CN111848802B

CN111848802B - Nano antibody of glypican 3 with outstanding thermal stability and preparation method thereof

Info

Publication number: CN111848802B
Application number: CN202010704194.9A
Authority: CN
Inventors: 王文义; 徐畅; 姜长安
Original assignee: Zhuhai Institute Of Advanced Technology Chinese Academy Of Sciences Co ltd
Current assignee: Zhuhai Institute Of Advanced Technology Chinese Academy Of Sciences Co ltd
Priority date: 2020-07-21
Filing date: 2020-07-21
Publication date: 2022-02-08
Anticipated expiration: 2040-07-21
Also published as: CN111848802A

Abstract

The invention relates to a nanometer antibody of glypican 3 with outstanding thermal stability and a preparation method thereof, and also relates to an amino acid sequence and a gene coding sequence of the nanometer antibody, and an expression vector and a host cell capable of expressing the nanometer antibody. The amino acid sequence of the nanometer antibody of GPC3 provided by the invention is shown as SEQ ID NO. 7, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 8. The nanometer antibody of GPC3 of the invention can specifically identify liver cancer cells highly expressed by GPC3 by binding with GPC3 expressed by cell membranes. The antibody has outstanding thermal stability, and can be used for GPC3 function research, and can also be used for development of diagnostic reagents and therapeutic drugs for hepatocellular carcinoma.

Description

Nano antibody of glypican 3 with outstanding thermal stability and preparation method thereof

Technical Field

The invention relates to a nano antibody of glypican 3 with outstanding thermal stability and a preparation method thereof, and also relates to a gene, an expression vector, a host cell and related applications for encoding the nano antibody, belonging to the technical field of biology.

Background

Hepatoma carcinoma has the name of "cancer king", which is the third highest cancer with high mortality rate. The number of new liver cancer cases and death cases in China account for more than half of the total number of the whole world. Hepatocellular carcinoma (HCC) is the predominant form of liver cancer, accounting for approximately 75%. Because of the hiding of early symptoms of liver cancer and the limitation of diagnosis means, most liver cancer patients are diagnosed in the late stage of liver cancer, while the late stage liver cancer is poor in prognosis and very difficult to treat. Therefore, early diagnosis of liver cancer is of great significance in improving the survival time of liver cancer patients. The targeted drug has strong targeting property and small side effect, and becomes one of the most promising drugs for liver cancer diagnosis and treatment. The marker protein of the liver cancer tumor cell is a common molecular target of targeted drugs.

Glypican 3(Glypican-3, GPC3) is a marker protein for hepatocellular carcinoma and is highly expressed specifically in hepatocellular carcinoma tumor cells. GPC3 is a heparan sulfate proteoglycan anchored to the cell membrane by Glycosylphosphatidylinositol (GPI) and is involved in regulating ontogeny, cell proliferation and differentiation. Normally, GPC3 is expressed only in fetal liver, and there is generally no or low expression of GPC3 in healthy adult liver. However, GPC3 is overexpressed in liver tissue of most hepatocellular carcinoma patients. The study shows that the sensitivity of GPC3 as a hepatocellular carcinoma marker protein is 70-94%, and the specificity is 86-100%. Therefore, GPC3 is a good target molecule for hepatocellular carcinoma targeting drugs. The specific antibody of the target molecule plays a targeting role in targeting drugs, specifically binds the drugs to the molecular target, and plays a diagnosis and/or treatment function. At present, monoclonal antibodies specifically recognizing GPC3 have been reported, however, in view of the limitations of low stability, complex preparation process, high cost and the like of the conventional antibodies, development of novel antibodies against GPC3 is urgently needed.

The Nanobody (Nb) is a Heavy chain Variable region (Variable domain of the Heavy chain of HcAbs, VHH) part of a Heavy chain antibody (HcAbs) naturally lacking a light chain, which exists in the serum of camelidae and cartilaginous fishes as a novel high-quality antibody. The nanobody has complete antigen binding capacity, is the smallest antigen binding fragment, has the molecular weight of 13-15kDa, and the size (the diameter of the crystal structure is 2.5 nanometers, and the length is 4 nanometers) is in the nanometer size. The structure of the nanobody is mainly divided into conserved Framework Regions (FRs) and sequence-variant Complementarity Determining Regions (CDRs), which are respectively responsible for maintaining the basic structure of the nanobody and determining specific binding with an antigen. The CDRs are divided into three independent regions, complementarity determining region 1(CDR1), complementarity determining region 2(CDR2), and complementarity determining region 3(CDR3), depending on their positions in the whole antibody. Compared with the traditional antibody, the nano antibody has smaller volume, can penetrate into the cancerated tissues more deeply, and can identify hidden antigen sites. In addition, the nano antibody has the advantages of strong specificity, small molecular weight, high solubility, high structural stability, strong binding force with antigen, strong tissue penetration capacity, low human immunogenicity, easy screening, easy preparation and the like, and can be applied to the research, diagnosis and treatment of diseases. The molecular imaging technology is very concerned due to the characteristics of non-invasiveness and high resolution, the nano antibody is a high-quality targeting molecule in molecular imaging, the nano antibody can be quickly positioned at a target site, and free nano antibody can be quickly discharged out of a body through a kidney, so that the signal to noise ratio of a specific signal is ensured, a high-resolution tumor anatomical image and target protein molecule positioning information are favorably obtained, and the early diagnosis efficiency of cancer is effectively improved. In tumor treatment, the nano antibody is coupled with a tumor drug and can be used for targeted drug delivery; in Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T), a nano antibody as a targeting molecule can accurately target T cells to tumor cells, thereby efficiently and accurately killing the tumor cells. Particularly, compared with the traditional antibody, the nano antibody has strong tissue penetration capability, and has a better application prospect in diseases related to solid tumors such as HCC. In addition, the nano-antibody can also carry out targeted tracing, functional manipulation and the like on the GPC3 protein, and is used for GPC3 related biomedical research.

Disclosure of Invention

The invention aims to provide a nanometer antibody of GPC3 and a preparation method thereof, provides effective targeting molecules for diagnosis and treatment of hepatocellular carcinoma, and also provides a gene, an expression vector, a host cell and related applications for encoding the nanometer antibody.

One technical solution for solving the above technical problems of the present invention is as follows: a nanobody of glypican 3 having outstanding thermostability, which nanobody comprises 3 complementarity determining regions, CDR1, CDR2 and CDR 3; wherein, the amino acid sequence of the CDR1 is shown as SEQ ID NO.1 (Gly Leu Glu Val Gln Trp Ser Asp), the amino acid sequence of the CDR2 is shown as SEQ ID NO.2 (Ser Trp Thr Pro Phe Phe Ile Ser), and the amino acid sequence of the CDR3 is shown as SEQ ID NO.3 (Ala Ser Gln His Ser His Ala Met Ala Met His Thr Lys Thr Leu Tyr).

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the nanobody further comprises four framework regions, FR1, FR2, FR3 and FR4, alternately linked to three complementarity determining regions; wherein the amino acid sequence of FR1 is shown as SEQ ID NO.13 (Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser), the amino acid sequence of FR2 is shown as SEQ ID NO.14 (Leu Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Cys Gly Ile), the amino acid sequence of FR3 is shown as SEQ ID NO.15 (Tyr Glu Asp Ser Val Lys Gly Arg Phe Thr Cys Ser Arg Asp Asp Ala Arg Asn Thr Val Tyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys), and the amino acid sequence of FR4 is shown as SEQ ID NO.16 (Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser).

Furthermore, the amino acid sequence of the nano antibody is shown as SEQ ID NO. 7

Another technical solution of the present invention for solving the above technical problems is as follows: a gene of the nanobody of glypican 3 with outstanding thermostability is encoded, comprising three nucleotide sequences respectively encoding 3 complementarity determining regions thereof, wherein the nucleotide sequence encoding CDR1 is shown in SEQ ID No.4 (ggtcttgagg tgcagtggtc ggat), the nucleotide sequence encoding CDR2 is shown in SEQ ID No.5 (agctggacgc cgttttttat tagt), and the nucleotide sequence encoding CDR3 is shown in SEQ ID No.6 (gcgtcgcagc atagtcatgc gatggctatg catactaaga cgctgtac).

Furthermore, the nucleotide sequence of the gene is shown as SEQ ID NO. 8

Another technical solution of the present invention for solving the above technical problems is as follows: an expression vector, wherein the expression vector can express the nano antibody or contain the gene.

Further, the expression vector is a phage or plasmid.

Another technical solution of the present invention for solving the above technical problems is as follows: a host cell comprising said expression vector.

Further, the host cell is Escherichia coli.

Another technical solution of the present invention for solving the above technical problems is as follows: a method for preparing a nanobody of the phosphatidylinositol glycan 3 nanobody with outstanding thermostability, which is screened by phage display technology, comprising the following steps:

(1) preparing GPC3 delta GPI-FLAG recombinant protein with GPI region removed and carboxyl terminal with FLAG label;

(2) incubating the nano antibody phage library with GPC3 delta GPI-FLAG recombinant protein, eluting phage combined with GPC3 delta GPI-FLAG recombinant protein and infecting host cells, re-incubating the phage generated by the elution and the GPC3 delta GPI-FLAG recombinant protein, performing a new round of screening, and repeating the screening for three times;

(3) selecting a single clone from a host cell containing phage plasmids obtained finally after screening for sequencing, regarding clones with the same sequences of three complementarity determining regions CDR1, CDR2 and CDR3 as the same clone strain, and selecting the clone strain with high repetition rate according to a sequencing result;

(4) connecting the gene segments of the selected nano antibody in the clone to an expression vector through PCR amplification, restriction enzyme digestion and T4 ligase connection, converting the expression vector to a protein prokaryotic expression strain, inoculating the protein prokaryotic expression strain to a culture medium for culture, collecting bacteria, and extracting and purifying periplasmic protein to obtain the nano antibody of GPC 3.

Another technical solution of the present invention for solving the above technical problems is as follows: the application of the nano-antibody of the glypican 3 with outstanding thermal stability can be used for functional research of GPC3 or development of hepatocellular carcinoma diagnostic reagents and therapeutic drugs, and comprises an analysis reagent for sub-cellular localization of GPC3 protein, a detection reagent for the protein level of GPC3, a hepatocellular carcinoma molecular imaging diagnostic reagent, a drug targeting delivery reagent, a chimeric antigen receptor T cell immunotherapy and development of nano-antibody drugs.

The invention has the beneficial effects that: the invention screens the nanometer antibody of GPC3 by the phage display technology, the nanometer antibody specifically binds to GPC3 positioned on the cell membrane, and the stability of the antibody is high. The invention utilizes prokaryotic expression to prepare the nano antibody, and has simple operation and low cost. As a target molecule of hepatocellular carcinoma, the nano antibody can be used for hepatocellular carcinoma molecular imaging diagnosis, drug targeted delivery, chimeric antigen receptor T cell immunotherapy or development of nano antibody drugs, and the nano antibody can also be used for GPC3 function research.

Drawings

FIG. 1 is a flow chart of construction of expression vector pLenti6/V5-GPC 3. delta. GPI-FLAG.

FIG. 2 shows the results of purification of the secretory protein GPC 3. delta. GPI-FLAG in the culture broth of the stable cell line using FLAG M2 magnetic beads, followed by immunoblotting with a FLAG antibody to confirm the target protein.

FIG. 3 is a flow chart of construction of a nanobody expression vector pET22 b-Nb-His.

FIG. 4 shows the results of detection of antigen-antibody binding using GPC3 co-immunoprecipitated nanobodies.

FIG. 5 shows the results of detection of antigen-antibody binding using Nanobody co-immunoprecipitation GPC 3.

FIG. 6 shows the results of immunofluorescent staining experiments to detect the co-localization of nanobodies with GPC 3.

FIG. 7 shows the result of specific binding between the nano-antibody and the liver cancer cells detected by immunofluorescence staining assay.

FIG. 8 shows the stability test results of the selected nanobody and known GPC3 nanobody HN3 and GPC3 monoclonal antibody GPC3-mAb detected by ELISA.

Detailed Description

The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.

EXAMPLE 1 preparation of GPC3 recombinant protein

Preparing GPC3 recombinant protein with FLAG tag at carboxyl terminal.

(1) F359S mutation was carried out based on the coding gene Sequence of GPC3 in NCBI (NCBI Reference Sequence: NM-004484.4) and the amino acid Sequence of GPC3 in UniProt (UniProt Reference Sequence: P51654-1), GPI region (1690 and 1740bp) was removed, and the fused Sequence of GPC 3. delta. GPI lacking GPI site and FLAG tag was artificially synthesized (Shenzhen Huada Genet science, Inc.), and EcoRI and NotI cleavage sites were carried at 5 'end and 3' end, respectively. The amino acid sequence coded by GPC3 delta GPI is shown as SEQ ID NO 9

The nucleotide sequence is shown as SEQ ID NO. 10

(2) GPC 3. delta. GPI-FLAG DNA fragment and pcDNA3.1 vector (intermediate vector) containing SfiI cleavage sites at both ends of the engineered polyclonal site were subjected to double digestion with restriction enzymes EcoRI and NotI (New England Biolabs Co.), and the obtained fragments were subjected to ligation reaction with T4 ligase (Thermo Fisher Scientific Co.) for sequencing and validation. GPC 3. delta. GPI-FLAG was ligated into the lentiviral packaging vector pLenti6/V5 by SfiI digestion to finally obtain the correct pLenti6/V5-GPC 3. delta. GPI-FLAG vector (see FIG. 1).

(3) pLenti6/V5-GPC 3. delta. GPI-FLAG and lentiviral packaging vectors pLP1, pLP2 and pVSVG were co-transfected into HEK293FT cells and the corresponding lentiviruses were obtained from the supernatant. The lentivirus is transduced into HEK293T cells, and cell strains which stably express GPC3 delta GPI-FLAG recombinant protein are obtained through antibiotic screening. The cell lines were incubated at 37 ℃ with 5% CO₂Culturing for 2 days under the above conditions, collecting the culture supernatant, and subjecting GPC 3. delta. GPI-FLAG recombinant protein to FLAGM2 magnetic beads (Sigma-Aldrich Co.) as described in the specificationAnd (5) purifying.

Example 2 Nanobody phage library screening for GPC3

(1) Coating antigen: GPC 3. DELTA. GPI-FLAG protein secreted into the culture supernatant was purified according to the FLAG M2 magnetic bead instructions to prepare magnetic beads coated with GPC 3. DELTA. GPI-FLAG protein and verified by Coomassie blue staining and immunoblotting. As shown in FIG. 2, the size of the purified protein is between 72 kD and 85kD as shown by the result of the Coomassie brilliant blue staining on the left image, and the purified protein has a single band, which indicates high purity. The right panel shows the result of immunoblotting detection of the FLAG-tagged antibody against GPC3 Δ GPI-FLAG recombinant protein, and shows that the protein size is consistent with the result of Coomassie brilliant blue staining, indicating that the purified protein is GPC3 Δ GPI-FLAG target protein. GPC3 protein has several glycosylation sites, and the trailing diffuse band should be due to glycosylation. The position of the band for the GPC3 Δ GPI-FLAG recombinant protein is indicated by an arrow. (2) Pretreatment of the nano antibody library: 1ml TBS buffer solution (pH7.4) containing 5% (g/ml) BSA and 0.1% Tween-20 was added to the Nanobody phage display library constructed in this laboratory to bring the phage in solution to about 1X 10¹¹pfu. After mixing, 20. mu.l of FLAG M2 magnetic beads were added and incubated for 60min at room temperature with rotation. (3) Binding of phage containing nanobodies to GPC 3: the pretreated nanobody phage solution was transferred to a new centrifuge tube, 20. mu.l of magnetic beads coated with GPC 3. delta. GPI-FLAG (GPC 3. delta. GPI-FLAG protein amount: about 2. mu.g) was added, and the mixture was incubated at room temperature for 60min by rotation. (4) Washing: the nanobody phage solution was discarded, and the beads were washed 3 times with a solution containing TBST (0.1% Tween-20). (5) And (3) elution: adding 100 μ l of 0.1M triethylamine solution into the centrifuge tube containing the magnetic beads, oscillating at normal temperature for 10min, adding 100 μ l of 1M Tris-HCl (pH 6.8), oscillating, mixing, and standing at room temperature for 5 min. (6) Infection: the eluate was added to 1ml of Escherichia coli SS320 culture medium (OD600 of 0.6) and incubated at 37 ℃ for 40 min. (7) Counting: centrifuging SS320 culture solution at 5000rpm for 4min, discarding supernatant, adding 200 μ l LB, mixing, collecting partial bacterial solution according to certain dilution, spreading on counting plate, and culturing overnight. (8) After counting, 1ml ddH is used₂O collecting the bacteria on the plate, taking a certain amount of bacteria liquid in 2 XYT (containing 2% Glucose),incubated at 37 ℃ for 15min at 220 rpm. (9) The helper phage M13KO7(New England Biolabs) was added, infected with SS320, incubated at 37 ℃ for 60min, and phages were prepared and purified for the next round of screening. (10) The collected phage nanobody library was subjected to the next round of screening three times in total according to the procedure of example 2.

EXAMPLE 3 preparation of Nanobodies

In example 2, after the third round of phage infection selection, E.coli SS320 was plated and phage plasmid-containing monoclonals were picked for sequencing. The gene sequences of the respective antibody clones were analyzed and aligned using Vector NTI software, and clones having the same sequence as the complementary regions CDR1, CDR2, and CDR3 were considered as the same clones. According to the sequencing result, one clone strain with high repetition rate is selected and marked as G8 clone, the shown DNA sequence is shown as SEQ ID NO. 8, and the coded amino acid sequence is shown as SEQ ID NO. 7. The nucleotide fragment of the selected G8 nano antibody is connected into an expression vector pET22b through PCR amplification, restriction enzyme digestion and T4 ligase connection, and the specific process is as follows: (1) designing PCR primers for amplifying the nucleotide fragments of the nanobody according to the sequencing result: an upstream primer catgactagt caagttcaat tagtc (SEQ ID NO:11), a downstream primer cgcggatcca agcttgaatt c (SEQ ID NO: 12). (2) A DNA fragment of the nanobody was PCR-amplified using a G8 phage plasmid as a template, and then digested with SpeI and EcoRI restriction enzymes (New England Biolabs), ligated into an expression vector pET22b using T4 ligase (Thermo Fisher Scientific Co.), and subjected to DNA sequencing to obtain a recombinant plasmid pET22b-G8-His containing a nucleotide sequence encoding G8 (see FIG. 3). (3) A monoclonal strain of the phage gene library of the nano-antibody without any screening after infecting escherichia coli SS320 is randomly picked, the nano-antibody expressed by the monoclonal strain is used as a Negative control antibody (NC), and a control nano-antibody expression vector pET22b-NC-His (see fig. 3) is constructed according to example 3(1) and example 3 (2). (4) The recombinant plasmids pET22b-G8-His and pET22b-NC-His are respectively transformed into escherichia coli BL21 to obtain engineering bacteria for expressing the His-tag-fused nano-antibody, the engineering bacteria are respectively inoculated into an LB culture medium, the culture is carried out at 37 ℃ and 220rpm until the OD600 value of a culture solution is 0.6, IPTG is added until the final concentration is 1mM, and then the induction expression is carried out at 18 ℃ and 220rpm for 12 hours. After the culture, the escherichia coli was collected and periplasmic protein was extracted, and the nano antibody carrying the His tag was purified according to the instruction of using His tag protein purification magnetic beads (suzhou beaver biomedical engineering limited).

Example 4GPC3 Co-immunoprecipitated Nanobody assay

(1) Magnetic beads bound to GPC3 Δ GPI-FLAG were obtained according to example 1. (2) The Nb-His-containing cell lysate was obtained as described in example 3, and the cell lysate was diluted to a 1 XBuffer W solution system using 10 XBuffer W (1M Tris/HCl, pH 8.0,1.5M NaCl,10mM EDTA), and 500. mu.l of the diluted cell lysate was added to 5. mu.l of magnetic beads bound to GPC 3. delta. GPI-FLAG, followed by incubation at 4 ℃ for 2 hours with rotation. (3) The beads were washed 3 times with BufferW, the wash solution was discarded, 10. mu.l of 1 XSDS gel loading buffer was added to each tube, shaken, mixed well and boiled for 10 min. (4) GPC3 and nano-antibody in the sample after co-immunoprecipitation were detected by protein immunoblotting (Western blot, WB).

The detection result is shown in fig. 4, wherein NC is a negative control nanobody, and Input on the left side is applied nanobody protein; the right side is immunoprecipitated nanobodies, using Blank FLAGM2 magnetic Beads (Blank Beads) without bound antigen as a control to exclude direct binding of nanobodies to the magnetic Beads. In the immunoblotting detection, the anti-His antibody is used to detect the nano antibody, and the intensity of the strip represents the amount of the nano antibody protein. It can be seen that: the negative control nano antibody NC is not combined with the magnetic beads; although trace G8 was bound to the blank FLAG M2 magnetic bead, the binding force of G8 to the GPC 3. delta. GPI-FLAG magnetic bead was greatly enhanced, indicating that the G8 nano antibody can bind GPC3 with high specificity.

Example 5G8 Nanobody Co-immunoprecipitation GPC3 assay

(1) Magnetic beads successfully bound with Nb-His were obtained according to example 3. (2) 5ml of the GPC 3. delta. GPI-FLAG secretion protein-containing culture solution obtained in example 1 was added, and the mixture was subjected to rotary incubation at 4 ℃ for 2 hours. (3) The culture medium was discarded, and the beads were washed 3 times with TBST. The wash solution was discarded and 10. mu.l SDS gel loading buffer was added to each tube, shaken, mixed and boiled for 10 min. (4) GPC3 and nanobodies were detected in the sample after co-immunoprecipitation using Western blotting.

The detection result is shown in FIG. 5, wherein NC is negative control nano antibody, the upper graph is Input, and GPC3 Δ GPI-FLAG recombinant protein added in each group of the experiment is shown; the middle graph shows the amount of the nano-antibody bound on each group of magnetic beads for experimental detection; the lower panel shows immunoprecipitated GPC3 Δ GPI-FLAG recombinant protein. The results show that: the negative control nanobody NC did not bind GPC3 Δ GPI-FLAG, whereas group G8 was able to efficiently precipitate GPC3 Δ GPI-FLAG, indicating that G8 binds GPC3 with high specificity.

Example 6 immunofluorescence staining experiment of Nanobody-recognized cell membrane surface GPC3

(1) To further confirm that nanobody G8 can recognize and bind to cell membrane-localized GPC3, we fused mCherry to the N-terminus of GPC3, constructed the relevant vector according to the method of example 1, and obtained HEK293T cell strain stably expressing mCherry-GPC3 by lentivirus packaging and transduction. (2) Fully digesting the subcultured cell strain by pancreatin to obtain a single cell suspension, and inoculating the single cell suspension to a cell climbing sheet. (3) The cell slide was rinsed with PBS (pH7.4, the same applies below) at 37 ℃ and fixed with 4% PFA (paraformaldehyde) at 37 ℃ for 10min, and rinsed with PBS. (4) Blocking was performed in 2% BSA/TBS for 30min at room temperature. (5) The nanobodies were diluted to 10. mu.g/mL with 2% BSA/TBS and incubated overnight at 4 ℃. (6) TBST was rinsed, and anti-HA (Cell signaling) diluted with 2% BSA/TBS was added thereto, followed by incubation at 37 ℃ for 2 hours. (7) TBST rinse, Alexa Fluor 488 fluorophore conjugated secondary antibody (Thermo Fisher Scientific Co.) diluted with 2% BSA/TBS was added and incubated at 37 ℃ for 1 h. (8) The nuclei were stained by TBST rinsing and incubation with 5. mu.g/mL DAPI for 2 min. And (5) after the slide is made, observing and photographing under a fluorescence confocal microscope.

The detection result is shown in fig. 6A, and the distribution of mCherry fluorescence signal indicates that mCherry-GPC3 is located in cell membrane and cytoplasm, while Alexa Fluor 488 fluorescence signal indicating the location of nanobody G8 only appears on cell membrane. Since no surfactant treatment was applied in this example, the cell membrane structure was not destroyed, and thus G8 bound only to the cell membrane localized GPC3 protein. The results of the analysis of the linear line region are shown in fig. 6B, the abscissa is the distance from the left end point on the linear line, the ordinate represents the intensity of the fluorescence signal, and it can be seen that the intensities of the two fluorescence signals mCherry and Alexa Fluor 488 change synchronously with the position variation, and the results of the calculation of the rectangular region show that the pearson correlation coefficient of the two fluorescence signals is 0.966 (fig. 6C). The results show that the nano antibody G8 and the mCherry-GPC3 with the cell membrane location have good co-location, and the antibody can specifically recognize and bind the GPC3 protein on the cell membrane.

Example 7 immunofluorescence staining experiment for recognizing liver cancer cells by nano antibody

To further verify whether the nanobody G8 specifically recognizes GPC 3-positive hepatoma cells, we performed immunofluorescence staining on a human hepatocellular carcinoma cell line HepG2 highly expressing GPC3 using the nanobody according to the method of example 6. Human epidermal squamous carcinoma cell line a431 not expressing GPC3 was used as a negative control cell line.

The detection result is shown in fig. 7, the negative control cell a431 has no fluorescence signal of the nanobody G8 detected on the cell membrane, but a stronger fluorescence signal can be detected on the cell membrane of HepG2, which indicates that G8 can specifically recognize and bind to the hepatocellular carcinoma cell.

Example 8 stability testing experiment of Nanobodies

The stability of the nano antibody G8, the known GPC3 nano antibody HN3 and the known GPC3 monoclonal antibody GPC3-mAb is detected by enzyme-linked immunosorbent assay.

(1) The antigenic protein GPC3 Δ GPI-FLAG was expressed and purified as in example 1. (2) Nanobody G8 was expressed and purified according to example 3. HN3 Nanobody was purchased from Creative Biolabs, and is HN3-hFc fusion antibody [ Feng M, Gao W, Wang R, et al.2013.therapeutic targeting collagen-3 via a formation-specific single-domain antibody in a heterocyclic antigen. PNAS 110(12): E1083-E1091 ]. GPC3-mAb was purchased from Chengdu Biotechnology, Inc. (3) The antibody concentrations of G8, HN3-hFc and GPC3-mAb were measured, and 2. mu.g of the antibody was put in a 1 XPBS (pH7.4) solution and treated at 4 ℃, 20 ℃, 37 ℃, 55 ℃, 70 ℃ and 90 ℃ for 2 hours, respectively. (4) Equal amounts of GPC 3. delta. GPI-FLAG protein were diluted in the coating solution, added to the plates and coated overnight at 4 ℃. (5) After Blocking with Blocking buffer (PBS, 0.05% Tween-20, 1% BSA), the above different temperature treated G8, HN3-hFc and GPC3-mAb antibodies were added to the plate and incubated at 37 ℃ for 1 hour. (6) After washing, a mouse His-tag antibody and a mouse hFc antibody were added to the G8 well and the HN3-hFc well, respectively, and incubated at 37 ℃ for 1 hour. GPC3-mAb microwells omitted this step. (7) After washing, a fresh dilution of HRP-conjugated enzyme-labeled secondary antibody (Thermo Fisher Scientific, 1:10000 dilution) was added and incubated at 37 ℃ for 30 min. (8) Adding TMB color development solution (Beijing Soilebao Tech., Ltd.), and incubating at 37 deg.C for 20min for color development. (9) After the development, a development stop solution (Beijing Solebao technologies Co., Ltd.) was added to each reaction well, and the light absorption value at 450nm in each reaction well was measured by an microplate reader.

As shown in FIG. 8, under the same conditions, the antigen binding ability of the mouse monoclonal antibody GPC3-mAb sharply decreased with increasing temperature, and at 70 ℃ the antigen binding ability decreased by 92.4% (with reference to the light absorption value at 4 ℃ at 450nm, the same applies below); the heat stability of the nano antibody HN3 reported in the literature is higher than that of GPC3-mAb, but the stability is still obviously reduced along with the temperature rise, the antigen binding capacity is reduced by 31.1% at 70 ℃, and is reduced by 53.2% at 90 ℃; the light absorption value of the nano antibody G8 in the invention does not change greatly with the temperature rise, the antigen binding capacity is only reduced by 5.5% at 70 ℃, and is reduced by 29.8% at 90 ℃, which shows that the thermal stability of G8 is higher than that of HN3 and GPC 3-mAb. The basic structure of the nano antibody is maintained by the framework region of the nano antibody, the protein stability of the nano antibody is influenced, and the amino acid sequences of the framework region FR1-4 of the nano antibody G8 obtained by screening are respectively shown as SEQ ID NO 13-16.

The above-mentioned embodiments are preferred embodiments of the present invention, and do not limit the present invention, and any other changes or modifications such as substitutions, simplifications, combinations, etc. without departing from the spirit and principles of the present invention are included in the protection scope of the present invention.

Sequence listing

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caagttcaat tagtcgagtc cggcggagct ctggtccagc ctggaggtag tctgcgttta 60

tcctgcgcag ccagcggtct tgaggtgcag tggtcggatc tccgctggta tcgccaggca 120

ccgggtaagg agcgcgaatg ggtatgcggt attagctgga cgccgttttt tattagttac 180

gaagacagcg ttaaagggcg ttttacttgt tcccgcgacg acgctcgtaa cacagtctat 240

ttacaattaa actcattaaa gcctgaagac acagcggtat attactgcgc gtcgcagcat 300

agtcatgcga tggctatgca tactaagacg ctgtactggg ggcagggcac gcaggtaacc 360

gttagctca 369

<210> 9

<211> 563

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu

1 5 10 15

Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp

20 25 30

Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly

35 40 45

Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val

50 55 60

Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys

65 70 75 80

Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala

85 90 95

Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln

100 105 110

Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala

115 120 125

Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe

130 135 140

Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp

145 150 155 160

Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro

165 170 175

Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu

180 185 190

Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe

195 200 205

Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln

210 215 220

Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile

225 230 235 240

Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu

245 250 255

Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys

260 265 270

Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly

275 280 285

Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu

290 295 300

Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu

305 310 315 320

Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys

325 330 335

Asn Ala Gly Lys Leu Thr Thr Thr Ile Gly Lys Leu Cys Ala His Ser

340 345 350

Gln Gln Arg Gln Tyr Arg Ser Ala Tyr Tyr Pro Glu Asp Leu Phe Ile

355 360 365

Asp Lys Lys Val Leu Lys Val Ala His Val Glu His Glu Glu Thr Leu

370 375 380

Ser Ser Arg Arg Arg Glu Leu Ile Gln Lys Leu Lys Ser Phe Ile Ser

385 390 395 400

Phe Tyr Ser Ala Leu Pro Gly Tyr Ile Cys Ser His Ser Pro Val Ala

405 410 415

Glu Asn Asp Thr Leu Cys Trp Asn Gly Gln Glu Leu Val Glu Arg Tyr

420 425 430

Ser Gln Lys Ala Ala Arg Asn Gly Met Lys Asn Gln Phe Asn Leu His

435 440 445

Glu Leu Lys Met Lys Gly Pro Glu Pro Val Val Ser Gln Ile Ile Asp

450 455 460

Lys Leu Lys His Ile Asn Gln Leu Leu Arg Thr Met Ser Met Pro Lys

465 470 475 480

Gly Arg Val Leu Asp Lys Asn Leu Asp Glu Glu Gly Phe Glu Ser Gly

485 490 495

Asp Cys Gly Asp Asp Glu Asp Glu Cys Ile Gly Gly Ser Gly Asp Gly

500 505 510

Met Ile Lys Val Lys Asn Gln Leu Arg Phe Leu Ala Glu Leu Ala Tyr

515 520 525

Asp Leu Asp Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro

530 535 540

Lys Asp Asn Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser

545 550 555 560

Pro Leu Lys

<210> 10

<211> 1689

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggccggga ccgtgcgcac cgcgtgcttg gtggtggcga tgctgctcag cttggacttc 60

ccgggacagg cgcagccccc gccgccgccg ccggacgcca cctgtcacca agtccgctcc 120

ttcttccaga gactgcagcc cggactcaag tgggtgccag aaactcccgt gccaggatca 180

gatttgcaag tatgtctccc taagggccca acatgctgct caagaaagat ggaagaaaaa 240

taccaactaa cagcacgatt gaacatggaa cagctgcttc agtctgcaag tatggagctc 300

aagttcttaa ttattcagaa tgctgcggtt ttccaagagg cctttgaaat tgttgttcgc 360

catgccaaga actacaccaa tgccatgttc aagaacaact acccaagcct gactccacaa 420

gcttttgagt ttgtgggtga atttttcaca gatgtgtctc tctacatctt gggttctgac 480

atcaatgtag atgacatggt caatgaattg tttgacagcc tgtttccagt catctatacc 540

cagctaatga acccaggcct gcctgattca gccttggaca tcaatgagtg cctccgagga 600

gcaagacgtg acctgaaagt atttgggaat ttccccaagc ttattatgac ccaggtttcc 660

aagtcactgc aagtcactag gatcttcctt caggctctga atcttggaat tgaagtgatc 720

aacacaactg atcacctgaa gttcagtaag gactgtggcc gaatgctcac cagaatgtgg 780

tactgctctt actgccaggg actgatgatg gttaaaccct gtggcggtta ctgcaatgtg 840

gtcatgcaag gctgtatggc aggtgtggtg gagattgaca agtactggag agaatacatt 900

ctgtcccttg aagaacttgt gaatggcatg tacagaatct atgacatgga gaacgtactg 960

cttggtctct tttcaacaat ccatgattct atccagtatg tccagaagaa tgcaggaaag 1020

ctgaccacca ctattggcaa gttatgtgcc cattctcaac aacgccaata tagatccgct 1080

tattatcctg aagatctctt tattgacaag aaagtattaa aagttgctca tgtagaacat 1140

gaagaaacct tatccagccg aagaagggaa ctaattcaga agttgaagtc tttcatcagc 1200

ttctatagtg ctttgcctgg ctacatctgc agccatagcc ctgtggcgga aaacgacacc 1260

ctttgctgga atggacaaga actcgtggag agatacagcc aaaaggcagc aaggaatgga 1320

atgaaaaacc agttcaatct ccatgagctg aaaatgaagg gccctgagcc agtggtcagt 1380

caaattattg acaaactgaa gcacattaac cagctcctga gaaccatgtc tatgcccaaa 1440

ggtagagttc tggataaaaa cctggatgag gaagggtttg aaagtggaga ctgcggtgat 1500

gatgaagatg agtgcattgg aggctctggt gatggaatga taaaagtgaa gaatcagctc 1560

cgcttccttg cagaactggc ctatgatctg gatgtggatg atgcgcctgg aaacagtcag 1620

caggcaactc cgaaggacaa cgagataagc acctttcaca acctcgggaa cgttcattcc 1680

ccgctgaag 1689

<210> 11

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

catgactagt caagttcaat tagtc 25

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cgcggatcca agcttgaatt c 21

<210> 13

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Gln Val Gln Leu Val Glu Ser Gly Gly Ala Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser

20 25

<210> 14

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Leu Arg Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Trp Val Cys

1 5 10 15

Gly Ile

<210> 15

<211> 37

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Tyr Glu Asp Ser Val Lys Gly Arg Phe Thr Cys Ser Arg Asp Asp Ala

1 5 10 15

Arg Asn Thr Val Tyr Leu Gln Leu Asn Ser Leu Lys Pro Glu Asp Thr

20 25 30

Ala Val Tyr Tyr Cys

35

<210> 16

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

Claims

1. A nanobody of glypican 3 having outstanding thermostability, which comprises 3 complementarity determining regions (CDR1, CDR2 and CDR3, respectively); wherein, the amino acid sequence of the CDR1 is shown in SEQ ID NO.1, the amino acid sequence of the CDR2 is shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 is shown in SEQ ID NO. 3.

2. The nanobody of glypican 3 having outstanding thermostability according to claim 1, which further comprises four framework regions alternately linked to three complementarity determining regions, FR1, FR2, FR3 and FR 4; wherein the amino acid sequence of FR1 is shown as SEQ ID NO.13, the amino acid sequence of FR2 is shown as SEQ ID NO.14, the amino acid sequence of FR3 is shown as SEQ ID NO.15, and the amino acid sequence of FR4 is shown as SEQ ID NO. 16.

3.The nanobody of glypican 3 having outstanding thermostability according to claim 2, characterized in that the amino acid sequence of the nanobody is shown in SEQ ID NO 7.

4. A gene encoding the Nanobody with glypican 3 having outstanding thermostability according to any one of claims 1 to 3, which comprises three nucleotide sequences each encoding 3 complementarity determining regions thereof, wherein a nucleotide sequence encoding CDR1 is shown in SEQ ID NO.4, a nucleotide sequence encoding CDR2 is shown in SEQ ID NO.5, and a nucleotide sequence encoding CDR3 is shown in SEQ ID NO. 6.

5. The gene of the nanobody of glypican 3 having outstanding thermostability according to claim 4, wherein the nucleotide sequence of the gene is shown in SEQ ID NO. 8.

6. An expression vector capable of expressing the nanobody of any one of claims 1 to 3 or containing the gene of claim 4 or 5.

7. A host cell comprising the expression vector of claim 6.

8. The host cell of claim 7, wherein the host cell is E.coli.

9. Use of the Nanobody of glypican 3 having outstanding thermostability according to any one of claims 1 to 3 for the preparation of a functional research reagent of glypican 3, for the preparation of a diagnostic reagent for hepatocellular carcinoma or for the preparation of a therapeutic drug for hepatocellular carcinoma, the functional research reagent comprising an analytical reagent for the subcellular localization of glypican 3 protein, the diagnostic reagent comprising a detection reagent for the level of glypican 3 protein, a diagnostic reagent for imaging hepatocellular carcinoma molecules, and the therapeutic drug comprising a drug targeting delivery reagent, a chimeric antigen receptor T cell immunotherapy drug, and a Nanobody drug.