CN107226858A - Interferon macromolecule combination IFN-PMPC preparation and its application - Google Patents

Abstract

The present invention discloses a kind of interferon macromolecule combination IFN PMPC preparation and its application.The invention provides a kind of method for preparing protein high molecular combination, comprise the following steps：By protein initiator combination, 2 methylacryoyloxyethyl phosphocholines, CuCl, CuCl₂, 1, Isosorbide-5-Nitrae, 7,10,10 hexamethyl triethylene tetramines carry out polymerisation in buffer solution, obtain protein high molecular combination；The protein initiator combination is that initiator and albumen are covalently attached obtained product.It is demonstrated experimentally that with the IFN PMPC combinations prepared by the present invention, yield is high, purifying is simple, site-specific sex modification, in the absence of immunogenicity, Bioactivity can be preferably remained, pharmacokinetics and bio distribution is significantly improved, and effectively strengthens therapeutic efficiency.

Description

Interferon macromolecule combination IFN-PMPC preparation and its application

Technical field

The invention belongs to biomedicine field, and in particular to prepare interferon macromolecule combination IFN-PMPC preparation and It is applied.

Background technology

Protein has been widely used for the multiple fields such as biological medicine development, targeted therapy and clinical diagnosis.Individually There are problems that half-life short, stability using protein.Protein is connected with macromolecule and prepares protein-high score Sub- combination, can effectively improve dissolubility, stability, pharmacokinetics and the therapeutic efficiency of protein and reduce Its immunogenicity.The synthetic method of traditional protein-macromolecule combination is by macromolecule and albumen well prepared in advance Matter is connected, often exist that conjugation sites are uncertain, efficiency is low, yield is poor, product is difficult to separate, quality control is poor, Activity such as is difficult to keep at many problems.

Interferon-' alpha ' 2 (IFN-α 2) is the potent inhibitor of virus replication and growth of tumour cell, has been employed successfully in Treat the disease such as virus hepatitis and cancer.But, IFN is very short through systemic injection administration Posterior circle half-life period, needs Want frequent drug administration and can be only achieved expected curative effect in higher concentrations, so as to cause some toxic side effects, while to patient Bring heavy financial burden.IFN is modified with polyethylene glycol (PEG), is to improve its pharmacokinetics and improve its to treat The effective measures of effect, are referred to as long-acting interferon.However, current PEG-IFN exist as reaction yield it is low, The drawbacks such as binding site and conjugation chemistry metering are difficult to control to, the serious reduction of bioactivity.It is immunized in addition, PEG is present After originality, multiple dosing, PEG antibody can be produced, accelerates its removing in vivo, changes the distribution of medicine in vivo And metabolism.Therefore, research and development reaction condition is gentle, step is simple, the site-specific sex modification side without immunogenicity Method is particularly important to interferon and other medical proteins.

The content of the invention

It is an object of the present invention to provide a kind of method for preparing protein-macromolecule combination.

The method that the present invention is provided, be it is following 1) or 2)：

1) method shown in comprises the following steps：

By protein-initiator combination, high polymer monomer, CuCl, CuCl₂, 1,1,4,7,10,10- hexamethyls Triethylene tetramine carries out polymerisation in buffer solution, obtains protein-macromolecule combination；

The protein-initiator combination is that initiator and albumen are covalently attached obtained product；

Buffer solution in shown method used in polymerisation is that pH value is the PBS aqueous solution that 7.4, concentration is 10mM, Specific formula is：2.684g Na₂HPO₄·12H₂O、0.34g NaH₂PO₄·2H₂O, 8.19g NaCl are dissolved in 1L water Obtained solution.

2) method shown in is to connect high-molecular compound and albumen by oligomerization glycine in the presence of initiator Connect, obtain protein-macromolecule combination；

The high-molecular compound is to be obtained by high polymer monomer polymerization.

In above two method,

The albumen is interferon, and the interferon is selected from interferon-' alpha ' or its fusion protein, interferon beta or its fusion egg In vain, interferon gamma or its fusion protein, interferon lambda or its fusion protein；

Or the albumen is specially the interferon alpha fusion protein in sequence table shown in sequence 2.

The high polymer monomer is 2- methylacryoyloxyethyl phosphocholines；

The high-molecular compound is poly- 2- methylacryoyloxyethyls phosphocholine.

The initiator is the initiator of atom transition free radical polymerization reaction, the fracture chain transfer polymerization of reversible addition one The initiator of initiator, the initiator of ring opening metathesis polymerization or ring opening polyaddition；

The structural formula of the initiator of the atom transition free radical polymerization reaction such as following formula 1：

Wherein, R is H or (CH₂)_nCH₃, n is >=1 integer；X is Cl or Br or I；M is >=1 integer；N is >=1 integer；

The atom transfer radical polymerization initiator is specially 2 bromo 2 methyl propionic acid 2- (2- (2- (2- amino second Amide groups) acetamido) acetamido) carbethoxy hydrochloride；

The initiator is covalently attached to the C-terminal or N-terminal of the albumen；

1) in the method shown in, the initiator is covalently attached to the C-terminal or N-terminal of the albumen；

Or the initiator is specifically covalently attached to the C-terminal of the albumen；

In the above method, 1) in the method shown in, the protein-initiator combination, the 2- metering systems Acyloxyethyl phosphocholine, the CuCl, the CuCl₂With the 1,1,4,7,10,10- hexamethyls triethylene four The mol ratio of amine is 1：200-10000：10-500：10-2000：10-4000；

The protein-initiator combination, the 2- methylacryoyloxyethyls phosphocholine, the CuCl, The CuCl₂Mol ratio with the 1,1,4,7,10,10- hexamethyl triethylene tetramines is specially 1：1000-4000： 10-500：10-2000：10-4000；

The coupling ratio of albumen and the initiator described in the protein-initiator combination is 1：1.

In the above method, the protein-initiator combination, the 2- methylacryoyloxyethyls phosphocholine, The CuCl, the CuCl₂Mol ratio with the 1,1,4,7,10,10- hexamethyl triethylene tetramines is 1：2000： 25：75：125.

1) in the method shown in, above-mentioned protein-initiator combination is C-terminal of the initiator in albumen, the two Combination is formed by forming amido link, is specifically prepared according to the method comprised the following steps：By albumen (IFN-LPETGGH₆Albumen), transpeptidase (Sortase A-H₆Albumen), 2- (2- (2- (2- glycyls amido) Acetamido) acetamido) ethyl 2 bromo 2 methyl propionic acid ester, CaCl₂It is 50mM in the concentration of pH 7.4 Mixed in the TrisHCl aqueous solution, reaction obtains interferon-initiator combination IFN-Br；It is above-mentioned IFN-LPETGGH₆Albumen, Sortase A-H₆Albumen, 2- (2- (2- (2- glycyls amido) acetamido) acetyl Amido) ethyl 2 bromo 2 methyl propionic acid ester, CaCl₂Mol ratio be 2：1：50：200.

It is above-mentioned 2) shown in method, comprise the following steps：The initiator and the high polymer monomer are occurred poly- Reaction is closed, obtains connecting the high molecular polymer of oligomerization glycine；Again by the macromolecule of the connection oligomerization glycine Polymer is covalently attached with the albumen, obtains protein-macromolecule combination.

It is above-mentioned 2) shown in method, the polymerisation be by initiator, 2- methylacryoyloxyethyls phosphocholine, CuCl、CuCl₂With 1, Isosorbide-5-Nitrae, 7,10,10- hexamethyl triethylene tetramines carry out polymerisation in buffer solution, are connected Connect the high molecular polymer of oligomerization glycine；The buffer solution that the polymerisation is used is that 7.4, concentration is for pH value The 10mM PBS aqueous solution；

It is described be covalently linked to by the protein, transpeptidase, the high molecular polymer of the connection oligomerization glycine, And CaCl₂Reacted in buffer solution, obtain protein-macromolecule combination；In the reaction of the covalent attachment Buffer solution is that the concentration of pH 7.4 is the 50mM TrisHCl aqueous solution.

It is above-mentioned 2) shown in method, in the polymerisation, the initiator, the 2- methacryloxypropyls second Base phosphocholine, the CuCl, the CuCl₂With rubbing for the 1,1,4,7,10,10- hexamethyl triethylene tetramines You are than being 1：200-10000：10-500：10-2000：10-4000；

Or the initiator, the 2- methylacryoyloxyethyls phosphocholine, the CuCl, the CuCl₂ Mol ratio with the 1,1,4,7,10,10- hexamethyl triethylene tetramines is specially 1：2000：25：75：125；

In the covalent attachment, the protein (IFN-LPETGGH₆Albumen), the transpeptidase (Sortase A-H₆ Albumen), it is described connection oligomerization glycine high molecular polymer (connection triglycine poly- 2- methacryloxypropyls Ethylphosphocholine) and CaCl₂Mol ratio be 2：1：40：200.

The buffer solution is that the concentration of pH 7.4 is the 50mM TrisHCl aqueous solution；

In the above method, the polymerisation is carried out under hypoxemia or atmosphere of inert gases；

The time of the polymerisation is 5 minutes to 24 hours, and the temperature of the polymerisation is 0-80 DEG C.

The protein prepared by the above method-macromolecule combination is also the scope of protection of the invention.

Above-mentioned protein-application of the macromolecule combination in antitumor or anti-virus product is prepared is also the present invention The scope of protection.

Another object of the present invention is to provide a kind of antitumor or anti-virus product.

The product that the present invention is provided, it includes above-mentioned protein-macromolecule combination.

The present invention is modified simultaneously by using biochemistry and macromolecular chemical technology in the C- ends away from interferon PMPC (poly- 2- metering systems are gone out by ATRP (ATRP) technology optimized efficient growth in situ Acyloxyethyl phosphocholine), produce site-specific and mono-modified interferon-macromolecule combination (i.e. IFN-PMPC). This ATRP technologies reaction condition in situ is gentle, yield is high, purification step is simple, cost is low, can effectively keep raw Thing activity simultaneously improves vitro stability, and a kind of general method is provided for protein modification.

Brief description of the drawings

Fig. 1 is the schematic diagram of interferon-macromolecule combination IFN-PMPC synthetic routes.

Fig. 2 is to obtain IFN-LPETGGH by affinity chromatography₆。

Fig. 3 is to obtain Sortase A-H by affinity chromatography₆。

Synthesis and purge process of the Fig. 4 for original position ATRP initiator As EBM.

Fig. 5 is that IFN-Br is synthesized and purge process analysis.

Fig. 6 is that MALDI-TOF analyzes IFN-LPETGGH₆With IFN-Br molecular weight.

Fig. 7 is the special sex modification situation that LC-MS/ESI analyzes IFN-Br.

Fig. 8 is that IFN-PMPC is synthesized and purge process.

Fig. 9 is gpc analysis IFN-PMPC.

Figure 10 passes through to show¹H NMR analyze IFN-PMPC combinations.

Figure 11 synthesizes IFN-PMPC check experiment for analysis original position ATRP.

Figure 12 is by the way that " grafting to " technologies obtain IFN-PMPC

Figure 13 is that SDS-PAGE analyzing proteins enzymes K handles IFN-PMPC.

Figure 14 is the hydration radius that DLS analyzes IFN-PMPC and IFN-α.

Figure 15 is the secondary structure that CD analyzes IFN-PMPC and IFN-α.

Figure 16 is the Bioactivity that mtt assay determines IFN-PMPC, PEG-IFN alpha-2a and IFN-α.

Figure 17 is the medicine that concubine's model analysis IFN-PMPC, PEG-IFN alpha-2a and IFN-α are eliminated using chamber in DAS softwares Thing dynamic metabolism situation.

Figure 18 is the distribution situation of IFN-PMPC, PEG-IFN alpha-2a and IFN-α in tumour and its hetero-organization.

Figure 19 is that IFN-PMPC, PEG-IFN alpha-2a, IFN-α and physiological saline suppress tumour growth situation.

Figure 20 is that nude mice injects the survivorship curve after IFN-PMPC, PEG-IFN alpha-2a, IFN-α and physiological saline.

Figure 21 is that nude mice injects the changes of weight situation after IFN-PMPC, PEG-IFN alpha-2a, IFN-α and physiological saline.

Figure 22 is tumour, heart, liver and kidney HE staining conditions after nude mice treatment end.

Figure 23 swashs for lactic dehydrogenase, creatine after nude mice injection IFN-PMPC, PEG-IFN alpha-2a, IFN-α and physiological saline The physiological indexes situation such as enzyme isoenzyme, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, creatinine and urea nitrogen.

Figure 24 is red blood cell, leucocyte, blood red egg after nude mice IFN-PMPC, PEG-IFN alpha-2a, IFN-α and physiological saline The physiological indexes situations such as white and blood platelet.

Embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.

Plasmid pET-25b (+) in following embodiments is Sangon Biotech (Shanghai) Co., Ltd.) limited company's product.

TB culture mediums in following embodiments are configured as follows：Peptone 12g, ferment are added into 900mL water 121 DEG C of autoclaving 15min after female extract 24g and glycerine 4mL, fully dissolving, the mixed liquor after sterilizing is cooled to 60 DEG C, then add 100mL sterilizings contains 170mmol/LKH₂PO₄With 0.72mol/L K₂HPO₄The aqueous solution.

People Burkitt ' s B lymphoma cells and Proliferation of Human Ovarian Cell (OVCAR-3) in following embodiments are purchased from Chinese Academy of Sciences's tumour cell storehouse.

RMPI-1640 culture mediums in following embodiments are Gibco Products.

Female athymic (Nude) nude mice in following embodiments is Beijing Vital River Experimental Animals Technology Co., Ltd. Product.Female athymic (Nude) nude mice hereinafter abbreviation nude mice.

Flow in following embodiments is as shown in Figure 1.

In following embodiments, Matrix-assisted laser desorption ionization (MALDI-TOF), enzyme mark are utilized The analysis means such as instrument, dynamic light scattering (DLS), circular dichroism spectra (CD) characterize IFN-PMPC and IFN molecule Amount, phase transition temperature, the hydration physical and chemical performance such as radius and secondary structure.From people's Burkitt ' s B lymthomas Cell, tests the ability of IFN-PMPC and IFN Bioactivity, i.e. its extracorporeal anti-tumor cell propagation；Make With nude mice model, the pharmacokinetics of test IFN-PMPC and IFN in vivo utilizes the medicines of DAS 3.0 Pharmacokinetic analysis software calculates pharmacokinetic parameter；Nude mouse tumor model is set up, is tested IFN-PMPC and IFN antitumous effect and drug distribution.

Quantitative experiment in following embodiments, is respectively provided with three repetitions, results averaged unless otherwise specified.

Embodiment 1, the method for preparing interferon macromolecule combination IFN-PMPC

First, interferon-initiator combination is prepared

1st, IFN-LPETGGH is prepared₆Fusion protein and transpeptidase Sortase A-H₆

1) preparation of recombinant bacterium

IFN-LPETGGH₆The amino acid sequence of fusion protein is sequence 2, the nucleotides sequence of its encoding gene in sequence table It is classified as sequence 1 in sequence table；

Transpeptidase Sortase A-H₆Amino acid sequence be sequence table in sequence 4, the nucleotide sequence of its encoding gene For sequence in sequence table 3；

Express IFN-LPETGGH₆The recombinant vector of fusion protein is by the IFN-LPETGGH shown in sequence 1₆Fusion protein The carrier obtained in Nde I and Eco the RI restriction enzyme sites of encoding gene insertion pET-25b (+) carrier, is named as pET-25b-IFN-LPETGGH₆；

Express Sortase A-H₆Recombinant vector be by the Sortase A-H shown in sequence 3₆Protein coding gene is inserted The carrier obtained in Nde I and Eco the RI restriction enzyme sites of pET-25b (+) carrier, is named as pET-25b-Sortase A-H₆。

Express IFN-LPETGGH₆The recombinant bacterium of fusion protein is that will express IFN-LPETGGH₆The recombinant vector of fusion protein Import in E.coli BL21Rosetta-gami (DE3) pLysS (Invitrogen) and obtain recombinant bacterium, be named as BL21/pET-25b-IFN-LPETGGH₆；

Express Sortase A-H₆Recombinant bacterium be will expression Sortase A-H₆Recombinant vector import E.coli BL21 (DE3) recombinant bacterium is obtained in pLysS (Novagen), is named as BL21/pET-25b-Sortase A-H₆。

2)IFN-LPETGGH₆The expression and purifying of albumen

By recombinant bacterium BL21/pET-25b-IFN-LPETGGH₆(contain 100 μ g/mL ammonia in 50mL TB culture mediums Parasiticin), 37 DEG C, concussion and cultivate is stayed overnight under the conditions of 250rpm.Transfer again within second day into the fresh TB cultures of 1L (it is contained among base in 2L shaking flask, ampicillin concentration is 100 μ g/mL) and carries out large-scale culture and induce Expression.Comprise the following steps that：First at 37 DEG C, concussion and cultivate 5h under the conditions of 200rpm, then by cultivation temperature 18 DEG C are set to, isopropyl-β-D-thiogalactoside (IPTG) is added, final concentration of 0.4mM cultivates 16h, Collect nutrient solution.Thalline is collected with 3000 × g centrifugal forces, culture supernatants is removed, obtains recombinant bacterium BL21/pET-25b-IFN-LPETGGH₆Thalline.

BL21/pET-25b-IFN-LPETGGH is resuspended with the ice-cold PBS of 30mL₆Thalline, then with Ultrasound Instrument in 4 DEG C of conditions Lower smudge cells, then centrifuges Escherichia coli breakdown products 15 minutes under 4 DEG C, 14000 × g centrifugal force. 2mL polyethyleneimines (PEI, 10%) are added in the supernatant of collection, are centrifuged 15 minutes again.Obtained supernatant After 0.45 μm of membrane filtration, pass through on AKTA protein purification systems (AKTA Purifier 10, GE) Nickel affinity chromatography post (HisTrap HP 5mL) purify, level pad be 10mM PBS, 500mM NaCl, 5% glycerine, elution buffer is that level pad adds the mixed liquor that 500mM imidazoles is obtained.Collect that eluting peak is corresponding washes De- liquid, and detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), stay about 21.1kDa's The corresponding eluent of target product, is IFN-LPETGGH₆Albumen.SDS-PAGE analyzes sample by containing 5% β-mercapto The Laemmli sample buffers of base ethanol are prepared, and concentration is 10 μ L at 1mg/mL, 95 DEG C after heating 5min Sample is loaded into prefabricated 10%SDS-PAGE gels, and 90min (electricity is run under vertical electrophoresis 80~100V voltages Swimming liquid be：25mM Tris, 250mM Glycine, 0.1%SDS).Gel is handled with Coomassie brilliant blue G-250 dyeing After observe pillar location.

IFN-LPETGGH obtained by elution₆Albumen removes imidazoles through desalting column (HiPrep 26/10Desalting), Displacement is into 50mM TrisHCl buffer solutions simultaneously.Purification of samples tests purity with SDS-PAGE, and uses light splitting light Degree meter method (NanoDrop 2000) determines the concentration of albumen.

As a result as shown in Fig. 2 A is logical nickel affinity chromatography purifying, UV monitoring linear elutions obtain target product IFN-LPETGGH₆；B is that SDS-PAGE analyzes IFN-LPETGGH₆Purifying situation；Show IFN-LPETGGH₆ Protein expression purifies situation with nickel affinity chromatography post.Cell before standard protein sample, loading is followed successively by from left to right to crack Liquid, nickel post flow through liquid, 50mM imidazoles and wash post and 500mM imidazoles elution destination protein, it can be seen that pass through Escherichia coli are expressed, nickel affinity chromatography post purifying acquisition purity ＞ 95% IFN-LPETGGH₆Albumen, yield Reach that 200mg/L cultivates bacterium solution, it can be seen that obtain about 21.1kDa target product.

3)Sortase A-H₆The expression and purifying of albumen

Using same method processing recombinant bacterium BL21/pET-25b-Sortase A-H₆, collect the corresponding elution of eluting peak Liquid, and detected with SDS-PAGE.As a result as shown in figure 3, A is logical nickel affinity chromatography purifying, UV monitorings are linear Afford target product Sortase A-H₆；B is SDS-PAGE analysis Sortase A-H₆Purifying situation；Display Sortase A-H₆Protein expression purifies situation with nickel affinity chromatography post.Be followed successively by from left to right standard protein sample, on Cell pyrolysis liquid, nickel post flow through liquid, 50mM imidazoles and wash post and 500mM imidazoles elution destination protein before sample, can To find out the target product for staying about 22.9kDa.

2nd, ATRP initiators are synthesized

2- Hydroxy-ethylamino formic acid tert-butyl alcohol esters (2.0g, 12.5mmol), N, N- diisopropyl ethyl amines (2.4 ML, 14mmol, 1.1 equivalent) dichloromethane (20ml) is dissolved in ice-water bath.Under less than 0 degrees celsius The bromo- 2- methyl propionyl bromides (1.25ml, 10mmol) of 2-, 15 minutes completion of dropping is added dropwise.After 30 minutes, remove Ice-water bath, and be kept stirring for 16 hours.Revolving removes solvent, and product purifies (dichloromethane with silicagel column：Acetic acid second Ester=1:1).Product is yellow oily liquid, 2 bromo 2 methyl propionic acid 2- (2- (tertbutyloxycarbonyl) amido) ethyl ester (C11H20BrNO4,2.9g, 74.7%)¹HNMR(400MHz,CDCl₃):δ4.827(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 bromo 2 methyl propionic acid 2- (2- (tertbutyloxycarbonyl) amido) ethyl ester (2.5g) is dissolved in 6M- hydrogen chloride second Acetate solution 50mL, is stirred 2 hours.After reaction terminates, filtering, product is white powder, the bromo- 2- methyl of 2- Propionic acid 2- amino ethyl ester hydrochlorides (C₆H₁₃BrClNO₂, 1.95g, 98%) and .ESI-mass m/z:210.3([M– Cl+Na]⁺),212.3([M–Cl+Na]⁺).

2- (2- (2- tertbutyloxycarbonyls) acetamido) acetamido) acetic acid (289mg, 1mmol), the bromo- 2- of 2- Methylpropanoic acid 2- amino ethyl esters hydrochloride (246mg, 1mmol), EDC (288mg, 1.5mmol), and N, N- Diisopropyl ethyl amine (175 μ l, 1mmol) is dissolved in chloromethanes (10ml), is stirred at room temperature 16 hours.Reaction knot Shu Hou, removes solvent, and residual white solid is once with 4 milliliters of water washings 2 times, 1 milliliter of ethanol washing 3 times, so Washed afterwards with 4 milliliters of ethyl acetate, obtain white powder product, 2 bromo 2 methyl propionic acid 2- (2- (2- (2- Tertbutyloxycarbonyl) acetamido) acetamido) acetamido) ethyl ester (C₁₅H₂₆BrN₃O₆, 269mg, 56%) and¹HNMR (400MHz,CDCl₃):δ4.261(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 bromo 2 methyl propionic acid 2- (2- (2- (2- tertbutyloxycarbonyls) acetamido) acetamido) acetamido) Ethyl ester (1.08g, 2.25mmol) is dissolved in 6M Hydrochloride/ethyl acetate 50ml, stirs 2 hours.Production Thing is white powder, 2 bromo 2 methyl propionic acid 2- (2- (2- (2- glycyls amido) acetamido) acetamides Base) carbethoxy hydrochloride, i.e. ATRP initiator As EBM (C₁₂H₂₂BrClN₄O₅, chemical structural formula as shown in Equation 1,0.84 G, 98%)¹HNMR(400MHz,D₂O):δ4.196(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 ATRP initiator As EBM in situ synthesis and purge process.Finally successfully obtain purity ＞ 95% Initiator A EBM, yield is 40.2%.

3rd, interferon-initiator combination is obtained by transpeptidase A enzymatics

By transpeptidase A enzymatics in IFN-LPETGGH₆C- ends introduce ATRP initiator A EBM, specifically such as Under：The above-mentioned IFN-LPETGGH that above-mentioned 1 is prepared₆Albumen, Sortase A-H₆Albumen, it is above-mentioned 2 prepare AEBM, CaCl₂Mixed in the concentration of pH 7.4 is the 50mM TrisHCl aqueous solution, reaction obtains interferon-initiator Combination IFN-α；

Above-mentioned IFN-LPETGGH₆Albumen, Sortase A-H₆Albumen, above-mentioned 2 AEBM, CaCl prepared₂Mole Than for 2：1：50：200.

Specific method is as follows：Contain IFN-LPETGGH in 10mL₆The 50mM TrisHCl of (200 μM) are molten (solute is IFN-LPETGGH to liquid₆, solvent is that pH value is that 7.4, concentration is the 50mM TrisHCl aqueous solution) Middle addition 5mM AEBM, contain 100 μM of transpeptidase A and 20mM CaCl with 10mL₂TrisHCl it is molten Liquid is mixed, and room temperature reaction is stayed overnight, and obtains reactant mixture.

Reactant mixture is purified with anion-exchange chromatography (HiTrap Capto Q 5mL), IFN-Br is obtained； The level pad used is purified for 20mM TrisHCl, pH 7.5；Elution buffer is to contain 1M NaCl The 20mM TrisHCl aqueous solution, pH 7.5；Pillar size is 5mL, and flow velocity is 2mL/min；Collection is washed The de- corresponding eluent in peak, and detected with SDS-PAGE, the about 20.5kDa corresponding eluent of target product is stayed, For interferon-initiator combination IFN-Br, and small molecular weight impurity is further removed through desalting column with PBS.This is anti- Answer efficiency ＞ 95%.

Interferon-initiator combination IFN-Br checking analysis is subjected to SDS-PAGE, as a result such as Fig. 5 institutes Show, it is shown that IFN-Br is synthesized and purge process analysis, wherein, A：AEX separation transpeptidase A catalysis IFN-LPETGGH₆The mixture reacted with AEBM, UV monitoring linear elutions obtain target product IFN-Br；B： SDS-PAGE analyzes IFN-LPETGGH₆IFN-Br result is obtained before and after being reacted with AEBM.From left to right successively For standard protein sample, SrtA-H₆、IFN-LPETGGH₆, reactant mixture, elution protein I FN-Br, flow through sample SrtA-H₆。

Purified product IFN-Br molecular weight MALDI _ TOFMS (MALDI-TOF) determine, instrument is 4800PlusMALDI-TOF/TOF^TMAnalyzer (AB SCIEX). Checking IFN-Br amino acid sequence and C- is analyzed with High Performance Liquid Chromatography-Electrospray Ionization Tandem Mass method (LC-MS/ESI) The special sex modification of end, instrument is Q-Exactive LC-MSs mass spectrograph (Thermo Scientific). Fig. 6 is that MALDI-TOF analyzes IFN-LPTEGGH₆With IFN-Br molecular weight, respectively 21105.8 and 20531.9, Meet with theoretical value 21105.9 and 20532.0.Fig. 7 is IFN-Br C- terminal peptide fragments EGSGGGGSLPETGGG (- Br) IP situation, A figures are LC-MS/ESI experiment values；B figures are theoretical expectation values, theoretical value with Experiment value coincide.Table 1 shows peptide fragment amino acid sequence after the IFN-Br trypsase decomposition that LC-MS/ESI is analyzed Row composition.Fig. 6, Fig. 7 and table 1 show being capable of site-directed quantitative acquisition interferon-initiator knot using transpeptidase A Fit IFN-Br, initiator pointed decoration is in the C- ends of IFN-α, interferon-initiator combination IFN-Br The coupling ratio of middle albumen and initiator is 1:1.

Table 1

2nd, interferon macromolecule combination IFN-PMPC preparation and sign

1st, interferon macromolecule combination IFN-PMPC preparation

Using ATRP technologies in above-mentioned interferon-initiator combination IFN-Br surface in situ synthesis PMPC, method Comprise the following steps：By interferon-initiator combination IFN-Br, MPC, CuCl, CuCl₂、1,1,4,7,10,10- Hexamethyl triethylene tetramine (HMTETA) and pH value are the PBS aqueous solution (the PBS formulas that 7.4, concentration is 10mM For：2.684g Na₂HPO4·12H₂O、0.34g NaH₂PO4·2H₂O, 8.19g NaCl are dissolved in 1L water), close Reacted under the conditions of closing, obtain interferon macromolecule combination IFN-PMPC；

Interferon-initiator combination IFN-Br, MPC, CuCl, CuCl₂, the second of 1,1,4,7,10,10- hexamethyls three The mol ratio of alkene tetramine (HMTETA) is 1：2000：25：75：150；

It is specific as follows：

Contain 40 μM of IFN-Br in 2.5mL PBS solution (solute is IFN-Br, solvent be pH value be 7.4, Concentration is 10mM PBS solution) the middle MPC for adding 100 μm of ol, leads to after nitrogen 15min, adds molten in advance Solution is in 1mL distilled waters and except the CuCl (2.5 μm of ol), CuCl of air₂(7.5 μm of ol) and 1, Isosorbide-5-Nitrae, 7,10,10- hexamethyl triethylene tetramines (HMTETA) (12.5 μm of ol) react 1h under confined conditions, Then terminating reaction in atmosphere is exposed, reactant mixture is obtained.

Reactant mixture is through anion-exchange column (HiTrap Capto Q column, GE Healthcare) point From IFN-PMPC is purified into, the purification step is same as described above：The level pad used is purified for 20mM TrisHCl, pH 7.5；Elution buffer is 20mM TrisHCl, 1M NaCl, pH 7.5, pillar size For 5mL, flow velocity is 2mL/min；The corresponding eluent of eluting peak is collected, and is detected with SDS-PAGE, about 60kDa is stayed The corresponding eluent of target product, be IFN-PMPC.

It is (quality/IFN-LPETGGH of IFN-PMPC combinations after purification to calculate yield formula₆Quality) * 100%, As a result such as table 2, IFN-PMPC yield is respectively 67.0%.

Table 2

Fig. 8 shows IFN-PMPC synthesis and purge process, wherein, A is that AEX separation original position ATRP is anti- The mixture answered, UV monitoring linear elutions obtain target product IFN-PMPC；B is that SDS-PAGE analyzes IFN-Br ATRP in situ grows macromolecule PMPC, is followed successively by from left to right：Standard specimen, IFN-LPETGGH₆, it is IFN-Br, anti- Answer mixture, IFN-PMPC after purification.Fig. 9 is gpc analysis IFN-PMPC combinations.Figure 10 passes through¹H NMR Analyze IFN-PMPC combinations.

In order to verify that PMPC is to be synthesized using the initiator on IFN-Br in C- ends, with three without initiator Glycine, which is connected on IFN, obtains IFN-α, has then carried out check experiment with above-mentioned identical condition.Figure 11 Show that analysis ATRP synthesizes IFN-PMPC check experiment, wherein, A：Gpc analysis IFN-α is synthesized IFN-PMPC check experiment.B：SDS-PAGE is analyzed, and standard protein sample, IFN-α are followed successively by from left to right and is entered Row ATRP reacts and IFN-α.Check experiment use with IFN-Br identicals reaction condition IFN-α is used for into Row ATRP reacts, and display IFN-α does not grow PMPC, illustrates that the polymerisation is only being connected to ATRP The IFN-Br of initiator C- ends are carried out, and in the absence of the side reaction on albumen other reactive groups.

3rd, interferon macromolecule combination IFN-PMPC another preparation method

In order to show the superiority of ATRP technologies in situ, " grafting to " technologies synthesis IFN-PMPC is utilized. Grafting to technologies include two steps：

1st, the poly- 2- methylacryoyloxyethyls phosphocholine PMPC of synthesis connection triglycine

According to 1 preparation process in above-mentioned two, by initiator A EBM, MPC, CuCl, CuCl₂、 1,1,4,7,10,10- hexamethyl triethylene tetramines (HMTETA) and pH value are the PBS water that 7.4, concentration is 10mM Solution, reacts in confined conditions, obtains connecting the PMPC of triglycine；Wherein, initiator A EBM, MPC, CuCl、CuCl₂, 1,1,4,7,10,10- hexamethyl triethylene tetramines (HMTETA) mol ratio be 1：2000：25： 75：125；

Above-mentioned reaction condition is 1 identical with above-mentioned two.

The impurity such as small molecule are removed using hyperfiltration process, the molecular weight for the PMPC for connecting triglycine is detected with GPC, About 60kDa.

2nd, PMPC is combined with IFN obtains interferon macromolecule combination IFN-PMPC

By transpeptidase A catalysis in IFN-LPETGGH₆C- ends introduce connection triglycine PMPC, specifically It is as follows：25 μM of IFN-LPETGGH that above-mentioned two are prepared₆Albumen, 12.5 μM of Sortase A-H₆Albumen, 500 μM PMPC the and 10mM CaCl of the above-mentioned 1 connection triglycine prepared₂, it is 50mM TrisHCl in the concentration of pH 7.4 Mixed in the aqueous solution, ambient temperature overnight is incubated, obtain incubation product and obtain interferon macromolecule combination by purifying IFN-PMPC；

Above-mentioned IFN-LPETGGH₆Albumen, Sortase A-H₆Albumen, PMPC, the CaCl for connecting triglycine₂Rub You are than being 2：1：40：200.

Above-mentioned incubation product through cation exchange column (HiTrapMacrocap SPcolumn, GE Healthcare, Pillar size is 5mL, and flow velocity is 2mL/min, and level pad is 20mM TrisHCl, pH 7.0；Elution Buffer solution is 20mM TrisHCl, 1M NaCl, pH 7.0) transpeptidase A is removed, eluent is handed over for cation Change product；

By cation exchange product through anion-exchange column (HiTrap Capto Q a column, GE Healthcare, pillar size is 5mL, and flow velocity is 2mL/min, and level pad is 20mM Tri sHCl, pH 8.0；Elution buffer is 20mM TrisHCl, 1M NaCl, pH 8.0) remove unreacted IFN-LPETGH₆ And PMPC, collection eluent is IFN-PMPC；Purity ＞ 95%, yield is 0.9%.Figure 12 is GPC and SDS-PAGE Analyze " grafting to " technologies synthesize IFN-PMPC, wherein, A figures are GPC technical Analysis " grafting To " technologies；B figures are SDS-PAGE analysis ion-exchange purification IFN-PMPC, are respectively from left to right：Albumen Mixture after mixture, first time cation exchange column after mixture, reaction 24h before standard specimen, reaction, Second of anion-exchange column after purification IFN-PMPC, utilize ATRP technologies in situ synthesis IFN-PMPC.As a result Show, " grafting to " technologies synthesis IFN-PMPC reaction yields are low, and purification difficult illustrates original for utilization Position ATRP technologies have that yield is high, purifying is simple, be easy to amplify and using etc. significant advantage.

4th, interferon macromolecule combination IFN-PMPC Physico-Chemical Characterization

IFN-PMPC (using the above-mentioned two combination IFN-PMPC prepared) molecular weight and polydispersity coefficient (PDI) Determined with GPC, instrument is Waters HPLC/GPC systems connection UV detectors (Waters 2489) With differential refraction detector (Waters 2414).Chromatographic column is Asahipak GS-520HQ and GS-320HQ Or GS-520HQ series connection, mobile phase is 50mM TrisHCl buffer solutions (pH 7.4), and testing conditions are 25 DEG C, Flow velocity 0.5mL/min.The standard curve of the narrow ditribution PEG standard samples generation of different molecular weight is used for calculating molecule Amount and PDI.In order to accurately calculate macromolecule P_MPCMolecular weight, first pass through proteasome degradation even with macromolecule in situ The protein of connection is characterized again.Concretely comprise the following steps：IFN-PMPC combinations (1mg/mL) and Proteinase K (0.5 Mg/mL) in 50mM TrisHCl, 2mM CaCl₂, 7.4,45 DEG C of incubation 12h of pH.Figure 13 shows IFN-PMPC Can be by proteinase K digestion IFN, then by GPC accurate characterization PMPC molecular weight, be followed successively by from left to right：Albumen Before standard specimen, IFN-PMPC, IFN-PMPC and Proteinase K reaction, IFN-PMPC and Proteinase K after reaction overnight, egg White enzyme K.By gpc analysis, the IFN-PMPC coefficient of dispersion is 1.43, and molecular weight is 57kDa.

On Malvern Zetasi zer Nano-zs90 IFN-PMPC is determined with dynamic light scattering (DLS) method Hydration radius.Sample is diluted in PBS, is handled before test through 0.22 μm of aperture membrane filtration. Tested through DLS, IFN-LPETGGH₆, IFN-Br and IFN-α hydration radius be 2.4nm, 2.3nm and 2.3nm, and the IFN-PMPC synthesized is 9.7nm.Figure 14 shows that DLS analyzes IFN-PMPC.

With NMR (¹H NMR) characterize protein-high molecular chemical constitution.IFN-PMPC samples are through cold It is lyophilized it is dry after, be dissolved in D2O, analyzed on JEOL ECX-400 400MHz nuclear magnetic resonance spectrometers. Pass through¹H NMR spectras, which are confirmed, successfully synthesizes PMPC on protein molecular.Figure 10, which is shown, to be passed through¹NMR points of H Analyse IFN-PMPC combinations.

IFN-PMPC secondary structure is measured with circular dichroism analysis of spectrum.Samples with water solution is diluted to 0.10 Mg/mL (1.0 μM), with Pistar π -180 (Appl ied Photophysics Co., Ltds) in 190-260 UV scanning analysis is carried out in nm wave-length coverages.Figure 15 shows circular dichroism chromatography IFN-LPETGGH₆、 IFN-Br, IFN-α, IFN-PMPC secondary structure.By circular dichroism analysis of spectrum, in 190-260nm Circular dichroism spectra in wave-length coverage all shows same 209/219nm bimodal curves, and with IFN-α curve weight It is folded good, show that macromolecule in situ is grown on protein to be had not significant impact to the secondary structure of protein molecule.

IFN-PMPC protein concentration is determined with bicinchoninic acid method (BCA), it is known that the bovine serum albumin(BSA) of concentration For standard sample, specific steps are to specifications.

Above-mentioned three IFN-PMPC prepared are with the above-mentioned two IFN-PMPC testing results prepared without significant difference.

4th, prepared by contrast method

Contain 40 μM of IFN-αs in 2.5mL PBS solution (solute is IFN-α, solvent be pH value be 7.4, Concentration is 10mM PBS solution) adding a certain amount of MPC, (mol ratio of MPC and IFN-α is respectively 1000:1) With the 11O μ L vitamin Cs aqueous solution (4Omg/mL), lead to after nitrogen 15min, add 10 μ L stannous chloride and The PMDETA of N, N, N`, N'`, N'` mono- (PMDTA) (concentration is respectively 100mM and 300mM) is mixed Thing liquid storage, reacts lh under confined conditions, then exposes terminating reaction in atmosphere, obtains reactant mixture.

Reactant mixture is through anion-exchange column (HiTrap Capto Q column, GE Healthcare) point Go out IFN-PMPC from purifying (purification process is the same), collect 8mL column volumes for interferon macromolecule combination IFN-PMPC (control group)；

IFN-PMPC (control group) is determined with SDS-PAGE detections and GPC, molecular weight is 59.1kDa.

Yield is calculated, formula is (to obtain quality/IFN-LPETGGH of IFN-PMPC combinations₆Quality) * 100%, As a result such as table 3, IFN-PMPC (control group) yield is 18.9%.

Table 3

As can be seen that two method of the present invention and the IFN-PMPC of the same molecular weight of three contrast method preparation Combination compares, and the yield of IFN-PMPC combinations prepared by two method of the invention is significantly larger than three control Method, illustrates that the method effect of the present invention is good.

The functional verification of embodiment 2, interferon macromolecule combination IFN-PMPC

1st, IFN-PMPC Bioactivity

(IFN-PMPC prepared by two method of selection example 2, i.e. molecular weight is by IFN-PMPC in the present invention 57kDa is characterized, lower same) antiproliferation using MTT methods determine.People is selected Burkitt ' s B lymphoma cells (Daudi B), because the cell has higher sensitivity to IFN-α 2.

Daudi B cells are containing 15%FBS, 50U/mL penicillin and 50 μ g/mL streptomysins Cultivated in RMPI-1640 after a period of time, certain density cell suspending liquid (50 μ L/ are inoculated with 96 orifice plates Hole, 7500 cells), by IFN-LPETGGH₆, IFN-Br, IFN-α, PEG-IFN alpha-2a and IFN-PMPC after purification Combination sample series are diluted, and each 50 μ L are added in 96 well culture plates, if negative control (being free of IFN-α 2) With blank control (containing only nutrient solution), 37 DEG C, 5%CO₂72h, plus the μ L/ holes of MTT lysates 20 are cultivated, Determine what cell after the absorption value of each hole 490nm wavelength, relatively more different sample treatments was bred after 3h with ELIASA Degree.

Figure 16 show MTT determine IFN-PMPC Bioactivity, table 4 show IFN-α, IFN-LPETGGH₆, IFN-Br, PEG-IFN alpha-2a and IFN-PMPC Bioactivity, its IC₅₀Respectively 10.79 Pg/mL, 10.78pg/mL, 11.18pg/mL, 175.00pg/mL and 20.02pg/mL, active conservation rate Respectively 100%, 100.09%, 96.50%, 6.17% and 53.90%.In summary, IFN-PMPC is anti-swollen in vitro Tumor activity is far above PEG-IFN alpha-2a, shows ATRP in situ without IFN activity is seriously reduced, and is internal antitumor work Property test provide foundation.

Table 4

Sample	IFN-α	IFN-LPETGGH6	IFN-Br	PEG-IFN alpha-2a	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

2nd, IFN-PMPC pharmacokinetics test

All zooperies are completed in the case where Tsing-Hua University instructs on every regulation of zoopery below.This hair Bright middle utilization Bal/c nude nude mice models are determined through tail vein injection IFN-α, PEG-IFN alpha-2a and IFN-PMPC The situation of interferon concentration changes with time in blood, and utilize DAS softwares to carry out data analysis.In medicine Before process phase, 9 Female nude mices, which are observed, is randomly divided into 3 groups after a period of time.It is quiet with 1mg/kg body weight dose tails Arteries and veins injection IFN-PMPC, PEG-IFN alpha-2a and unmodified IFN-α tester, then setting time point (1,5,15, 30min, 1,3,6,24,48,72 and 96h) tail vein take the μ L of blood 20 (heparin tube in advance use liquaemin (river The biochemical medical limited company products of Su Wanbang) infiltrate and dry), 1h is stored at room temperature, in 4 DEG C, 3000 × g Under upper plasma is collected by centrifugation, be stored in 80 DEG C of low temperature refrigerators of ﹣.With humanIFN-α's 2ELISA kits (PBL Interferon source) content of IFN-α 2 in serum is determined according to specification.Utilize the medicines of DAS 3.0 Pharmacokinetic analysis software calculates the pharmacokinetic parameter of IFN-PMPC, PEG-IFN alpha-2a and IFN-α.

Table 5 and Figure 17 respectively illustrate IFN-PMPC, PEG-IFN alpha-2a and the IFN-α pharmacokinetic in nude mice Situation.IFN distribution half-lifes (t1/2 α) elimination half-life period (t1/2 β) is respectively 0.42h and 1.58 H, after administration 5 minutes, the concentration of interferon is to quickly fall to less than the 50% of predose, 24h in blood The 0.1% of the not enough initial concentration of residual concentration afterwards.And the concentration of IFN-PMPC in vivo is gradually decreased, at the beginning of it Half-life period beginning and elimination half-life period are respectively 2.04h and 51.62h, are 6.2 times and 34.7 of IFN-α respectively Times.IFN-PMPC area under the drug-time curve (AUC0- ∞) is 194.1 times of IFN-α.Result above table It is bright, compared with unmodified IFN-α, the elimination half-life period of IFN-PMPC combinations and internal average residence time It is obviously prolonged, Drug-time curve area is dramatically increased, and clearance rate is significantly reduced, has with PEG-IFN alpha-2a close internal Circulating half-life.

Table 5

3rd, IFN-PMPC distributions situation

In the present invention tumour after administration 2h, 24h and 4d is determined using the nude mice for having transplanted ovarian cancer cell The concentration of the interferon of middle residual.Proliferation of Human Ovarian Cell (OVCAR-3) is containing 10%FBS, 50U/mL disks Cultivated in the RMPI-1640 culture mediums of Buddhist nun XiLin and 50 μ g/mL streptomysins after a period of time, use trypsase Digestion is peeled off, and is washed through PBS, is resuspended in the RMPI-1640 culture mediums without above-mentioned additive and and BD In the isometric mixtures of Matrigel Matrix, 0.2mL single cell suspensions (5 × 10⁶Individual cell) it is inoculated in Dorsal sc at nude mice left hind femur, culture forms 100-200mm after 30 days³The solid tumor lump of size. Nude mice is randomly divided into 3 groups, IFN-PMPC, PEG-IFN alpha-2a, IFN-α are squeezed into nude mouse with tail vein injection, Dosage is 10 μ g/20g body weight.It is administered after 2h, 24h and 4d and puts to death nude mice, collection heart, The histoorgans such as kidney, liver, spleen, lungs, pancreas, stomach, muscle, small intestine and tumour.Use Extraction buffer (PBS EDTA containing 1mM, 0.5%Tri ton X-100,0.5% NaTDC, 1mM PMSF, by 1:100 The protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich) of dilution proportion) it is broken Afterwards, centrifuging and taking supernatant extract solution.IFN concentration is quantitative determined with ELISA method in tissue.

Figure 18 is accumulation situations of the IFN-PMPC in each tissue, illustrates IFN-PMPC in heart, kidney, liver Can effectively it be accumulated in dirty, spleen, lungs, pancreas, stomach, muscle, small intestine and tumour.Wherein, A figures be IFN-PMPC, PEG-IFN alpha-2a, IFN-α the accumulation situation in tumour, it can be seen that the concentration of interferon in IFN-PMPC group tumours Compared with the concentration of interferon in IFN group tumours, injection 2h is 12.9 times, injection 24h is 158.2 times, injection IFN-PMPC still has 20% residual after 4d.B figures are IFN-PMPC, PEG-IFN alpha-2a, IFN-α in its hetero-organization The situation of accumulation.Show, IFN-PMPC can make interferon in tumor group effectively using penetrating and retention effect is strengthened Aggregation, the Increased Plasma Half-life in blood circulation are knitted, so as to improve interferon bioavilability in vivo and antitumor Effect.

4th, antitumor activity in nude mice model test IFN-PMPC bodies

IFN-PMPC in vivo bioactivity is determined with animal transplanting tumor experimental method.As stated above, OVCAR-3 cells are inoculated in dorsal sc at nude mice left hind femur, and culture forms solid tumor lump behind 3 weeks days (~20mm³), so as to set up nude mouse tumor model.26 nude mices are divided into 4 groups, IFN-PMPC, PEG-IFN alpha-2a And reference substance (IFN-α is positive control, and physiological saline is negative control) squeezes into nude mouse with tail vein injection It is interior, dosage be 20 μ g/20g body weight, inject weekly once, until physiological saline group, IFN-α group and The nude mice of PEG-IFN alpha-2a group is all dead.Nude mice death in this experiment includes natural death and euthanasia, euthanasia Refer to nude mouse tumor growth more than 500mm³Or Body weight loss is more than 15%, the execution of barbital sodium class medicine is injected.Often Observe nude mice survival condition and tumour growth situation week, dynamic measurement nude mice body weight and gross tumor volume are with the time Change.

To detect IFN-PMPC toxicity, after being administered three times, mouse is put to death, and collect tumour, heart, liver and kidney It is dirty, it is fixed in 4% formalin, HE dyeing is carried out by standard method after section, observes the histology shape of organ State.Terminate after treatment, blood taken by eyeball, measurement lactic dehydrogenase, creatine kinase isozyme, glutamic-pyruvic transaminase, The basic physiological index such as glutamic-oxalacetic transaminease, creatinine, urea nitrogen, red blood cell, leucocyte, blood platelet, hemoglobin.

Test result indicates that (Figure 19 and Figure 20), during experiment, the gross tumor volume of the nude mice of physiological saline group quickly increases Greatly, inject the 42nd day, nude mouse tumor volume is more than 500mm³, median survival is only 35 days；During experiment, IFN- The gross tumor volume of the nude mice of α groups gradually increases, and injects the nude mouse tumor volume of the 49th day IFN group also above 500mm³, Median survival is 38 days, does not embody obvious antitumor activity；During experiment, the tumour body of the nude mice of PEG-IFN alpha-2a group Product gradually increases, and injects the nude mouse tumor volume of the 63rd day IFN group also above 500mm³, median survival is 60 days, There is certain antitumor activity；During experiment, the gross tumor volume of the nude mice of IFN-PMPC groups does not change substantially, meanwhile, The mouse tumor for having 75% is disappeared, and 1 mouse tumor is no longer grown up, and only 1 mouse tumor slowly increases, until Gross tumor volume is just more than 500mm within 93 days³.Data above shows that IFN-PMPC can effectively suppress the growth of tumour, With extraordinary internal antitumor activity, even better than presently commercially available medicine PEG-IFN alpha-2a.

During experimental result is also shown that experiment, the nude mice body weight of IFN-PMPC groups is increased slightly (Figure 21), shows IFN-PMPC does not have obvious side effect to nude mice.

Figure 22 is the tumour of mouse, heart, liver, the Histological section of kidney after being administered 3 times, it can be seen that note Penetrate after IFN-PMPC, hole occurs in mouse tumor cell gap, endochylema and karyomorphism be not obvious, meronecrosis, Bulk cell membrane comes off, obvious with control group difference.Meanwhile, the cellular morphology of heart, liver and kidney is complete, nothing Obvious meronecrosis, with control group Histological Study no significant difference.Figure 23 and Figure 24 are respectively to terminate small after treatment The biochemistry and physiochemical indice of mouse, compared with the nude mice of physiological saline group, IFN-α group and PEG-IFN alpha-2a group, IFN-PMPC groups The lactic dehydrogenase of nude mice, creatine kinase isozyme, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, creatinine, urea nitrogen, The basic physiological index such as red blood cell, leucocyte, blood platelet, hemoglobin does not have marked difference.Data above shows IFN-PMPC will not cause overt toxicity to nude mice intracorporeal organ.

The method original position ATRP synthetic technologys yield of the present invention is high, and purifying is simple, it is easy to industrialize, the interference of preparation Element-P^MPCCombination has higher bioactivity storage rate, the half-life period significantly extended and effective tumor inhibition effect, More excellent effect is shown compared with marketed drugs PEG-IFN alpha-2a In vitro and in vivo activity.ATRP synthetic technologys are expected substitution PEGylation turns into modified protein medicine to improve medicine stability, improve pharmacokinetics and strengthen therapeutic efficiency New method.

Claims (10)

1. a kind of method for preparing protein-macromolecule combination, be it is following 1) or 2)：

1) method shown in comprises the following steps：

By protein-initiator combination, high polymer monomer, CuCl, CuCl₂, 1,1,4,7,10,10- hexamethyls Triethylene tetramine carries out polymerisation in buffer solution, obtains protein-macromolecule combination；

The protein-initiator combination is that initiator and albumen are covalently attached obtained product；

2) method shown in is to connect high-molecular compound and albumen by oligomerization glycine in the presence of initiator Connect, obtain protein-macromolecule combination；

The high-molecular compound is to be obtained by high polymer monomer polymerization.

2. according to the method described in claim 1, it is characterised in that：The albumen is interferon, the interferon Selected from interferon-' alpha ' or its fusion protein, interferon beta or its fusion protein, interferon gamma or its fusion protein, interferon lambda Or its fusion protein；

Or the albumen is specially the interferon alpha fusion protein in sequence table shown in sequence 2；

The high polymer monomer is 2- methylacryoyloxyethyl phosphocholines；

The high-molecular compound is poly- 2- methylacryoyloxyethyls phosphocholine.

3. method according to claim 1 or 2, it is characterised in that：The initiator is that atom transfer is free The initiator of base polymerisation, the initiator of the fracture chain transfer polymerization of reversible addition one, the initiator of ring opening metathesis polymerization Or the initiator of ring opening polyaddition；

The structural formula formula 1 specific as follows of the initiator of the atom transition free radical polymerization reaction：

Wherein, R is H or (CH₂)_nCH₃, n is >=1 integer；X is Cl or Br or I；M is >=1 integer；N is >=1 integer；

The initiator of the atom transition free radical polymerization reaction is especially specially 2- (2- (2- (2- glycyls amido) Acetamido) acetamido) ethyl 2 bromo 2 methyl propionic acid ester.

4. according to any described method in claim 2-3, it is characterised in that：

1) in the method shown in, the initiator is covalently attached to the C-terminal or N-terminal of the albumen；

Or the initiator is specifically covalently attached to the C-terminal of the albumen；

The protein-initiator combination, the 2- methylacryoyloxyethyls phosphocholine, the CuCl, The CuCl₂Mol ratio with the 1,1,4,7,10,10- hexamethyl triethylene tetramines is 1：200-10000： 10-500：10-2000：10-4000；

The coupling ratio of albumen and the initiator described in the protein-initiator combination is 1：1；

Or the protein-initiator combination, the 2- methylacryoyloxyethyls phosphocholine, the CuCl, The CuCl₂Mol ratio with the 1,1,4,7,10,10- hexamethyl triethylene tetramines is specially 1：2000：25： 75：125.

5. according to any described method in claim 1-4, it is characterised in that：2) method shown in, including Following steps：Polymerisation is occurred into for the initiator and the high polymer monomer, obtains connecting oligomerization glycine High molecular polymer；The high molecular polymer of the connection oligomerization glycine is covalently attached with the albumen again, obtained To protein-macromolecule combination.

6. method according to claim 5, it is characterised in that：The polymerisation is by initiator, 2- Methylacryoyloxyethyl phosphocholine, CuCl, CuCl₂With 1,1,4,7,10,10- hexamethyl triethylene tetramines slow Polymerisation is carried out in fliud flushing, obtains connecting the high molecular polymer of oligomerization glycine；

It is described be covalently linked to by the protein, transpeptidase, the connection oligomerization glycine high molecular polymer And CaCl₂Reacted in buffer solution, obtain protein-macromolecule combination.

7. method according to claim 6, it is characterised in that：In the polymerisation, the initiator, The 2- methylacryoyloxyethyls phosphocholine, the CuCl, the CuCl₂With the 1,1,4,7,10,10- The mol ratio of hexamethyl triethylene tetramine is 1：200-10000：10-500：10-2000：10-4000；

Or the initiator, the 2- methylacryoyloxyethyls phosphocholine, the CuCl, the CuCl₂ Mol ratio with the 1,1,4,7,10,10- hexamethyl triethylene tetramines is specially 1：2000：25：75：125；

In the covalent attachment, the protein, the transpeptidase, the polyphosphazene polymer of the connection oligomerization glycine Compound and CaCl₂Mol ratio be 2：1：40：200.

8. according to any described method in claim 1-7, it is characterised in that：

The polymerisation is carried out under hypoxemia or atmosphere of inert gases；

The time of the polymerisation is 5 minutes to 24 hours, and the temperature of the polymerisation is 0-80 DEG C.

9. protein-macromolecule the combination prepared by any methods describeds of claim 1-8；

Or the protein-application of the macromolecule combination in antitumor or anti-virus product is prepared.

10. a kind of antitumor or anti-virus product, its protein-macromolecule for including described in claim 9 is combined Body.