CN111320694A

CN111320694A - Nano antibody GN2 composed of variable region of heavy chain antibody and preparation method and application thereof

Info

Publication number: CN111320694A
Application number: CN202010094232.3A
Authority: CN
Inventors: 段斯亮; 于声; 桂雄
Original assignee: Guangxi University of Science and Technology
Current assignee: Guangxi University of Science and Technology
Priority date: 2020-02-15
Filing date: 2020-02-15
Publication date: 2020-06-23
Anticipated expiration: 2040-02-15
Also published as: CN111320694B

Abstract

The invention relates to a nanobody GN2 consisting of the variable region of a heavy chain antibody; the variable region of the heavy chain antibody comprises an epitope-complementary region selected from the group consisting of CDR1, CDR2 and CDR3 and their homologues and a framework region selected from the group consisting of FR1, FR2, FR3 and FR4 and their homologues. The invention also relates to a preparation method and application of the nano antibody GN 2. The nano antibody GN2 of the invention can specifically bind with the liver cancer cell of high expression GPC3 protein, inhibit the proliferation of the liver cancer cell, and has the advantages of small molecular weight, stable structure, good heat resistance, high affinity, convenient storage and transportation, weak immunogenicity to human body, strong penetrating power to tumor tissue, easy expression and genetic engineering transformation, and is especially suitable for being used as a diagnostic reagent or a therapeutic antibody. The method can retain more antigen epitopes, and has the advantages of high carrier quality, good enzyme digestion effect, high connection efficiency, low self-connection efficiency and low cost.

Description

Nano antibody GN2 composed of variable region of heavy chain antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a nano antibody consisting of a variable region of a heavy chain antibody, and a preparation method and application thereof.

Background

Glypican 3(GPC3) is a glycoprotein anchored to the cell surface by Glycosylphosphatidylinositol (GPI) and belongs to the heparan sulfate glycoprotein (HSPG) family. GPC3 is associated with the occurrence and presence of liver cancer. GPC3 is highly expressed in the tissues and serum of liver cancer patients, but not in normal liver tissues or serum. GPC3 may have better utility in detecting early stage liver cancer than alpha-fetoprotein. Therefore, GPC3 has become an auxiliary diagnostic marker and therapeutic target for liver cancer.

The monoclonal antibody has wide application prospect in diagnosis and treatment of diseases. However, due to the constraints of high production cost, strong immunogenicity, large molecular weight, poor tumor tissue penetration and other properties, monoclonal antibodies need to be further improved in various aspects such as cost, stability, affinity and the like.

There is a need in the art for safe and effective antibody reagents or antibody drugs which are targeted to bind GPC3 for diagnosing and treating GPC 3-related disorders (such as liver cancer), and a method for screening the nanobody. The present invention satisfies this need.

Disclosure of Invention

To address the above-described needs in the art and to solve the above-described problems in the prior art, a first aspect of the present invention provides a nanobody GN2, wherein:

the nanobody GN2 consists of the variable region of a heavy chain antibody;

the variable region of the heavy chain antibody includes a complementary-to-epitope (CDR) region and a Framework Region (FR);

the framework regions are selected from the group consisting of FR1, FR2, FR3 and FR4 and amino acid sequences having homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, further preferably not less than 99% thereto, and the epitope-complementary regions are selected from the group consisting of CDR1, CDR2 and CDR3 and amino acid sequences having homology of not less than 80%, preferably not less than 90%, more preferably not less than 95%, further preferably not less than 99% thereto;

the amino acid sequence of the CDR1 has a sequence shown in SEQ ID NO.1, the amino acid sequence of the CDR2 has a sequence shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 has a sequence shown in SEQ ID NO. 3; the amino acid sequence of FR1 has a sequence shown in SEQ ID NO. 4; the amino acid sequence of FR2 has a sequence shown as SEQ ID NO. 5; the amino acid sequence of FR3 has a sequence shown in SEQ ID NO. 6; the amino acid sequence of FR4 has the sequence shown in SEQ ID NO. 7.

The nano antibody GN2 provided by the invention can be specifically bound with GPC3 protein.

In some preferred embodiments, the amino acid sequence of nanobody GN2 is shown as SEQ id No.8 in the sequence listing.

In a second aspect, the present invention provides a polynucleotide encoding the nanobody GN2 of the first aspect of the present invention.

In some preferred embodiments, the nucleotide sequence is represented by SEQ ID NO.9 of the sequence Listing.

In a third aspect, the present invention provides a recombinant vector comprising a polynucleotide according to the second aspect of the present invention operably linked to a regulatory sequence, or a nucleotide sequence comprising a codon lacking 1 to 5 amino acid residues and/or a nucleotide sequence having a missense mutation of 1 to 5 base pairs in the nucleotide sequence according to the second aspect of the present invention.

In a fourth aspect, the present invention provides a host cell comprising a recombinant vector according to the third aspect of the invention.

In a fifth aspect, the present invention provides a pharmaceutical composition, comprising nanobody GN2 of the first aspect of the present invention and a pharmaceutically acceptable carrier, preferably, the pharmaceutical composition is used for detecting and/or treating liver cancer, more preferably, the liver cancer is small liver cancer.

In a sixth aspect, the present invention provides a kit comprising the nanobody GN2 of the first aspect of the present invention, preferably, the pharmaceutical composition is used for detecting and/or treating liver cancer, more preferably, liver cancer is small liver cancer.

The present invention provides in a seventh aspect a method for the preparation of the nanobody GN2 of the first aspect of the invention, the method comprising the steps of:

(1) immunizing alpaca with GPC3 protein;

(2) extracting total RNA using peripheral blood from the immunized alpaca;

(3) constructing a nano antibody gene library by using the total RNA;

(4) performing affinity panning on a nano antibody gene library by using magnetic beads coupled with streptavidin and biotinylated GPC3 protein, embedding an ELISA pore plate by using immunoglobulin G (IgG) obtained by separating and purifying alpaca serum by using a protein A agarose gel chromatographic column, and identifying positive clones by using sandwich phase ELISA;

(5) and selecting a positive clone strain with the highest phase ELISA signal to induce and express a nano antibody (named GN2), and separating and purifying to obtain the nano antibody GN 2.

In some preferred embodiments, in step (1), the GPC3 protein is a eukaryotic HEK293 expressed protein; the alpaca is an adult healthy alpaca; and immunization was performed using subcutaneous multiple injections.

In other preferred embodiments, in step (2), lymphocytes from the peripheral blood of the immunized alpaca are used to extract total RNA.

In other preferred embodiments, in step (3), the construction of the nanobody gene library comprises the steps of:

synthesis of cDNA: reverse transcribing the total RNA to synthesize a cDNA strand;

b. amplification of the alpaca heavy chain antibody variable region genes: using the cDNA chain as a template, and respectively using two pairs of primers to obtain variable region (VHH) genes of an alpaca heavy chain antibody IgG2 and a heavy chain antibody IgG3 through PCR amplification, wherein the two pairs of primers comprise a first pair of primers consisting of an HS primer and a Hanti1 primer and a second pair of primers consisting of a primer HS primer and a Hanti2 primer;

c. ligation of the VHH gene to a vector: ligating the VHH gene into a plasmid vector and purifying to recover the ligation product comprising the recombinant vector; preferably, the ligation is performed using a Kit based on the principle of homologous recombination, such as the ClonExpressUltra One Step Cloning Kit (Vazyme biotechco., Ltd) of naught, the plasmid vector is pComb3X vector, and the purification recovery is performed using a PCR product purification Kit;

d. transformation of the recombinant vector: transforming the ligation product into escherichia coli by an electric shock transformation method and detecting the library capacity; preferably, the escherichia coli is ER2738 escherichia coli; more preferably, the shock transformation is performed by: for the mixture of ER2738 e.coli and ligation products, gently stir 2-3 cycles with Tip head to avoid bubble formation, add to a cooled 1.0mm electroporation cuvette, gently flick the electroporation cuvette to allow the mixture to fully enter the electroporation zone, immediately place the electroporation apparatus, and perform electroporation conditions: 1400-1600V, 200-400 omega, 10 muF, 3.5-4.5 milliseconds, adding 975 muL preheated recovery culture medium immediately, blowing up and down the mixed cells for three times, transferring the mixed cells into a bacterial culture tube at 37 ℃, 250rpm, and performing recovery culture for 1 h; more preferably, the detection is performed by diluting 10. mu.L of the bacterial solution^-1、10^-3、10^-5、10^-7After the amplification, plating on a plate of LB culture medium containing ampicillin;

e. phage display of the gene bank: d, resuscitating in the step dThe cultured transformed bacteria are transferred into 200mL SB culture medium containing ampicillin and tetracycline for culture, the culture is carried out at 37 ℃ and 250rpm until the bacteria log phase, and the titer is 10¹²pfu M13KO7 helper phage, standing at 37 ℃ for 30min for infection, shaking at 220-250rpm for 1h, adding kanamycin to a final concentration of 70 mu g/mL and shaking overnight, centrifuging overnight at 13000rpm for 10min at 4 ℃ the next day, taking supernatant, adding 5 × PEG/NaCl, carrying out centrifugation after ice bath for 2-4h, suspending the obtained precipitate in a protective buffer (PBS solution containing 1 × protease inhibitor, 0.02% sodium azide and 0.5% BSA), filtering with a 0.22 mu M filter membrane, subpackaging and standing at-80 ℃ to obtain the nano antibody phage gene library.

In other preferred embodiments, in step (4), the panning is repeated 3-4 times; preferably, the identification is performed by: after panning, selecting clones for culturing, embedding an ELISA pore plate by using protein A agarose gel chromatography column to separate and purify alpaca immunoglobulin G, identifying positive clones by sandwich phase ELISA, extracting clone plasmid with the highest ELISA signal value for sequencing, and obtaining the nucleotide sequence of the nano antibody GN2 and the amino acid sequence of the nano antibody GN 2.

In other preferred embodiments, in step (5), the expression and purification are performed by: extracting plasmids by using strains corresponding to the positive clones, transferring the plasmids into escherichia coli Top10F', selecting monoclonal culture, performing induced expression, collecting bacteria lysis bacteria, purifying by using a nickel column, and washing by using imidazole to obtain a nanometer antibody GN2 of the anti-GPC 3 protein.

In other preferred embodiments, in step (3), the vector is a pComb3X vector; the HS primer used for PCR amplification has a sequence shown as SEQ ID NO. 10; the Hanti1 primer has a sequence shown in SEQ ID NO. 11; and/or the Hanti2 primer has a sequence shown in SEQ ID NO. 12.

In other preferred embodiments, in step (4), the panning and sandwich phase ELISA comprises the following sub-steps:

① washing streptavidin-magnetic beads twice with TBST, adding blocking liquid, and vibrating to block, wherein the first round of screening is BSA blocking liquid, the second round of screening is skimmed milk blocking liquid, and the BSA blocking liquid and the skimmed milk blocking liquid are alternately used;

② adding the phage library into the same sealing liquid for vibration sealing to remove impurities;

③ placing the sealed magnetic beads on a magnetic frame, sucking and removing the supernatant, and adding TBST for cleaning;

④ adding the washed magnetic beads into a binding buffer solution and biotinylated GPC3 protein, then performing oscillation binding, and then adding an impurity-removed phage library for oscillation binding, wherein the binding buffer solution comprises Tris-HCl, NaCl and EDTA;

⑤ placing the reaction solution into a magnetic frame, sucking and removing the supernatant, adding TBST for cleaning, adding glycine-hydrochloric acid into magnetic beads, oscillating for combination, placing into the magnetic frame, sucking out the supernatant, and immediately adding Tris-HCl to neutralize the eluted product to obtain a first product;

⑥ performing titer determination and amplification on the first product to obtain first product amplification solution and second product amplification solution for next round of panning;

⑦ the first product amplification solution was used to put into a second round of panning, the second product amplification solution was put into a third round of screening, the second round of biotinylated GPC3 protein usage was reduced to 6 μ L, and the third round of biotinylated GPC3 protein usage was reduced to 1.5 μ L;

⑧ selecting clone from the plate screened in three rounds, selecting the monoclonal with sterilized toothpick, inoculating to the plate, adding ampicillin resistant culture medium, shake culturing at 37 deg.C and 250rpm for 5-6h, adding M13KO7 for infection, standing at 37 deg.C for 30min, shake culturing at 37 deg.C and 250rpm for 1h, adding kanamycin, overnight culturing at 37 deg.C and 250rpm, centrifuging at 6000rpm for 15min the next day, collecting supernatant, adding 3% skimmed milk, removing impurities, and standing at 4 deg.C for phase ELISA detection.

⑨ immunoglobulin G in the serum of purified alpaca was isolated using a protein A Sepharose column and embedded in ELISA plates, and positive clones were identified by sandwich phase ELISA.

Protein A Sepharose affinity chromatography column separates and purifies anti-GPC 3 immunoglobulin G (IgG). On the 7 th day after the last immunization, blood is collected from the peripheral blood of the alpaca, and the blood is centrifuged for 10min at 2000-3000rpm, and anti-GPC 3 serum is obtained by separation; after dilution of the serum by multiple ratios, the anti-GPC 3 serum titers were measured; preparing a binding buffer (0.02mol/L sodium phosphate, pH7.0), an elution buffer (0.1mol/L glycine-hydrochloric acid, pH2.7), a neutralization buffer (1mol/L Tris-HCl, pH9.0), wherein the above buffers are sterilized by a 0.45 mu m filter before being used, the binding buffer is 0.02mol/L sodium phosphate with pH7.0, the elution buffer is 0.1mol/L glycine-hydrochloric acid solution with pH2.7, and the neutralization buffer is 1mol/L Tris-HCl solution with pH 9.0; equilibrating the protein a sepharose affinity chromatography column with 10-20mL binding buffer; then adding the serum sample into the balanced chromatographic column, and then adding 15-20mL of binding buffer solution; adding 5-8mL of elution buffer solution, and collecting an elution part; performing SDS-PAGE gel electrophoresis on the eluate, and detecting the purity and the concentration; simultaneously plating GPC3 protein, and detecting the biological activity of the collected IgG by using ELISA;

⑩ the anti-GPC 3 immunoglobulin separated in the previous step is diluted to 5-10 μ g/mL with coating buffer, each well is 100 μ L, adding pore plate, coating overnight at 4 ℃, after washing the plate the next day, adding GPC3 protein, incubating for 1-2h at 37 ℃, after washing the plate adding 300 μ L5% skimmed milk blocking liquid to seal each well for 1h, after washing the plate and drying, adding the supernatant of the clone culture in step ⑧, shaking and combining for 1h at 150-.

In an eighth aspect, the present invention provides the use of the nanobody GN2 of the first aspect of the present invention or the polynucleotide of the second aspect of the present invention or the recombinant vector of the third aspect of the present invention or the host cell of the fourth aspect of the present invention in the preparation of a medicament or a kit for detecting and/or treating liver cancer. Preferably, the detection is selected from the group consisting of diagnostic agent immunoassay, flow assay, cellular immunofluorescence assay.

The invention has the following technical effects:

(1) the nanobody GN2 has a small molecular weight of only 15 kDa.

(2) The nano antibody GN2 has stable structure, good heat resistance, high affinity and high resistance to high temperature and high denaturing agent and is easy to store and transport.

(3) Has weak immunogenicity to human body and strong penetrating power to tumor tissues.

(4) The GN2 nano antibody has the function of resisting the proliferation of liver cancer cells, is easy to express and is easy to be modified by genetic engineering, and is particularly suitable to be used as a diagnostic reagent or a therapeutic antibody.

(5) According to the method, the GPC3 protein expressed by the eukaryotic cell (HEK293 cell) is adopted for immunization, the protein expressed by the HEK293 cell is closer to the natural conformation of the protein than the protein expressed by the prokaryotic cell, more epitopes are reserved, alpaca is stimulated to generate high-titer antibodies, and the diversity of a gene library is ensured.

(6) The invention adopts a plurality of pairs of primers to amplify the heavy chain variable region (VHH) gene of the heavy chain antibody, and improves the diversity of the amplified VHH gene.

(7) The phagemid vector is amplified by adopting a host lacking methyltransferase, so that a high-quality vector can be prepared, and the enzyme digestion effect is ensured.

(8) And the homologous recombinase is utilized to increase the connection efficiency and reduce the self-connection efficiency of the vector.

(9) By adopting the liquid phase panning method, more epitopes can be exposed, the probability of blocking some epitopes due to antigen embedding in the solid phase screening process is reduced, and the comprehensive screening of more epitope antibodies is realized.

(10) In sandwich phase ELISA (sandwich phage enzyme-linked immunosorbent assay), a protein A agarose gel chromatographic column is used for separating and purifying alpaca immunoglobulin G embedded ELISA pore plate to screen positive clones, so that the cost is saved, and the phenomenon that when GPC3 protein is embedded and directly adsorbed on a plate, some hydrophobic epitopes are adsorbed on the plate is avoided, so that the probability of combining a phage library with the phage library is reduced, and the positive clones are lost.

The nano antibody GN2 and related products thereof can be used for preparing medicines for diagnosing and treating liver cancer, and the gene of the coded nano antibody GN2 or plasmid containing the gene or recombinant cells of the recombinant plasmid containing the gene can be used for preparing diagnostic reagents for immunoassay, flow assay and cellular immunofluorescence assay and medicines for treating liver cancer.

Drawings

FIG. 1 is a graph of alpaca (Vicugna pacos) immune serum titer detection. The abscissa is the serum dilution factor and the ordinate is the corresponding absorbance OD450 value. The alpaca serum is positive according to the ratio of positive serum to negative serum (positive/negative) being more than or equal to 2.1, and the titer of the alpaca serum after visible immunity can reach 1: 512000. the immune effect is good, and a large amount of antibodies are generated in the alpaca body.

FIG. 2 shows the amplification product of VHH gene. In the figure, lane 1 shows the VHH gene of IgG3 which is an amplification product of HS and Hanti1, and lane 2 shows the VHH gene of IgG2 which is an amplification product of HS and Hanti 2. Lane 3 is a molecular weight standard.

FIG. 3 is an Sfi I restriction map of pComb3X phagemid vector, lane 1 of FIG. 3 shows the result of Sfi I restriction of plasmid extracted after amplification of Escherichia coli DH5 α transformed with pComb3X phagemid vector, and the result of Sfi I restriction of plasmid extracted because the plasmid is methylated, lane 2 of FIG. 3 shows the result of Sfi I restriction of plasmid extracted after amplification of Escherichia coli C2925 transformed with pComb3X phagemid vector, E.coli C2925 shows methylase-deficient bacteria, and the plasmid pComb3X is not methylated, so that Sfi I restriction is successful, and lane M shows the molecular weight standard.

FIG. 4 is a test of the efficiency of the ligation of pComb3X phagemid vector and VHH gene. A. The homologous recombinase is used, the connection efficiency of the vector and the VHH gene is increased, 28 randomly selected clones are positive clones, and the vector self-connection efficiency is 0%. B. Using T4DNA ligase, 28 clones were randomly selected, 10 were vectors without VHH gene ligation, and the vector self-ligation efficiency was 35%.

FIG. 5 library capacity was evaluated by gradient dilution. A. Constructing a library of the library by using a homologous recombinase to connect the pComb3X vector and the VHH gene; 10 μ L of the library were diluted to 10^-4、10^-5、10^-6Number of clones after plating, 10^-6The number of plate clones was 30, so that the library volume of the VHH gene library was 3.0 × 10⁹CFU/mL. B. Using T4 ligase to connect the pComb3X vector and the VHH gene to construct a library volume of the library; 10 μ L of the library were diluted to 10^-3、10^-4、10^-5Number of clones after plating, 10^-5The number of plate clones was 29, so that the library size of the VHH gene library was 2.9 × 10⁸CFU/mL. From this, it was found that the library capacity of the gene library constructed by linking the VHH gene and the vector with the homologous recombinase was ten-fold higher than that of the gene library constructed by linking the VHH gene and the vector with T4DNA ligase.

FIG. 6 shows the results of positive clones identified by sandwich phase ELISA. FIG. A is a schematic diagram of the mechanism of a sandwich phase ELISA. Graph b. results of absorbance values for phase ELISA. Of the 46 clones selected, 38 were positive clones with a positive rate of 82.6%.

FIG. 7 is an SDS-PAGE protein electrophoresis of GPC3 nanobody GN2, shown in lane M: a molecular weight marker; lane 1: flowing cell lysate through the flow-through solution of the nickel column; lane 2: nanobody GN 2. The molecular weight of the GPC3 nanobody GN2 is approximately 15 kDa.

FIG. 8 is a graph of the results of a thermal stability experiment for GN2 nano-antibody. FIG. 8 shows that the nanobody structure is stable and still remains active after 2h treatment at 90 ℃ but GPC3-mAb has lost activity after 2h treatment at 80 ℃. Compared with GPC3-mAb, the nano antibody of the invention has stable structure and good heat resistance.

FIG. 9 is an SDS-PAGE electrophoresis of GN2-Luc fusion protein. Lane M in the figure: a molecular weight standard; lane 1: purified GN2-Luc fusion protein. The GN2-Luc fusion protein has a molecular weight of about 35 kDa.

FIG. 10 shows the establishment of a sandwich ELISA method based on the fusion protein GN 2-Luc. Panel A is a fluorescence intensity curve showing the optimum detection range of 53.87ng/mL-218.76ng/mL for GPC3 protein (i.e., between the EC20-EC80 range in the figure, where EC20, EC80 correspond to GPC3 protein levels at 20% and 80% of the most intense fluorescence, respectively). The content range of serum GPC3 of the liver cancer patient is as follows: 150ng/mL-300ng/mL (GASTROENTEROLOGY 2003; 125: 89-97). Therefore, the detection method is suitable for diagnosing liver cancer. FIG. B is a schematic diagram of the method.

FIG. 11 shows flow cytometry for binding of GN2 to HepG2, a target liver cancer cell. A. Negative control group (Blank control); B. after being blocked by a monoclonal antibody (9C2) bound to the N terminal of GPC3 protein, the binding rate of GN2 and liver cancer cells is high; the binding rate of GN2 and liver cancer cells after the C-terminal monoclonal antibody (SP86) of the protein of GPC3 is blocked; D. no block, the binding rate of GN2 to hepatoma cells.

Fig. 12 is a graph showing the binding of the nanobody GN2 to the hepatoma cell line HepG2(GCP3 positive), and the laser confocal results show that the nanobody GN2 is bound to the cell membrane of HepG2 cell line. The nano antibody GN2 is shown to be capable of specifically binding GPC3 positive cells, namely the nano antibody GN2 specifically binds GPC3 protein.

FIG. 13GN2 Nanobody inhibited the proliferation of GPC3 positive hepatoma cells. The figure shows that GN2 nano antibody can inhibit the proliferation of GPC3 positive liver cancer cells Hep3B, HepG2 and Huh7, and the inhibition efficiency is respectively as high as 49.99%, 54.7% and 42.9%. And GN2 has no inhibition effect on the proliferation of GPC3 negative hepatoma cells Sk-Hep1 and Bel 7404. The GN2 nano antibody can inhibit the proliferation of GPC3 positive liver cancer cells, and can be used as an anti-liver cancer antibody medicament for treating liver cancer.

Detailed Description

The invention will be further illustrated by means of the following examples, without however restricting its scope to these examples.

Example 1: preparation of Nanobody GN2

The preparation process comprises the following steps:

(1) immunizing alpaca:

emulsifying GPC3 protein expressed by 1mg eukaryotic HEK293 cells and Freund's complete adjuvant, wherein the total amount is 2mL, and performing primary immunization on a healthy adult alpaca by adopting a subcutaneous multipoint injection mode; on day 15, 0.5mg of GPC3 protein was emulsified with freund's complete adjuvant (2 mL total) and a second immunization was performed by subcutaneous multiple injections; thereafter, 0.5mg of GPC3 protein was emulsified with Freund's incomplete adjuvant every 7 days to obtain a total of 2mL of emulsified injection for immunization for the next immunization. The total immunization is 6 times, and the blood sampling detection titer is carried out on the 7 th day after each immunization. After the serum titer is detected, 100mL of peripheral blood is collected. The inventor finds that the protein expressed by the HEK293 cell eukaryotic cell is closer to the natural conformation of the protein than the protein expressed by the prokaryotic cell, more epitopes are reserved, alpaca is stimulated to generate high-titer antibodies (figure 1), and the diversity of a gene library is ensured.

(2)100mL of peripheral blood was used to extract lymphocytes for RNA extraction:

using LeukoLOCK^TM(ThermoFisher) Total RNA was extracted according to the protocol.

(3) Construction of a nano antibody gene library:

the construction of the nano antibody gene library comprises the following steps:

a. synthesis of the cDNA strand: reference to

III First-Strand Synthesis System for RT-PCR instructions 8. mu.g of RNA was reverse transcribed to synthesize cDNA strands;

b. amplification of the alpaca VHH gene: and (b) performing 4 PCR reactions by using the cDNA chain of the step a as a template and using primers HS and Hanti1 to amplify the VHH gene of the alpaca heavy chain antibody IgG 3. The VHH gene of the alpaca heavy chain antibody IgG2 was amplified by 6 PCR reactions using primers HS and Hanti2 (FIG. 2). The inventor finds that the VHH genes are derived from heavy chain antibody IgG2 and heavy chain antibody IgG3, so that the VHH genes amplified by the two pairs of primers comprise the VHH gene of IgG2 and the VHH gene of IgG3, and the diversity of gene libraries constructed by the VHH-P1 and VHH-P2 (only the VHH gene of IgG2 is amplified) used in other research institutions is more abundant.

The reaction system is as follows: 2 μ L of cDNA; HS primer 1.3. mu.L; hanti1 primer or Hanti2 primer 1.3 μ L; taqenzyme Mix 44.4. mu.L; reaction procedure: 94 ℃ for 3 min; 94 ℃, 30s, 55 ℃, 30s, 72 ℃, 1min, 32 cycles; 72 ℃ for 5 min. The PCR product was analyzed by electrophoresis, and the PCR product (about 500bp single band) was recovered by cutting the gel.

C, carrying out enzyme digestion linearization treatment on the pComb3X phagemid vector Sfi I: the inventor finds that because Sfi I enzyme is sensitive to methylated DNA, the host bacteria for vector amplification select Escherichia coli C2925, Escherichia coli C2925 lacks non-specific endonuclease I (endA1) activity, lacks methyltransferase, NNA sequence is not methylated, can be used for amplifying high-quality pComb3X plasmid, and does not influence the enzyme digestion effect of Sfi I. Thus, E.coli C2925 competent cells were purchased, and the pComb3X phagemid vector was transformed into E.coli C2925 competent cells. Adding 1 μ L of pComb3X phagemid vector into melted competent cell of Escherichia coli C2925, standing in ice water for 10-20min, heat-shocking at 42 deg.C for 60-90s, ice-cooling for 2min, adding SOC culture medium at 25-37 deg.C, culturing at 37 deg.C and 250rpm for 1 h. 200. mu.L of the bacterial solution was applied to ampicillin-resistant plates and incubated overnight at 37 ℃. The next day, single colonies were picked up to 37 ℃ in ampicillin-resistant LB medium and cultured overnight at 250rpm, and plasmids were extracted using a plasmid extraction kit. The cleavage reaction was performed in a 0.2 μ L PCR tube: mu.g of plasmid pComb3X, 4. mu.L of Sfi I enzyme, 4. mu.L of LCutSmart Buffer were taken and 50. mu.L of deionized water was supplemented with ultrapure water. The mixture was placed in a PCR machine and digested at 50 ℃ for 12h (FIG. 3). The next day, taking the enzyme digestion product for electrophoresis, cutting the gel, recovering large fragments, and dissolving in ultrapure deionized water.

d. According to the principle of homologous recombination, the VHH gene was ligated to pComb3X vector using Clonexpress UltraOne Step Cloning Kit, pComb3X vector large fragment (recovered from Sfi I after digestion) 200ng, VHH gene 60ng, 2 × Clonexpress Mix 5 μ L, ultrapure deionized water supplemented to 10 μ L, left at 50 ℃ for 5 minutes, and immediately cooled on ice after reaction.A PCR product purification Kit was used to purify and recover the ligated product.

e. Electric shock transformation of the recombinant vector: d, adding 3 mu L of the purified ligation product obtained in the step d into 50 mu L of escherichia coli ER2738 electrotransformation competent cells, slightly stirring 1-2 circles by using a Tip head to avoid generating bubbles, adding the electrotransfer system into a cooled 1.0mm electrotransfer cup, slightly throwing the electrotransfer cup by a wrist to enable the cells to sink to the bottom of the electrotransfer cup, and immediately putting the cells into an electrotransfer under electrotransfer conditions: 1400 ℃ 1600V, 200 ℃ 400. omega., 10. mu.F, 3.5-4.5 ms, 975 is added immediatelyBlowing up and down three times of uniformly mixed cells by using microliter of preheated recovery culture medium, transferring the cells into a bacterial culture tube, performing recovery culture at 37 ℃ and 250rpm for 1 h; diluting 1 μ L of bacterial liquid 10^-1、10^-3、10^-5、10^-7The library capacity was checked after doubling (ampicillin resistant LB). The ligation efficiency was verified by colony PCR of 28 clones randomly picked from the plate (figure 4). The primers for colony PCR were: primer HS and primer Hback (SEQ ID NO. 13).

f. Gene bank rescue: transferring the transformed bacteria obtained by the recovery culture in the step e into 200mL of SB culture medium containing ampicillin and tetracycline for culture, culturing at 37 ℃, culturing at 250rpm until the bacteria log phase, and adding 10¹²pfu M13KO7 helper phage is kept standing at 37 ℃ for 30min for infection, added with kanamycin (70 mu g/mL of final concentration) for shaking for 1h and shaken overnight, the next day, the overnight bacteria are centrifuged (13000rpm, 4 ℃) for 10min, the supernatant is taken and added with 5 × PEG/NaCl, and after ice bath for 2h, centrifugation is carried out, the obtained precipitate is resuspended in a protective buffer (PBS solution containing 1 × protease inhibitor, 0.02% sodium azide and 0.5% BSA), filtered by a 0.22 mu M filter membrane and placed at-80 ℃ in a subpackage way, thus obtaining the nano antibody phage gene library.

(4) GPC3 nanobody GN2 was panned and identified: and f, performing affinity panning on the phage library obtained in the step f by using streptavidin-coupled magnetic beads to obtain a first product (output), measuring the titer of 10 mu L of product, performing second and third rounds of screening after amplifying the rest products, selecting 96 clones from a culture plate obtained in the third round of screening, performing overnight culture at 37 ℃, and identifying positive clones by phase ELISA. The inventor finds that compared with a solid-phase screening method for coating antigen protein, magnetic bead liquid-phase screening can expose more epitopes, reduces the probability of blocking some epitopes due to antigen embedding during solid-phase screening, and realizes comprehensive screening of more epitope antibodies.

The affinity panning method is as follows:

a. taking 200 mu L streptomycin affinity magnetic beads, washing the magnetic beads twice by using 1mL TBST, adding 1mL of blocking solution (3% BSA is used for the first round of screening, 3% skimmed milk is used for the second round of screening, and 3% BSA and 3% skimmed milk are alternately used), and carrying out shaking blocking at the temperature of 4 ℃ and the speed of 160rpm for 1 h;

b. adding the phage library into the same sealing solution, and carrying out vibration sealing and impurity removal at the temperature of 4 ℃, 150 and 160 rpm;

c. centrifuging the closed magnetic beads at low speed (2000-;

d. adding the cleaned magnetic beads into 200 mu L of magnetic bead binding buffer solution (20mM pH7.5Tris-HCl, 0.5M NaCl,1mM EDTA) and 30 mu L of biotinylated GPC3 protein, carrying out shaking binding at 4 ℃, 130-150rpm for 30min, adding an impurity-removed phage library, and carrying out shaking binding at 4 ℃, 150-160rpm for 1 h;

e. centrifuging the reaction solution at low speed (2000 plus 3000rpm) for 30s, removing the supernatant, adding 1mL TBST for washing for 10 times, adding 200. mu.L of glycine-hydrochloric acid with pH 2.2 into magnetic beads, oscillating and combining at 150 plus 200rpm for 15min, centrifuging at low speed (2000 plus 3000rpm) for 30s, immediately adding 1.6. mu.L of Tris-HCl with pH 9.1 into the supernatant to neutralize the eluted product, thus obtaining a first product (output);

f. and e, measuring the titer of 10 mu L of the product obtained in the step e, and amplifying the rest products by the following amplification method: adding the eluted product into 3-5mL of logarithmic phase-grown ER2738 bacteria, standing and incubating at 37 ℃ for 30-45 minutes, adding into 5mL of 37 ℃ preheated SB medium containing 200 ug ampicillin and 60 ug tetracycline resistance, shaking at 37 ℃ and 250rpm for 1 hour, supplementing 500 ug ampicillin, continuing shaking at 37 ℃ and 250rpm for 1 hour, adding 10¹²pfu M13KO7 helper phage, standing at 37 ℃ for 30min, transferring to 91mL of pre-warmed SB medium containing 9.4mg ampicillin and 920. mu.g tetracycline, shaking at 220rpm and 250rpm for 1h, adding kanamycin to a final concentration of 70. mu.g/mL and shaking overnight, the next day, overnight bacteria were centrifuged at 13000rpm for 10min at 4 ℃, supernatant was taken and 5 × PEG/NaCl was added, and after 2-4h in ice bath, the obtained pellet was resuspended in 0.01M PBS buffer for the next round of panning.

g. The first product amplification solution is used for feeding into a second round of elutriation; and similarly, the second round of product amplification solution is put into a third round of screening. The second and third rounds of biotinylated GPC3 protein use were sequentially reduced in volume to 6. mu.L, 1.5. mu.L.

h. 46 clones were selected from the three rounds of plates for sandwich Phage Elisa identification of positive clones.

(5) Purified alpaca immunoglobulin G was isolated using a protein a sepharose chromatography column and embedded in ELISA plates and positive clones were identified by sandwich phase ELISA (figure 14). The positive clones were identified by sandwich Phage Elisa as follows:

a. protein A Sepharose affinity chromatography column separates and purifies anti-GPC 3 immunoglobulin G (IgG). 50 ml of alpaca peripheral blood is collected on the 7 th day after the last immunization, centrifuged for 10min at 3000rpm of 2000-. anti-GPC 3 serum titers were determined after dilution of the sera by fold. A binding buffer (0.02mol/L sodium phosphate, pH7.0), an elution buffer (0.1mol/L glycine-hydrochloric acid, pH2.7), and a neutralization buffer (1mol/L Tris-HCl, pH9.0) were prepared, and the above buffers were sterilized with a 0.45 μm filter before use. Cutting off the bottom tip of the column and removing the lid, placing on a rack, and equilibrating the column with 10-20mL of binding buffer; slowly adding the serum sample, and adding 15-20mL of binding buffer solution; 5-8mL of elution buffer was added and the eluted fractions were collected. And (5) carrying out SDS-PAGE gel electrophoresis on the eluate, and detecting the purity and the concentration. GPC3 protein was plated simultaneously and the collected IgG biological activity was measured using ELISA.

b. The anti-GPC 3 immunoglobulin isolated in the previous step was diluted to 5-10. mu.g/mL with coating buffer at 100. mu.L per well, added to a 96-well plate, and coated overnight at 4 ℃. After washing the plate the next day, adding GPC3 protein, and incubating for 1-2h at 37 ℃; after washing the plate, each well was sealed for 1h by adding 300. mu.L of 5% skim milk blocking solution. After the plate is washed and patted dry, the plate is placed at 4 ℃ for standby. Selecting 46 monoclonals with sterilized toothpick, inoculating to 96 deep-well culture plate, culturing 800 μ L ampicillin resistance culture medium per well at 37 deg.C and 250rpm under shaking for 5-6 hr to obtain bacterial solution OD600 of about 0.6-0.8, and adding 10 bacteria per well¹¹pfu M13KO7, left to stand at 37 ℃ for 30min, cultured at 37 ℃ with shaking at 250rpm for 1h, added with kanamycin to a final concentration of 70. mu.g/mL, and cultured at 37 ℃ overnight at 250 rpm. Centrifuging at 6000rpm for 15min, collecting supernatant, adding 3% skimmed milk, removing impurities, adding into Elisa96 pore plate at 4 deg.C, and shaking at 160rpm at room temperature of 150-. Washing with plate washing machine for 3 times, adding secondary antibody against M13, shaking and combining at room temperature of 150-TMB color analysis was performed 30min after pm shaking (FIG. 6).

(6) GPC3 nanobody GN2 expression and purification: extracting plasmids from the ELISA strain with the strongest positive signal obtained in the step (5) by using a plasmid kit (provided by Qiagen company) for transforming escherichia coli Top10F', and selecting a monoclonal for overnight culture at 37 ℃; according to the following steps of 1: 100 percent, adding into 100mL SB culture medium, culturing for 3-4h at 37 ℃ until OD600 reaches 0.6-0.8; adding IPTG (final concentration of 0.5mmol/L), and inducing expression at 26 ℃ overnight; centrifuging at 10000rpm for 10min in the morning the next day, collecting thallus, lysing with bacterial protein extraction reagent (B-PER) at room temperature for 30min, lysing bacteria to release protein, centrifuging again (12000rpm for 15min), collecting supernatant, adding into nickel column, binding at 4 deg.C for 3-6h, washing 4 column volumes with 20mmol/M imidazole, and collecting 5mL of imidazole washing solution of 50mmol/L and 100mmol/L respectively to obtain nanometer antibody GN2 (FIG. 7).

And (3) selecting the positive clone obtained in the step (4) for sequencing to obtain the nucleotide sequence (shown as SEQ ID No.9 in the sequence table) of the nano antibody GN2, and further obtaining the amino acid sequence (shown as SEQ ID No.1 in the sequence table) of the nano antibody GN2 according to a codon table.

Example 2: nanobody GN2 thermostability experiments:

GPC3 protein, 1. mu.g/mL GPC3 protein, 100. mu.L per well were coated overnight at 4 ℃ in ELISA plates. After three PBST washes, 300. mu.L of 5% skim milk was added to each well and blocked for 1 hour at 37 ℃. The Nanobody GN2 of the present invention and the commercial GPC3 monoclonal antibody (GPC3-mAb, Invitrogen) were added to each well at different temperatures (4 ℃,37 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃) for 2 hours. Add 100. mu.L per well and after incubation for 1 hour at room temperature, wash the plate 3 times with PBST.

GN2 group, because GN2 antibody HAs HA tag, HA-mAb labeled with HRP enzyme (SANTACRUZ Co.) was added to each well, incubated at room temperature for 40 minutes, washed with PBST 3 times, developed with TMB for 10 minutes, and stopped with 2M sulfuric acid, and then ultraviolet absorbance (OD450 value) at 450nm was measured with a microplate reader (see FIG. 8).

In the GPC3-mAb group, an anti-mouse IgG secondary antibody (SANTA CRUZ) labeled with HRP enzyme was added, and after incubation at room temperature for 30 minutes, the plate was washed with PBST 3 times, followed by addition of TMB for color development for 10 minutes, and after terminating the reaction with 2M sulfuric acid, ultraviolet absorbance (OD450 value) at 450nm was detected with a microplate reader (see FIG. 8).

Example 3: establishment of sandwich ELISA method for detecting serum GPC3 protein by GN 2-luciferase fusion protein based on GN2 nanometer antibody:

(1) the GN2 gene was amplified. Using the plasmid obtained in the step (4) of example 1 as a template, amplifying GN2 nucleotide sequence by PCR; the PCR product is subjected to enzyme digestion by using Nco I and Sfi I, and the enzyme digestion product is purified and recovered by using the PCR product purification kit.

(2) The fusion gene GN2-Luc was constructed. Synthesizing a fluorescent reporter gene, namely a Nano-luciferase gene (Nano-luciferase, Luc for short) (the amino acid sequence of the fluorescent protein reporter gene luciferase is shown as SEQ ID NO. 14): subcloning the fragment between Not I and SalI sites of a vector pET22b, double digesting the vector pET22b containing the fluorescence reporter gene nano-luciferase by Nco I and Sfi I, and cutting gel to recover a pET22b vector skeleton sequence containing the luciferase gene; and (3) connecting the vector framework sequence recovered in the step (2) and the PCR product purified in the step (1) under the action of T4 ligase to construct a fusion gene GN2-Luc, transforming escherichia coli BL21(DE3) competent cells by the connecting product, selecting positive clones with correct sequencing, and performing induced expression.

(3) And (3) expression and purification of the fusion protein GN 2-Luc. Positive clones were selected and cultured overnight at 37 ℃ and 220rpm in 4mL of SB medium containing ampicillin resistance. Overnight cultures were taken the following day according to 1: 100 into 200mL SB medium containing ampicillin resistance, 37 ℃, 220rpm culture until OD600 reaches about 0.6, adding IPTG (final concentration of 0.5mM, 220rpm, overnight induction expression fusion protein GN 2-Luc. centrifugation in the afternoon of the next day (10000rpm, 10min) to collect thalli, using bacterial protein extraction reagent (B-PER) to lyse for 30min at room temperature, lysing bacteria to release protein, centrifuging again (12000rpm, 15min), collecting supernatant, adding nickel column, binding for 3-6h at 4 ℃, washing 4 column volumes with 20mmol/M and 40mmol/M imidazole, collecting 5mL 100mmol/L imidazole washing solution, namely, the fusion protein GN2-Luc (figure 9) is obtained, the inventors find that the nano antibody has a simple structure and is easier to construct by genetic engineering operation, and the fusion protein GN2-Luc is obtained by prokaryotic expression in the invention.

(4) Establishment of a sandwich ELISA method based on the fusion protein GN 2-Luc. Commercial GPC3 polyclonal antibody (bio-techne Biol.) was diluted to 3. mu.g/mL with coating buffer, 100. mu.L per well, added to a 96-well plate, and coated overnight at 4 ℃. The next day, PBST was used as eluent, and after 3 washes with a plate washer, 300. mu.L of 5% skim milk was added and blocked for 1 h. The plate washer is used for 3 times, GPC3 protein with different concentrations is added, and the mixture is shaken and combined for 1h at the room temperature of 150-. The plate was washed 3 times with a plate washer, 0.2. mu.g/mL of the fusion protein GN2-Luci was added, shaking was performed at 150. mu.g/mL and 160rpm at room temperature for 0.5H, the plate was washed 5 times with a plate washer, and after patting the 96-well plate, 100. mu.L of luciferase substrate Coelenterazine-H (5. mu.g/mL) was added to each well, and the fluorescence signal was immediately detected using a plate reader (FIG. 10).

Example 4: flow cytometry for detecting binding efficiency and binding epitope of nano antibody GN2 and target cell HepG2

HepG2 cells highly expressing GPC3 were selected as target cells, and the binding efficiency of GN2 to GPC3 antigen was examined. Meanwhile, in order to detect whether the GN2 nanobody binds to the N-terminus or the C-terminus of the GPC3 protein, the blocking experiment was performed using a GPC3 monoclonal antibody (9C2) that binds to the N-terminus of the GPC3 protein and a GPC3 monoclonal antibody (SP86) that binds to the C-terminus of the GPC3 protein, respectively, in this experiment.

Collection 1 × 10⁵-10⁶HepG2 cells were resuspended in 500. mu.L PBS (2% BSA) in three groups. The first group, the N-terminal group is blocked, and GPC3 monoclonal antibody (9C2) is added into HepG2 cells; second, blocking C end group, adding GPC3 monoclonal antibody (SP86) into HepG2 cell; third, no blocking group, PBS control was added. After incubation for 30min binding, cells were washed 3 times with PBS, and GN2 nanobodies were added to each of the three groups, incubated at 4 ℃ for binding for 30-40min, and slowly shaken at 150-160rpm to prevent non-specific binding. After washing the cells with PBS, HA-Tag (C29F4) rabbitmab (PE Conjugate) was added to the cell culture, incubated at 4 ℃ for 30 minutes, the cells were washed and analyzed by flow cytometry (fig. 11).

Example 5: fluorescence imaging observation of targeted binding of nanobody GN2 to GPC3 positive hepatoma cells:

specific binding of nanometer antibody GN2 and GPC3 liver cancer cell is observed by confocal scanning fluorescence microscope, and the specific method comprises adding nanometer antibody GN2(1 μ g) into 1-5 × 10⁶HepG2 liver cancer cells (GPC3 positive) were incubated at 4 ℃ for 20-40min in the dark, washed with PBS 2 times, then incubated at 4 ℃ for 20-40min with 5. mu.l of PE anti-HA-tagged antibody (PE anti-Hatagatoside) (abcam, Clone:20A12), washed with PBS 2-3 times, and then the sample was placed in a confocal scanning fluorescence microscope to observe the specific binding of the Nanobody GN2 to HepG2 liver cancer cells (FIG. 12).

Example 6: inhibition of nanometer antibody GN2 on GPC3 positive hepatoma cell proliferation:

culturing cells with 24-well plate, and controlling the number of cells per well to be about 2-3 × 10⁴Nanobody GN2 (20. mu.M) was added while using human immunoglobulin hIgG as a control; the liver cancer cell lines selected GPC3 positive liver cancer cells (Hep3B, HepG2 and Huh-7 cells) and GPC3 negative liver cancer cells (SK-Hep1 and Bel-7404). After 4-5 days of incubation, MTT solution (5mg/mL) was added to each well and incubation continued for 4h, the supernatant was carefully aspirated and discarded, 150. mu.L DMSO was added to each well, the crystals were fully dissolved by shaking for 10min, and the 490nm wavelength was measured using a microplate reader (FIG. 13).

TABLE 1 sequence of the amplified products or primers used in the examples.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Guangxi university of science and technology

<120> a nano antibody GN2 composed of variable region of heavy chain antibody, preparation method and application thereof

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Claims

1. A nanobody GN2 consisting of the variable region of a heavy chain antibody, characterized by:

the nanobody GN2 consists of the variable region of a heavy chain antibody;

the variable region of the heavy chain antibody comprises an epitope-complementing region and a framework region;

the framework region is selected from the group consisting of FR1, FR2, FR3 and FR4 and amino acid sequences having homology of not less than 80%, and the epitope-complementary region is selected from the group consisting of CDR1, CDR2 and CDR3 and amino acid sequences having homology of not less than 80%;

the amino acid sequence of the CDR1 has a sequence shown in SEQ ID NO.1, the amino acid sequence of the CDR2 has a sequence shown in SEQ ID NO.2, and the amino acid sequence of the CDR3 has a sequence shown in SEQ ID NO. 3; the amino acid sequence of FR1 has a sequence shown in SEQ ID NO. 4; the amino acid sequence of FR2 has a sequence shown in SEQ ID NO. 5; the amino acid sequence of FR3 has a sequence shown in SEQ ID NO. 6; the amino acid sequence of FR4 has a sequence as shown in SEQ ID NO. 7;

more preferably, the amino acid sequence of the nanobody GN2 is shown as SEQ ID No.8 in the sequence table.

2. A polynucleotide encoding nanobody GN2 of claim 1 or 2; preferably, the nucleotide sequence is shown as SEQ ID NO.9 in the sequence table.

3. A recombinant vector comprising the polynucleotide of claim 2 operably linked to a regulatory sequence or comprising a nucleotide sequence of the nucleotide sequence of claim 2 lacking codons for 1-5 amino acid residues and/or having missense mutations of 1-5 base pairs, or a host cell comprising the recombinant vector.

4. A pharmaceutical composition comprising nanobody GN2 of claim 1 and a pharmaceutically acceptable carrier, preferably for use in the detection and/or treatment of liver cancer, more preferably, the liver cancer is small liver cancer.

5. A kit, comprising: (a) the nanobody GN2 of claim 1; (b) and/or a fusion protein comprising the nanobody GN2 and a luciferase amino acid sequence, preferably, the luciferase amino acid sequence has a sequence shown as SEQ ID NO.14, more preferably, the kit is used for detecting and/or treating liver cancer, and more preferably, the liver cancer is small liver cancer.

6. A method for preparing nanobody GN2 of claim 1, comprising the steps of:

(1) immunizing alpaca with GPC3 protein;

(2) extracting total RNA using peripheral blood from the immunized alpaca;

(3) constructing a nano antibody gene library by using the total RNA;

(4) performing affinity panning on a nano antibody gene library by using magnetic beads coupled with streptavidin and biotinylated GPC3 protein, embedding an ELISA pore plate by using immunoglobulin G obtained by separating and purifying alpaca serum by using a protein A agarose gel chromatographic column, and identifying positive clones by sandwich phase ELISA;

(5) and selecting the positive clone strain with the highest phase ELISA signal to induce and express the nano antibody, and separating and purifying to obtain the nano antibody GN 2.

7. The method of claim 6, wherein:

in the step (1), the GPC3 protein is a protein expressed by eukaryotic cells (HEK 293); the alpaca is an adult healthy alpaca; and/or immunization is performed using subcutaneous multi-site injection;

in step (2), total RNA is extracted by using lymphocytes from peripheral blood of immunized alpaca;

in the step (3), the construction of the nano antibody gene library comprises the following steps:

b. amplification of the alpaca heavy chain antibody variable region genes: using the cDNA chain as a template, and respectively using two pairs of primers to obtain variable region genes of an alpaca heavy chain antibody IgG2 and a heavy chain antibody IgG3 through PCR amplification, wherein the two pairs of primers comprise a first pair of primers consisting of an HS primer and a Hanti1 primer and a second pair of primers consisting of a primer HS primer and a Hanti2 primer;

c. linking the heavy chain antibody variable region genes to a vector: ligating the heavy chain antibody variable region gene into a phagemid vector and purifying to recover a ligation product comprising a recombinant vector; preferably, the ligation is performed by using a kit based on the principle of homologous recombination, the phagemid vector is pComb3X vector, and the purification recovery is performed by using a PCR product purification kit;

d. transformation of the recombinant vector: transforming the ligation product into escherichia coli by an electric shock transformation method and detecting the library capacity; preferably, the escherichia coli is ER2738 escherichia coli; more preferably, the shock transformation is performed by: for the mixture of ER2738 e.coli and ligation products, gently stir 2-3 cycles with Tip head to avoid bubble formation, add to a cooled 1.0mm electroporation cuvette, gently flick the electroporation cuvette to allow the mixture to fully enter the electroporation zone, immediately place the electroporation apparatus, and perform electroporation conditions: 1400-1600V, 200-400 omega, 10 muF, 3.5-4.5 milliseconds, adding 975 muL preheated recovery culture medium immediately, blowing up and down the mixed cells for three times, transferring the mixed cells into a bacterial culture tube at 37 ℃, 250rpm, and performing recovery culture for 1 h; further preferably, the detection is performed by: diluting 1 μ L of bacterial liquid 10^-1、10^-3、10^-5、10^-7After the amplification, plating on a plate of LB culture medium containing ampicillin;

e. phage display of the gene bank: transferring the transformed bacteria obtained by the recovery culture in the step d into 200mL of SB culture medium containing ampicillin and tetracycline for culture, culturing at 37 ℃, culturing at 250rpm until the bacteria log phase, and adding the bacteria with the titer of 10¹²pfu M13KO7 helper phage, 37 ℃ standing infection for 30min, 220 and 250rpm shake culture for 1h, adding kanamycin to a final concentration of 70 μ g/mL and shake culture overnight; the next day, overnight bacteria were centrifuged at 13000rpm for 10min at 4 deg.C and harvestedAdding 5 × PEG/NaCl into the clear liquid, carrying out ice bath for 2-4h, then carrying out centrifugation, suspending the obtained precipitate in a protective buffer (PBS solution containing 1 × protease inhibitor, 0.02% sodium azide and 0.5% BSA), filtering the precipitate by using a 0.22 mu m filter membrane, subpackaging and placing at-80 ℃ to obtain a nano antibody phage display library;

in step (4), the panning is repeated 3-4 times; preferably, the identification is performed by: after the elutriation is finished, selecting clones for culturing; separating and purifying immunoglobulin G in alpaca serum by using a protein A agarose gel chromatography column, embedding an ELISA pore plate, identifying positive clones by sandwich phase ELISA, extracting positive clone plasmids with the highest ELISA signal value for sequencing, and obtaining a nucleotide sequence of a nano antibody GN2 and an amino acid sequence of a nano antibody GN 2;

in step (5), the expression and purification are carried out by: extracting plasmids by using strains corresponding to the positive clones, transferring the plasmids into escherichia coli Top10F', selecting monoclonal culture, performing induced expression, collecting bacteria lysis bacteria, purifying by using a nickel column, and washing by using imidazole to obtain a nanometer antibody GN2 of the anti-GPC 3 protein.

8. The method of claim 6 or 7, wherein in step (3), the phagemid vector is a pComb3X vector; the HS primer used for PCR amplification has a sequence shown as SEQ ID NO.10, the Hanti1 primer has a sequence shown as SEQ ID NO.11, and the Hanti2 primer has a sequence shown as SEQ ID NO. 12.

9. The method according to any one of claims 6 to 8, wherein in step (4) the panning and sandwich phase ELISA comprises the sub-steps of:

⑧ selecting clone from the plate screened by three rounds, selecting monoclonal with sterilized toothpick, inoculating to the plate, adding ampicillin resistance culture medium, shake culturing at 37 deg.C and 250rpm for 5-6h, adding M13KO7 for infection, standing at 37 deg.C for 30min, shake culturing at 37 deg.C and 250rpm for 1h, adding kanamycin, overnight culturing at 37 deg.C and 250rpm, centrifuging at 6000rpm for 15min the next day, collecting supernatant, adding 3% skimmed milk, removing impurities, and standing at 4 deg.C for phase ELISA detection;

⑨ separating and purifying immunoglobulin G in alpaca serum by using a protein A agarose gel chromatography column, embedding an ELISA pore plate, identifying positive clones by sandwich phase ELISA, separating and purifying anti-GPC 3 immunoglobulin G by using the protein A agarose gel affinity chromatography column, collecting blood of alpaca peripheral blood on the 7 th day after the last immunization, centrifuging at 3000rpm of 2000-3000rpm for 10min, separating to obtain anti-GPC 3 serum, detecting the titer of the anti-GPC 3 serum after diluting the serum by multiple proportions, preparing a binding buffer solution, an elution buffer solution and a neutralization buffer solution, wherein the buffer solution is a Tris-Hcl solution which is 0.02mol/L and pH7.0 and is 0.1mol/L and pH2.7, the neutralization buffer solution is a 1mol/L and pH9.0, balancing the protein A gel affinity column by using the binding buffer solution of 10-20mL, adding a sample into the balanced serum, adding 15-20mL of the serum into the balanced chromatography column, adding 5-20mL of the neutralization buffer solution into the Tris-Hcl solution of pH9.0, collecting an IgG-HCl solution, and collecting an IgG-HCl eluate by using a GPC gel electrophoresis gel chromatography column, and collecting the IgG concentration, and collecting the IgG, wherein the IgG is detected by using a biological gel electrophoresis detection reagent, and detecting the IgG concentration of the IgG is 3;

⑩ diluting the immunoglobulin G separated in the previous step with coating buffer solution to 5-10 μ G/mL, adding 100 μ L of each well, adding a pore plate, coating overnight at 4 ℃, washing the plate the next day, adding GPC3 protein, incubating at 37 ℃ for 1-2h, adding 300 μ L of 5% skimmed milk blocking solution to each well after washing the plate, sealing for 1h, washing the plate, beating, adding the supernatant of the clone culture obtained in the step ⑧, shaking and combining at room temperature of 150-.

10. Use of nanobody GN2 of claim 1 or nucleotide of claim 2 or recombinant vector of claim 3 or host cell comprising the recombinant vector in the preparation of a medicament or kit for detecting and/or treating liver cancer; preferably, the detection is selected from the group consisting of diagnostic agent immunoassay, flow assay, cellular immunofluorescence assay.