阅读说明：本技术 Tk1抗体、试剂盒及其用途 (TK1 antibody, kit and application thereof ) 是由 李劲 张波 李惠军 周际 艾伦·何 斯文·斯库格 于 2021-01-06 设计创作，主要内容包括：本发明公开了一种TK1抗体、试剂盒及其应用。TK1抗体包含重链可变区,重链可变区包含SEQ ID NO：1氨基酸序列的重链CDR1、SEQ ID NO：2氨基酸序列的重链CDR2以及SEQ ID NO：3氨基酸序列的重链CDR3；以及轻链可变区,轻链可变区包含SEQ ID NO：4氨基酸序列的轻链CDR1、SEQ ID NO：5氨基酸序列的轻链CDR2以及SEQ ID NO：6氨基酸序列的轻链CDR3。本发明制备了肝细胞肿瘤特异性抗体,其特异性结合肝肿瘤细胞且不阻断正常细胞的正常生存活力,亲和力强且表达稳定,能有意义地降低人肝肿瘤细胞增殖的速率,增加细胞凋亡频率。本发明的抗体可以用作诊断或治疗用,是一种新的阻断肿瘤细胞增殖的有效靶向治疗药物,特别是肝癌。(The invention discloses a TK1 antibody, a kit and application thereof. The TK1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 amino acid sequence of heavy chain CDR1, SEQ ID NO: 2 and the heavy chain CDR2 of SEQ ID NO: 3 amino acid sequence of heavy chain CDR 3; and a light chain variable region comprising SEQ ID NO: 4 amino acid sequence of light chain CDR1, SEQ ID NO: 5 amino acid sequence light chain CDR2 and SEQ ID NO: 6 amino acid sequence light chain CDR 3. The invention prepares the specific antibody of the liver cell tumor, which is specifically combined with the liver tumor cell without blocking the normal viability of normal cells, has strong affinity and stable expression, can meaningfully reduce the proliferation rate of the human liver tumor cell and increase the apoptosis frequency of the cell. The antibody of the invention can be used for diagnosis or treatment, and is a novel effective targeted therapeutic drug for blocking tumor cell proliferation, in particular to liver cancer.)

1. A TK1 antibody, comprising:

a heavy chain variable region comprising SEQ ID NO: 1 amino acid sequence of heavy chain CDR1, SEQ ID NO: 2 and the heavy chain CDR2 of SEQ ID NO: 3 amino acid sequence of heavy chain CDR 3; and

a light chain variable region comprising SEQ ID NO: 4 amino acid sequence of light chain CDR1, SEQ ID NO: 5 amino acid sequence light chain CDR2 and SEQ ID NO: 6 amino acid sequence light chain CDR 3.

2. The TK1 antibody of claim 1, wherein the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7.

3. The TK1 antibody of claim 1, wherein the antibody comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

4. A nucleic acid encoding the antibody of any one of claims 1-3.

5. The nucleic acid of claim 4, wherein the nucleic acid comprises a first nucleic acid encoding the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and (b) 9.

6. The nucleic acid of claim 4, wherein the nucleic acid comprises a second nucleic acid encoding the light chain variable region, wherein the second nucleic acid comprises the amino acid sequence of SEQ ID NO: 10, and (b) a coding sequence of 10.

7. A vector comprising the nucleic acid of claim 4.

8. A host cell comprising the nucleic acid of claim 4 or the vector of claim 7.

9. A kit comprising the antibody of any one of claims 1-3.

10. Use of an antibody according to any one of claims 1-3 in the manufacture of a medicament/composition for the treatment of liver cancer.

Technical Field

The invention relates to the field of immunology, and particularly relates to a TK1 antibody, a kit and application thereof.

Background

Liver cancer is one of the common malignant tumors in China, and has more than 50 ten thousand new cases each year, and the treatment efficiency is low, so that almost the same number of death rates are caused. Hepatocellular carcinoma accounts for about 80% of all liver cancers and is rarely treated. Their 5-year survival rate is only about 10%, and survival times after diagnosis are usually less than 6 months.

As a method for treating liver cancer, there are surgical hepatectomy, radio wave ablation, ethanol injection, microwave coagulation necrosis and the like for visible lesions, but these local therapies are not suitable in some cases.

Currently, chemotherapy used more commonly is intra-arterial injection (infusion) therapy of CDDP, ADM or 5-FU for patients with advanced cancer who cannot adapt to the above-mentioned therapy and hepatic artery embolization surgery, or patients who relapse after these treatments. However, the liver injection chemotherapy using the combination of lipiodol and the above drugs, which is an oily contrast agent, has been reported to have a significant effective rate of 13.0% and an effective rate of 30.0%; chemotherapy that excludes the combination of hepatic arterial thrombosis therapy and lipiodol has been reported to have a significant effective rate of only 2.5% and an effective rate of only 3.1%, with poor therapeutic efficacy. Furthermore, there is a problem that such a combination therapy causes complications such as ulcer. As described above, there is no standard treatment method which can stably ensure a certain degree of therapeutic effect on liver cancer. Particularly, intra-arterial injection has problems in terms of manipulation techniques and patient burden, and development of chemotherapy as a substitute for this therapy is desired. Further, since liver cancer is highly resistant to chemotherapy, it is difficult to achieve temporary tumor stabilization, i.e., prolongation of life without tumor proliferation, even by treatment.

Therefore, there is still a need in the art to develop a biological diagnostic and therapeutic means capable of specifically binding to liver tumor cells and thereby blocking proliferation of liver tumor cells.

Disclosure of Invention

In order to solve the problems, the invention provides an antibody, a kit and application thereof. The antibody can specifically bind to liver tumor cells, thereby blocking liver tumor cell proliferation.

In a first aspect, the invention provides a TK1 antibody comprising a heavy chain variable region comprising SEQ ID NO: 1 amino acid sequence of heavy chain CDR1, SEQ ID NO: 2 and the heavy chain CDR2 of SEQ ID NO: 3 amino acid sequence of heavy chain CDR 3; and a light chain variable region comprising SEQ ID NO: 4 amino acid sequence of light chain CDR1, SEQ ID NO: 5 amino acid sequence of light chain CDR2 and SEQ ID NO: 6 amino acid sequence light chain CDR 3.

The TK1 antibody disclosed by the invention can be specifically combined with liver tumor cells, has strong affinity and stable expression, can be used for significantly reducing the proliferation rate of human liver tumor cells and increasing the apoptosis frequency. Thus, mass production of the TK1 antibody of the invention will provide patients with more stable neutralizing antibodies, and with higher affinity.

Preferably, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 7.

Preferably, the antibody comprises a light chain variable region comprising SEQ ID NO: 8.

In a second aspect, the invention provides a nucleic acid encoding the above antibody.

Preferably, the nucleic acid comprises a first nucleic acid encoding a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9, and (b) 9.

Preferably, the nucleic acid comprises a second nucleic acid encoding a light chain variable region, the second nucleic acid comprising the amino acid sequence of SEQ ID NO: 10, and (b) a coding sequence of 10.

In a third aspect, the present invention provides a vector comprising the nucleic acid described above.

In a fourth aspect, the present invention provides a host cell comprising the above-described nucleic acid or the above-described vector.

In a fifth aspect, the invention provides a kit comprising the antibody described above.

In a sixth aspect, the invention provides a use of the above antibody in the preparation of a medicament/composition for treating liver cancer.

In conclusion, the invention has the following beneficial effects: the invention prepares the specific antibody of the liver cell tumor, which is specifically combined with the liver tumor cell without blocking the normal viability of normal cells, has strong affinity and stable expression, can meaningfully reduce the proliferation rate of the human liver tumor cell and increase the apoptosis frequency of the cell. The antibody of the invention can be used for diagnosis or treatment, and is a novel effective targeted therapeutic drug for blocking tumor cell proliferation, in particular to liver cancer.

Drawings

FIG. 1 is a schematic drawing showing a Western Blot (Western Blot/WB) of lysates of HT29 TK1+ and 143B TK-cells;

FIG. 2 is an SDS-PAGE electrophoresis of purified TK1 antigen;

FIG. 3 is a schematic diagram showing a statistically significant decrease in tumor cell viability (p < 0.05) of hepatoma cells Hep G2 cultured after 72 hours of treatment with different concentrations of a thymidine kinase 1 monoclonal antibody (SSTK mouse hybridoma cell strain 43 #);

FIG. 4 is a schematic diagram showing a statistically significant decrease in tumor cell viability (p < 0.05) of hepatoma cell Hep 3B cultured after 72 hours of treatment with different concentrations of a thymidine kinase 1 monoclonal antibody (SSTK mouse hybridoma cell strain 43 #);

FIG. 5 is a schematic representation of immunohistochemically stained colon malignancy with the 43# monoclonal antibody TK 1;

FIG. 6 is a schematic representation of immunohistochemically stained colon malignancy with the 43# monoclonal antibody TK 1;

FIG. 7 is a schematic representation of immunohistochemically stained breast malignancy with the 43# monoclonal antibody TK 1.

Detailed Description

The invention will be further illustrated with reference to specific examples. It will be understood by those of ordinary skill in the art that these examples are provided merely to illustrate the manner in which the invention may be practiced so as to enable those of ordinary skill in the art to better understand the invention and are not intended to limit the scope of the invention to these particular examples.

In the present application, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., an antibody or antigen-binding fragment thereof will not exhibit any significant binding to other molecules than its specific binding partner.

The antibody of the present invention means any antibody capable of specifically binding TK1, including natural, or partially or wholly synthetically produced polypeptides or polypeptide fragments. The antibodies of the invention also encompass any polypeptide or protein comprising an antigen-binding fragment of an antibody. Antibody fragments comprising an antigen binding fragment are, for example, Fab ', F (ab ') 2, scFv, Fv, dAb, Fd ', bispecific antibody molecules, or multispecific antibody molecules, or the like. The antibody can be monoclonal antibody or polyclonal antibody, can be of murine or human or rabbit or sheep or other animal origin, or can be genetically engineered antibody, such as human antibody, humanized antibody, chimeric antibody or single chain antibody. In a specific embodiment, the antibody of the invention is a monoclonal antibody, e.g., a murine monoclonal antibody.

It will be apparent to those skilled in the art that monoclonal and other antibodies can be taken and recombinant DNA techniques used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. For example, DNA encoding the variable region of an immunoglobulin may be linked to the constant region, or Complementarity Determining Regions (CDRs) of an antibody may be introduced into the constant region plus framework regions of a different immunoglobulin. In addition, yeast cells, hybridomas, or other cells that produce antibodies can also be subjected to genetic mutations or other alterations that may or may not alter the binding specificity of the produced antibodies.

Thymidine kinase 1(TK1) (ATP: thymidine-5' -phosphotransferase, EC2.7.1.21) is the only kinase that phosphorylates thymidine (TdR) to thymidine monophosphate (dTMP) via the salvage pathway, which is further phosphorylated to thymidine diphosphate (dTDP) and thymidine triphosphate (dTTP), providing the dTTP required for DNA synthesis, ensuring DNA synthesis. Thus, TK1 is one of the key enzymes closely involved in the regulation of DNA synthesis, and is closely associated with the rate of cell proliferation, also referred to as a specific enzyme in the S phase of the cell cycle. Thymidine (TdR) is one of the important precursors for DNA synthesis. The intracellular concentration of TdR has been shown to correlate with cell viability. The activity/concentration of the TK1 enzyme in the cell can be detected by the TdR/TTP concentration method. The TK1 gene has been considered as a suicide gene. The activity is inhibited and the cell proliferation rate is reduced.

Thymidine kinase 1 is a cell cycle dependent enzyme, is closely related to cell proliferation, is the first serological cell proliferation marker, and can monitor the abnormal proliferation speed of cells. The kit is mainly applied to serological detection and immunohistochemical detection at present, is suitable for physical examination, precancerous lesion assessment and the like, and can be used for predicting prognosis survival rate, predicting recurrence risk rate, assessing risk degree of canceration process and the like.

Tumors are a chronic, long-lasting, abnormally proliferative disease. Multiple genetic mutations associated with cell growth regulation result in uncontrolled growth of normal cells, abnormal proliferation of cells, and eventual progression to malignant tumors for up to 10-30 years. Finding targets associated with tumor proliferation is a key point. In the research, the inventor of the application finds that TK1 can be used as a credible marker for evaluating the proliferation of tumor cells, and has important value in the aspect of evaluating the proliferation rate of human tumors. TK1 is not only expressed in cytoplasm, but also appears in outer cell membrane, and the appearance of TK1 in the outer membrane is related to tumor malignancy.

TK1 is a cell cycle dependent enzyme, closely related to cell proliferation. In contrast, the inventor of the application selects recombinant human TK1 as an immune antigen, and successfully prepares a specific mouse anti-human thymidine kinase 1 monoclonal antibody (derived from an SSTK mouse hybridoma cell line 43#) through strict screening and identification. Further, the inventors of the present application have unexpectedly found that the murine anti-human thymidine kinase 1 monoclonal antibody prepared by the method can be used for treating tumors, especially liver tumors. Therefore, the mouse anti-human thymidine kinase 1 monoclonal antibody can be regarded as a novel targeted drug, can block the proliferation of human tumor cells, and will be an effective targeted therapeutic drug for treating liver tumors in the future. Based on this finding, the present invention has been completed.

The invention successfully screens and obtains a mouse hybridoma cell strain 43#, and develops a specific mouse anti-human TK1 monoclonal antibody. The cell strain is sent to a China center for type culture collection to carry out the preservation of patent programs, the patent preservation number is CCTCC NO: C2019267, the preservation address is China, Wuhan university, and the cell strain is classified and named as SSTK mouse hybridoma cell strain 43#, and the date of receipt by the China center for type culture collection is 2019, 11 and 28 days.

All liver tumor cell lines of the invention were purchased from the China center for type culture Collection [ Hep G2, catalog number GDC 0024; hep 3B, catalog number GDC 0070).

To confirm the specificity used for this antibody, we used cell lysates of cultured human colon carcinoma TK1 positive strain (HT29 TK1+) and human osteosarcoma TK1 negative cell strain (143B TK-) in the identification process, and verified cell lysates of HT29 TK1+ strain by gel electrophoresis/Western Blot (Western Blot) identification, showing a distinct electrophoretic band specific to TK1, without non-specific immunological cross-reaction, but cell lysates of 143B TK-did not show any electrophoretic band of TK1 (FIG. 1). TK1 cell immunohistochemical staining identification method verifies cell in HT29 TK1+ strain, and shows that TK1 has different degrees of specific TK1 brown yellow staining in cytoplasm, but cell of 143B TK-strain has no specific TK1 brown yellow staining. The antibody prepared from the mouse hybridoma cell strain 43# has high specificity.

Preparation example 1:

the method comprises the following steps: preparation of TK1 antigen

Inoculation: the TK1 full-length gene sequence was synthesized into the vector pET21a-TK1-C6His, pET21a-TK1 BL21 expression strain was inoculated from-80 ℃ in 5ml of ampicillin-resistant LB medium in a refrigerator and cultured overnight at 37 ℃ and 220rpm (the expression strain did not melt at the time of inoculation).

Inducing in a large amount: the next day BL21(DE3) was inoculated in the ratio 1:200 into 1000ml ampicillin-resistant TB medium. Culturing at 37 deg.C and 220rpm, cooling the flask in ice-water mixture when the bacteria enter logarithmic phase (OD about 0.6, when cultured bacteria just look opaque, cloud state), adding IPTG at final concentration of 1mM, and inducing at 25 deg.C for 15 hr.

Collecting the expression bacteria: the bacterial liquid was centrifuged at 6000rpm for 15min to remove the supernatant, 0.9% physiological saline was used to resuspend the cells (200ml/L expression cells), and the supernatant was centrifuged at 6000 rpm. The cells were either frozen at-80 ℃ or disrupted for purification.

And (3) crushing thalli: disruption buffer (50mM Tris-Cl pH8.0, 500mM NaCl, 10mM imidazole, 1% TritonX-100, 1xPMSF) to 15ml:1g bacteria ratio heavy suspension. Putting the mixture into a beaker, putting the mixture into an ice water bath, crushing the mixture in an ultrasonic cell crusher, using a phi 10 amplitude transformer, working for 3 seconds, intermittent working for 3 seconds, working for 25 minutes in total and working at 350 watts. Transferring the broken solution into a 50ml high-speed centrifuge tube, and centrifuging for 20 minutes by a high-speed refrigerated centrifuge at 13000 rpm. Centrifuging supernatant, filtering at 0.45 μm/0.22 μm, and loading on column.

Loading: taking GE Ni-sepharose 2.5ml/1000ml expression bacteria, washing with pure water for three times by 10 times of column volume, and balancing by using a balancing solution of 50mM Tris-Cl pH8.0, 500mM NaCl, 10mM imidazole and 1% TritonX-100 by 10 times of column volume. The filler was mixed with the crushed and filtered sample solution and mixed for 2 hours on a 360 ° shaker at 4 ℃. Then taking out the mixture at 4000rpm and centrifuging the mixture for 5 minutes at normal temperature, carefully sucking out the supernatant, adding sodium azide for storage, and transferring the filler to a purification column.

Leaching at 4 ℃:

eluting 20 times of column volume by 50mM Tris-Cl pH8.0, 500mM NaCl, 10mM imidazole and 1% TritonX-100;

eluting 10 times of column volume by 50mM Tris-Cl pH8.0, 500mM NaCl, 20mM imidazole and 1% TritonX-100;

50mM Tris-Cl pH8.0, 500mM NaCl, 30mM imidazole, 10 column volumes eluted.

Elution at 4 ℃:

eluent mother liquor 50mM Tris-Cl pH8.0, 500mM NaCl, 20% glycerol.

Eluent 1: the eluent mother liquor +50mM imidazole, three column volumes were eluted, one tube collected for each column volume.

Eluent 2: the eluent mother liquor +100mM imidazole, three column volumes were eluted, one tube collected for each column volume.

Eluent 3: eluent stock +150mM imidazole, three column volumes were eluted, one tube was collected for each column volume.

Eluent 4: the eluent mother liquor +300mM imidazole, eluted eight column volumes, one tube collected per column volume.

SDS-PAGE electrophoresis detects the eluted fractions, and the purer fractions collected from the elution are pooled (see FIG. 2).

Step two: animal immunization: mixing the prepared TK1 antigen with Freund's complete adjuvant, emulsifying (antigen 100ul + adjuvant 100 ul/mouse), mixing the antigen and adjuvant 1:1 in a 2ml round-bottom centrifuge tube, fixing, connecting with electric motor, and binding the steel wire with one end of the motor being cyclized. The circular ring head extends into the centrifugal tube, is 2 mm away from the bottom, is connected with a power supply, and the motor runs at a high speed to drive the circular ring to stir and emulsify the antigen. After about 30 minutes, the stirring was stopped, the centrifuge tube was inverted, and the emulsifying effect was achieved if the mixture did not flow out. The bottom of the centrifuge tube was pierced with a needle and a 2ml syringe plunger pushed the emulsified antigen from the top of the centrifuge tube into the syringe from the bottom to prepare for immunization. Four 6-week-old Balb/c female mice were initially immunized. Balb/c female mice were boosted after the TK1 antigen prepared above was mixed well with Freund's incomplete adjuvant. Serum titer was measured, and TK1 antigen prepared above was diluted with PBS (see Table 1 for results), and mice with good immunoreactivity were selected for intraperitoneal challenge immunization with antigen at 25ug antigen/mouse. Three days later splenocytes were taken for cell fusion. The immunization schedule is shown in table 2.

And (3) ELISA titer detection: 20170206 murine Tri-immune titer, envelope recombinant TK 11 ug/ml, primary anti-mouse serum 1h, secondary antibody Jackson HRP-goat anti-mouse secondary antibody 1:1W 1 h.

TABLE 1

Injecting: the positive control hole is an anti-TK 1 mouse monoclonal antibody, the concentration is 0.1ug/ml, and the positive control hole is used for determining whether the test sample of the whole experiment is negative or not due to experiment operation or reagent addition errors.

TABLE 2

Step three:

cell fusion and screening:

myeloma cell culture: the myeloma cells used in the laboratory are SP2/0 derived from BALB/c, the collected cells are suspended with 1ml of PBS when the cells grow to the logarithmic phase, and 6-8 weeks of healthy mice are selected to be injected subcutaneously in multiple points, and tumor cells are taken out and separated about 12 days (large-scale expansion culture and freezing are needed, and the separation process of the tumor cells is the same as that of spleen cells). Upon fusion, the cells were placed in logarithmic growth phase.

1. Sp20 cells were revived more than one week earlier.

2. The state of the cells is adjusted according to the number of the cells and the growth rate until the cells are round and smooth, the state is better, and the growth rate is more than 1:10 after every two days of passage.

3. Cell fusion was collected when sp20 status was good.

Note: tumor cells were cultured with 20% fetal calf serum (20% FCS + DMEM + desmethyl antibody) and washed 2 times with serum-free DMEM before cell fusion was collected.

Isolation of spleen lymphocytes:

1. a1.5 ml test tube was prepared in the clean bench. 1ml of serum-free medium, two 3.5cm culture dishes, 2ml of serum-free medium (with a small amount of antibiotics), two 15ml centrifuge tubes, one of which is added with 10ml of serum-free medium, surgical instruments (high pressure moist heat sterilization), silk screen, pipettor (1ml) and a pipette tip.

2. 1.5ml tubes were taken to the animal room. Immunized BALB/c mice were taken, blood was collected from the eyes, and serum was isolated as a positive control serum for antibody detection. Meanwhile, the mice were sacrificed by cervical dislocation, soaked in 75% alcohol for 5 minutes, fixed on a wax tray, and then the upper skin of the spleen was cut off, and the spleen was removed with forceps and placed in a 1.5ml test tube.

3. The spleen was transferred to one of the 3.5cm petri dishes in a clean bench, fat and connective tissue on the spleen were removed, washed once, a silk mesh was laid out on the petri dish lid, and the spleen was lightly crushed and placed in the middle of the silk mesh. Folding the silk net twice, sucking the serum-free culture medium by a pipettor, slightly blowing off, grinding by a grinding rod to make the splenic lymphocytes into single cell suspension through the silk net, and collecting the single cell suspension in a 15ml centrifuge tube. Centrifuge at 800rpm for 5 minutes.

Preparing feeder cells:

isolation of lymphocytes in the thymus

1. A1.5 ml test tube was prepared in the clean bench. Adding 1ml of serum-free medium, adding two 3.5cm culture dishes, adding 2ml of serum-free medium (adding a small amount of antibiotic gentamicin), adding two 15ml centrifuge tubes, adding 10ml of serum-free medium into one centrifuge tube, adding surgical instruments (high pressure moist heat sterilization), adding a silk screen, adding a pipette (1ml) and adding a pipette tip.

2. Female mice below 4 weeks of age were taken and sacrificed by cervical dislocation. Soaking in 75% alcohol for 5min, fixing on wax plate, cutting off chest skin, opening chest cavity, picking up thymus, and placing in 1.5ml test tube.

3. The thymus gland is transferred to one of the 3.5cm culture dishes in the super clean bench, washed once, a silk net is spread on the cover of the culture dish, and the thymus gland is placed in the middle of the silk net. Folding the silk net twice, sucking the serum-free culture medium by a pipette, slightly blowing the serum-free culture medium away, grinding the serum-free culture medium by a grinding rod to enable the thymic lymphocytes to penetrate through the silk net to prepare single cell suspension, and collecting the single cell suspension in a 15ml centrifuge tube. Centrifuge at 800rpm for 5 minutes.

Cell fusion:

1. counting the lymphocytes and myeloma cells respectively, wherein SP2/0 is counted after being diluted by 10 times by serum-free DMEM, and the lymphocytes are counted after being diluted by 100 times by the serum-free DMEM; with SP 2/0: b cell ═ 1: 2-1: 10 were mixed in a 50ml centrifuge tube and mixed well.

2. Centrifuging at 800rpm for 5-10 min, and sucking the supernatant as clean as possible.

3. And (5) slightly tapping the bottom of the fusion tube to ensure that the precipitated cells are loose and uniform.

4. 1ml of 50% PEG4000(pH 8.0) at 37 ℃ was added dropwise over 1 minute by a pipette while gently rotating the fusion tube.

5. After the PEG addition was completed, the mixture was left to stand for about 1 minute.

6. Sucking 1ml of serum-free culture medium preheated to 37 ℃ by a water bath pot by using a pipette, dripping the serum-free culture medium into the fusion tube within one minute, and finishing adding the rest stop solution within 10 minutes and gradually increasing the speed.

7. 800rpm for 5 minutes; the supernatant was discarded.

8. Adding 500ul HAT, 500ul glutamine, 10ml Fetal Bovine Serum (FBS), suspending the feeder cells gently, adding MC semisolid culture medium to constant volume of 50ml, and mixing.

9. The cells were separated into 3.5cm dishes of about 2ml each, and the dishes were placed in a sterilized wet box and cultured in a 5% CO2 incubator at 37 ℃.

Primary subcloning:

the colonies were picked from the MC medium dish and plated in 96-well plates, and cultured with 20% HT for 2-3 days before the first ELISA assay, and wells with high cell status were selected for the first dilution.

ELISA assay fusion transfer plate & first subclone dilution plate:

1. the 96-well plate is labeled with antigen, coating date.

2. Coating: recombinant TK1 protein was diluted with 1 XCBS and added to 96-well plates at 0.05ug/100 ul/well overnight at 4 ℃.

3. Washing the plate: washing with washing solution for 1 time or directly throwing off coating solution.

4. And (3) sealing: 200 ul/well at 37 ℃ for 1 hour.

5. Washing the plate: washed 1 time with wash solution.

6. Primary antibody (cell culture supernatant): fusion plates 100 ul/well, dilution plates 100 ul/well. And 2 holes in the lower right corner of the 96-well plate are used as controls, positive holes are TK1 monoclonal antibody 057M 1:10000 diluted, and negative holes are added with culture medium. Incubate at 37 ℃ for 1 hour.

Plate washing for 3 times

8. Adding a secondary antibody: jackson secondary antibody was diluted 1:20000 with diluent at 100 ul/well and incubated at 37 ℃ for 1 hour.

9. Washing the plate for 3 times, and developing: the single-component TMB developing solution is placed in a thermostat at 37 ℃ for 10-15 min at 100 uL/hole.

10. Termination reaction detection of OD: adding 50 uL/hole stop solution to turn yellow; and measuring the light absorption values of 450nm and 630nm by using a microplate reader (450 nm is selected as a detection wavelength, and 630nm is selected as a reference wavelength).

11. Data processing: after original data are stored, selecting positive wells with OD values larger than 3.0, selecting 50 wells with the highest fusion plate value (about 5 wells are selected on average on each plate) to enter into first subcloning dilution, and selecting the wells with good states, the highest values and few cells according to detection results to enter into second subcloning dilution.

Subcloning:

after the cells diluted for the second time form cell clusters (5-6 days, the number of cells is more than 50), the microscope is used for plate picking, monoclonal and polyclonal are distinguished, after the labeling is respectively carried out, the ELISA detection for the second time is carried out (the detection step is the same as the above detection step). And (4) selecting the monoclonals with high positive values and good states, and performing cell counting limiting dilution.

Cell dilution protocol was as follows (1.6 cells/well &0.8 cells/well, 96-well plates in half each)

1. The cells were transferred by blow-up to sterilized 0.6ml EP tubes and left to count.

2. Aspirate 10. mu.l of cell suspension cell counting plate for counting. If the number of cells is large, the cells should be diluted once and counted again, and the total number in the counting region should be controlled to be within 50.

3. Diluted to 100 cells/10 ul (104 cells/ml). For example, the cell count is 40X 104 cells/ml if the count is 40, and the cell concentration is 104 cells/ml if 5. mu.l of the cell suspension is added to 195. mu.l of DMEM.

4. To 15ml of the culture medium was added 12. mu.l of the cell suspension diluted in step 3, and the mixture was added to a half block of 96-well cell culture plate at 200. mu.l/well, i.e., 1.6 cells/well. The remaining 5ml of media was supplemented and added to the remaining half of the 96 well cell culture plate at 200. mu.l/well, 0.8 cells/well.

Step four: antibody identification and expanding culture and freezing storage: the method comprises the following steps of (1) detecting the total positive of a cell plate reaching a fixed strain standard by ELISA (enzyme-linked immunosorbent assay) (the total positive of the cells in the holes with the cells and the difference of OD (optical density) height values is not more than 0.5), selecting two monoclonal holes with the best cell state, supplementing liquid, blowing the cells away at the same time, and transferring the cells to a 24-hole plate after the cells grow to 80% full; and (5) collecting cell supernatant until 80% of the cell supernatant is full, and performing WB detection and subtype detection. Transferring the cells to a 6-hole plate, transferring the cells to a 10cm culture dish through a 6cm culture dish, culturing the cells until the cells grow to 80% full, wherein the WB result is positive, performing amplification culture on the cells in two batches, and performing cryopreservation on the cells in four dishes (15 ml of cell supernatant is collected and is detected by rabbit anti-TK 1 multi-antibody sandwich HT29 lysate and 143B TK-lysate to react with natural TK1), and using the rest cells for monoclonal antibody production.

The positive cell strain is subjected to amplification culture and frozen storage, cell supernatant is detected by Western Blot of TK1 positive cell line (HT29 TK1+) lysate and negative cell line (143B TK-) lysate, and clones with specific Western Blot detection bands and clean backgrounds are subjected to serum-free medium spinner flask culture to produce antibodies (see figure 1).

Step five: immunohistochemical identification: as shown in FIGS. 5-7, TK1 expressed positive colon tumor tissue and breast tumor tissue sections, anti-TK 1 antibody produced was diluted and incubated as primary antibody, Elivision TM plus Polymer HRP (Mouse/Rabbit) IHC Kit of Michhol, Fuzhou was used as secondary antibody for reaction, and 43# clone with specific cytoplasmic reaction and clean background was selected after DAB color development and hematoxylin counterstaining.

Example 1

The method comprises the following steps: the liver tumor cell lines Hep G2 and Hep 3B were thoroughly mixed with 0.1. mu.g/ml monoclonal antibody containing SSTK mouse hybridoma cell line 43# (2X 10)⁴) With a 0.22um sterile filter head (cat #: SLGP033RB) were filter sterilized in a biosafety cabinet and cultured for 72h in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog number FS500) and no antibiotics or other reagents;

step two: liver tumor cell lines Hep G2, Hep 3b0.22um were filter sterilized and then cultured for 72h in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog No. FS500) and no antibiotics or other agents;

step three: cell viability assay (Cell Counting Kit-8, MedChemexpress)

And (3) activity calculation: tumor cell viability (%) was ═ 100% (absorbance of experimental wells-absorbance of blank medium control)/(absorbance of untreated control wells-absorbance of blank medium control) ×

Example 2

The method comprises the following steps: the liver tumor cell lines Hep G2 and Hep 3B were thoroughly mixed with 1. mu.g/ml monoclonal antibody containing SSTK mouse hybridoma cell line 43# (2X 10)⁴) Culturing for 72h in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog No. FS500) and no antibiotics or other reagents;

step two: the liver tumor cell lines Hep G2, Hep 3B were cultured for 72h in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog number FS500) and no antibiotics or other agents;

step three: cell viability assay (Cell Counting Kit-8, MedChemExpress) viability calculation: tumor cell viability (%) was (absorbance of experimental wells-blank medium control absorbance)/(absorbance of untreated control wells-blank medium control absorbance) × 100%

Example 3

The method comprises the following steps: the liver tumor cell lines Hep G2 and Hep 3B were thoroughly mixed with 10. mu.g/ml monoclonal antibody containing SSTK mouse hybridoma cell line 43# (2X 10)⁴) Culturing for 72h in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog No. FS500) and no antibiotics or other reagents;

step two: the liver tumor cell lines Hep G2, Hep 3B were cultured for 72h in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog number FS500) and no antibiotics or other agents;

step three: cell viability assay (Cell Counting Kit-8, MedChemExpress) viability calculation: tumor cell viability (%) was (absorbance of experimental wells-blank medium control absorbance)/(absorbance of untreated control wells-blank medium control absorbance) × 100%

Example 4

The method comprises the following steps: the liver tumor cell lines Hep G2 and Hep 3B were thoroughly mixed with 100. mu.g/ml monoclonal antibody containing SSTK mouse hybridoma cell line 43# (2X 10)⁴) Culturing for 72h in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog No. FS500) and no antibiotics or other reagents;

step two: the liver tumor cell lines Hep G2, Hep 3B were cultured for 72h in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog number FS500) and no antibiotics or other agents;

step three: cell viability assay (Cell Counting Kit-8, MedChemExpress) viability calculation: tumor cell viability (%) was (absorbance of experimental wells-blank medium control absorbance)/(absorbance of untreated control wells-blank medium control absorbance) × 100%

Comparative example 1

The method comprises the following steps: liver tumor cell lines Hep G2, Hep 3B were cultured in MEM Alpha basic cell culture medium (GIBCO, catalog C12571500BT) containing 15% fetal bovine serum (ExCell Bio, catalog number FS500) and no antibiotics or other agents, respectively, for 72 h;

step two: cell viability assay (Cell Counting Kit-8, MedChemexpress)

And (3) activity calculation: tumor cell viability (%) was ═ 100% (absorbance of experimental wells-absorbance of blank medium control)/(absorbance of untreated control wells-absorbance of blank medium control) ×

The experimental results are as follows:

72-hour activity inhibition of 43# antibody on liver tumor cell Hep G2

72-hour activity inhibition of 43# antibody on liver tumor cell Hep 3B

72-hour activity inhibition of 43# antibody on liver tumor cell Hep G2

72-hour activity inhibition of 43# antibody on liver tumor cell Hep 3B

The viability data calculated according to the formula shows that the viability of the antibody # 43 at 0ug/ml for 72 hours is 100% compared with the control, and gradually decreases as the antibody concentration increases from 0.1 to 100ug/ml, with the lowest viability at 100 ug/ml.

The above description is only for explaining the present invention, and it is not limited to the present invention, and those skilled in the art can make modifications to the present embodiment as necessary without inventive contribution, but all of them are protected by patent law within the scope of the claims of the present invention.