CN102344938A

CN102344938A - Experimental technique for large-scale production of recombinant soluble human stem cell factor and immunoglobulin Fc segment fusion protein (sSCF-Fc) in mammalian cell

Info

Publication number: CN102344938A
Application number: CN2011102743768A
Authority: CN
Inventors: 柴笑梅; 朱月珍; 杨宣武; 周慧芳; 王小娟; 江永海
Original assignee: ABZYME BIOTECHNOLOGY Inc
Current assignee: ABZYME BIOTECHNOLOGY Inc
Priority date: 2011-09-16
Filing date: 2011-09-16
Publication date: 2012-02-08

Abstract

The invention relates to a technique for transferring Fc segment fusion gene (sSCF-Fc) of human stem cell factor and human immunoglobulin G1 (IgG1) into a mammalian cell for large-scale production of a protein product of the gene, and aims to establish a method for large-scale production of sSCF-Fc protein with biological activity by using the mammalian cell. The experimental technique meets the overall requirements of reducing production cost, simplifying process flow and improving product yield and quality in industrial production. By adopting the method, a large amount of sSCF-Fc protein with high purity and high biological activity can be obtained through normal large-scale cell culture. The technique is applied to large-scale production of the sSCF-Fc protein; and the cost can be obviously reduced, the process flow can be simplified, and the product yield and quality can be improved.

Description

The experimental technique of mammalian cell scale operation recombinant soluble human stem cell factor and immunoglobulin Fc fragment fusion rotein (sSCF-Fc)

Technical field

The present invention relates to a kind of Fc section fusion gene (sSCF-Fc) and change the technology of mammalian cell over to its protein product of scale operation with human stem cell factor and human normal immunoglobulin IgGl.

Background technology

1.SCF biological action and application prospect

STEM CELL FACTOR (stem cell factor, SCF), claim again the KIT part (KIT Ligand, KITLG) or mast cell growth factor (Mast cell growth factor MGF), is a kind of and proto-oncogene c-Kit product bonded glycoprotein (CD117).SCF through variable shearing can form transmembrane and two kinds of forms of solubility protein product, the two is biologically active all, in the processes such as formation of profit and other disease take place, develop and invade for body development, tumour, plays an important role.

SCF is most important for the hemoposieis in the embryo development procedure.SCF instructs hemopoietic stem cell to get into hematopoieticmicroenviron-ment, and keeps the survival ability and the regenerative power of hemopoietic stem cell.The SCF of solubility or cell surface is through combining to influence mastocyte, hemopoietic forebody cell, sexual cell and the melanocytic differentiation and the function of effect with its cell surface receptor c-Kit.The tissue that all blood vessels take place such as marrow, tire liver etc. all have SCF to express.There is research to show that SCF or its expression of receptor defective can cause macrocytic anemia disease even death.

SCF also plays an important role in the spermatogeny process.SCF is the key adjusting molecule that spermary forms and grows, and itself and receptors bind activated signal path are all being brought into play important regulatory role for propagation, migration, reduction division and the apoptosis thereof of sexual cell.The formation of the time that SCF expresses and zone and sexual cell and the path of migration are consistent, and phase also has identity with the forming process of sexual cell during its receptor expression.The SCF system is not limited only to embryonic stage to the regulation and control of people's androgone, all brings into play regulating and controlling effect in a different manner in pubescence and spermatogenetic subsequently whole process.SCF and acceptor disappearance thereof can cause infertility.

SCF is extremely important for melanocytic location in growth course.The pigmentation of body can produce melanic melanophore control by one type.SCF instructs it to be divided into melanophore from central nervous system arrival skin or hair specific region through the Kit acceptor that is combined into the melanophore surface.In addition, SCF is for the last eventually melanophore survival of breaking up and the adjusting no less important of propagation in the adult.SCF table defective can cause piebaldism.

The SCF abnormal expression is in close relations with some tumour also.Compare with normal hepatic tissue, the proteic expression of SCF and c-Kit obviously reduces in the liver cancer tissue; After the patient with esophageal carcinoma gastroesophagostomy, the SCF expression level obviously reduces than the normal people in its stomach mucous membrane and the remaining oesophagus; SCF expression level and positive rate significantly improve in the cholangiocarcinoma patients body, and it is expressed and tumor grade is proportionate, and SCF expresses maybe be closely related with the generation of cholangiocarcinoma, and have participated in the profit process of invading of tumour.At present, SCF has been used for some treatment of diseases.SCF is used for early stage diagnosis or evaluating prognosis as the important detection index of related neoplasms; In addition, SCF also can be used as the novel targets in the oncotherapy, has significance for the blocking-up diffusion transfer organized towards periphery of tumour cell or the recurrence of tumour.

In addition, the success of the large scale culturing of external hemopoietic stem cell or other multipotential stem cells is that organ transplantation provides possibility.SCF is a cytokine indispensable in the stem cell culturing process; With SCF and other cytokine (like IL-3; IL-6; IL-1; TPO; GM-CSF etc.) but the stem cell in synergy amplification in vitro marrow or peripheral blood source; Like hemopoietic stem cell, endothelial progenitor cells, neural stem cell; Mescenchymal stem cell etc.; These stem cell direct feedbacks are to the patient body; Or external evoked its carry out tissue differentiation, and the organ transplantation of inducing formation in the patient body, is used for the treatment of leukemia, tumour or the big area surface of a wound.

At present, SCF also plays a role in other treatment of diseases process.The mobilization that SCF and other chemotherapeutics synergy leukaemic can improve hemopoietic forebody cell; SCF can be used for reducing the thrombocytopenia that produces in nonsmall-cell lung cancer and the breast cancer chemotherapy process.Preparation high reactivity, high purity, hypotoxic reorganization SCF albumen are to produce reagent for clinical diagnosis, the treatment prerequisite with SCF medicine and organ transplantation, for it provides offers additional possibilities and vast market prospect in aspect research such as scientific research, clinical diagnosis and treatment and application.

2.Fc Expression of Fusion Protein

The Fc fusion rotein of immunoglobulin IgG is meant at gene level the Fc fragment gene of goal gene with IgG is linked to each other and the recombinant protein of expression; The structural domain that has target protein and immunoglobulin (Ig) simultaneously; Thereby given the recombinant protein many new characteristics, expanded the application of albumen at scientific research, pharmacy and aspect such as clinical.

In the process of expression of recombinant proteins, the Fc recombinant protein is easy to combine Protein A, thereby has simplified proteic purge process; The Fc fragment can be carried out methods such as streaming, immunohistochemical methods and co-immunoprecipitation easily and detected, and can be used for interested gene studies; In the experiment in vitro, the Fc fusion rotein can be used for antagonism or the activating cells surface receptor is used for studying receptor-ligand reaction and signal path; In addition, the Fc recombinant protein can also be applied to display technique of bacteriophage, as as the human whole antibody of antigen prepd storehouse.

In addition, the Fc fragment has also given recombinant protein some other characteristics, has increased it in the using value at pharmacy and clinicing aspect.Add the constant region of heavy chain in the fusion rotein, its transformation period obviously prolongs, and has prolonged its transformation period in vivo; The Fc section can be passed placenta, conjugated complement and mediated cytotoxicity that complement relies on etc.; The hinge area structural domain that recombinant protein contains makes it form dimer or polymer, has strengthened its antigen bonding force.More than these characteristics expanded the potential applicability in clinical practice of fusion rotein aspect the treatment of infectious diseases, autoimmune disorder and graft-rejection.

3. present Research

The sSCF-Fc recombinant protein of producing in the world basically all is to adopt intestinal bacteria or yeast expression system to obtain at present, and protein product is not high without glycosylation or degree of glycosylation, and active and content of toxins is difficult to satisfy the purpose of clinical diagnosis and treatment.Giving birth to the sSCF-Fc recombinant protein with mammalian cell is production scientific research and clinical important channel with SCF, significant for new bio technical development that promotes the clinical diagnosis link and clinical medicine exploitation.

Summary of the invention

The present invention is intended to set up the proteic method of sSCF-Fc that has biologic activity with mammalian cell scale operation.In industrial production, satisfy the general requirement that production cost reduces, simplifies technical process, improves product production and quality.

The present invention includes the preparation transfection with plasmid and PEI, plasmid transfection 293T cell and expression, protein purification and etc. four steps.As transfection reagent, it can combine double-stranded DNA efficiently as " proton sponge " with PEI, it is brought in the purpose cell, protein product can be in the of short duration time (generally can express the time in a week) thus in obtain a mistake property expression and obtain a large amount of albumen.

The used 293T cell of the present invention is that the industrial production people is clinical in the most common a kind of cell of albumen.Not only expression level is high; Native protein is similar in protein translation post-treatment mode such as glycosylation etc. and the body; And there is not a potential biological hazard property; Thereby self excretory albumen has seldom been simplified follow-up purge process, is particularly suitable for the scale operation of products such as secretor type cytokine or chemokine.And this cell can produce in serum free medium through domestication, is essential for reducing industrial cost.Cell density can improve tens of times even thousands of times, has improved protein yield greatly.

Adopt present method, can cultivate the sSCF-Fc albumen that obtains a large amount of high purity high biological activities through common mass cell.This technology is applied to the proteic scale operation of sSCF-Fc, can be significantly reduced to this, simplify technical process, improves product production and quality.

Description of drawings

Figure is the sSCF-Fc recombinant protein electrophoresis result figure after SDS-PAGE identifies Protein A affinity chromatography column purification

Embodiment

1. transfection is with the preparation of plasmid

With removing the required sSCF-Fc expression of gene plasmid pR-sSCF-Fc that is connected with of the big extraction reagent kit of endotoxic plasmid (available from Qiagen company) preparation transfection.Method is seen specification sheets.With 200 μ l TE damping fluids dissolving plasmid, ultraviolet spectrophotometer is surveyed its concentration.

2. transfection is with the preparation of PEI

1) in the beaker of 500ml, pours the ultrapure water that 450ml Milli-Q prepares into;

2) the linear PEI (available from Poly Science company) of balance weighing 500mg adds in the beaker, and magnetic stirring apparatus constantly stirs;

3) dropwise add the dense HCl of 12M for preparing in advance and make its pH＜2.0 (approximately needing 800 μ l).Stirred PEI solution about 2-3 hour;

4) add the 10M NaOH solution of preparing in advance pH was transferred to for 7.0 (approximately needing 500 μ l).With the Milli-Q ultrapure water PEI solution is settled to 500ml.

5) 0.22-μ m membrane filtration PEI solution is distributed into the 1ml/ pipe, and-20C preserves.

3. plasmid transfection 293T cell

1) preceding 1 day of transfection (about 24 hours), with the 293T cell with nonreactive, contain 10% foetal calf serum the DMEM perfect medium resuspended, according to 5 * 10 ⁵/ ml spreads in the 150mm culture dish, and every bottle adds 25mL, overnight incubation in the 5%CO2 incubator;

2) transfection same day, required Opti MEM substratum (or phosphate buffered saline buffer) is preheating to 25 °-37 ℃.Dissolving on ice connects SCF-Fc expression of gene plasmid and PEl;

3) in the Ep of 5ml pipe, add 2.5ml Opti MEM/PBS, add the plasmid DNA (ultraviolet spectrophotometer survey its concentration after, add elution buffer adjustment concentration) that 43.75 μ g are connected with the sSCF-Fc gene, vortex gently on vibrator to 1ug/ul;

4) PEI of adding 262.5 μ L 1mg/ml vibrates 3 times each 3 seconds immediately on vibrator.Under the room temperature mixed solution was hatched 15 minutes;

5) take out the cell of spreading culture dish in advance, the DNA/PEI mixed solution added in the cell culture fluid,, jiggle the culturing bottle mixing; Cell is put into incubator to be continued to cultivate.

4. expression product is collected and purifying

1) transfection is after 6 hours, with Tissue Culture Dish fully in the substratum sucking-off, add the 25ml serum free medium (serum free media, SFM).Adding the Sodium.alpha.-ketopropionate continued 4mM L-glutaminate and 1mM simultaneously cultivates;

2) changed liquid respectively and added fresh SFM and the Sodium.alpha.-ketopropionate of L-glutaminate and 1mM in 2 days and 4 days after the transfection.Collecting cell supernatant (noting cell not being removed), 12, the centrifugal 10min of 000rpm/min removes dead cell and cell debris;

3) with supernatant and isopyknic combination the/rinsing damping fluid (0.15M NaCl, 20mM Na ₂HPO ₄, pH8.0).Meanwhile, with ultrapure water washing protein purification post, wash pillar with 1ml combination/rinsing damping fluid again;

4) Resin of absorption 1ml ProteinA adds purification column, and liquid is wherein flow to end; Add 5mL combination/rinsing damping fluid balance pillar, making its flow velocity is 1ml/min;

5) the supernatant liquor upper prop after will diluting, the adjusting flow velocity is 0.5ml/min;

6) 20mL combination/rinsing damping fluid is washed post, and the adjusting flow velocity is 2ml/min;

7) use 4 * 1ml 0.1M elution buffer (glycine, pH 2.5) eluted protein product at last, collect liquid with the 1.5ml centrifuge tube, the Tris-C1 that adds 110ul 1M pH 8.5 in the pipe in advance regulates the pH value.Ultraviolet spectrophotometer and SDS-PAGE survey protein concentration and purity is seen accompanying drawing.

Claims

1. one kind has the proteic method of biologic activity SCF-Fc with mammalian cell scale operation, it is characterized in that this method has the SCF-Fc albumen of biologic activity with mammalian cell scale operation through following steps:

The preparation transfection with plasmid and PEI, plasmid transfection 293T cell and expression, protein purification and etc. four steps.

2. the SCF-Fc albumen that has biologic activity with mammalian cell scale operation as claimed in claim 1 is characterised in that described SCF-Fc is through preparing purifying in the mammalian cell of stably manufactured.

3. the SCF-Fc albumen that has biologic activity with mammalian cell scale operation as claimed in claim 1 is characterised in that the soluble proteins that is expressed as secreted of described SCF-Fc.

4. the SCF-Fc albumen that has biologic activity with mammalian cell scale operation as claimed in claim 1 is characterised in that described SCF-Fc is applied to research and clinical diagnosis as biotechnological formulation.