CN107226858B - Preparation and application of interferon high-molecular conjugate IFN-PMPC - Google Patents

Abstract

The invention discloses a preparation method and application of an interferon polymer conjugate IFN-PMPC. The invention provides a method for preparing a protein-macromolecule combination, which comprises the following steps: mixing protein-initiator combination, 2-methacryloyloxyethyl phosphorylcholine, CuCl₂Carrying out polymerization reaction on 1,1,4,7,10, 10-hexamethyl triethylene tetramine in a buffer solution to obtain a protein-polymer combination; the protein-initiator combination is a product obtained by covalently connecting an initiator and protein. Experiments prove that the IFN-PMPC combination prepared by the invention has high yield, simple purification, site-specific modification and no immunogenicity, can better retain in-vitro biological activity, greatly improves the pharmacokinetics and biological distribution and effectively enhances the treatment effect.

Description

Preparation and application of interferon high-molecular conjugate IFN-PMPC

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to preparation and application of an interferon high-molecular combination IFN-PMPC.

Background

Proteins have been widely used in various fields such as biomedical imaging, targeted therapy, and clinical diagnosis. The single use of protein has the problems of short half-life, poor stability and the like. The protein is connected with the macromolecule to prepare the protein-macromolecule combination, which can effectively improve the solubility, the stability, the pharmacokinetics and the treatment efficacy of the protein and reduce the immunogenicity thereof. The traditional synthesis method of the protein-polymer combination is to connect a pre-prepared polymer with a protein, and has the problems of uncertain coupling sites, low efficiency, poor yield, difficult separation of products, poor quality control, difficult maintenance of activity and the like.

Interferon-alpha 2 (IFN-alpha 2) is a potent inhibitor of viral replication and tumor cell growth and has been successfully used in the treatment of viral hepatitis and cancer. However, IFN has a very short circulating half-life after systemic administration, and requires frequent administration and high concentration to achieve the desired therapeutic effect, resulting in some toxic side effects and also causing a heavy economic burden on patients. Modifying IFN with polyethylene glycol (PEG) is an effective measure to improve its pharmacokinetics and therapeutic efficacy, and is called long-acting interferon. However, the current pegylated interferons have disadvantages such as low reaction yield, difficult control of binding site and coupling stoichiometry, and severely reduced biological activity. In addition, PEG has immunogenicity, and after multiple administrations, PEG antibodies can be generated, so that the clearance of the PEG antibodies in a body is accelerated, and the distribution and metabolism of the medicine in the body are changed. Therefore, the development of site-specific modification methods with mild reaction conditions, simple steps and no immunogenicity is particularly important for interferons and other pharmaceutical proteins.

Disclosure of Invention

An object of the present invention is to provide a method for producing a protein-polymer conjugate.

The method provided by the invention is 1) or 2):

1) the method shown comprises the following steps:

combining protein-initiator, high molecular monomer and CuCl、CuCl₂Carrying out polymerization reaction on 1,1,4,7,10, 10-hexamethyl triethylene tetramine in a buffer solution to obtain a protein-polymer combination;

the protein-initiator combination is a product obtained by covalently connecting an initiator and protein;

in the method, the buffer solution used for the polymerization reaction is a PBS aqueous solution with the pH value of 7.4 and the concentration of 10mM, and the specific formula is as follows: 2.684g Na₂HPO₄·12H₂O、0.34g NaH₂PO₄·2H₂O, 8.19g NaCl in 1L water solution.

2) The method comprises the steps of connecting a high molecular compound with protein through oligoglycine under the action of an initiator to obtain a protein-high molecular combination;

the polymer compound is obtained by polymerizing a polymer monomer.

In the two methods described above, the first and second methods,

the protein is interferon, and the interferon is selected from interferon alpha or fusion protein thereof, interferon beta or fusion protein thereof, interferon gamma or fusion protein thereof, and interferon lambda or fusion protein thereof;

or the protein is specifically interferon alpha fusion protein shown in a sequence 2 in a sequence table.

The high molecular monomer is 2-methacryloyloxyethyl phosphorylcholine;

the macromolecular compound is poly 2-methacryloyloxyethyl phosphorylcholine.

The initiator is an initiator for atom transfer radical polymerization reaction, an initiator for reversible addition-fragmentation chain transfer polymerization, an initiator for ring-opening ectopic polymerization or an initiator for ring-opening addition polymerization;

the structural formula of the initiator for atom transfer radical polymerization is shown as the following formula 1:

wherein R is H or (CH)₂)_nCH₃N is an integer not less than 1; x is Cl or Br or I; m is an integer greater than or equal to 1; n is an integer of 1 or more;

the atom transfer radical polymerization initiator is 2- (2- (2- (2-aminoacetamido) acetamido) ethyl 2-bromo-2-methylpropionate hydrochloride;

the initiator is covalently linked to the C-terminus or N-terminus of the protein;

1) in the method shown, the initiator is covalently linked to the C-terminus or N-terminus of the protein;

or the initiator is specifically covalently linked to the C-terminus of the protein;

in the method according to 1) above, the protein-initiator combination, the 2-methacryloyloxyethyl phosphorylcholine, the CuCl, or the CuCl₂And the molar ratio of the 1,1,4,7,10, 10-hexamethyltriethylene tetramine is 1: 200-10000: 10-500: 10-2000: 10-4000;

the protein-initiator combination, the 2-methacryloyloxyethyl phosphorylcholine, the CuCl₂And the molar ratio of the 1,1,4,7,10, 10-hexamethyl triethylene tetramine is specifically 1: 1000-4000: 10-500: 10-2000: 10-4000;

the coupling ratio of the protein to the initiator in the protein-initiator combination is 1: 1.

in the above method, the protein-initiator combination, the 2-methacryloyloxyethyl phosphorylcholine, the CuCl, and the CuCl₂And the molar ratio of the 1,1,4,7,10, 10-hexamethyltriethylene tetramine is 1: 2000: 25: 75: 125.

1) in the method, the protein-initiator combination is formed by forming an amide bond between the initiator and the protein at the C end of the protein, and the method comprises the following steps: protein (IFN-LPETGGH)₆Protein), transpeptidase (Sortase A-H)₆Protein), 2- (2- (2- (2-aminoacetamido) acetamido) ethyl 2-bromo-2-methylpropionate, CaCl₂Mixing the mixture in a Tris-HCl aqueous solution with the pH value of 7.4 and the concentration of 50mM, and reacting to obtain an interferon-initiator combination IFN-Br; as described aboveIFN-LPETGGH₆Protein, Sortase A-H₆Protein, 2- (2- (2- (2-aminoacetamido) acetamido) ethyl 2-bromo-2-methylpropionate, CaCl₂In a molar ratio of 2: 1: 50: 200.

the method shown in 2) above, comprising the steps of: carrying out polymerization reaction on the initiator and the high molecular monomer to obtain a high molecular polymer connected with oligoglycine; and then, covalently connecting the high molecular polymer connected with the oligoglycine with the protein to obtain a protein-high molecular combination.

In the method shown in the above 2), the polymerization reaction is carried out by adding an initiator, 2-methacryloyloxyethyl phosphorylcholine, CuCl₂Carrying out polymerization reaction with 1,1,4,7,10, 10-hexamethyl triethylene tetramine in a buffer solution to obtain a high molecular polymer connected with oligoglycine; the buffer solution adopted by the polymerization reaction is a PBS (phosphate buffer solution) water solution with the pH value of 7.4 and the concentration of 10 mM;

the covalent linkage is formed by linking the protein, the transpeptidase, the high-molecular polymer linked with the oligoglycine and CaCl₂Reacting in a buffer solution to obtain a protein-macromolecule combination; the buffer in the covalent coupling reaction was 50mM Tris-HCl aqueous solution at pH 7.4.

In the method according to 2) above, the initiator, the 2-methacryloyloxyethyl phosphorylcholine, the CuCl, or the CuCl is used in the polymerization reaction₂And the molar ratio of the 1,1,4,7,10, 10-hexamethyltriethylene tetramine is 1: 200-10000: 10-500: 10-2000: 10-4000;

or the initiator, the 2-methacryloyloxyethyl phosphorylcholine, the CuCl₂And the molar ratio of the 1,1,4,7,10, 10-hexamethyl triethylene tetramine is specifically 1: 2000: 25: 75: 125;

in the covalent linkage, the protein (IFN-LPETGGH)₆Protein), said transpeptidase (Sortase A-H)₆Protein), the high-molecular polymer connected with the oligoglycine (poly-2-methacryloyloxyethyl phosphorylcholine connected with triglycine) and CaCl₂In a molar ratio of 2: 1: 40：200。

The buffer solution is a Tris-HCl aqueous solution with the pH value of 7.4 and the concentration of 50 mM;

in the above method, the polymerization reaction is carried out under a low oxygen or inert gas atmosphere;

the time of the polymerization is 5 minutes to 24 hours, and the temperature of the polymerization is 0 to 80 ℃.

The protein-polymer conjugate prepared by the above method is also within the scope of the present invention.

The application of the protein-polymer combination in preparing anti-tumor or anti-virus products is also within the protection scope of the invention.

It is another object of the invention to provide an anti-tumor or anti-viral product.

The invention provides a product comprising the protein-polymer conjugate.

The invention utilizes biochemical and polymer chemical technologies to modify the C-terminal far away from the interferon and efficiently grows PMPC (poly-2-methacryloyloxyethyl phosphorylcholine) in situ by an optimized Atom Transfer Radical Polymerization (ATRP) technology to generate a site-specific and mono-modified interferon-polymer conjugate (namely IFN-PMPC). The in-situ ATRP technology has the advantages of mild reaction conditions, high yield, simple purification steps and low cost, can effectively maintain the biological activity and improve the in vitro stability, and provides a universal method for modifying proteins.

Drawings

FIG. 1 is a schematic diagram of the synthetic route of IFN-PMPC.

FIG. 2 shows the IFN-LPETGGH obtained by nickel column affinity chromatography₆。

FIG. 3 shows the recovery of Sortase A-H by nickel column affinity chromatography₆。

FIG. 4 shows the synthesis and purification of in situ ATRP initiator AEBM.

FIG. 5 shows the analysis of the IFN-Br synthesis and purification process.

FIG. 6 shows MALDI-TOF analysis of IFN-LPETGGH₆And the molecular weight of IFN-Br.

FIG. 7 shows specific modifications of IFN-Br analyzed by LC-MS/ESI.

FIG. 8 shows the IFN-PMPC synthesis and purification process.

FIG. 9 shows the GPC analysis of IFN-PMPC.

FIG. 10 is a schematic view showing the passing of¹H NMR analysis of IFN-PMPC binding.

FIG. 11 is a control experiment for analyzing the in situ ATRP synthesis of IFN-PMPC.

FIG. 12 shows the IFN-PMPC acquisition by the "grafting to" technique

FIG. 13 shows SDS-PAGE analysis of proteinase K-treated IFN-PMPC.

FIG. 14 shows the hydration radii of IFN-PMPC and IFN- α for DLS analysis.

FIG. 15 shows the secondary structure of IFN-PMPC and IFN- α in CD analysis.

FIG. 16 shows the in vitro biological activity of IFN-PMPC, pirocin and IFN- α measured by MTT assay.

FIG. 17 is a graph showing the pharmacokinetic profile of IFN-PMPC, pirocin and IFN- α using the atrioventricular elimination biventricular model in the DAS software.

FIG. 18 shows the distribution of IFN-PMPC, pyroxin and IFN- α in tumors and other tissues.

FIG. 19 shows the inhibition of tumor growth by IFN-PMPC, pyroxin, IFN- α and saline.

FIG. 20 is a graph showing the survival curves of nude mice injected with IFN-PMPC, pirocin, IFN- α and physiological saline.

FIG. 21 shows the change of body weight of nude mice injected with IFN-PMPC, pyroxin, IFN- α and physiological saline.

FIG. 22 shows HE staining of tumor, heart, liver and kidney after treatment of nude mice.

FIG. 23 shows the variation of physiological indexes such as lactate dehydrogenase, creatine kinase isoenzyme, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, creatinine, and urea nitrogen after injecting IFN-PMPC, pirocin, IFN-alpha, and normal saline into nude mice.

FIG. 24 shows the change of physiological indexes such as erythrocytes, leukocytes, hemoglobin and platelets after administration of IFN-PMPC, pirocin, IFN- α and physiological saline.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The plasmid pET-25b (+) in the following examples is a product of Industrial bioengineering (Shanghai) Inc.

The TB medium in the following examples was prepared as follows: adding peptone 12g, yeast extract 24g and glycerol 4mL into 900mL water, fully dissolving, autoclaving at 121 deg.C for 15min, cooling the sterilized mixture to 60 deg.C, and adding 100mL sterilized 170mmol/LKH₂PO₄And 0.72mol/L K₂HPO₄An aqueous solution of (a).

Human Burkitt's B lymphoma cells and human ovarian cancer cells (OVCAR-3) in the examples described below were purchased from the national academy of sciences tumor cell Bank.

The RMPI-1640 medium in the examples below is a product of Gibco.

The female athymic (Nude) Nude mice in the following examples are products of experimental animal technology ltd, viton, beijing. Female athymic (Nude) Nude mice are hereinafter abbreviated Nude mice.

The flow in the following example is shown in FIG. 1.

In the following examples, the physicochemical properties of IFN-PMPC and IFN, such as molecular weight, phase transition temperature, hydration radius, secondary structure, etc., were characterized by analytical means such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), microplate reader, Dynamic Light Scattering (DLS), Circular Dichroism (CD), etc. Selecting human Burkitt's B lymphoma cells, testing IFN-PMPC and IFN in vitro biological activity, namely the ability of the IFN to resist the proliferation of tumor cells in vitro; testing pharmacokinetics of IFN-PMPC and IFN in vivo by using a nude mouse model, and calculating pharmacokinetic parameters by using DAS 3.0 pharmacokinetic analysis software; establishing a nude mouse tumor model, and testing the anti-tumor effect and the drug distribution of IFN-PMPC and IFN.

In the following examples, three replicates were set up unless otherwise specified, and the results averaged.

Example 1 method for preparing IFN-PMPC as interferon polymer conjugate

First, preparing interferon-initiator combination

1. Preparation of IFN-LPETGGH₆Fusion protein and transpeptidase Sortase A-H₆

1) Preparation of recombinant bacterium

IFN-LPETGGH₆The amino acid sequence of the fusion protein is a sequence 2 in a sequence table, and the nucleotide sequence of the coding gene is a sequence 1 in the sequence table;

transpeptidase Sortase A-H₆The amino acid sequence of (A) is sequence 4 in the sequence table, and the nucleotide sequence of the coding gene is sequence 3 in the sequence table;

expression of IFN-LPETGGH₆The recombinant vector of the fusion protein is IFN-LPETGGH shown in sequence 1₆The vector obtained by inserting the fusion protein coding gene into Nde I and Eco RI enzyme cutting sites of pET-25b (+) vector is named as pET-25b-IFN-LPETGGH₆；

Expression of Sortase A-H₆The recombinant vector is a Sortase A-H shown in a sequence 3₆The vector obtained by inserting the protein coding gene into Nde I and Eco RI enzyme cutting sites of pET-25b (+) vector is named as pET-25b-Sortase A-H₆。

Expression of IFN-LPETGGH₆The recombinant strain of the fusion protein is to express IFN-LPETGGH₆Coli BL21Rosetta-gami (DE3) pLysS (Invitrogen) to obtain recombinant bacteria named BL21/pET-25b-IFN-LPETGGH₆；

Expression of Sortase A-H₆The recombinant strain is to express Sortase A-H₆The recombinant vector is introduced into E.coli BL21(DE3) pLysS (Novagen) to obtain a recombinant strain named as BL21/pET-25b-Sortase A-H₆。

2)IFN-LPETGGH₆Expression and purification of proteins

Recombinant bacteria BL21/pET-25b-IFN-LPETGGH₆The cells were cultured overnight at 37 ℃ and 250rpm in 50mL of TB medium (containing 100. mu.g/mL ampicillin). Switching to 1L the next dayLarge-scale cultivation and induction of expression were carried out in fresh TB medium (ampicillin concentration 100. mu.g/mL in 2L flasks). The method comprises the following specific steps: first, shake culture was carried out at 37 ℃ and 200rpm for 5 hours, then the culture temperature was set to 18 ℃ and isopropyl-. beta. -D-thiogalactoside (IPTG) was added to a final concentration of 0.4mM, and culture was carried out for 16 hours, and the culture broth was collected. Centrifuging at 3000 Xg centrifugal force to collect thallus, removing upper layer culture solution to obtain recombinant bacteria BL21/pET-25b-IFN-LPETGGH₆And (3) bacteria.

Resuspend BL21/pET-25b-IFN-LPETGGH with 30mL ice-cold PBS₆The cells were disrupted at 4 ℃ by an ultrasonic instrument, and the disrupted product of Escherichia coli was centrifuged at 14000 Xg for 15 minutes at 4 ℃. To the collected supernatant was added 2mL of polyethyleneimine (PEI, 10%), and centrifuged again for 15 minutes. The resulting supernatant was filtered through a 0.45 μm filter and then purified by a nickel affinity column (HisTrap HP 5mL) on an AKTA protein purification system (AKTA Purifier 10, GE) using 10mM PBS, 500mM NaCl, 5% glycerol as an equilibration buffer and 500mM imidazole as an elution buffer. Collecting eluate corresponding to the elution peak, detecting with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and retaining eluate corresponding to about 21.1kDa target product, which is IFN-LPETGGH₆A protein. SDS-PAGE analysis samples are prepared by Laemmli sample buffer solution containing 5% beta-mercaptoethanol, the concentration is 1mg/mL, after heating for 5min at 95 ℃,10 mu L of samples are loaded into a prefabricated 10% SDS-PAGE gel, and vertical electrophoresis is carried out for 90min under the voltage of 80-100V (electrophoresis solution is 25mM Tris, 250mM Glycine and 0.1% SDS). The gel was stained with Coomassie blue G-250 and the band positions were observed.

Eluting the obtained IFN-LPETGGH₆The protein was subjected to Desalting column (HiPrep 26/10 desaling) to remove imidazole and replaced in 50mM Tris HCl buffer. The purified samples were tested for purity by SDS-PAGE and the protein concentration was determined spectrophotometrically (NanoDrop 2000).

The result is shown in FIG. 2, wherein A is the purification by nickel affinity chromatography and the linear elution is monitored by UV to obtain the target product IFN-LPETGGH₆(ii) a B is SDS-PAGE analysis of IFN-LPETGGH₆Purification of(ii) a condition; shows IFN-LPETGGH₆Protein expression and nickel affinity chromatography column purification. Protein standard sample, cell lysate before sample loading, nickel column flow-through liquid, 50mM imidazole washing column and 500mM imidazole eluting target protein are sequentially arranged from left to right, and it can be seen that IFN-LPETGGH with purity of more than 95% is obtained by expression of escherichia coli and purification of nickel affinity chromatography column₆The yield of protein reaches 200mg/L of culture solution, and the target product of about 21.1kDa is obtained.

3)Sortase A-H₆Expression and purification of proteins

The recombinant bacterium BL21/pET-25b-Sortase A-H is treated by the same method₆And collecting eluent corresponding to the elution peak, and detecting by SDS-PAGE. The result is shown in figure 3, wherein A is the target product Sortase A-H obtained by nickel affinity chromatography purification and UV monitoring linear elution₆(ii) a B is SDS-PAGE analysis of Sortase A-H₆Purification conditions; shows that Sortase A-H₆Protein expression and nickel affinity chromatography column purification. From left to right, the protein standard, the cell lysate before loading, the nickel column flow-through, the 50mM imidazole column and the 500mM imidazole elute the target protein, and it can be seen that the target product of about 22.9kDa is remained.

2. Synthesis of ATRP initiators

Tert-butyl 2-hydroxyethylcarbamate (2.0g,12.5mmol), N, N-diisopropylethylamine (2.4mL,14mmol,1.1 equiv.) were dissolved in dichloromethane (20mL) in an ice-water bath. 2-bromo-2-methylpropanoyl bromide (1.25ml,10mmol) was added dropwise at below 0 ℃ over 15 minutes. After 30 minutes, the ice-water bath was removed and stirring was maintained for 16 hours. The solvent was removed by rotary evaporation and the product was purified on a silica gel column (dichloromethane: ethyl acetate ═ 1: 1). The product was a yellow oily liquid, 2- (2- (tert-butoxycarbonyl) amino) ethyl 2-bromo-2-methylpropionate (C11H20BrNO4,2.9g, 74.7%).¹HNMR(400MHz,CDCl₃):4.827(s,1H),4.243(m,2H),3.424(m,2H),1.945(s,6H),1.447(s,9H).ESI-mass m/z:332.1([M+Na]⁺),334.1([M+Na]⁺).

2- (2- (tert-Butoxycarbonyl) amino) ethyl 2-bromo-2-methylpropionate (2.5g) was dissolved in 50mL of a 6M ethyl hydrogen chloride solution in ethyl acetate, and the mixture was stirred for 2 hours. After the reaction is finishedFiltered, and the product is white powder, 2-bromo-2-methylpropanoic acid 2-aminoethyl ester hydrochloride (C)₆H₁₃BrClNO₂,1.95g,98％).ESI-mass m/z:210.3([M–Cl+Na]⁺),212.3([M–Cl+Na]⁺).

2- (2- (2-tert-Butoxycarbonyl) acetamido) acetic acid (289mg,1mmol), 2-aminoethyl 2-bromo-2-methylpropionate hydrochloride (246mg,1mmol), EDC (288mg,1.5mmol), and N, N-diisopropylethylamine (175. mu.l, 1mmol) were dissolved in methyl chloride (10ml) and stirred at room temperature for 16 hours. After the reaction was completed, the solvent was removed, and the residual white solid was washed once with 4ml of water 2 times, 1ml of ethanol 3 times, and then with 4ml of ethyl acetate to obtain a white powdery product, 2- (2- (2- (2-tert-butoxycarbonyl) acetamido) ethyl 2-bromo-2-methylpropionate (C)₁₅H₂₆BrN₃O₆,269mg,56％).¹HNMR(400MHz,CDCl₃):4.261(t,2H),3.909(s,2H),3.871(s,2H),3.779(s,2H),3.544(t,2H),1.938(s,6H),1.449(s,9H).ESI-mass m/z:481.2([M+H]⁺),483.1([M+H]⁺),503.2([M+Na]⁺),505.1([M+Na]⁺).

2- (2- (2- (2-tert-Butoxycarbonyl) acetamido) ethyl 2-bromo-2-methylpropionate (1.08g,2.25mmol) was dissolved in 50ml of a 6M ethyl acetate hydrochloride solution and stirred for 2 hours. The product was a white powder, 2- (2- (2- (2-aminoacetamido) acetamido) ethyl 2-bromo-2-methylpropionate hydrochloride, the ATRP initiator AEBM (C)₁₂H₂₂BrClN₄O₅The chemical structural formula is shown as formula 1, 0.84g, 98%).¹HNMR(400MHz,D₂O):4.196(t,2H),3.938(s,2H),3.815(s,2H),3.794(s,2H),3.456(t,2H),1.813(s,6H).ESI-mass m/z:381.1([M–Cl+H]⁺),383.0([M–Cl+H]⁺)。

Formula 1 is as follows:

FIG. 4 shows the synthesis and purification of the in situ ATRP initiator AEBM. Finally, the initiator AEBM with the purity of more than 95% is successfully obtained, and the yield is 40.2%.

3. Obtaining interferon-initiator combinations by enzymatic catalysis with transpeptidase A

Catalysis at IFN-LPETGGH by transpeptidase A₆The ATRP initiator AEBM is introduced into the C-terminal, and the concrete steps are as follows: IFN-LPETGGH prepared by the method 1₆Protein, Sortase A-H₆Protein, AEBM and CaCl prepared in 2₂Mixing in 50mM Tris-HCl aqueous solution with the pH value of 7.4, and reacting to obtain interferon-initiator combination IFN-alpha;

IFN-LPETGGH as described above₆Protein, Sortase A-H₆Protein, AEBM and CaCl prepared in 2₂In a molar ratio of 2: 1: 50: 200.

the specific method comprises the following steps: in 10mL of a solution containing IFN-LPETGGH₆(200. mu.M) of 50mM Tris-HCl solution (solute IFN-LPETGGH)₆Solvent 50mM Tris-HCl aqueous solution at pH 7.4) with 5mM AEBM, and 10mL of a mixture containing 100. mu.M transpeptidase A and 20mM CaCl₂Mixing with Tris-HCl solution, reacting overnight at room temperature to obtain reaction mixture.

Purifying the reaction mixture by anion exchange chromatography (HiTrap Capto Q5 mL) to obtain IFN-Br; the equilibrium buffer used for purification was 20mM Tris & HCl, pH 7.5; the elution buffer is 20mM Tris-HCl aqueous solution containing 1M NaCl, pH 7.5; the size of the column is 5mL, and the flow rate is 2 mL/min; and collecting eluent corresponding to the elution peak, detecting by SDS-PAGE, reserving eluent corresponding to the target product with about 20.5kDa, which is an interferon-initiator combination IFN-Br, and further removing small molecular impurities by using PBS through a desalting column. The reaction efficiency is more than 95%.

The interferon-initiator conjugate IFN-Br was validated by SDS-PAGE and the results are shown in fig. 5, which shows the analysis of the IFN-Br synthesis and purification process, wherein a: AEX separation of transpeptidase A catalyzed IFN-LPETGGH₆Carrying out UV monitoring on the mixture reacted with AEBM to carry out linear elution to obtain a target product IFN-Br; b: SDS-PAGE analysis of IFN-LPETGGH₆IFN-Br results were obtained before and after reaction with AEBM. From left to right, the protein standard sample and SrtA-H are sequentially arranged₆、IFN-LPETGGH₆Reaction mixture, elution protein IFN-Br, flow-through sample SrtA-H₆。

The molecular weight of the purified IFN-Br was determined by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) using 4800plusMALDI-TOF/TOF^TMAnalyzer (AB SCIEX). The amino acid sequence of IFN-Br and the specific modification of C-terminal were verified by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/ESI) analysis using a Q-active liquid mass spectrometer (Thermo Scientific). FIG. 6 shows MALDI-TOF analysis of IFN-LPTEGGH₆And IFN-Br molecular weights, 21105.8 and 20531.9, respectively, in agreement with theoretical values 21105.9 and 20532.0. FIG. 7 shows the distribution of C-terminal peptide EGSGGGGSLPETGGG (-Br) isotopes of IFN-Br, wherein graph A shows the experimental values of LC-MS/ESI; and B, the diagram is a theoretical predicted value, and the theoretical value is consistent with the experimental value. Table 1 shows the amino acid sequence composition of the peptide fragment after trypsin decomposition of IFN-Br by LC-MS/ESI analysis. FIG. 6, FIG. 7 and Table 1 show that the IFN-initiator conjugate IFN-Br can be obtained quantitatively in a fixed point manner by using the transpeptidase A, the initiator is fixed point modified at the C-terminal of the IFN-alpha, and the coupling ratio of the protein in the IFN-initiator conjugate IFN-Br to the initiator is 1: 1.

TABLE 1

Preparation and characterization of interferon macromolecule combination IFN-PMPC

1. Preparation of interferon macromolecule combination IFN-PMPC

In-situ synthesis of PMPC on the surface of the interferon-initiator combination IFN-Br by ATRP technology comprises the following steps: combining interferon-initiator combinations IFN-Br, MPC, CuCl ₂1,1,4,7,10, 10-Hexamethyltriethylenetetramine (HMTETA) and 10mM PBS aqueous solution (PBS formulation: 2.684g Na) with pH 7.4 (PBS formulation: 10 mM)₂HPO4·12H₂O、0.34g NaH₂PO4·2H₂Dissolving O and 8.19g NaCl in 1L of water), and reacting under a closed condition to obtain an interferon polymer combination IFN-PMPC;

interferon-initiator combinations IFN-Br, MPC, CuCl₂And the molar ratio of 1,1,4,7,10, 10-hexamethyl triethylene tetramine (HMTETA) is 1: 2000: 25: 75: 150;

the method comprises the following specific steps:

mu. mol of MPC was added to 2.5mL of a PBS solution containing 40. mu.M IFN-Br (solute IFN-Br, solvent pH 7.4, 10mM PBS solution) under nitrogen for 15min, and CuCl (2.5. mu. mol), CuCl which had been previously dissolved in 1mL of double distilled water and had been deaerated, was added₂(7.5. mu. mol) and 1,1,4,7,10, 10-Hexamethyltriethylenetetramine (HMTETA) (12.5. mu. mol) under a closed condition for 1 hour, and then exposed to air to terminate the reaction, thereby obtaining a reaction mixture.

IFN-PMPC was isolated and purified from the reaction mixture by anion exchange column (HiTrap Capto Q column, GE Healthcare) using the same procedure as described above: the equilibrium buffer used for purification was 20mM Tris & HCl, pH 7.5; the elution buffer solution is 20mM Tris-HCl, 1M NaCl, pH 7.5, the size of the column is 5mL, and the flow rate is 2 mL/min; and collecting eluent corresponding to the elution peak, detecting by SDS-PAGE, and reserving the eluent corresponding to the target product of about 60kDa as IFN-PMPC.

The calculated yield is expressed as (mass of IFN-PMPC conjugate after purification/IFN-LPETGGH)₆Mass) of 100%, the results are shown in table 2, and the IFN-PMPC yields are 67.0% respectively.

TABLE 2

FIG. 8 shows the synthesis and purification of IFN-PMPC, where A is AEX separation in situ ATRP reaction mixture and UV monitoring linear elution gives the target product IFN-PMPC; b is SDS-PAGE analysis, IFN-Br in-situ ATRP grows macromolecule PMPC, and the following are sequentially performed from left to right: standard sample, IFN-LPETGGH₆IFN-Br, reaction mixture, purified IFN-PMPC. FIG. 9 is a schematic view ofGPC analyzed IFN-PMPC binding. FIG. 10 is a schematic view of¹H NMR analysis of IFN-PMPC binding.

To verify that PMPCs were synthesized at the C-terminus using an initiator on IFN-Br, IFN-. alpha.was obtained by linking triglycine to IFN without an initiator, and then a control run was performed using the same conditions as described above. FIG. 11 shows a control experiment for the analysis of the IFN-PMPC synthesis by ATRP, in which A: GPC analysis IFN-alpha synthesis IFN-PMPC control experiment. B: SDS-PAGE analysis, ATRP reaction and IFN-alpha are carried out on the protein standard sample and the IFN-alpha from left to right. The control experiment used the same reaction conditions as IFN-Br for the ATRP reaction on IFN- α, showing that IFN- α did not grow PMPC, indicating that the polymerization was only carried out at the C-terminus of IFN-Br linked to the ATRP initiator, and that no side reactions on other reactive groups of the protein were present.

Thirdly, another preparation method of interferon macromolecule combination IFN-PMPC

To show the superiority of the ATRP technique in situ, IFN-PMPC was synthesized using the "grafting to" technique. The Grafting to technique comprises two steps:

1. synthesis of Triglycine-linked poly-2-methacryloyloxyethyl phosphorylcholine (PMPC)

The initiators AEBM, MPC, CuCl were prepared according to the preparation procedure of 1 in the two above₂Reacting 1,1,4,7,10, 10-hexamethyl triethylene tetramine (HMTETA) and PBS aqueous solution with the pH value of 7.4 and the concentration of 10mM under a closed condition to obtain PMPC connected with triglycine; wherein, the initiators AEBM, MPC, CuCl and CuCl₂And the molar ratio of 1,1,4,7,10, 10-hexamethyl triethylene tetramine (HMTETA) is 1: 2000: 25: 75: 125;

the reaction conditions are the same as those in 1 of the above two.

Impurities such as small molecules are removed by an ultrafiltration method, and the molecular weight of the triglycine-linked PMPC is measured by GPC and is about 60 kDa.

2. Combining PMPC and IFN to obtain IFN-PMPC polymer conjugate

Catalysis at IFN-LPETGGH by transpeptidase A₆The PMPC connected with triglycine is introduced into the C-terminal of (A), and the specific steps are as follows: prepared by the two methods25μM IFN-LPETGGH₆Protein, 12.5 mu MSortase A-H₆Protein, 500. mu.M Triglycine ligated PMPC prepared as described in 1 above and 10mM CaCl₂Mixing in a Tris-HCl aqueous solution with the pH value of 7.4 and the concentration of 50mM, incubating overnight at normal temperature to obtain an incubation product, and purifying to obtain an interferon macromolecule combination IFN-PMPC;

IFN-LPETGGH as described above₆Protein, Sortase A-H₆Protein, triglycine linked PMPC, CaCl₂In a molar ratio of 2: 1: 40: 200.

removing the transpeptidase A from the incubation product by a primary cation exchange column (HiTrapPicap SPcolumn, GE Healthcare, column size 5mL, flow rate 2mL/min, equilibration buffer 20mM Tris HCl, pH 7.0; elution buffer 20mM Tris HCl, 1M NaCl, pH 7.0), wherein the elution solution is a cation exchange product;

the cation-exchanged product was passed through a primary anion exchange column (HiTrap Capto Q column, GE Healthcare, column size 5mL, flow rate 2mL/min, equilibration buffer 20mM Tri s. HCl, pH 8.0; elution buffer 20mM Tris. HCl, 1M NaCl, pH 8.0) to remove unreacted IFN-LPETGH₆And PMPC, collecting the eluent as IFN-PMPC; purity > 95% and yield 0.9%. FIG. 12 is a diagram of the synthesis of IFN-PMPCs by the "shifting to" technique analyzed by GPC and SDS-PAGE, wherein, a diagram A is the "shifting to" technique analyzed by GPC; panel B shows SDS-PAGE analysis of ion exchange purified IFN-PMPC, from left to right: protein standard sample, mixture before reaction, mixture after 24h reaction, mixture after first cation exchange column purification, IFN-PMPC after second anion exchange column purification, and IFN-PMPC synthesized by using in-situ ATRP technology. The results show that the IFN-PMPC synthesized by the technology of 'grafting to' has low reaction yield and difficult purification, and the in-situ ATRP technology has the remarkable advantages of high yield, simple purification, easy amplification, application and the like.

Physicochemical characterization of interferon-polymer conjugate IFN-PMPC

The molecular weight and polydispersity index (PDI) of IFN-PMPC (using the conjugate IFN-PMPC prepared above) were determined by GPC using a Waters HPLC/GPC system coupled to a UV detector (Watt)ers 2489) and a differential refractive detector (Waters 2414). The chromatographic column is formed by connecting Asahipak GS-520HQ and GS-320HQ or GS-520HQ in series, the mobile phase is 50mM Tris & HCl buffer solution (pH 7.4), the detection condition is 25 ℃, and the flow rate is 0.5 mL/min. Standard curves generated from narrow distribution PEG standards of different molecular weights were used to calculate molecular weight and PDI. To accurately calculate the polymer P_MPCThe molecular weight of (a) is characterized by degrading the protein coupled with the in-situ polymer by protease. The method comprises the following specific steps: IFN-PMPC conjugate (1mg/mL) and proteinase K (0.5mg/mL) in 50mM Tris-HCl, 2mM CaCl₂Incubation at 45 ℃ for 12h, pH 7.4. FIG. 13 shows that IFN-PMPC can be degraded by proteinase K IFN, and PMPC molecular weight is accurately characterized by GPC, from left to right: protein standard sample, IFN-PMPC and proteinase K before reaction, IFN-PMPC and proteinase K after reaction overnight, proteinase K. By GPC analysis, the IFN-PMPC had a dispersion coefficient of 1.43 and a molecular weight of 57 kDa.

The hydration radius of IFN-PMPC was determined by Dynamic Light Scattering (DLS) method on a Malvern Zetasi zer Nano-zs 90. Samples were diluted in PBS buffer and filtered through a 0.22 μm pore filter before testing. DLS test shows that IFN-LPETGGH₆IFN-Br and IFN-alpha had hydration radii of 2.4nm,2.3nm and 2.3nm, whereas the synthetic IFN-PMPC was 9.7 nm. FIG. 14 shows the DLS analysis of IFN-PMPC.

Using nuclear magnetic resonance apparatus (¹H NMR) to characterize the chemical structure of the protein-macromolecule. IFN-PMPC samples were lyophilized, dissolved in D2O and analyzed on a JEOL ECX-400400 MHz NMR spectrometer. By passing¹The H NMR spectrum confirmed successful synthesis of PMPC on the protein molecule. FIG. 10 shows the passage of¹H NMR analysis of IFN-PMPC binding.

The secondary structure of IFN-PMPC was determined by circular dichroism analysis. The sample was diluted to 0.10mg/mL (1.0. mu.M) with an aqueous solution and analyzed by UV scanning using a Pistar π -180 (applied Photophysics, Inc.) over the wavelength range of 190-260 nm. FIG. 15 shows circular dichroism chromatography analysis of IFN-LPETGGH₆IFN-Br, IFN-alpha, IFN-PMPC. Through circular dichroism spectrum analysis, circular dichroism spectrums within the wavelength range of 190-260nm all show the same 209/219nm double peaksThe curves, which overlap well with the IFN-alpha curves, indicate that in situ macromolecule growth on protein has no significant effect on the secondary structure of protein molecules.

The protein concentration of IFN-PMPC was determined by the bicinchoninic acid method (BCA) using bovine serum albumin of known concentration as standard, following the protocol.

The detection results of the IFN-PMPC prepared in the three steps are not obviously different from those of the IFN-PMPC prepared in the two steps.

Fourth, preparation of control method

After adding a certain amount of MPC (MPC to IFN-alpha molar ratio of 1000:1) and 11O. mu.L of an aqueous solution of vitamin C (4Omg/mL) to 2.5mL of a PBS solution containing 40. mu.M IFN-alpha (solute as IFN-alpha and solvent as PBS solution with pH 7.4 and concentration of 10 mM), introducing nitrogen for 15min, 10. mu.L of a mixture of cuprous chloride and N, N, N' Pentamethyldiethylenetriamine (PMDTA) (concentration as 100mM and 300mM, respectively) was added to the mixture to store the mixture, reacting the mixture under sealed conditions for lh, and then exposing the mixture to air to terminate the reaction, thereby obtaining a reaction mixture.

Separating and purifying the reaction mixture by an anion exchange column (HiTrap Capto Q column, GE Healthcare) to obtain IFN-PMPC (the purification method is the same as the previous method), and collecting 8mL of column volume of IFN-PMPC (a control group) which is an interferon high-molecular conjugate;

IFN-PMPC (control) was detected by SDS-PAGE and determined by GPC, and had a molecular weight of 59.1 kDa.

The yield was calculated as (mass of IFN-PMPC conjugate/IFN-LPETGGH)₆Mass) of IFN-PMPC (control) was found to be 18.9% as shown in table 3.

TABLE 3

As can be seen, compared with the IFN-PMPC conjugate with the same molecular weight prepared by the third control method, the yield of the IFN-PMPC conjugate prepared by the second method is far higher than that of the third control method, and the method has good effect.

Example 2 functional verification of IFN-PMPC as interferon-Polymer conjugate

1. In vitro biological Activity of IFN-PMPC

In the present invention, the anti-cell proliferation activity of IFN-PMPC (IFN-PMPC prepared by the method of the second embodiment of the present invention, i.e., characterized by a molecular weight of 57kDa, hereinafter the same) was determined by MTT method. Human Burkitt's B lymphoma cells (Daudi B) were selected because of their high sensitivity to IFN-. alpha.2.

After Daudi B cells were cultured in RMPI-1640 containing 15% FBS, 50U/mL penicillin and 50. mu.g/mL streptomycin for a certain period of time, a cell suspension (50. mu.L/well, 7500 cells) was seeded in a 96-well plate at a certain concentration to mix IFN-LPETGGH₆IFN-Br, IFN-alpha, pirocin and purified IFN-PMPC conjugate samples were serially diluted and 50. mu.L each was added to a 96-well plate, negative control (no IFN-alpha 2) and blank control (medium only) were set at 37 ℃ with 5% CO₂Culturing for 72h, adding 20 μ L/well of MTT solution, measuring the absorbance of 490nm wavelength of each well with enzyme labeling instrument after 3h, and comparing the cell proliferation degree after different samples are treated.

FIG. 16 shows the in vitro biological activity of IFN-PMPC in MTT assay, and Table 4 shows IFN-. alpha.and IFN-LPETGGH₆In vitro biological Activity of IFN-Br, piroxin and IFN-PMPC, IC thereof₅₀10.79pg/mL, 10.78pg/mL, 11.18pg/mL, 175.00pg/mL, and 20.02pg/mL, respectively, with retention of activity of 100%, 100.09%, 96.50%, 6.17%, and 53.90%, respectively. In conclusion, the in vitro anti-tumor activity of IFN-PMPC is much higher than that of pirocin, which indicates that the in situ ATRP does not seriously reduce the activity of IFN, and provides a basis for the in vivo anti-tumor activity test.

TABLE 4

Sample (I)	IFN-α	IFN-LPETGGH6	IFN-Br	Paluoxin	IFN-PMPC
						IC50(pg/mL)	10.79	10.78	11.18	175.00	20.02
Relative Activity (%)	100	100.09	96.50	6.17	53.90

2. Pharmacokinetic testing of IFN-PMPC

All of the following animal experiments were performed under the guidelines of the various regulations of the university of Qinghua regarding animal experiments. In the invention, a Bal/c nude mouse model is used for injecting IFN-alpha, peroxin and IFN-PMPC through tail vein, the change condition of the interferon concentration in blood along with time is determined, and DAS software is used for data analysis. Prior to the drug treatment period, 9 female nude mice were randomly divided into 3 groups after a period of observation. Injecting IFN-PMPC, peroxin and unmodified IFN-alpha control substance into tail vein at 1mg/kg body weight, then taking 20 μ L of tail vein blood (blood collection tube soaked with heparin sodium (product of Wyowa Bomban Biochemical medicine Co., Ltd.) in advance and oven-dried) at set time point (1,5,15,30min,1,3,6,24,48,72 and 96h), standing at room temperature for 1h, centrifuging at 4 deg.C and 3000 Xg, collecting upper layer plasma, and storing in a low temperature refrigerator at-80 deg.C. IFN-. alpha.2 content in serum was determined using a human IFN-. alpha.2 ELISA kit (PBL interferon source) according to the instructions. Pharmacokinetic parameters for IFN-PMPC, peroxin and IFN- α were calculated using DAS 3.0 pharmacokinetic analysis software.

Table 5 and FIG. 17 show the pharmacokinetics of IFN-PMPC, pirocin and IFN- α in nude mice, respectively. IFN distribution half-life (t1/2 alpha) elimination half-life (t1/2 beta) is 0.42h and 1.58h respectively, and after 5 minutes of administration, the concentration of interferon in blood rapidly drops to below 50% of the initial dose, and after 24 hours, the residual concentration is less than 0.1% of the initial concentration. The IFN-PMPC concentration in vivo is gradually reduced, and the initial half-life and elimination half-life are respectively 2.04h and 51.62h, and respectively 6.2 times and 34.7 times of IFN-alpha. The area under the drug-time curve (AUC0- ∞) of IFN-PMPC was 194.1 times that of IFN-alpha. The results show that compared with unmodified IFN-alpha, the elimination half-life period and the in vivo average retention time of the IFN-PMPC combination are obviously prolonged, the curve area during drug administration is obviously increased, the clearance rate is obviously reduced, and the in vivo circulation half-life period is similar to that of peroxin.

TABLE 5

3. IFN-PMPC in vivo tissue distribution

In the present invention, the concentration of interferon remaining in the tumor after 2h, 24h and 4d administration was measured using nude mice transplanted with ovarian cancer cells. Human ovarian carcinoma cells (OVCAR-3) in RMPI-16 containing 10% FBS, 50U/mL penicillin and 50. mu.g/mL streptomycin40 for a period of time, trypsinized and stripped, washed with PBS, resuspended in RMPI-1640 medium without the above supplements and in an equal volume mixture with BD Matrigel Matrix, 0.2mL of a single cell suspension (5X 10)⁶Individual cell) is inoculated to the dorsal subcutaneous part of the left hind limb femur of a nude mouse, and 100-200mm is formed after 30 days of culture³Solid tumor masses of size. Nude mice were randomly divided into 3 groups, and IFN-PMPC, pirocin, IFN- α were injected into nude mice by tail vein injection at a dose of 10 μ g/20g body weight. Nude mice were sacrificed 2h, 24h and 4d after administration, and tissues and organs such as heart, kidney, liver, spleen, lung, pancreas, stomach, muscle, small intestine and tumor were collected. After disruption with extraction buffer (PBS containing 1mM EDTA, 0.5% Tri ton X-100, 0.5% sodium deoxycholate, 1mM PMSF, protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich) diluted 1: 100), the supernatant was centrifuged. The IFN concentration in the tissue was quantified by ELISA.

FIG. 18 is a graph of IFN-PMPC accumulation in various tissues, demonstrating that IFN-PMPC can be efficiently accumulated in heart, kidney, liver, spleen, lung, pancreas, stomach, muscle, small intestine, and tumor. Wherein, the A picture is the accumulation condition of IFN-PMPC, peroxin and IFN-alpha in the tumor, and it can be seen that, compared with the concentration of interferon in the IFN group tumor, the concentration of interferon in the IFN-PMPC group tumor is 12.9 times for 2h injection, 158.2 times for 24h injection, and IFN-PMPC still has 20% residue after 4d injection. The B picture shows the accumulation of IFN-PMPC, pyroxin and IFN-alpha in other tissues. The IFN-PMPC can effectively utilize and enhance permeation and retention effects, so that interferon is gathered in tumor tissues, and the half-life period of the interferon in blood circulation is prolonged, thereby improving the bioavailability and the anti-tumor efficacy of the interferon in vivo.

4. Testing of IFN-PMPC in vivo antitumor Activity in nude mouse model

The in vivo biological activity of IFN-PMPC was determined by animal transplantation tumor assay. According to the method, OVCAR-3 cells are inoculated to the subcutaneous back of the left hind limb femur of a nude mouse and cultured for 3 days to form solid tumor masses (20 mm)³) Thus establishing a nude mouse tumor model. 26 nude mice were divided into 4 groups, IFN-PMThe PC, the pyroxin and a reference substance (IFN-alpha is a positive control, and physiological saline is a negative control) are injected into the nude mice by tail vein injection, the administration dose is 20 mu g/20g of body weight, and the injection is performed once per week until all the nude mice of the physiological saline group, the IFN-alpha group and the pyroxin group die. The death of the nude mice in this experiment included natural death and euthanasia, which means that the tumor growth of the nude mice exceeded 500mm³Or weight loss of more than 15% by injection of barbiturate. The survival status and the tumor growth status of the nude mice were observed every week, and the changes of the nude mice body weight and the tumor volume with time were dynamically measured.

To test for toxicity of IFN-PMPC, mice were sacrificed after three administrations, and tumors, hearts, livers and kidneys were collected, fixed in 4% formaldehyde solution, sectioned and examined for histological morphology of organs by HE staining using standard methods. After the treatment is finished, blood is taken from eyeballs, and basic physiological indexes such as lactate dehydrogenase, creatine kinase isoenzyme, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, creatinine, urea nitrogen, red blood cells, white blood cells, platelets, hemoglobin and the like are measured.

The experimental results show (FIGS. 19 and 20) that the tumor volume of the mice in the saline group rapidly increased during the experiment, and the tumor volume of the mice exceeded 500mm on the 42 th day of injection³Median survival was only 35 days; during the experiment, the tumor volume of the IFN-alpha group of the nude mice gradually increases, and the tumor volume of the IFN-alpha injection day 49 of the nude mice exceeds 500mm³The median survival time is 38 days, and no obvious antitumor activity is embodied; during the experiment, the tumor volume of the nude mice in the peroxin group gradually increased, and the tumor volume of the nude mice in the IFN injection day 63 group also exceeded 500mm³The median survival time is 60 days, and the compound has certain anti-tumor activity; during the experiment, the tumor volume of the nude mice of IFN-PMPC group is not changed basically, meanwhile, 75% of the tumors of the mice disappear, 1 tumor of the mice does not grow any more, only 1 tumor of the mice slowly increases, and the tumor volume does not exceed 500mm until 93 days³. The data show that IFN-PMPC can effectively inhibit the growth of tumors, has very good in-vivo anti-tumor activity, and is even superior to the currently marketed medicine peroxin.

The results also showed that the mice in the IFN-PMPC group gained slightly weight during the experiment (FIG. 21), indicating that IFN-PMPC had no significant side effects in the mice.

FIG. 22 is a histological section of tumor, heart, liver and kidney of mice after 3 times of administration, and it can be seen that after IFN-PMPC injection, cavities appear in the tumor cell gaps of the mice, the morphology of cytoplasm and nucleus is not obvious, cells are necrotic, large cell membranes are shed, and the difference with the control group is obvious. Meanwhile, the cell morphology of the heart, the liver and the kidney is complete, no obvious cell necrosis exists, and no obvious difference exists between the cell morphology and the histological morphology of the control group. FIGS. 23 and 24 are the biochemical and blood indices of mice after the treatment was completed, respectively, and there was no significant difference in the basic physiological indices of lactate dehydrogenase, creatine kinase isoenzyme, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminase, creatinine, urea nitrogen, erythrocytes, leukocytes, platelets, hemoglobin, etc. of the IFN-PMPC group compared with the saline group, the IFN- α group, and the Paroxin group. The above data indicate that IFN-PMPC does not cause significant toxicity to the organs in the nude mice.

The method has the advantages of high yield of the in-situ ATRP synthetic technology, simple purification and easy industrialization, and the prepared interferon-P^MPCThe combination has higher biological activity preservation rate, obviously prolonged half-life period and effective tumor inhibition effect, and shows more excellent effect compared with the in-vivo and in-vitro activity of the marketed medicine peroxin. ATRP synthetic technology is expected to replace PEG to be converted into a new method for modifying protein drugs so as to improve drug stability, improve pharmacokinetics and enhance treatment efficacy.

Claims (3)

1. A method for preparing a protein-polymer conjugate, comprising the steps of:

combining protein-initiator combination IFN-Br, polymer monomer MPC, CuCl and CuCl₂Carrying out polymerization reaction on 1,1,4,7,10, 10-hexamethyl triethylene tetramine in a buffer solution to obtain a protein-polymer combination IFN-PMPC;

the protein-initiator combination IFN-Br is catalyzed by transpeptidase A in IFN-LPETGGH₆Introducing ATRP initiator AEBM to the C-terminal of (1);

the structural formula of the AEBM is shown as the following formula 1:

the protein-initiator conjugate IFN-Br, the polymeric monomer MPC, the CuCl₂And the molar ratio of the 1,1,4,7,10, 10-hexamethyltriethylene tetramine is 1: 2000: 25: 75: 125;

the PMPC is a high molecular compound obtained by polymerizing a high molecular monomer MPC;

the IFN is interferon selected from interferon alpha, interferon beta, interferon gamma or interferon lambda.

2. The method of claim 1, wherein:

the protein IFN-LPETGGH₆The amino acid sequence of (A) is sequence 2 in the sequence table.

3. The method according to claim 1 or 2, characterized in that:

the polymerization reaction is carried out under the atmosphere of low oxygen or inert gas;

the time of the polymerization is 5 minutes to 24 hours, and the temperature of the polymerization is 0 to 80 ℃.

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