CN103751796A - Straight-chain polyethylene glycol-recombinant porcine interferon alpha conjugate, and preparing method and application thereof - Google Patents
Straight-chain polyethylene glycol-recombinant porcine interferon alpha conjugate, and preparing method and application thereof Download PDFInfo
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- CN103751796A CN103751796A CN201310526045.8A CN201310526045A CN103751796A CN 103751796 A CN103751796 A CN 103751796A CN 201310526045 A CN201310526045 A CN 201310526045A CN 103751796 A CN103751796 A CN 103751796A
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Abstract
The invention discloses a polyethylene glycol-recombinant porcine interferon alpha conjugate and a preparing method thereof. The preparation method comprises the steps: under a weakly alkaline condition, coupling polyethylene glycol with amino of a recombinant porcine interferon alpha, and purifying to obtain the target product. The modification rate of the polyethylene glycol modified recombinant porcine interferon alpha is about 50%, the purity is about 97% or more, the in-vivo retention time is long, and the blood plasma half-life period reaches 16-38 hours. The conjugate can be applied in preparation of animal antiviral drugs.
Description
Technical field
The present invention relates to biological medicine and chemical field, be specifically related to a kind of preparation method of carrying the recombinant porcine alpha interferon polyethylene glycol conjugation thing of labelled protein, with and in the purposes for the preparation of in antivirus veterinary medicine.
Background technology
PEG(Polyethylene Glycol) be a kind of neutrality, water solublity, avirulent polymer.It is a few by FDA, ratified can be used for the polymer in body surface and body, in food, cosmetics, individual health care and pharmaceuticals industry extensive use.The characteristic of PEG is disclosed by its behavior soluble in water, and the PEG molecule of long-chain in aqueous solution has hyperhydrated (being that water molecules arrives its surface).The key property of PEG is can be attached to other molecules and surface is upper, and a biology mixes, protectiveness shell is provided.This protectiveness shell has reduced the removing of (as human body) these materials in biosystem, greatly reduces the absorption of protein, cell and antibacterial, has reduced the clearance rate of kidney.PEG is solvable in water and many organic solvents, and in aqueous solution, it can form two-phase system with other polymer together as glucosan.Insoluble in as hexane at ether and carbohydrate.The chemical characteristic that the water solublity of bifunctional PEG, avirulence, high flexibility and feature are clear and definite, makes it become the cross-linked material of desirable modified protein series products.The coupling of PEG and interferon has been widely used in for clinical.As Pegylation human interferon-alpha-2 β of Schering Plough company exploitation (trade name, wear happy can, PegIntron); (trade name sends sieve first, PegasyS) etc. to Pegylation human interferon-alpha-2 α of Roche Holding Ag's exploitation.Verified interferon after coupled, biologic activity major part is retained, and immunne response greatly reduces simultaneously, and serum half-life has also greatly obtained prolongation.
Animal alpha-interferon has good prevention effect to animal viral disease.The bright and beautiful teach problem group of the king of microbiology teaching and research room of Medical University Of Anhui adopts technique for gene engineering to clone and expressed porcine alpha-type interferon, and laboratory qualified products are made to lyophilized injectable powder is sent to Duo Jia pig farm, Anhui Province and carries out clinical trial.Result proves, the recombinant porcine alpha interferon of this chamber development can effectively be treated pig canine viral disease.To treatment pig virus diarrhoea effective percentage, be 83.5%, cure rate is 63.9%(Chinese invention patent CN101289666 Α).This project is in industrialization process.On the basis of this project, recombinant porcine alpha interferon is carried out to pegylation, to obtain durative action preparation, thus prolong drug in vivo circulating half-life, reduce clearance rate in body, reduce administration number of times, reduce animal irritability, heighten the effect of a treatment, reduce drug cost.This project of exploitation as novel veterinary drug has important learning value and application prospect, has higher Social benefit and economic benefit simultaneously.
Summary of the invention
The object of the invention is exactly for providing a kind of straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate, preparation method and application thereof.
The present invention is achieved by the following technical solutions:
Straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate, it has following structural formula:
In formula, mPEG is polyglycol chain, and Protein is that N end carries labelled protein recombinant porcine alpha interferon (TRX-HIS-Interferon).
Straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate, described interferon is that N end carries labelled protein Recombinant Swine interferon (TRX-HIS-Interferon, Mw=39KD).
Straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate, described polyalkylene glycol moiety is that molecular weight is the straight chain Polyethylene Glycol of 2-30KD.
A preparation method for straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate, comprises the following steps:
(1) interferon is diluted to 0.1mg/ml with the potassium phosphate buffer of 5mM pH3.0-5.0;
(2) add the interferon of amount of calculation, with NaOH solution, regulate pH value to 8.0, ice bath is preserved, the amount of substance that is 1:20 by interferon: mPEG adds the mPEG-SC of 1/4 amount, under 0-4 ℃ of condition, react, at interval of 2h, add subsequently the mPEG-SC of 1/4 amount, continue reaction 4-8h after adding, described mPEG-SC has following structure:
(3) add the glycine cessation reaction of 0.75 Μ;
(4) get above-mentioned reactant liquor a little, by SDS-PAGE, calculate response rate, carry out subsequently purification, described purification comprises the following steps:
(1) above-mentioned reactant liquor is doubly measured to the sodium-acetate buffer dilution that volume, concentration are 50mM pH7.2 with 5-10, then by carboxymethyl cellulose post on diluent (Waterman CM-52), with the sodium-acetate buffer of 5 times of volume pH7.2, wash after post, with the sodium-acetate buffer eluting containing 0.5M NaCl, collect the eluent containing PEG-interferon, concentrated;
(2) with Superdex200, to obtaining PEG-interferon in (1), be further purified, the sodium-acetate buffer that eluent is pH7.2 (containing 0.2M NaCl), collects the eluent containing PEG-interferon, concentrated.
Straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate, this medicine can be used in to be prepared in antivirus veterinary medicine.
Advantage of the present invention is:
(1) the polyethyleneglycol modified recombinant porcine alpha interferon that carries labelled protein of the present invention can keep the antiviral activity of interferon.Its plasma clearance is low, and Half-life in vivo is long, and liver accumulating capability is strong, therefore as antivirus veterinary medicine, than the recombinant porcine alpha interferon of unmodified, has larger superiority.
(2) the present invention obtains mono-modified Polyethylene Glycol-the carry recombinant porcine alpha interferon of labelled protein by controlling reaction condition (comprise the addition of dressing agent and add mode, reaction temperature, time pH of buffer and ionic strength etc.).Combine plurality of color spectrometry simultaneously and carry out the highly purified polyethyleneglycol modified recombinant porcine alpha interferon that carries labelled protein of separated preparation (in reactant mixture each component again different isoelectric point, IP and molecular weight).
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE electrophoresis detection collection of illustrative plates of embodiment 1, wherein: Band1-standard molecular weight albumen (Maker); Band2-Ν end carries the recombinant porcine alpha interferon (Μ w=39KD) of labelled protein; Band3-reactant mixture; Band4-straight chain Polyethylene Glycol-the carry recombinant porcine alpha interferon conjugate (Μ w=59KD) of labelled protein
The specific embodiment
Straight chain Polyethylene Glycol-the carry preparation of the recombinant porcine alpha interferon of labelled protein
Interferon is diluted to 0.1mg/ml with the potassium phosphate buffer of 5mM pH3.0-5.0; The interferon that adds amount of calculation, with NaOH solution, regulate pH value to 8.0, the amount of substance that is 1:20 by interferon: mPEG adds the mPEG-SC(Mw=20KD of 1/4 amount), under 0-4 ℃ of condition, react, at interval of 2h, add subsequently the mPEG-SC of 1/4 amount, after adding, continue reaction 4h, add the glycine 1ml cessation reaction of 0.75 Μ; The sodium-acetate buffer dilution that is 50mM pH7.2 by above-mentioned reactant liquor by 5 times of amount volumes, concentration subsequently, then go up carboxymethyl cellulose post (Waterman CM-52), with the sodium-acetate buffer of 5 times of volume pH7.2, wash after post, with the sodium-acetate buffer eluting containing 0.5M NaCl, collect the eluent containing PEG-interferon, concentrated, SDS-PAGE electrophoresis detection.With Superdex200, to obtaining PEG-IFN in (1), be further purified, eluent is the sodium-acetate buffer (containing 0.2M NaCl) of pH7.2, and the uv absorption that is 280nm with wavelength detects, and collects the eluent containing PEG-interferon, concentrated, SDS-PAGE electrophoresis detection.
The detection of embodiment 2:SDS-PAGE electrophoresis method to product
Each concentrated eluting peak is adopted to SDS-PAGE electrophoresis detection, separation gel 10%, concentrated glue 5%.After electrophoresis finishes, colloid is fixed to (fixative: 30% methanol, 5% acetic acid) room temperature and shaken dyeing 1h(dyeing liquor: 0.05% Coomassie brilliant blue, 30% methanol, 5% acetic acid); Finally colloid is moved into room temperature in destaining solution (destaining solution: 1% glutaraldehyde, 30% methanol, 5% acetic acid) and shake immersion until band is clear.
Embodiment 3: the recombinant porcine alpha interferon to Polyethylene Glycol after purification-carry labelled protein can suppress 100TCID in WISH cell
50the propagation result of VSV virus carry out titration;
WISH cell is processed with the modified interferon of various dose, after 24h, inhaled and abandon, then inoculate 100TCID respectively
50vSV virus; Result shows that the recombinant porcine alpha interferon after polyethyleneglycol modified can obviously suppress the cytopathy that VSV causes, by titration, the mono-modified its lytic activity of gained retains and reached more than 25%.
Embodiment 4: Half-life in vivo is measured
Choose respectively 22 of the SD rats that body weight is close, be divided at random two groups, 11 of the recombinant porcine alpha interferon groups of the Polyethylene Glycol of embodiment 1-carry labelled protein, the interferon after the modification of intramuscular injection 50 μ g/kg body weight; Carry 11 of the recombinant porcine alpha interferon groups of labelled protein, the interferon of intramuscular injection 50 μ g/kg body weight, different time points eye socket is got blood, with 2500 revs/min, centrifugal removal serum ,-20 ℃ of preservations, after all serum has been collected, use ELISA test kit to survey the content of sample in blood plasma, the half-life is that in blood, sample size is down to half time of peak.The recombinant porcine alpha interferon concentration that found that unmodified reaches rapidly maximum, at 1.5h, is down to half; And recombinant porcine alpha interferon concentration after polyethyleneglycol modified reaches maximum at 2h, 25h is down to half.Therefore, the polyethyleneglycol modified recombinant porcine alpha interferon half-life in animal body obviously extends, and is 15 times of left and right of the recombinant porcine alpha interferon of unmodified.
Claims (5)
1. straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate, is characterized in that it has following structural formula:
In formula, mPEG is polyglycol chain, and Protein is that N end carries labelled protein recombinant porcine alpha interferon (TRX-HIS-Interferon).
2. a kind of straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate according to claim 1, is characterized in that described interferon is that N end carries labelled protein recombinant porcine alpha interferon (TRX-HIS-Interferon, Mw=39KD).
3. a kind of straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate according to claim 1, is characterized in that described polyalkylene glycol moiety is that molecular weight is the straight chain Polyethylene Glycol of 2-30KD.
4. a preparation method for straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate as claimed in claim 1, is characterized in that comprising the following steps:
(1) interferon is diluted to 0.1mg/ml with the potassium phosphate buffer of 5mM pH3.0-5.0;
(2) add the interferon of amount of calculation, with NaOH solution, regulate pH value to 8.0, ice bath is preserved, the amount of substance that is 1:20 by interferon: mPEG adds the mPEG-SC of 1/4 amount, under 0-4 ℃ of condition, react, at interval of 2h, add subsequently the mPEG-SC of 1/4 amount, continue reaction 4-8h after adding, described mPEG-SC has following structure:
(3) add the glycine cessation reaction of 0.75 Μ;
(4) get above-mentioned reactant liquor a little, by SDS-PAGE, calculate response rate, carry out subsequently purification, described purification comprises the following steps:
(1) above-mentioned reactant liquor is doubly measured to the sodium-acetate buffer dilution that volume, concentration are 50mM pH7.2 with 5-10, then by carboxymethyl cellulose post on diluent (Waterman CM-52), with the sodium-acetate buffer of 5 times of volume pH7.2, wash after post, with the sodium-acetate buffer eluting containing 0.5M NaCl, collect the eluent containing PEG-interferon, concentrated;
(2) with Superdex200, to obtaining PEG-interferon in (1), be further purified, the sodium-acetate buffer that eluent is pH7.2 (containing 0.2M NaCl), collects the eluent containing PEG-interferon, concentrated.
5. straight chain Polyethylene Glycol-recombinant porcine alpha interferon conjugate according to claim 1, is characterized in that this medicine can be used in to prepare in antivirus veterinary medicine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104262480A (en) * | 2014-09-28 | 2015-01-07 | 重庆理工大学 | Construction and modification method of recombinant porcine long-acting-alpha interferon and preparation method of lyophilized injection |
| CN105380911A (en) * | 2015-12-07 | 2016-03-09 | 宋宏婷 | Preparation method for lyophilized agent of swine interferon |
| CN105496972A (en) * | 2015-12-07 | 2016-04-20 | 宋宏婷 | Preparation method of chicken interferon lyophilized preparation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1966527A (en) * | 2005-11-16 | 2007-05-23 | 中国科学院过程工程研究所 | Polyethylene glycol-interferon coupler and its preparation method |
| CN102229667A (en) * | 2011-06-03 | 2011-11-02 | 北京伟嘉人生物技术有限公司 | Polyethylene glycol modified porcine alpha-interferon, preparation method and application thereof |
-
2013
- 2013-10-29 CN CN201310526045.8A patent/CN103751796A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1966527A (en) * | 2005-11-16 | 2007-05-23 | 中国科学院过程工程研究所 | Polyethylene glycol-interferon coupler and its preparation method |
| CN102229667A (en) * | 2011-06-03 | 2011-11-02 | 北京伟嘉人生物技术有限公司 | Polyethylene glycol modified porcine alpha-interferon, preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 蔡家利等: "重组猪IFNα的构建及原核可溶性表达研究", 《重庆理工大学学报（自然科学）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104262480A (en) * | 2014-09-28 | 2015-01-07 | 重庆理工大学 | Construction and modification method of recombinant porcine long-acting-alpha interferon and preparation method of lyophilized injection |
| CN105380911A (en) * | 2015-12-07 | 2016-03-09 | 宋宏婷 | Preparation method for lyophilized agent of swine interferon |
| CN105496972A (en) * | 2015-12-07 | 2016-04-20 | 宋宏婷 | Preparation method of chicken interferon lyophilized preparation |
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Effective date of registration: 20161027 Address after: 241000 Anhui city of Wuhu Province Branch Center D Park Economic and Technological Development Zone 101, Room 102 Applicant after: Anhui JiuChuan Biotechnology Co., Ltd. Address before: 230032 Medical University Of Anhui, Hefei, Anhui No. 81 Applicant before: Wang Mingli Applicant before: Li Zeng |
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