CN101450214A - Transferrin-frog-egg ribonuclease coupler and production method and use thereof - Google Patents
Transferrin-frog-egg ribonuclease coupler and production method and use thereof Download PDFInfo
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- CN101450214A CN101450214A CNA2007101718921A CN200710171892A CN101450214A CN 101450214 A CN101450214 A CN 101450214A CN A2007101718921 A CNA2007101718921 A CN A2007101718921A CN 200710171892 A CN200710171892 A CN 200710171892A CN 101450214 A CN101450214 A CN 101450214A
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Abstract
The invention discloses a transferrin-frogspawn ribalgilase conjugate which belongs to the biological medicine field. The conjugate includes one transferrin and 1-5 frogspawn ribalgilase. The invention also discloses a method for preparing the conjugate, an use and a pharmaceutical composition containing the conjugate. Many tumour cells high express transferrin receptor, and transferrin is easy to be endocytosis after combining with the receptor, accordingly the conjugate is easy to enter into cytolymph than dissociative frogspawn ribalgilase and has stronger and special ability for killing tumour cell.
Description
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to a transferrin (Tf) -frogspawn ribonuclease (Onconase) conjugate as well as a preparation method and application thereof.
Background
Frog egg ribonuclease (Onconase is also known as P30protein, Ranpirnase, simply called Onc) is a nuclease isolated from oocytes and early embryos of Rana pipiens (Rana pipiens), and belongs to the RNaseA superfamily. Research shows that Onc enters cells through endocytosis, selectively degrades tRNA in cytoplasm, inhibits protein synthesis, and further inhibits cell proliferation and causes cell apoptosis. In vitro experiments show that Onc has antiproliferative and cytotoxic effects on various tumor cells, such as: human prostate cancer cells, pancreatic cancer cells, leukemia cells, and the like. In vivo experiments of mouse models prove that the Onc can inhibit tumor growth, prolong the survival time of mice and enhance the effects of various antitumor drugs.
At present, Onc has already entered three-phase clinic as a drug for resisting malignant mesenchymal cell tumor, and may be a potential antitumor drug with good prospect. However, because the protein is a non-human protein, certain immune reaction and side reaction (such as kidney injury) can still be generated when the protein is used in a human body, and meanwhile, the Onc also has certain killing effect on some normal cells, and the specific killing effect of the Onc on tumor cells needs to be enhanced to overcome the defects, so that the efficiency of the Onc entering the tumor cells is improved. Therefore, there is still a need to improve the ability of onco to specifically enter tumor cells to reduce its side effects.
Disclosure of Invention
The invention aims to provide a transferrin (Tf) -frog egg ribonuclease (Onconase) conjugate.
The invention also aims to provide a preparation method and application of the transferrin-frogspawn ribonuclease conjugate.
In a first aspect of the invention there is provided an isolated transferrin (Tf) -frog egg ribonuclease (Onconase) conjugate, said conjugate comprising 1 transferrin and 1 to 5 (preferably 2 to 4; more preferably 2 to 3) frog egg ribonucleases conjugated to said transferrin, said conjugate having a molecular weight of 89 to 137 KD.
In another preferred embodiment, the transferrin has the amino acid sequence of SEQ ID NO: 2; or the frog egg ribonuclease has the nucleotide sequence shown in SEQ ID NO: 4.
In another preferred embodiment, the conjugate is prepared by the following method:
(1) conjugating transferrin to frog egg ribonuclease using N-hydroxysuccinimide 3- (2-pyridyldithio) propionate (SPDP) and 2-Iminothiolane & HCl (also known as Traut's reagent); and
(2) separating to obtain the transferrin-frogspawn ribonuclease conjugate.
In a second aspect of the present invention, there is provided a method for preparing the transferrin-frogspawn ribonuclease conjugate, the method comprising:
(1) coupling transferrin to frog egg ribonuclease using N-hydroxysuccinimide 3- (2-pyridyldithio) propionate and 2-Iminothiolane & HCl; and
(2) separating to obtain the transferrin-frogspawn ribonuclease conjugate.
In another preferred example, the method comprises:
(a) contacting transferrin with N-hydroxysuccinimide 3- (2-pyridyldithio) propionate to obtain transferrin treated with N-hydroxysuccinimide 3- (2-pyridyldithio) propionate;
(b) contacting the frog egg ribonuclease with 2-Iminothiolane & HCl to obtain frog egg ribonuclease protein treated by the 2-Iminothiolane & HCl;
(c) mixing the N-hydroxysuccinimide 3- (2-pyridyldithio) propionate-treated transferrin obtained in step (a) with the 2-Iminothiolane-HCl-treated frog egg ribonuclease protein obtained in step (b) such that transferrin and frog egg ribonuclease are coupled; and
(d) collecting the conjugate with molecular weight of 89-137 kD.
In another preferred embodiment, free transferrin and Onc are removed by ion exchange to collect conjugates with molecular weights of 89-137 kD.
In another preferred embodiment, in step (a), the molar ratio of transferrin to N-hydroxysuccinimide 3- (2-pyridyldithio) propionate is 1 (5-10).
In another preferred embodiment, in step (b), the molar ratio of frog egg ribonuclease to 2-Iminothiolane HCl is 1 (5-10).
In another preferred embodiment, in step (c), the molar ratio of the N-hydroxysuccinimide 3- (2-pyridyldithio) propionate-treated transferrin obtained in step (a) to the 2-Iminothiolane & HCl-treated frog egg ribonuclease protein obtained in step (b) is 1 (1-4).
In another preferred embodiment, the molar ratio of the N-hydroxysuccinimide 3- (2-pyridyldithio) propionate-treated transferrin obtained in step (a) to the 2-Iminothiolane & HCl-treated frog egg ribonuclease protein obtained in step (b) is about 1: 3.
In the third aspect of the invention, the use of the transferrin-frogspawn ribonuclease conjugate is provided for preparing a composition for inhibiting tumors.
In another preferred embodiment, the tumor is a tumor that highly expresses transferrin receptor.
In a fourth aspect of the present invention, there is provided a tumor-inhibiting composition comprising:
(i) an effective amount of the transferrin-frog egg ribonuclease conjugate; and
(ii) a pharmaceutically acceptable carrier.
In a fifth aspect of the invention, there is provided a method of inhibiting the growth of tumour cells in vitro (preferably non-therapeutically), said method comprising treating tumour cells with said transferrin-frogspawribonuclease conjugate.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.
Drawings
FIG. 115% SDS-PAGE shows the gel image of the protein of the invention. Wherein,
FIG. 2(a) 15% SDS-PAGE gel electrophoresis shows the protein gel image of the effect of the coupling of transferrin and frog egg ribonuclease. Wherein,
FIG. 2(b) 10% SDS-PAGE gel electrophoresis further shows the molecular weight distribution of transferrin and frogspawn ribonuclease coupled protein. Wherein,
FIG. 3 is a graph showing the effect of MTT method on detecting the cytotoxicity of Tf-Onc conjugates and Onc on mouse myeloma cells SP2/0, which were obtained by using SPDP and 2-Iminothiolane & HCl as coupling agents.
FIG. 4 is a graph showing the cytotoxicity effect of Tf-Onc conjugate and Onc on mouse myeloma cells SP2/0 obtained by using SPDP alone as a coupling agent in MTT method detection.
FIG. 5 is a graph showing the effect of MTT method on detecting the cytotoxicity of Tf-Onc conjugate and Onc on human neuroblastoma SH-SY5Y obtained by using SPDP and 2-Iminothiolane & HCl as coupling agents.
Detailed Description
Aiming at the defects that the specificity of the frog egg ribonuclease to tumor cells is not high and some normal cells are killed and killed in the prior art, the inventor firstly prepares a highly purified transferrin-frog egg ribonuclease conjugate through extensive research and repeated selection and test. In the conjugate, transferrin can enter tumor cells through the cell body transport function mediated by transferrin receptors, and the rana chensinensis ribonuclease is brought into the tumor cells to play a role in killing the tumor cells. In the conjugate of the invention, 1-5 frog egg ribonuclease molecules are coupled on each transferrin molecule, so the molecule has moderate size and is easy to penetrate the cell membrane of tumor cells and enter the cells. The present invention has been completed based on this finding.
The main point of the invention is that transferrin is used as a drug carrier to transport Onc to the surface of the tumor cells, so that the endocytosis of Onc can be enhanced, the specificity of Onc can be improved, the tumor treatment range of Onc can be enlarged, the drug effect can be enhanced, and the side effect can be reduced.
The invention is mainly based on the characteristics of a plurality of tumor cells with vigorous metabolism and high transferrin receptor expression (usually, the tumor cells express 10,000 to 100,000 TfR1, and the TfR1 has low expression or no expression detected on normal cells, see ZHONG MING QIAN et al, Targeted drug delivery via the transfer receptor-mediated end cytosis pathway. Pharmacol Rev 54: 561-. Therefore, the conjugate can be specifically combined on tumor cells, greatly improves the efficiency of the frog egg ribonuclease entering the tumor cells and the specificity of killing the tumor cells, enhances the killing effect on the tumor cells and reduces the side effect of the frog egg ribonuclease. The conjugates of the invention are particularly suitable for the treatment of a variety of tumors that highly express the transferrin receptor type, such as, but not limited to, the treatment of brain tumors, leukemia, and the like.
Transferrin
Transferrin (Tf) is a glycoprotein having a molecular weight of about 77kD and capable of binding to iron ions. Transferrin bound to iron ions can specifically bind to its receptor (TfR 1) to mediate the intracellular transport of iron ions. TfR1 is highly expressed on tumor cells and therefore can transport more transferrin into tumor cells.
In the present invention, the transferrin used may be naturally occurring, e.g. it may be isolated or purified from an animal. In addition, the transferrin may also be artificially produced, for example, recombinant transferrin may be produced according to conventional genetic engineering recombinant techniques.
Any suitable transferrin may be used in the present invention. The transferrin comprises full-length transferrin or a biologically active fragment thereof. Preferably, the amino acid sequence of transferrin can be identical to SEQ ID NO: 2 are substantially identical.
The amino acid sequence of transferrin formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. Transferrin or a biologically active fragment thereof comprises a portion of a conservative amino acid substitution sequence, wherein said amino acid substitution sequence does not affect its activity or retains a portion of its activity. Appropriate substitutions of amino acids are well known in the art and can be readily made and ensure that the biological activity of the resulting molecule is not altered. These techniques allow one of skill in the art to recognize that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/Cummingspub. Co. P224.
Any biologically active fragment of transferrin may be used in the present invention. Herein, a biologically active fragment of transferrin is meant to be a polypeptide that still retains all or part of the function of full-length transferrin. Typically, the biologically active fragment retains at least 50% of the activity of full-length transferrin. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length transferrin.
Modified or improved transferrin can also be employed in the present invention, for example, transferrin modified or improved to promote its half-life, effectiveness, metabolism, and/or potency of the protein. The modified or modified transferrin or gene may be different from naturally occurring transferrin or gene in that it inhibits tumor cells without other adverse effects or toxicity. That is, any variant that does not affect the biological activity of transferrin or the biological function of the gene can be used in the present invention.
The transferrin may be obtained from a commercially available source, and as an example of the present invention, the transferrin is obtained from Calbiochem.
Frog egg ribonuclease
The frog egg ribonuclease is an enzyme which is useful for inhibiting tumor cells, can selectively degrade tRNA in cytoplasm, inhibit protein synthesis and further inhibit cell proliferation and cause cell apoptosis.
In the present invention, the frog egg ribonuclease to be used may be naturally occurring, for example, it may be isolated or purified from an animal. In addition, the frog egg ribonuclease can also be artificially prepared, for example, recombinant frog egg ribonuclease can be produced according to the conventional genetic engineering recombination technology. Preferably, recombinant frog egg ribonucleases can be used in the present invention.
Any suitable frog egg ribonuclease may be used in the present invention. The frog egg ribonuclease includes full-length frog egg ribonuclease or its bioactive fragment. Preferably, the amino acid sequence of the frog egg ribonuclease can be identical to the amino acid sequence shown in SEQ ID NO: 4 are substantially identical.
The amino acid sequence of the frog-egg ribonuclease formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. The frog egg ribonuclease or the bioactive fragment thereof comprises a part of conservative amino acid substitution sequence, and the amino acid substitution sequence does not affect the activity or keeps partial activity. Appropriate substitutions of amino acids are well known in the art and can be readily made and ensure that the biological activity of the resulting molecule is not altered. These techniques allow one of skill in the art to recognize that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. See Watson et al Molecular Biology of The Gene, fourth edition, 1987, The Benjamin/Cummings Pub. Co. P224.
Any biologically active fragment of frog egg ribonuclease can be used in the present invention. Herein, the biologically active fragment of frog egg ribonuclease means a polypeptide which still retains all or part of the functions of the full-length frog egg ribonuclease. Typically, the biologically active fragment retains at least 50% of the activity of the full length frog egg ribonuclease. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length frog egg ribonuclease.
The present invention may also employ a modified or improved frog egg ribonuclease, for example, a frog egg ribonuclease modified or improved to extend its half-life, improve its stability or enhance its ability to kill tumor cells. The modified or improved frog egg ribonuclease or gene can be different from the naturally-occurring frog egg ribonuclease or gene in certain points, but can also kill tumor cells without causing other adverse effects or toxicity. That is, any modification which does not affect the biological activity of the frog egg ribonuclease or the biological function of the gene can be used in the present invention.
As an example of the invention, the frog egg ribonuclease is expressed by recombination, the total length of the coding gene of the frog egg ribonuclease is 315bp, and the coding gene codes a polypeptide with 104 AA, and the gene is shown as SEQ ID NO: 3, and the encoded protein is shown as SEQ ID NO: 4, respectively.
The frog egg ribonuclease can be produced by conventional genetic engineering methods, and generally comprises the following steps:
(1) transforming or transducing a suitable host cell with a recombinant expression vector comprising a gene encoding a frogspawn ribonuclease;
(2) a host cell cultured in a suitable medium;
(3) isolating and purifying the protein from the culture medium or the cells.
Conjugates
The invention provides a transferrin-frogspawn ribonuclease conjugate, the molecular weight of the conjugate is 89-137KD, and 1-5 frogspawn ribonucleases are coupled on each transferrin molecule.
The inventor finds in research that molecules which can enter cells through a cell membrane need to meet a certain size, if too many frogspawn ribonuclease molecules are coupled on transferrin, the molecular structure is too large, the combination of the transferrin and a transferrin receptor on a tumor cell membrane is influenced, and the effect is not ideal; if multiple transferrins are coupled to one or more frogspawn ribonucleases, the resulting molecules are too bulky to penetrate the vessel wall into the tumor tissue. Therefore, in the process of preparing the conjugate, the obtained conjugate needs to be controlled to contain one transferrin (about 77KD) and 1 or several (preferably 1-5) frogspawn ribonuclease (about 12KD) so that the conjugate can smoothly enter tumor cells and exert good effect of killing the tumor cells.
In view of the size of the conjugate and the efficacy of killing tumor cells, it is preferable to conjugate 2-4 frog egg ribonucleases per transferrin molecule; more preferably, 2-3 frog egg ribonucleases are coupled to each transferrin molecule.
The invention also provides the application of the transferrin-frogspawn ribonuclease conjugate in preparing a composition for inhibiting tumors (tumor cells). Preferably, the tumor is a tumor highly expressing transferrin receptor; the higher the expression level of the tumor transferrin receptor, the better the effect of the conjugate of the invention.
Method for preparing conjugate
The inventor finds that the transferrin-frogspawn ribonuclease conjugate prepared by using SPDP as a coupling agent has large randomness, different structures, large difference of molecular weight, is not beneficial to purification, and has low proportion of the conjugate entering tumor cells.
The present inventors have conducted long-term studies and improvements to find a method for preparing a transferrin-frogspawn ribonuclease conjugate which meets the requirements, comprising:
(1) coupling transferrin to frog egg ribonuclease using N-hydroxysuccinimide 3- (2-pyridyldithio) propionate (SPDP) and 2-Iminothiolane & HCl; and
(2) separating to obtain the transferrin-frogspawn ribonuclease conjugate.
As a preferred mode of the present invention, the method comprises:
(a) contacting transferrin with N-hydroxysuccinimide 3- (2-pyridyldithio) propionate (SPDP) to obtain N-hydroxysuccinimide 3- (2-pyridyldithio) propionate-treated transferrin;
(b) contacting the frog egg ribonuclease with 2-Iminothiolane & HCl to obtain frog egg ribonuclease protein treated by the 2-Iminothiolane & HCl;
(c) mixing the N-hydroxysuccinimide 3- (2-pyridyldithio) propionate-treated transferrin obtained in step (a) with the 2-Iminothiolane-HCl-treated frog egg ribonuclease protein obtained in step (b) such that transferrin and frog egg ribonuclease are coupled; and
(d) collecting the conjugate with molecular weight of 89-137 kD.
Transferrin is treated with N-hydroxysuccinimide 3- (2-pyridyldithio) propionate and coupled with 2-Iminothiolane-treated frog egg ribonuclease protein, and as a result, most of the resulting coupled products have a molecular weight of 89-137kD, i.e., most of the conjugates contain 1 transferrin molecule (about 77kD) and 1-5 frog egg ribonuclease molecules (about 12kD for 1 frog egg ribonuclease molecule). The conjugate has good capacity of entering tumor cells and killing the tumor cells. In addition, the conjugate is stable in structure and relatively concentrated in molecular weight in a certain range, so that the subsequent purification is facilitated.
In a preferred embodiment of the present invention, the molar ratio of transferrin to N-hydroxysuccinimide 3- (2-pyridyldithio) propionate as a coupling agent in the treatment with the coupling agent is 1 (5-10), and the molar ratio of frogspawn ribonuclease to 2-Iminothiolane HCl as a coupling agent is 1 (5-10), and the treatment in this ratio can finally provide a more desirable coupling product.
As a preferred mode of the invention, in step (c), the molar ratio of the N-hydroxysuccinimide 3- (2-pyridyldithio) propionate-treated transferrin obtained in step (a) to the 2-Iminothiolane-treated frog egg ribonuclease protein obtained in step (b) is 1 (2-4); more preferably, the molar ratio is about 1: 3.
As a preferred mode of the present invention, after the transferrin is treated with SPDP, in order to remove impurities and retain the treated transferrin, conventional purification methods can be used for purification, including but not limited to: chromatography, dialysis. In addition, after the frog egg ribonuclease is treated by 2-Iminothiolane & HCl, or after transferrin treated by the coupling agent is mixed with the frog egg ribonuclease treated by the coupling agent and coupling reaction is carried out, the purified frog egg ribonuclease can also be purified to obtain a purer intermediate product or product.
As a preferred mode of the present invention, in step (d), the free transferrin and Onc are removed by strong cation exchange chromatography to collect the conjugate with a molecular weight of 89-137 kD. The ion exchange method has high yield and high purity.
The identification of the conjugate product can be carried out by techniques commonly used in the art, such as electrophoresis, by observing the position and size of the electrophoretic band to determine whether the conjugate obtained is desired.
Pharmaceutical composition
The present invention also provides a composition for inhibiting a tumor, the composition comprising: (i) an effective amount (e.g., 0.00001-50uM) of the transferrin-frogspawn ribonuclease conjugate of the invention; and (ii) a pharmaceutically acceptable carrier.
As used herein, an ingredient that is "pharmaceutically or dietetically acceptable" is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Sufficient details regarding pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991). Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, glycerin and sorbitol. In addition, auxiliary substances, such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers, such as albumin and the like, may also be present in these carriers.
The compositions may be formulated into a variety of dosage forms suitable for mammalian administration including, but not limited to: injection, capsule, tablet, emulsion, and suppository.
The composition of the invention can be directly used for killing tumor cells. In addition, it may be used in combination with other therapeutic agents or adjuvants.
The main advantages of the invention are:
(1) in the conjugate, transferrin can bring the frogspawn ribonuclease into tumor cells through the pint receptor-mediated pinocytosis, thereby enhancing the killing capacity and specificity of the frogspawn ribonuclease on the tumor cells.
(2) In the conjugate of the invention, 1-5 frog egg ribonuclease molecules are coupled on each transferrin molecule, so the molecule has moderate size and is easy to penetrate the cell membrane of tumor cells and enter the cells.
(3) The conjugate of the invention is basically not mixed with unconjugated free transferrin and frog egg ribonuclease, and ensures that the effect of the drug is completely generated by the conjugate of the invention.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.
Example 1 Onc protein preparation
1. Construction of Onc-expressing plasmids
According to SEQ ID NO: 3, synthesizing the coding sequence of the Onc protein by the complete sequence, and designing primers at two ends as follows:
the 5' primer is: CCCAGGACTGGCTGACTTTCCA (SEQ ID NO: 5);
the 3' primer is: AAAGTCGACTCAGCAAGAACCAACACC (SEQ ID NO: 6);
taking the synthesized gene sequence as a template, and taking the sequence shown in SEQ ID NO: 5 and SEQ ID NO: 6 as a primer, carrying out PCR amplification, carrying out enzyme digestion on an amplification product by SalI, and cloning the amplification product between MscI and SalI sites of a pET22b (+) vector (Novagen) to obtain an expression plasmid capable of expressing Onc.
Onc protein expression
Coli BL21(DE3) was transformed with the expression plasmid constructed above, and the resulting clones were picked overnight in 50ml of LB medium and cultured overnight at 37 ℃. The following day, 1:20 transfer of 1LTB medium, culture at 37 deg.C to OD600When the concentration of IPTG was increased to 0.5mM, induction was carried out for 3.5 hours at 4 ℃ and 6000rpm for 10 minutes, and the cells were collected and used for the next inclusion body collection and purification.
3. Inclusion body washing
Suspending the collected cells in a buffer SSB _ A (20mM Tris-HCl, 10mM EDTA, pH8.0), and disrupting the cells with an ultrasonicator; centrifuging at 12000rpm for 10 min at 4 deg.C, and collecting inclusion body; suspension of the inclusion bodies in a WIBB1(20mM Tris-HCl, pH8.0, 10mM EDTA, 2% Triton X-100 and 2M urea, pH8.0) solution, washing with shaking, centrifugation at 12000rpm for 10 minutes at 4 ℃ and collection of the inclusion bodies, and repeating this step three times; suspending the inclusion bodies in a WIBB2(20mM Tris-HCl, 10mM EDTA, pH8.0) solution, washing with shaking (assisted with low-power ultrasonic waves), centrifuging at 12000rpm for 10 minutes at 4 ℃ and collecting the inclusion bodies; this step was repeated 2-3 times.
4. Inclusion body solubilization and renaturation
Dissolving the inclusion body obtained from 1L of the bacterial solution in 6ml of DIBB (20mM Tris-HCl, 7M guanidine hydrochloride, 10mM DTT, 10mM EDTA, pH8.0) solution, charging nitrogen gas, and standing at room temperature for 5 h; centrifuging at 4 deg.C and 12000rpm for 15min, collecting supernatant, and quantifying protein to 20 mg/ml; 1ml of the inclusion body solution was quickly diluted into 50ml of DB (20mM acetic acid) solution, centrifuged at 12000rpm for 30min at 4 ℃ and the precipitate discarded; removing the denaturant by DB dialysis; adding 20 XB (100mM Tris-AcOH, 100mM NaCl, 3.0mM reduced glutathione, 0.6mM oxidized glutathione, pH8.0) to the solution and adjusting pH to 8.0, centrifuging at 12000rpm for 30min at 4 ℃ to remove the precipitate; renaturation is carried out for 36-48 hours at 4 ℃ in a renaturation buffer RB solution, and protein precipitates generated in the renaturation process are removed after 12000 minutes at 4 ℃; after desalting the supernatant by dialysis, the protein solution was stored at 4 ℃ until use.
5. Purifying by cation exchange column chromatography
And (3) quantifying the protein solution with good renaturation and then loading the protein solution on a strong cation exchange column.
(1) The column was SP Sepharose (2X 2cm, Pharmacia), the mobile phases were LB (10mM phosphate buffer (pH6.0)) and WB (1M NaCl, 10mM phosphate buffer (pH6.0)), and the flow rate was 1 ml/min.
The elution method comprises the following steps: 30 column volumes were eluted with 100% WB and collected in fractions and samples containing protein were assayed by Coomassie Brilliant blue G250. The protein-containing sample was collected and desalted with a dialysate 10mM phosphate buffer (pH6.0) and then used.
(2) Quantitatively passing the desalted sample solution through a strong cation exchange column. The column was a pre-packed MonoS (HR5/5, 1ml, pharmacia) column, and the mobile phases were LB (10mM phosphate buffer (pH6.0)) and WB (1M NaCl, 10mM phosphate buffer (pH6.0)) at a flow rate of 1 ml/min.
The elution method comprises the following steps: the column volume was eluted with 0-100% WB for 20 column volumes and collected in steps, and protein peaks corresponding to around 20% were collected.
The products of each stage of the Onc protein preparation (including total bacterial protein before induction (lane 1), total bacterial protein after induction for 2.5 hours (lane 2), inclusion body of bacterial after induction (lane 3), Onc after renaturation and purification (lane 4), etc.) were analyzed by conventional 15% SDS-PAGE, and the results are shown in FIG. 1.
Example 2 transferrin Tf and ribonuclease Onc conjugate preparation
A. Coupling Using SPDP and 2-Iminothiolane
Tf (from CALBIOCHEM, lot No. Cat: 616397, powder form, amino acid sequence shown in SEQ ID NO: 2) was dissolved in PBS (pH7.4) to a final concentration of 20mg/ml, 50. mu.l (equivalent to 10 times the amount of Tf in terms of molar ratio) of 50mM SPDP was added per ml, mixed rapidly, reacted for 30min, separated using a desalting column HiTrap desaling (5X 5ml, Pharmacia) mobile phase PBS (pH7.4), collected stepwise, and 10% SDS-PAGE was detected to leave an egg sample for use.
2. The Onc protein prepared and purified as described above was dissolved in 20mM phosphate buffer (pH6.0) to a final concentration of 1mg/ml, and 16.67. mu.l of 50mM 2-Iminothiolane. HCl (Promega, corresponding to about 10 times the molar ratio of Onc) was added thereto, followed by rapid mixing, reaction for 1 hour, and dialysis to remove small molecules.
Etc. 3, Onc and Tf in a molar ratio of 3: 1 mix the two and stay at 4 ℃ overnight.
4. The overnight samples were loaded onto a molecular sieve column Superdex 7510/300 GL (pharmacia) and the mobile phase 20mM NaHPO4And (4) separating and removing the small molecular protein at the pH value of 6.0, and reserving the large molecular protein mixture for later use.
5. The macromolecular protein mixture was loaded onto an ion exchange column Mono S (HR5/5, 1ml, Pharmacia). Mono S was equilibrated with 10mM phosphate buffer (pH6.0), eluted with a 0-1M NaCl gradient at a flow rate of 1ml/min, and unreacted Tf was separated off while eluting the conjugate, and the conjugate product was concentrated.
6. Reduced and non-reduced treated samples were identified by 15% SDS-PAGE and 10% SDS-PAGE: the samples were mixed with reducing reagent (0.1M Tris-HCl (pH6.8), 20% glycerol, 4% SDS, 0.005% (W/V) bromophenol blue, 10% 2-mercaptoethanol) and non-reducing reagent (0.1M Tris-HCL (pH6.8), 20% glycerol, 4% SDS, 0.005% (W/V) bromophenol blue) in equal volume ratio, respectively, to prepare protein electrophoretic samples for electrophoretic identification.
The results are shown in FIG. 2, in which FIG. 2(a) is a 15% SDS-PAGE gel, and lane 1 is a non-reduced Onc purified product; lane 2 is pure non-reduced Tf; lane 3 is non-reduced Onc-Tf; lane 4 is pure reduced Onc; lane 5 is pure reduced Tf; lane 6 is reduced Onc-Tf; lane 7 is a protein molecular weight standard. FIG. 2(b) is 10% SDS-PAGE gel electrophoresis further showing the molecular weight distribution of transferrin and frog egg ribonuclease-coupled protein.
B. Coupling with SPDP alone
In this example, SPDP alone was used to couple Tf and Onc proteins.
Tf and Onc proteins were diluted to protein solutions of 20mg/ml and 1mg/ml, respectively, and the pH was adjusted to 7.4 for use as described above.
1. 1ml of each sample reacts with SPDP with 10 times of molecular excess respectively, and the mixture is quickly and uniformly mixed to obtain Onc-PDP, and the Onc-PDP is placed at room temperature for 30min and then dialyzed to remove small molecules.
2. Adding DTT with the final concentration of 2mM to reduce the Onc-PDP, and standing for 1h at room temperature; DTT was removed by dialysis with PBS (pH6.0).
3. BCA assay protein, the mole ratio of 3: 1 Onc was mixed with Tf and left overnight at 4 ℃.
The detection result of the coupling product shows that the coupling method obtains a larger ratio of high polymers (namely, the coupling between a plurality of Tf molecules (more than 2), or between a plurality of Tf molecules (more than 2) and a plurality of Onc molecules (more than 1)).
Example 3 comparison of the cytotoxic Effect of Onc-Tf and Onc on myeloma cells SP2/0
A. Conjugates prepared as described in example 2
Myeloma SP2/0 cells (purchased from ATCC) were cultured in RPMI 1640(GIBICO) medium containing 10% calf serum, penicillin (100 units/ml), streptomycin (100. mu.g/ml). Culturing in 96-well plate, adding 90. mu.l culture medium (5X 10)4Cells/ml), CO2(5%) after 12 hours of incubation in an incubator, the medium was removed and washed once with pre-chilled PBS, and 90. mu.l of serum-free medium (penicillin (100 units/ml), streptomycin (100. mu.g/ml) RPMI 1640) was added. Protein samples (Onc-Tf conjugate prepared as described in example 2, item A) were added in 10. mu.l volume to the control group, PBS was added, and after 2 hours of incubation, an equal volume of 20% calf serum was added. Followed byAfter 70 hours of post-culture, the cell viability was examined by the conventional MTT method.
As shown in FIG. 3, it can be seen that the toxic effect of Onc-Tf on myeloma cells is significantly stronger than Onc, 2000-fold higher than Onc.
B. Conjugates prepared using item B of example 2
Myeloma SP2/0 cells were cultured in RPMI 1640(GIBICO) medium containing 10% calf serum, penicillin (100 units/ml), streptomycin (100. mu.g/ml). Culturing in 96-well plate, adding 90. mu.l culture medium (5X 10)4cells/ml)，CO2(5%) culture was performed for 12 hours in an incubator, the culture medium was removed, washed once with pre-cooled PBS, and then 90. mu.l of serum-free culture medium (penicillin (100 units/ml), streptomycin (100. mu.g/ml) RPMI 1640) was added. Protein samples (Onc purified as prepared in example 1 and Onc-Tf conjugate as prepared in example 2) were added in 10. mu.l at the set concentration gradient, PBS was added to the control group, and after 2 hours of incubation, an equal volume of 20% calf serum was added. After 70h of subsequent incubation, cell viability was measured by conventional MTT assay.
The results are shown in FIG. 4, which indicates that the Onc-Tf conjugate prepared as described in item B has only about 10-fold higher toxic effects on myeloma cells than Onc.
Example 4 comparison of the cytotoxic Effect of Onc-Tf and Onc on neuroblastoma SH-SY5Y
Neuroblastoma SH-SY5Y (ATCC) was cultured in DMEM (GIBICO) medium containing 10% fetal bovine serum, penicillin (100 units/ml), streptomycin (100. mu.g/ml). Culturing in 96-well plate, adding 90. mu.l culture medium (5X 10)4Cells/ml), CO2(5%) after 12h incubation in the incubator, the medium was removed and washed once with pre-chilled PBS, and 90. mu.l serum-free medium (penicillin (100 units/ml), streptomycin (100. mu.g/ml) DMEM) was added. Protein samples (Onc purified as prepared in example 1 and Onc-Tf conjugate as prepared in example 2) were added in 10. mu.l at the set concentration gradient, PBS was added to the control group, and after 2 hours of incubation, an equal volume of 20% fetal bovine serum was added. After 70h of subsequent incubation, cell survival was determined by MTT assayAnd (4) rate.
As shown in FIG. 5, it can be seen that the toxic effect of Onc-Tf on neuroblastoma is significantly stronger than that of Onc, at least 100 times higher than that of Onc.
Example 5 composition
1ml of the Onc-Tf conjugate obtained in example 2, item A, was formulated in 100ml of normal physiological saline to obtain a composition containing the Onc-Tf conjugate.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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<400>6
Claims (10)
1. An isolated transferrin-frogspawn ribonuclease conjugate, wherein the conjugate comprises 1 transferrin and 1-5 frogspawn ribonucleases conjugated to the transferrin, and the molecular weight of the conjugate is 89-137 kD.
2. The conjugate of claim 1, wherein the transferrin has the amino acid sequence of SEQ id no: 2; or
The frog egg ribonuclease has the nucleotide sequence shown in SEQ ID NO: 4.
3. The conjugate of claim 1, which is prepared by the following method:
(1) coupling transferrin to frog egg ribonuclease using N-hydroxysuccinimide 3- (2-pyridyldithio) propionate and 2-Iminothiolane & HCl; and
(2) separating to obtain the transferrin-frogspawn ribonuclease conjugate.
4. A method of producing the transferrin-frogspawribonuclease conjugate of claim 1, comprising:
(1) coupling transferrin to frog egg ribonuclease using N-hydroxysuccinimide 3- (2-pyridyldithio) propionate and 2-Iminothiolane & HCl; and
(2) separating to obtain the transferrin-frogspawn ribonuclease conjugate.
5. The method of claim 4, wherein the method comprises:
(a) contacting transferrin with N-hydroxysuccinimide 3- (2-pyridyldithio) propionate to obtain transferrin treated with N-hydroxysuccinimide 3- (2-pyridyldithio) propionate;
(b) contacting the frog egg ribonuclease with 2-Iminothiolane & HCl to obtain frog egg ribonuclease protein treated by the 2-Iminothiolane & HCl;
(c) mixing the N-hydroxysuccinimide 3- (2-pyridyldithio) propionate-treated transferrin obtained in step (a) with 2-Iminothiolane-HCl-treated frog egg ribonuclease obtained in step (b) such that transferrin and frog egg ribonuclease are coupled; and
(d) collecting the conjugate with molecular weight of 89-137 kD.
6. The method of claim 5,
in step (a), the molar ratio of transferrin to N-hydroxysuccinimide 3- (2-pyridyldithio) propionate is 1: 5-10; or
In step (b), the molar ratio of the frog egg ribonuclease to 2-Iminothiolane HCl is 1 to (5-10); or
In step (c), the molar ratio of the N-hydroxysuccinimide 3- (2-pyridyldithio) propionate-treated transferrin obtained in step (a) to the 2-Iminothiolane & HCl-treated frog egg ribonuclease protein obtained in step (b) is 1: 1-4.
7. Use of the transferrin-frogspawn ribonuclease conjugate according to claim 1, for the preparation of a tumor inhibiting composition.
8. The use according to claim 7, wherein the tumour is a tumour which highly expresses transferrin receptor.
9. A composition for inhibiting a tumor, said composition comprising:
(i) an effective amount of the transferrin-frogspawribonuclease conjugate of claim 1; and
(ii) a pharmaceutically acceptable carrier.
10. A method of inhibiting the growth of tumor cells in vitro, the method comprising treating tumor cells with the transferrin-frogspawribonuclease conjugate of claim 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107513107A (en) * | 2016-06-15 | 2017-12-26 | 上海南方模式生物科技发展有限公司 | Antineoplastic amalgamation protein and its preparation method and application |
| CN109942715A (en) * | 2019-04-02 | 2019-06-28 | 河南师范大学 | A kind of recombinant fusion protein for targeted therapy of tumor and its preparation method and application |
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| US5529775A (en) * | 1988-04-06 | 1996-06-25 | Alfacell Corporation | Compositions comprising ONCONASE(™) and cisplatin, melphalan, or doxorubicin HCl |
| DE69731463T2 (en) * | 1996-02-21 | 2005-10-27 | The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services | RECOMBINANT RIBONUCLEASE PROTEINS |
| US20030099629A1 (en) * | 1999-03-11 | 2003-05-29 | Immunomedics, Inc. | Recombinant onconase and chemical conjugates and fusion proteins of recombinant onconase |
| CN1762489A (en) * | 2005-06-16 | 2006-04-26 | 广西医科大学 | Transferrin (Tf)-superoxide dismutase (SOD) conjugate and preparation process |
| CN100430417C (en) * | 2006-07-07 | 2008-11-05 | 广西医科大学 | Preparation method of transferrin (Tf)-nerve growth factor (NGF) conjugate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107513107A (en) * | 2016-06-15 | 2017-12-26 | 上海南方模式生物科技发展有限公司 | Antineoplastic amalgamation protein and its preparation method and application |
| CN107513107B (en) * | 2016-06-15 | 2022-03-15 | 上海南方模式生物科技股份有限公司 | Anti-tumor fusion protein and preparation method and application thereof |
| CN109942715A (en) * | 2019-04-02 | 2019-06-28 | 河南师范大学 | A kind of recombinant fusion protein for targeted therapy of tumor and its preparation method and application |
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