complementarity determining region CDR, is characterized in that,

The complementary determining region CDRs comprise CDR1 shown in SEQ ID No.4, CDR2 shown in SEQ ID No.8, and CDR3 shown in SEQ ID No.12;

The framework region FRs are selected from the group consisting of FR1, FR2, FR3 and FR4:

FR1 shown in the amino acid sequence of SEQ ID No. 15;

FR2 shown in the amino acid sequence of SEQ ID No. 19;

FR3 shown in the amino acid sequence of SEQ ID No. 23;

FR4 shown in the amino acid sequence of SEQ ID No. 27.

2. anti-recombinant human basic fibroblast growth factor nanobody according to claim 1, is characterized in that:

Its amino acid sequence is SEQ ID NO.32.

3 . The nucleotide molecule encoding the anti-recombinant human basic fibroblast growth factor nanobody according to claim 2 , wherein the sequence of the nucleotide molecule is SEQ ID NO.38. 4 .

4. a recombinant expression vector, is characterized in that:

The nucleotide sequence encoding the anti-recombinant human basic fibroblast growth factor nanobody according to any one of claims 1 to 2 is included.

5. a recombinant engineering cell, is characterized in that:

Contains the recombinant expression vector of claim 4 .

6. The application of the anti-recombinant human basic fibroblast growth factor nanobody according to any one of claims 1 to 2 in the preparation of a drug for the treatment of tumor antibody or a cancer diagnosis kit or reagent, characterized in that: the tumor is melanoma.