CN108642070B

CN108642070B - Recombinant human Fc antibody for specifically inducing tumor cell apoptosis and preparation method and application thereof

Info

Publication number: CN108642070B
Application number: CN201810319683.5A
Authority: CN
Inventors: 赵洪礼
Original assignee: Shenyang Jinshi Biopharmaceutical Co ltd
Current assignee: Shenyang Jinshi Biopharmaceutical Co ltd
Priority date: 2018-04-11
Filing date: 2018-04-11
Publication date: 2022-03-15
Anticipated expiration: 2038-04-11
Also published as: WO2019196531A1; CN108642070A

Abstract

The invention provides a recombinant human Fc antibody for specifically inducing tumor cell apoptosis and a preparation method and application thereof, and the key points are as follows: directly combining HC2 and HC3 region gene segments of an Fc segment of an adult IgG antibody by an artificial synthesis method, and simultaneously synthesizing a VP3 gene capable of specifically inducing tumor cell apoptosis to construct a single-chain antibody gene containing Fc-linker-VP 3; subcloning the gene fragment into a pPIC9K vector and transfecting a Pichia pastoris system GS115 strain, or subcloning the gene fragment into a mammalian cell expression vector and expressing recombinant human Fc antibody protein in a mammalian cell; the recombinant human Fc antibody has the effects of activating complement activity and inducing ADCC effect, has strong specificity and the effect of inducing tumor cell apoptosis, and can be used for treating malignant tumors.

Description

Recombinant human Fc antibody for specifically inducing tumor cell apoptosis and preparation method and application thereof

Technical Field

The invention belongs to antibody pharmacy and application thereof in malignant tumor, in particular to a recombinant human Fc antibody for specifically inducing tumor cell apoptosis, a preparation method thereof and application of a product obtained by the preparation method in treating malignant tumor.

Background

The human immune system is an important organ for preventing various diseases in the body from happening and developing, and is responsible for 4 immune functions of immune defense, immune surveillance, immune tolerance and immune regulation. Antibodies are one of the most important molecules that perform these 4 major functions. The antibody molecule is immunoglobulin produced after the body is stimulated by antigenic substance, and it can combine with corresponding antigen specificity and eliminate antigen by means of other immunological function, so that it has the important function of preventing diseases. The antibody molecule consists of 4 peptide chains and has 2 functional regions, namely, an amino terminal which is specifically combined with an antigen is called as an F (ab ') segment, and the F (ab') segment has different molecular structures and is also called as a variable region (V region) due to different antigens; and the carboxy-terminal, immunomodulatory function, called the Fc fragment, which is relatively conserved among organisms, also known as the constant region (C-region). Antibodies are classified into 5 isotypes, i.e., IgM, IgG, IgA, IgD, and IgE, according to the antigenicity of the constant regions; even the molecular structures of the same class of igs are different, and the same class of igs can be divided into different subclasses, for example, human iggs can be classified into IgG1, IgG2, IgG3 and IgG 4. Although the antibody Fc fragment does not specifically bind to an antigen, it is indispensable in exerting an immune function. After the V region of the antibody is combined with the antigen, various antigen-antibody combination reactions can occur in vitro by means of the action of the C region, which is powerful for the detection and function judgment of the antigen or the antibody, and can neutralize toxin, block pathogen invasion, clear pathogenic microorganisms and the like in vivo. The main functions of the Fc fragment are: complement activation function, Fc receptor binding function, and placental and mucosal crossing function. The antibody further exerts an immunoregulatory function by binding to an Fc receptor and antibody-dependent cell-mediated cytotoxicity (ADCC), which is one of the important functions of the body to clear pathogens and tumor cells. The Fc receptor in human body is divided into 3 minutes, widely distributed on the surface of various immune cells and somatic cells in the body, plays an important immunoregulation function, and simultaneously the half-life period of the antibody in the body plays an important role.

In recent years, with the rapid development of monoclonal antibody technology and genetic engineering technology, a large number of humanized antibodies, genetically engineered antibodies and antigen-binding Fc fusion proteins, commonly referred to as Fc antibodies, (Fcabs,) have emerged. The successful development of the antibodies provides powerful and accurate diagnosis and treatment means for various difficult and complicated diseases, more than 350 antibodies are approved to be applied to clinic, and the economic benefit is obtained by hundreds of billions of yuan every year. In particular, a large number of monoclonal or humanized antibodies against tumor cells have been developed. However, most of the anti-tumor antibodies clinically applied at present are directed against dominant antigens on the surface of tumor cells, such as CD antigen series, but not tumor cell specific antigens, because few tumor specific antigens can be proved at present, the treatment effect of the antibodies is influenced to a great extent, and the toxic and side effects of the antibodies are increased. At present, the prepared anti-tumor antibody has defects on the premise of lacking tumor specific antigens. In addition, the current antibody development focuses on the function of the V region, and ignores the function of the C region. The clinical Fcabs preparation is antigen blocking, such as etanercept (Enbrel) for rheumatoid arthritis, which blocks TNF receptor to exert therapeutic effect, and the Fc fragment of the antibody only plays roles of stabilizing protein and increasing half-life, but does not further play a role of immunoregulation.

As is well known, cancer treatment is a worldwide problem, and the conventional methods such as surgery, radiotherapy, chemotherapy and the like cannot cure the cancer at present, so that the search for more effective treatment means is one of the important research fields in the world. Therefore, the research and development of novel biological agents with therapeutic effects have important social significance and economic significance.

Domestic and foreign researches prove that the chicken anemia virus VP3 protein has specific killing effect on various tumor cells and abnormal transformed cells of human, is named as tumor specific apoptosis factor (Apoptin), and is expected to become a novel biological agent for effectively treating cancers. VP3 induces tumor cell apoptosis is characterized in that it is independent of the action pathway of tumor suppressor gene-53, and is not inhibited by the overexpression of apoptosis suppressor gene (Bcl-2), but VP3 is not structural protein of chicken anemia virus, and is very unstable in vitro, thus limiting large-scale production; in addition, due to the intergeneric difference, VP3 has no related protein receptor on the surface of human cell membrane, so VP3 can not be combined with human cells, and enters tumor cells, causing tumor cell apoptosis. The principle of inducing apoptosis by VP3 is that it enters cells and activates the enzyme (mainly caspase) related to apoptosis to induce apoptosis. The two technical problems severely limit the industrialization and clinical application of VP 3. In order to solve the above theoretical problems, we have obtained a technique (patent number: ZL 201010107675.8) for obtaining VP3 protein in vitro stably in batch by fusion expression of VP3 and GST, and solved the technical problem of obtaining VP3 protein in vitro in large quantities; meanwhile, folic acid is chemically connected to VP3 protein, so that folic acid can enter tumor cells independently, and the theoretical problem that VP3 cannot enter human tumor cells independently due to species difference is solved (patent number: ZL 201010107682.8). On the basis of solving the two technical problems, VP3 is further improved, and is fused with Fc fragment of human antibody IgG1 to develop the recombinant human Fc antibody capable of specifically inducing tumor cell apoptosis, the recombinant human Fc antibody not only increases the half-life period in vivo, but also utilizes the Fc fragment binding function of Fc receptor, the induced DACC function and cell crossing function, the complement activating function and the VP3 specific tumor cell apoptosis inducing function, so that the recombinant human Fc antibody has the triple anti-tumor biological functions and can be produced and applied in large scale.

Disclosure of Invention

The invention aims to provide a preparation method of a recombinant human Fc antibody which is efficiently expressed in eukaryotic cells, has good stability and high purity and can specifically induce tumor cell apoptosis, and an application of a product obtained by the method in anti-tumor treatment. The recombinant human Fc antibody provided by the invention can be combined with tumor cells and specifically induces the apoptosis of the tumor cells so as to fulfill the aim of clinically treating the tumors.

The technical scheme of the invention is as follows: on the basis of constructing an Fc-Linker-VP3 fusion protein (recombinant human Fc antibody) expression vector pPIC9K vector, a technical method for purifying the recombinant human Fc antibody is further established, so that the recombinant human Fc antibody is easy to dissolve and stable in vitro and has high purity; can be combined with tumor cells and induce the apoptosis of the tumor cells, and becomes a novel recombinant human Fc antibody.

The novel recombinant human Fc antibody gene provided by the invention has a nucleotide sequence shown as SEQ1 in a sequence table;

the novel recombinant human Fc antibody amino acid provided by the invention has an amino acid sequence shown as SEQ5 in a sequence table;

the preparation method of the recombinant human Fc antibody provided by the invention comprises the following steps:

(1) directly obtaining a gene fragment of a recombinant human Fc antibody by an artificial synthesis method, constructing an Fc-Linker-VP3 gene, and subcloning the Fc-Linker-VP3 gene to a pPIC9K expression vector; expressing protein by using a yeast system GS115 strain, and performing secretory expression by using a carrier signal peptide;

(2) directly obtaining a gene fragment of a recombinant human Fc antibody by an artificial synthesis method, constructing an Fc-Linker-VP3 gene, subcloning the Fc-Linker-VP3 gene to a mammalian cell expression vector pATX2 vector, transfecting a mammalian cell CHO cell strain by a liposome method for expression, screening a high-efficiency expression cell strain, and expressing recombinant human Fc antibody protein;

(3) fermenting, efficiently expressing and purifying the recombinant human Fc antibody yeast strain;

(4) fermenting, efficiently expressing and purifying a recombinant human Fc antibody CHO cell strain;

(5) the recombinant human Fc antibody induces the apoptosis of tumor cells.

(1) The method for constructing the recombinant human Fc antibody pPIC9K vector and the construction method of the engineering strain comprise the steps of directly and artificially synthesizing an Fc-Linker-VP3 fragment, and directionally subcloning the fragment to EcoRI and NotI enzyme cutting sites of the pPIC9K vector, as shown in figure 1; conventionally preparing competent cells of a yeast system GS115 strain; adding 50ug DNA into 20ul water, directly adding DNA into a competent cell tube in a frozen state, and water-bathing at 37 deg.C for 5min, and mixing for 2 times; taking out the conversion tube, adding 1.5ml of buffer B, completely mixing, and carrying out water bath at 30 ℃ for 1 hour; centrifuging the sample at 1500g for 10min at room temperature, removing the supernatant, resuspending the cells with 1.5ml Buffer C, centrifuging again, resuspending the cells with 0.2ml Buffer C, coating the contents of the tube on a selective growth plate, incubating for 3-4 days at 30 ℃, and screening the geneticin highly resistant clones.

Solution preparation:

buffer A1M sorbitol, 10mM bicine (bicine) glycine) pH8.35, 3% (v/v) ethylene glycol; buffer B40% (w/v) polyethylene glycol 1000, 0.2M N-bis (hydroxyethyl) glycine pH8.35;

buffer C0.15M NaCl, 10mM bicine (hydroxyethyl) pH 8.35;

filter sterilized and stored at-20 ℃. Fresh reagent grade DMSO, unopened or freshly prepared, was stored at-70 ℃ until use.

(2) The construction of recombinant human Fc antibody mammalian cell expression vector pATX2 vector and CHO cell transfection method comprises directly artificially synthesizing Fc-Linker-VP3 fragment, and directionally subcloning into expression plasmid pATX2 EcoRI and NotI restriction enzyme sites; transfection method of CHO cells: removing an original culture medium of CHO cells, adding about 5mL of fresh culture medium, adding 150 parts of DNA/DOTAP mixture, slightly shaking a culture bottle to uniformly distribute the culture medium, culturing in a cell culture box at 37 ℃ for 3-10 h, then replacing fresh complete 1640 culture medium, adding G418(800 mu G/mL) for screening after 48h, simultaneously setting untransfected cells and empty carrier cells as negative controls in an experiment, culturing in serum-free 1640 culture medium, collecting supernatant every 24h for each experimental group, filtering by using a 0.45 mu m filter, and storing the supernatant at-20 ℃; collecting cell culture supernatant for purification and screening the high-efficiency expression CHO cell strain.

(3) The strain fermentation, high-efficiency expression and purification steps of the recombinant human Fc antibody are as follows: taking a single clone bacterial strain with high-efficiency expression, inoculating the single clone bacterial strain into a 250ml shake flask containing 25ml of MGY, BMG or BMGY, culturing at the temperature of 28-30 ℃ and the rpm of 250-300 until the single clone bacterial strain is OD₆₀₀= 2-6 (about 12-20 hours); transferring the above 25ml culture medium to 5L shake flask containing 1L MGY, BMG or BMGY, shaking at high speed (250-300 rpm) at 28-30 deg.C, and culturing to logarithmic growth phase (OD)₆₀₀=2 to 5); centrifuging the mixture for 5min at room temperature of 1500-3000 g by using a sterilized centrifuge tube to collect cells; resuspending cells to OD with MM, BMM or BMMY₆₀₀Induction of =1.0 (2-6L); subpackaging the culture medium to N5L partition shake flasks of 1L per flask, covering the bottle openings with 8 layers of sterilization gauze, and placing into a shaking table at 28-30 ℃ for continuous culture; adding methanol for 1 time every 24 hours until reaching the optimal induction time, wherein the final concentration is 0.5%; centrifuging at 1500-3000 g for 5min at room temperature, keeping the supernatant, and precooling at 4 ℃ to purify the protein for later use. Slowly adding saturated ammonium sulfate with the same volume into the culture supernatant, mixing, standing at 4 deg.C for more than 4 hr, centrifuging at 4 deg.C for 20min to collect precipitate, dissolving the precipitate with 100ml of balance solution, dialyzing with 2000 balance solution for more than 12 hr, and changing overnight; sample Re-equilibrationStoring the anion chromatographic column with well balanced liquid or at-20 ℃ for later use, eluting by using balanced liquid containing 0.1mol/L, 0.2mol/L, 0.5mol/L and 1mol/L NaCl respectively, collecting elution peaks, carrying out SDS electrophoresis identification, and taking a protein part with the molecular weight of 39 kD; balancing the Separose12 chromatographic column by using a balancing solution; loading the concentrated 39kD protein fraction at a rate of 3% of the bed volume at a flow rate of 2 ml/min; collecting step by step; after SDS electrophoresis identification, taking the protein part with the molecular weight of 39kD and high purity as a recombinant human Fc antibody; storing at-20 ℃ for identification.

Solution preparation:

YPD or YPED: yeast Extract Peptone Dextrose Medium (1L)1% Yeast Extract 2% Peptone 2% Dextrose (glucose)

1 dissolving 10gYE, 20g PEP in 900ml water, adding 20g agar powder when preparing plate

2 high pressure for 20min

3 addition of 100ml of 10 XD

YPD-geneticin plates: 1% yeast extract, 2% peptone, 2% glucose, 2% various amounts of geneticin 100mg/ml geneticin: a30 ml stock solution of 100mg/ml geneticin was prepared with sterile water, filtered to sterilize, and stored at-20 ℃. Used to prepare plates containing different final concentrations of geneticin.

MGY and MGYH (minor glycerol medium. + -. histidine) 1L: 1.34% YNB 1% Glycerol 4X 10-5% Biotin. + -. 0.004% histidine

1 to 800ml of water, 100ml of 10 XYNB, 2ml of 500 XB, 100ml of 10GY

2 culturing his4 strain, adding histidine (named MGYH), adding 10ml of 100 XH stock solution, storing at 4 ℃,

saturated ammonium sulfate: prepared according to the following proportion by taking 1000ml as a unit

Ammonium sulfate 1000g or more

Balance liquid: 20mmol of PB buffer (pH 7.2) in 1000ml was prepared in the following proportions

Sodium dihydrogen phosphate 2H₂O 3.12g

Disodium hydrogen phosphate 12H₂O 7.16g

EDTA 0.37g

(4) The culture and purification steps of the recombinant human Fc antibody CHO cell strain in (1) are as follows: expressing the CHO cell strain with high efficiency, culturing in a serum-free 1640 culture medium for 24h, collecting the supernatant, filtering with a 0.45-micron filter, slowly adding saturated ammonium sulfate with the same volume into the supernatant, uniformly mixing, standing at 4 ℃ for more than 4 hours, centrifuging at 4 ℃ for 20min by 5000g, collecting the precipitate, dissolving the precipitate with 100ml of balance solution, dialyzing the balance solution 2000 for more than 12 hours, and changing the middle for one time; the sample is put on an anion chromatographic column balanced by the balance liquid or is stored for standby at the temperature of minus 20 ℃, the balance liquid containing 0.1mol/L, 0.2mol/L, 0.5mol/L and 1mol/LNaCl is respectively used for elution, elution peaks are collected, SDS electrophoresis identification is carried out, and the protein part containing the molecular weight of 39kD is taken; balancing the Separose12 chromatographic column by using a balancing solution; loading the concentrated 39kD protein fraction at a rate of 3% of the bed volume at a flow rate of 2 ml/min; collecting step by step; after SDS electrophoresis identification, the protein part with the molecular weight of 39kD and high purity is collected to be the recombinant human Fc antibody.

Solution preparation:

serum-free 1640 medium

Ammonium sulfate 1000g or more

Sodium dihydrogen phosphate 2H₂O 3.12g

Disodium hydrogen phosphate 12H₂O 7.16g

EDTA 0.37g

(5) The recombinant human Fc antibody product is used for inducing tumor cell apoptosis, and in vitro cultured cells are taken as examples: inoculating the tumor cells into a 96-well cell culture plate, and culturing overnight; adding recombinant human Fc antibodies with different concentrations, continuing to culture for 24 hours, determining apoptosis by an annexin V method, and setting a normal cell control in the test. As a result, more than 31.45% of the tumor cells were apoptotic by the recombinant single-chain antibody at 6 g/L.

(6) The recombinant human Fc antibody product is used for anti-tumor treatment by animal experimentsFor example, nude mice are inoculated subcutaneously with tumor cells until the tumor grows to 150mm³On the left and right, mice are randomly grouped, two kinds of recombinant single-chain antibodies with different dosages of high and low and normal saline (a control group) are injected subcutaneously respectively, every other day for 20 days continuously, and experimental results show that the recombinant single-chain antibodies have obvious tumor inhibition effect on tumor cells, and the tumor volume proliferation of the treatment group is obviously slow compared with that of the control group.

Drawings

The invention is further described below with reference to the accompanying drawings.

FIG. 1 shows the construction scheme of expression vector for directional cloning of artificially synthesized Fc-Linker-VP3 fragment to pPIC9K vector EcoRI and NotI restriction enzyme sites.

FIG. 2 shows the results of expression and purification of recombinant human Fc antibody, in which 12, 3, 4 and 5 are purified recombinant human Fc antibody; 6 is molecular weight standard, 97.0, 66.2, 43.0, 31.0 and 14.3kDa from big to small, and 7 is recombinant human Fc antibody protein expressed by fermentation.

FIG. 3 shows the results of expression and purification of recombinant human Fc antibody, in which 1 is the molecular weight standard, 97.0, 66.2, 43.0, 31.0, 14.3kDa from large to small, and 2 and 3 are purified recombinant human Fc antibody; and 4 is recombinant human Fc antibody protein expressed by CHO cells.

FIG. 4 inhibition of proliferation of breast cancer MCF-7 cells by recombinant human Fc antibody in vitro, as indicated by: the ordinate represents the cell proliferation inhibition rate; the abscissa is the concentration gradient of the recombinant human Fc antibody.

FIG. 5 shows the apoptosis induction of recombinant human Fc antibody against breast cancer MCF-7 cells, which are respectively marked as: 1.2 and 3, the concentrations of the recombinant human Fc antibody are respectively as follows: 0 g/L3 g/L and 6 g/L.

FIG. 6 shows the therapeutic effect of recombinant human Fc antibody on breast cancer in nude mice. The designations in the figures are respectively: the top panel is a line graph of tumor volume and the bottom panel is a solid photograph of the tumor.

Detailed Description

(1) Directionally cloning the Fc-Linker-VP3 fragment to EcoRI and NotI enzyme cutting sites of a pPIC9K vector, as shown in figure 1; conventionally preparing yeast system GS115 strain competent cells, adding 50ug DNA into 20ul water, directly adding DNA into the competent cell tube in frozen state, water-bathing at 37 deg.C for 5min, and mixing for 2 times; taking out the conversion tube, adding 1.5ml Buffer B, completely mixing, and carrying out water bath at 30 ℃ for 1 hour; the samples were centrifuged at 1500g for 10min at room temperature, the supernatant was removed, the cells were resuspended with 1.5ml Buffer C, centrifuged, the cells were resuspended with 0.2ml Buffer C and plated on selective growth plates, incubated at 30 ℃ for 3-4 days, and geneticin-highly resistant clones were selected (FIG. 1).

(2) Fermentation, high-efficiency expression and purification of the recombinant human Fc antibody are specifically as follows:

(2.1) experimental material YPD-plate: MGY and MGYH medium, saturated ammonium sulfate, equilibrium: 20mmol PB buffer (pH7.2), anion chromatography column, Sepharose 12 chromatography column;

(2.2) the experimental method comprises the steps of taking a single clone bacterial strain with high-efficiency expression, inoculating the single clone bacterial strain into a 250ml shake flask containing 25ml of MGY, BMG or BMGY, culturing at the temperature of 28-30 ℃ and the rpm of 250-300 to OD₆₀₀= 2-6 (about 12-20 hours); transferring the above 25ml culture medium to 5L shake flask containing 1L MGY, BMG or BMGY, shaking at high speed (250-300 rpm) at 28-30 deg.C, and culturing to logarithmic growth phase (OD)₆₀₀=2 to 5); centrifuging the mixture for 5min at room temperature of 1500-3000 g by using a sterilized centrifuge tube to collect cells; resuspending cells to OD with MM, BMM or BMMY₆₀₀Induction of =1.0 (2-6L); subpackaging the culture medium to N5L partition shake flasks of 1L per flask, covering the bottle openings with 8 layers of sterilization gauze, and placing into a shaking table at 28-30 ℃ for continuous culture; adding methanol for 1 time every 24 hours until reaching the optimal induction time, wherein the final concentration is 0.5%; centrifuging at 1500-3000 g for 5min at room temperature, keeping the supernatant, and precooling at 4 ℃ to purify the protein for later use. Slowly adding saturated ammonium sulfate with the same volume into the culture supernatant, mixing, standing at 4 deg.C for more than 4 hr, centrifuging at 4 deg.C for 20min to collect precipitate, dissolving the precipitate with 100ml of balance solution, dialyzing with 2000 balance solution for more than 12 hr, and changing overnight; the sample is put on an anion chromatographic column balanced by the equilibrium liquid or stored at the temperature of minus 20 ℃ for standby, the equilibrium liquid containing 0.1mol/L, 0.2mol/L, 0.5mol/L and 1mol/LNaCl is respectively used for elution, elution peaks are collected, and SDS electrophoresis identification is carried outTaking a protein part with the molecular weight of 39 kD; balancing the Separose12 chromatographic column by using a balancing solution; loading the concentrated 39kD protein fraction at a rate of 3% of the bed volume at a flow rate of 2 ml/min; collecting step by step; after SDS electrophoresis identification, taking the protein part with the molecular weight of 39kD and high purity as a recombinant human Fc antibody; storing at-20 ℃ for identification;

(2.3) according to the experimental result, the purity of the protein is measured by an SDS electrophoresis method or a high performance liquid chromatography method, the purity is over 95 percent, the molecular weight is 39kD, and the content of the protein is measured by an ultraviolet spectrophotometer and is about 0.5 mg/ml. (FIG. 2)

(3) Construction, culture and purification of recombinant human Fc antibody mammalian cell strain

(3.1) Experimental materials: chinese hamster ovary cells (CHO cells), liposome DOTAP, and complete 1640 culture medium;

(3.2) test method the artificially synthesized gene fragment "Fc-linker-VP 3" was subcloned into the mammalian cell expression vector pATX2 vector, and mammalian cell CHO was transfected by liposome method. Transfection method of CHO cells: removing an original culture medium of CHO cells, adding a fresh culture medium L of about 5m, adding a DNA/DOTAP mixture 150 times, slightly shaking a culture bottle to uniformly distribute the culture medium, culturing in a cell culture box at 37 ℃ for 3-10 h, then replacing a fresh complete 1640 culture medium, adding G418(800 mu G/mL) for screening after 48h, simultaneously setting untransfected cells and empty carrier cells as negative controls in the experiment, culturing in a serum-free 1640 culture medium, collecting supernatant every 24h for each experimental group, filtering by using a 0.45 mu m filter, and storing the supernatant at-20 ℃; the IgG1-VP3 fusion protein in the supernatant was purified as follows; slowly adding saturated ammonium sulfate with the same volume into the culture supernatant, mixing, standing at 4 deg.C for more than 4 hr, centrifuging at 4 deg.C for 20min to collect precipitate, dissolving the precipitate with 100ml of balance solution, dialyzing with 2000 balance solution for more than 12 hr, and changing overnight; the sample is put on an anion chromatographic column balanced by the balance liquid or is stored for standby at the temperature of minus 20 ℃, the balance liquid containing 0.1mol/L, 0.2mol/L, 0.5mol/L and 1mol/LNaCl is respectively used for elution, elution peaks are collected, SDS electrophoresis identification is carried out, and the protein part containing the molecular weight of 39kD is taken; balancing the Separose12 chromatographic column by using a balancing solution; loading the concentrated 39kD protein fraction at a rate of 3% of the bed volume at a flow rate of 2 ml/min; collecting step by step; after SDS electrophoresis identification, taking the protein part with the molecular weight of 39kD and high purity as a recombinant human Fc antibody; storing at-20 ℃ for identification;

(3.3) the protein purity is measured by SDS electrophoresis or high performance liquid chromatography, the purity is above 95%, the molecular weight is 39kD, and the protein content is measured by an ultraviolet spectrophotometer and is about 0.5 mg/ml. (FIG. 3)

(4) Recombinant human Fc antibody in vitro cell culture and in vitro proliferation-toxicity experiment

(4.1) Experimental Material human Breast cancer MCF-7 cells were purchased from China academy of sciences type culture Collection cell Bank, cultured in DMEM/F12 medium containing 10% fetal bovine serum, and cultured at 37 ℃ in 5% CO₂Growing the film on the wall under the condition of saturated humidity;

(4.2) preparation of Single cell suspension of breast cancer MCF-7 cells by Experimental method, adding the suspension into 96-well plate, adjusting the number of cells per well to 3 × 10⁴One per ml. After culturing the cells in a 96-well plate for 24 hours, the recombinant human Fc antibody was added to a final concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20g/L in a final volume of 200. mu.l in each of 3 wells, and a zero-adjustment well (medium alone) and a control group (DMSO was added to a final concentration of 0.1%) were set. Adding 10ul of CCK-8 reagent and 100ul of culture medium after culturing for 24h, uniformly mixing, incubating for 24h, detecting the absorbance OD value of each well under 450nm by using an enzyme-labeling instrument, and calculating the cell Inhibition Rate (IR) of different drug concentrations by taking the average value of the OD values of 3 multiple wells of each concentration as the average OD value of each concentration: cell inhibition (%) = (Ac-As)/(Ac-Ab) × 100%. As: the experimental hole contains cells, culture medium, CCK-8 and toxic substances; ac: control wells containing cells + medium + CCK-8; ab: blank wells containing medium + CCK-8. Half maximal Inhibitory Concentration (IC) was calculated using Graphpad Prism software₅₀A value);

(4.3) results of the experiment, the final concentration of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20g/L recombinant human Fc antibody treated the breast cancer MCF-7 cells for 24h, and the inhibition rates of the cells are respectively as follows: 13.4 percent, 32.2 percent, 49.7 percent, 63.6 percent, 80.1 percent, 89.7 percent,91.2%, 93.1%, 93.9%, 94.1%. Compared with a control group, the recombinant human Fc antibody can obviously inhibit the proliferation of breast cancer MCF-7 cells (P)<0.05) and in the presence of a certain concentration dependence, IC 24h₅₀It was 6.192 g/L. The above experimental data are all from three different experiments. (FIG. 4).

(5) Recombinant human Fc antibody in vitro tumor cell culture-apoptosis experiment

(5.1) Experimental Material human Breast cancer MCF-7 cells were purchased from China academy of sciences type culture Collection cell Bank, cultured in DMEM/F12 medium containing 10% fetal bovine serum at 37 ℃ with 5% CO₂Growing the film on the wall under the condition of saturated humidity;

(5.2) Experimental method breast cancer MCF-7 cells were inoculated into culture flasks, and the concentrations of the recombinant human Fc antibody added to the test groups were 3g/L and 6g/L, respectively, with reference to the test results of CCK-8. Terminating the culture after 24h, digesting the cells with pancreatin to prepare a single cell suspension, and adjusting the cell concentration to 1 × 10⁶Taking 1ml of cells per ml, centrifuging at 1000 r/min and 4 ℃ for 5min, removing supernatant, adding 1ml of cold PBS, gently shaking to suspend the cells, and repeatedly centrifuging for 2 times. Resuspending the cells in 400ul Binding Buffer (Binding Buffer), adding 5ul of annexin V labeled by fluorescein isothiocyanate (F1TC), reacting for 15 min at 4 ℃ in a dark place, adding 10ul of PI, gently mixing, incubating for 5min at 4 ℃, and detecting apoptosis by using a flow cytometer;

(5.3) the experimental result shows that the early and late apoptosis rate and necrosis rate of the cells in the experimental group are obviously higher than those in the control group. 0. The experimental groups of 3 and 6g/L recombinant human Fc antibodies had early apoptosis rates of 2.42%, 17.14% and 31.45%, and late apoptosis and cell necrosis rates of 1.30%, 2.73% and 9.26%, respectively (FIG. 5).

(6) Anti-tumor effect of recombinant human Fc antibody in vivo: the experiment was committed to completion of the research institute for chemotherapy of osaka cancer, japan.

(6.1) test materials: the male nude mice (with the weight of 18-20 g) and the human cancer breast cancer (H185 strain) are all raised and preserved by the institute;

(6.2) Experimental method human Breast cancer H185 Strain tissue piece 5mm³Inoculating to nude miceSubcutaneous right axilla, all animals were routinely housed until the tumor package grew to 150mm³Thereafter, the treatment was randomly divided into a recombinant human Fc antibody treatment group of 30. mu.g/ml, a recombinant human Fc antibody treatment group of 50. mu.g/ml and a physiological saline group, 7/group. Treatment groups: the right axilla of the nude mice are treated by subcutaneous injection of recombinant human Fc antibody at 30 mug/mL and 50 mug/mL (0.1 mL)/mouse, and the saline groups are injected with equal volume of saline respectively. Injecting each group of nude mice every other day for 1 time; observing the general living condition of the mouse and the size of the tumor every day, measuring the body weight every other day, killing the mouse after 3wk cervical vertebra dislocation after inoculation, separating the tumor tissue and measuring the tumor volume;

(6.3) Experimental results general conditions of nude mice were tested, and after inoculation of tumor cells, each group of mice was fed normally, and the general physiological conditions within 1wk of inoculation were not changed significantly. After 1wk, compared with a treatment group, the mice in the normal saline group have reduced food intake, emaciation, weight loss, tarnished fur and obviously increased tumor masses; while the treated mice were generally in good condition. Killing the mice alive 32 days later, taking tumor tissues to measure the tumor volume, wherein the tumor inhibition rates of the treatment groups of 30 mug/ml and 50 mug/ml are 61.6% and 64.6% respectively; the tumor inhibition rate difference was significant (p <0.01) (see figure 6).

Attached table: effect of recombinant human Fc antibodies on tumor volume and weight of Breast cancer in nude mice

Note: **: p <0.01

The invention has the beneficial effects that: the recombinant human Fc antibody can be efficiently expressed in eukaryotic cells, has good stability, high purity and high activity, has obvious treatment effect, particularly has no special requirements on production equipment when being implemented, is suitable for large-scale and industrialization, and has important significance for overcoming tumors and prolonging the life of human beings.

Sequence listing

<110> Zhao hong gift

<120> recombinant human Fc antibody for specifically inducing tumor cell apoptosis, and preparation method and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1100

<212> DNA

<213> anemia virus of Chicken (Chicken anemia virus)

<400> 1

gaattcatgg acaagaccca cacctgccct ccctgccccg cccctgagct gctgggcggc 60

cccagcgtgt tcctgtttcc accaaagcca aaggatcagc tgatgatctc caggacccct 120

gaggtgacct gcgtggtggt ggacgtgagc cacgaggacc ctgaggtgaa gtttaattgg 180

tacgtggatg gcgtggaggt gcacaatgcc aagacaaagc cacgcgagga gcagtacaat 240

tccacctaca gggtggtgtc cgtgctgaca gtgctgcacc aggactggct gaatggcaag 300

gagtacaagt gtaaggtgtc caataaggcc ctgcctgccc ctatcgagaa gaccatcagc 360

aaggccaagg gccagccacg cgagccacag gtgtacacac tgcctcctag ccgggaggag 420

atgaccaaga accaggtgtc cctgacctgt ctggtgaagg gcttctaccc tagcgacatc 480

gccgtggagt gggagtccaa cggccagcca gagaataatt acaagacaac accacctgtg 540

ctggattccg atggctcctt cttcctgtac tccaagctga cagtggataa gtcccgctgg 600

cagcagggca atgtgttctc ctgttccgtg ctgcacgagg ccctgcacaa tcactacaca 660

cagaagtccc tgtccctgtc ccctggcaag tgagcggccg cggatccccc gacctcgacg 720

gcggcggaag caacgctctc caagaagata ctccacccgg accatcaacg gtgttcaggc 780

caccaacaag ttcacggccg ttggaaaccc ctcactgcag agagatccgg attggtatcg 840

ctggaattac aatcactcta tcgctgtgtg gctgcgcgaa tgctcgcgct cccacgctaa 900

gatctgcaac tgcggacaat tcagaaagca ctggtttcaa gaatgtgcag gacttgagga 960

ccgatcaacc caagcctccc tcgaagaagc gatcctgcga cccctccgag tacagggtaa 1020

gcgagctaaa agaaagcttg attaccacta ctcccagccg accccgaacc gcaagaaggc 1080

gtataagact gtaagtcgac 1100

<210> 2

<211> 710

<212> DNA

<213> anemia virus of Chicken (Chicken anemia virus)

<400> 2

gacaagaccc acacctgccc tccctgcccc gcccctgagc tgctgggcgg ccccagcgtg 60

ttcctgtttc caccaaagcc aaaggatcag ctgatgatct ccaggacccc tgaggtgacc 120

tgcgtggtgg tggacgtgag ccacgaggac cctgaggtga agtttaattg gtacgtggat 180

ggcgtggagg tgcacaatgc caagacaaag ccacgcgagg agcagtacaa ttccacctac 240

agggtggtgt ccgtgctgac agtgctgcac caggactggc tgaatggcaa ggagtacaag 300

tgtaaggtgt ccaataaggc cctgcctgcc cctatcgaga agaccatcag caaggccaag 360

ggccagccac gcgagccaca ggtgtacaca ctgcctccta gccgggagga gatgaccaag 420

aaccaggtgt ccctgacctg tctggtgaag ggcttctacc ctagcgacat cgccgtggag 480

tgggagtcca acggccagcc agagaataat tacaagacaa caccacctgt gctggattcc 540

gatggctcct tcttcctgta ctccaagctg acagtggata agtcccgctg gcagcagggc 600

aatgtgttct cctgttccgt gctgcacgag gccctgcaca atcactacac acagaagtcc 660

ctgtccctgt cccctggcaa gtgagcggcc gcggatcccc cgacctcgac 710

<210> 3

<211> 12

<212> DNA

<213> anemia virus of Chicken (Chicken anemia virus)

<400> 3

ggcggcggaa gc 12

<210> 4

<211> 369

<212> DNA

<213> anemia virus of Chicken (Chicken anemia virus)

<400> 4

aacgctctcc aagaagatac tccacccgga ccatcaacgg tgttcaggcc accaacaagt 60

tcacggccgt tggaaacccc tcactgcaga gagatccgga ttggtatcgc tggaattaca 120

atcactctat cgctgtgtgg ctgcgcgaat gctcgcgctc ccacgctaag atctgcaact 180

gcggacaatt cagaaagcac tggtttcaag aatgtgcagg acttgaggac cgatcaaccc 240

aagcctccct cgaagaagcg atcctgcgac ccctccgagt acagggtaag cgagctaaaa 300

gaaagcttga ttaccactac tcccagccga ccccgaaccg caagaaggcg tataagactg 360

taagtcgac 369

<210> 5

<211> 352

<212> PRT

<213> anemia virus of Chicken (Chicken anemia virus)

<400> 5

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

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

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys Leu Gly Gly Gly Ser Met Asn Ala Leu Gln Glu Asp Thr

225 230 235 240

Pro Pro Gly Pro Ser Thr Val Phe Arg Pro Pro Thr Ser Ser Arg Pro

245 250 255

Leu Glu Thr Pro His Cys Arg Glu Ile Arg Ile Gly Ile Ala Gly Ile

260 265 270

Thr Ile Thr Leu Ser Leu Cys Gly Cys Ala Asn Ala Arg Ala Pro Thr

275 280 285

Leu Arg Ser Ala Thr Ala Asp Asn Ser Glu Ser Thr Gly Phe Lys Asn

290 295 300

Val Gln Asp Leu Arg Thr Asp Gln Pro Lys Pro Pro Ser Lys Lys Arg

305 310 315 320

Ser Cys Asp Pro Ser Glu Tyr Arg Val Ser Glu Leu Lys Glu Ser Leu

325 330 335

Ile Thr Thr Thr Pro Ser Arg Pro Arg Thr Ala Arg Arg Arg Ile Arg

340 345 350

<210> 6

<211> 227

<212> PRT

<213> anemia virus of Chicken (Chicken anemia virus)

<400> 6

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

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

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Lys

225

<210> 7

<211> 4

<212> PRT

<213> anemia virus of Chicken (Chicken anemia virus)

<400> 7

Gly Gly Gly Ser

1

<210> 8

<211> 121

<212> PRT

<213> anemia virus of Chicken (Chicken anemia virus)

<400> 8

Met Asn Ala Leu Gln Glu Asp Thr Pro Pro Gly Pro Ser Thr Val Phe

1 5 10 15

Arg Pro Pro Thr Ser Ser Arg Pro Leu Glu Thr Pro His Cys Arg Glu

20 25 30

Ile Arg Ile Gly Ile Ala Gly Ile Thr Ile Thr Leu Ser Leu Cys Gly

35 40 45

Cys Ala Asn Ala Arg Ala Pro Thr Leu Arg Ser Ala Thr Ala Asp Asn

50 55 60

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

65 70 75 80

Pro Lys Pro Pro Ser Lys Lys Arg Ser Cys Asp Pro Ser Glu Tyr Arg

85 90 95

Val Ser Glu Leu Lys Glu Ser Leu Ile Thr Thr Thr Pro Ser Arg Pro

100 105 110

Arg Thr Ala Arg Arg Arg Ile Arg Leu

115 120

Claims

1. A recombinant human Fc fusion protein for specifically inducing tumor cell apoptosis is characterized in that:

(1) the recombinant human Fc antibody gene fragment is directly obtained by a synthetic method, the complete nucleic acid sequence of the recombinant human Fc antibody gene fragment is SEQ ID No. 1, and the SEQ ID No. 1 sequence consists of the following three gene fragments: the gene fragment of human IgG H chain Fc fragment has the sequence of SEQ ID No. 2; the sequence of the linked fragment gene is SEQ ID No. 3; VP3 gene fragment with sequence SQE ID No. 4;

(2) the complete amino acid sequence of the recombinant human Fc antibody is SEQ ID No. 5, and the sequence consists of the following 3 amino acid fragments: the amino acid fragment of IgG H chain Fc fragment has the sequence of SEQ ID No. 6; the amino acid sequence of the link fragment is SEQ ID No. 7; VP3 amino acid fragment, sequence SQE ID No: 8.

2. The recombinant human Fc fusion protein for specifically inducing apoptosis in tumor cells according to claim 1, wherein: the recombinant human Fc antibody gene fragment is subcloned into a yeast expression vector and expressed in yeast cells; or subcloning into a mammalian cell expression vector and expressing in a mammalian cell.

3. The method for preparing the recombinant human Fc fusion protein for specifically inducing apoptosis in tumor cells according to claim 2, wherein:

(1) constructing, fermenting and recovering recombinant human Fc antibody yeast engineering bacteria, subcloning an artificially synthesized gene fragment 'Fc-linker-VP 3' into a yeast expression vector pPIC9K vector, transforming a Pichia pastoris system GS115 strain, screening a high-efficiency expression cell strain, and expressing recombinant human Fc antibody protein;

(2) purifying recombinant human Fc antibody protein, slowly adding equal volume of saturated ammonium sulfate into fermentation culture supernatant, mixing, standing at 4 deg.C for more than 4 hr, centrifuging at 4 deg.C for 20min to 5000g, collecting precipitate, dissolving the precipitate with 100ml of balance solution, dialyzing with 2000 balance solution for more than 12 hr, and changing the middle overnight; the sample is put on an anion chromatographic column balanced by the balance liquid or is stored for standby at the temperature of minus 20 ℃, the balance liquid containing 0.1mol/L, 0.2mol/L, 0.5mol/L and 1mol/LNaCl is respectively used for elution, elution peaks are collected, SDS electrophoresis identification is carried out, and the protein part containing the molecular weight of 39kD is taken; balancing the Separose12 chromatographic column by using a balancing solution; loading the concentrated 39kD protein fraction at a rate of 3% of the bed volume at a flow rate of 2 ml/min; collecting step by step; after SDS electrophoresis identification, collecting a protein part with the molecular weight of 39kD and high purity, namely the recombinant human Fc antibody;

(3) the purified recombinant human Fc antibody is identified by measuring the protein purity by SDS electrophoresis or high performance liquid chromatography, the purity is more than 95 percent, the molecular weight is 39kD, and the protein content is measured by an ultraviolet spectrophotometer to be 0.5 mg/ml.

4. The method for preparing the recombinant human Fc fusion protein for specifically inducing apoptosis in tumor cells according to claim 2, wherein:

(1) constructing, fermenting and recovering a recombinant human Fc antibody mammalian cell strain, subcloning a artificially synthesized gene fragment 'Fc-linker-VP 3' into a mammalian cell expression vector pATX2, transfecting a mammalian cell CHO cell strain by a liposome method for expression, screening a high-efficiency expression cell strain, and expressing recombinant human Fc antibody protein;

(2) purifying recombinant human Fc antibody protein, taking CHO cell culture supernatant, slowly adding saturated ammonium sulfate with the same volume, uniformly mixing, standing at 4 ℃ for more than 4 hours, centrifuging 5000g at 4 ℃ for 20min, collecting precipitate, dissolving the precipitate with 100ml of balance solution, dialyzing the balance solution 2000 for more than 12 hours, and changing the middle overnight; the sample is put on an anion chromatographic column balanced by the balance liquid or is stored for standby at the temperature of minus 20 ℃, the balance liquid containing 0.1mol/L, 0.2mol/L, 0.5mol/L and 1mol/LNaCl is respectively used for elution, elution peaks are collected, SDS electrophoresis identification is carried out, and the protein part containing the molecular weight of 39kD is taken; balancing the Separose12 chromatographic column by using a balancing solution; loading the concentrated 39kD protein fraction at a rate of 3% of the bed volume at a flow rate of 2 ml/min; collecting step by step; after SDS electrophoresis identification, collecting a protein part with the molecular weight of 39kD and high purity, namely the recombinant human Fc antibody;

(3) the purified recombinant human Fc antibody is identified by measuring the protein purity by SDS electrophoresis or high performance liquid chromatography, the purity is more than 96 percent, the molecular weight is 39kD, and the protein content is measured by an ultraviolet spectrophotometer to be 0.5 mg/ml.

5. Use of a recombinant human Fc fusion protein specifically inducing apoptosis in tumor cells according to claim 1 or 2 for the preparation of a medicament for the treatment of malignant tumors.