CN104293735A - Cell line for preparing Col17-IgG1Fc fusion protein and application thereof - Google Patents
Cell line for preparing Col17-IgG1Fc fusion protein and application thereof Download PDFInfo
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Abstract
The invention discloses a cell line for preparing a Col17-IgG1Fc fusion protein and application thereof. The invention provides a 293T cell line BPKU in stable transfection by COL17NC16A-IgG1Fc plasmids, and the cell line has a preservation number of CGMCCNo.7801. The invention also protects the application of BPKU to preparation of Col17-IgG1Fc fusion protein, application of Col17-IgG1Fc fusion protein to preparation of drugs for the treatment of bullous pemphigoid. The existing drug rituximab for the treatment of bullous pemphigoid has the killing effect on all B cells and causes severe side effect. However, the Col17-IgG1Fc fusion protein has specific killing effect on COL17NC16A specific B cells in the patients with bullous pemphigoid function, can avoid killing other B cells, and has extremely low side effect. The cell line BPKU provided by the invention has the performance of high efficiency production of Col17NC16A-IgG1Fc fusion protein. The invention has great value to the treatment of bullous pemphigoid.
Description
Technical field
The present invention relates to a kind of clone for the preparation of Col17-IgG1Fc fusion rotein and application thereof.
Background technology
Bullous pemphigoid (Bullous pemphigoid, BP) is bullous disease under modal cutaneous autoimmune epidermis, mainly occurs in the elderly.With skin, clinical manifestation occurs that pruritic erythema, tension blister and bleb are for feature, mucosa lesions is more rare.Can be there is serious Electrolyte imbalance, hypoproteinemia in patient with severe symptoms, infection even septicemia occurs, can threat to life.At present the first-line treatment of this disease is system application glucocorticosteroid and/or immunosuppressor, and it is hemorrhage that life-time service may cause infection, thrombus, osteoporosis, diabetes, hypertension, digestive tract ulcer are bored a hole, the health and lives of serious threat patient.The Minocycline HCl promoted gradually in recent years and niacinamide conjoint therapy can obviously reduce treatment side effect, but still have a considerable amount of patient insensitive to above-mentioned conventional treatment, or occur the recurrence without obvious inducement.Analyze the previously defect for the treatment of means in curative effect and side effect, largely not special with its therapy target relevant; Most critical link not for BP morbidity is intervened.Therefore further investigate by means of to BP pathogenesis, screening key link wherein, as specific treatment target spot, may develop medicine that is more efficient, low toxicity.
The selectively targeted treatment development with biotechnological formulation being representative in recent years rapidly, provides new thinking for solving existing deficiency in current BP treatment.Because humoral immunization has mediated the principal pathogenetic process of BP, B cell synthesis pathogenic autoantibodies is the key morbidity link of BP, therefore utilize with B cell be attack target spot medicine---Rituximab (Rituximab) kills and wounds B cell, the generation of autoantibody capable of blocking, thus the object reaching treatment BP.Rituximab is the anti-CD-20 monoclonal antibody of gene recombination synthesis, can specifically in conjunction with B cell surface specific antigens c D20, the Fc receptors bind of its cell surface such as Fc section and NK cell, cytotoxic T cell, scavenger cell, can induction of antibodies dependent cells mediation cellulotoxic effect (ADCC), simultaneously by the cellulotoxic effect (CDC) that antibody Fc section activating complement relies on, thus reach the effect of killing and wounding B cell.Multiple case retrospective research has reported the successful experience utilizing rituximab treatment BP.The curative effect of this targeted therapy and specificity significantly improve all comparatively before.But, kill and wound all B cell non-specificly and body can be caused to lack B cell completely within a certain period of time, add the risk of severe infections.Removing completely and rebuilding of B cell also may make the B cell immunological memory of patient impaired.Some patients fails to respond to any medical treatment to this kind or occurs recurrence, and the pathologic B cell that prompting produces autoantibody may not be completely removed.
Summary of the invention
The object of this invention is to provide a kind of clone for the preparation of Col17-IgG1Fc fusion rotein and application thereof.
The invention provides the 293T clone BPKU of a kind of COL17NC16A-IgG1Fc plasmid stabilisation transfection, be called for short clone BPKU, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 25th, 2013 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), its deposit number is CGMCC No.7801.
The present invention goes back the application of Cell protection system BPKU in preparation Col17-IgG1Fc fusion rotein; The protein of described Col17-IgG1Fc fusion rotein for being made up of the aminoacid sequence shown in sequence in sequence table 1.
The present invention also protects a kind of method preparing described Col17-IgG1Fc fusion rotein, comprises the steps: culturing cell system BPKU and collects culture supernatant, is the solution containing described Col17-IgG1Fc fusion rotein.DMEM incomplete substratum can be adopted when cultivating described clone.Described method also can comprise the steps: to get described culture supernatant, the centrifugal 10min of 12000rpm, and collect supernatant liquor and add in 50kD ultra-filtration centrifuge tube, 4 DEG C, the centrifugal 15min of 4000g, obtain the protein concentrated solution containing described Col17-IgG1Fc fusion rotein.
The present invention goes back the application of Cell protection system BPKU in the medicine for the preparation for the treatment of bullous pemphigoid.
The present invention also protects a kind of medicine being used for the treatment of bullous pemphigoid, and its activeconstituents is the Col17-IgG1Fc fusion rotein that the above method prepares.
The present invention also protects the application of described Col17-IgG1Fc fusion rotein in the medicine for the preparation for the treatment of bullous pemphigoid.
The present invention also protects a kind of medicine being used for the treatment of bullous pemphigoid, and its activeconstituents is described Col17-IgG1Fc fusion rotein.
The present invention also protects described Col17-IgG1Fc fusion rotein and the gene of described Col17-IgG1Fc fusion rotein of encoding.Described gene specifically can as shown in the sequence 2 of sequence table.
Existing bullous pemphigoid medicine Rituximab can not distinguish COL17NC16A specific b cells, all has lethal effect, so side effect is larger to all B cell.Col17-IgG1Fc fusion rotein has the effect of the COL17NC16A specific b cells in specific killing patients with bullous pemphigoid body, and can avoid killing and wounding other B cell, side effect is extremely low.Clone BPKU provided by the invention, has the performance of High-efficient Production Col17-IgG1Fc fusion rotein.The present invention has substantial worth for the treatment of bullous pemphigoid.
Accompanying drawing explanation
Fig. 1 is the structural representation of plasmid pFUSE-hIgGle4-Fc2.
Fig. 2 is the SDS-PAGE electrophorogram of the protein concentrated solution that each re-treatment obtains.
Fig. 3 is the western blot result of the protein concentrated solution that in embodiment 1, first re-treatment step 2 obtains.
Fig. 4 is the cells were tested by flow cytometry result of the sorting positive cell liquid of first BP patient in embodiment 2.
Fig. 5 is the result of first BP patient in the step one of embodiment 3.
Fig. 6 is the result of first BP patient in the step 2 of embodiment 3.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.This experiment is followed " Declaration of Helsinki ", obtains the approval of Ethics Committee of Peking University First Hospital, and patient and Healthy Volunteers all endorsed Informed Consent Form.
Lipofectamine2000：Invitrogen（11668-019）。50kD ultra-filtration centrifuge tube: Sartorius.Horseradish peroxidase-labeled goat anti-human IgG antibodies: health is century.Horseradish peroxidase-labeled rabbit anti goat igg antibody: health is century.Goat anti human IgG(H+L) antibody: health is century.Anticoagulant heparin pipe (green pipe vacuum test tube): BD company.D-Hank liquid (not calcium-magnesium-containing ion): Beijing ancient cooking vessel state is prosperous.Erythrocyte cracked liquid (Type B): TIANDZ company.Blood cell counting plate: refinement medical apparatus factory.Magnetic bead sorting damping fluid: Miltenyi Biotec.CD19 magnetic bead: Miltenyi Biotec.CD19-PE:BD company.HEK293T cell: Beijing consonance cell bank.LS type cell sorting post: Miltenyi Biotec.APEX?Antibody?Labling?Kit：Invitrogen。Rituximab: Roche.Plasmid pFUSE-hIgGle4-Fc2(structural representation is shown in Fig. 1): Invivogen company, Catalog#pfc2-hg1e4.The incomplete substratum of DMEM: Gibico.DMEM perfect medium: add foetal calf serum in the incomplete substratum of DMEM, makes its volume percent content be 10%.PBS damping fluid (pH7.4,0.01mol/L): get NaCl8g, KCl0.2g, NaH
2pO
41.44g and KH
2pO
40.24g, dissolves with distilled water and is settled to 1000ml.
BP (Bullous Pemphigoid): bullous pemphigoid.ADCC (Antibody Dependent Cell Cytotoxicity): Antibody-dependent cell cytotoxicity effect.CDC (Complement Dependent Cytotoxicity): the cytotoxicity of Complement Dependent.PBMC (Peripheral Blood Mononuclear Cell): peripheral blood mononuclear cell.
The acquisition of embodiment 1, clone BPKU
One, the structure of recombinant plasmid
1, the double chain DNA molecule shown in sequence 2 of composition sequence table.
2, with step 1 synthesize double chain DNA molecule for template, with F1 and R1 composition primer pair carry out pcr amplification, reclaim pcr amplification product.
F1：5’-CCG
GAATTCGAGGAGGTGAGGAAGCTGAA-3’；
R1：5’-GGA
AGATCTTCCTCGGAGATTTCCATT-3’。
3, use the pcr amplification product of restriction enzyme EcoRI and BglII double digestion step 2, reclaim digestion products.
4, with restriction enzyme EcoRI and BglII double digestion plasmid pFUSE-hIgGle4-Fc2, about 4172bp carrier framework is reclaimed.
5, the digestion products of step 3 is connected with the carrier framework of step 4, obtains recombinant plasmid
pFUSE-hIgG1e4-Fc2-Col17。According to sequencing result, structrual description carries out to recombinant plasmid pFUSE-hIgG1e4-Fc2-Col17 as follows: between EcoRI and the BglII restriction enzyme site of plasmid pFUSE-hIgGle4-Fc2, insert the sequence 2 of sequence table from the double chain DNA molecule shown in 5 ' end the 1 to 234 Nucleotide.The fusion dna molecule shown in sequence 2 of the encoding sequence formation sequence table of IgG1Fc fragment in the double chain DNA molecule shown in sequence 2 of sequence table and plasmid pFUSE-hIgGle4-Fc2, the protein shown in sequence 1 of expressed sequence table.In the protein shown in sequence 1 of sequence table, from N-terminal the 1 to 78 amino acids residue composition Col17 fragment (also known as COL17NC16A fragment or COL17NC16A epitope), the 81 to 307 amino acids residue composition IgG1Fc fragment.By the polypeptide called after Col17-IgG1Fc fusion rotein shown in the sequence 1 of sequence table, by the DNA molecular called after Col17-IgG1Fc fusion gene shown in the sequence 2 of sequence table.
The progress of BP pathogenesis aspect confirms that the autoantibody for the NC16A epi-position of COL17 molecule in hemidesmosome is the most important pathogenic antibody causing BP.From BP patient's blood, isolate the specific B cell of COL17NC16A, these B cell can secrete the antibody of anti-COL17NC16A in vitro.Therefore the COL17NC16A specific b cells in patient body can be used as the crucial target spot for the treatment of BP.Col17-IgG1Fc fusion rotein, is combined with NC16A specific b cells by COL17NC16A epitope, and utilize Fc section induction CDC and the ADCC effect of IgG molecule, targeting kills and wounds pathologic B cell simultaneously.
Two, the acquisition of reconstitution cell
5 re-treatments are set, proceed as follows respectively:
1,24h before transfection, by collected by trypsinisation HEK293T cell, by 6 × 10
5individual plating cells, in 10cm Tissue Culture Dish, adds 10ml DMEM perfect medium, in 37 DEG C containing 5%CO
2incubator in incubated overnight, liquid (namely replacing medium to the incomplete substratum of 5ml DMEM) is changed in transfection for first 2 hours.
2, get 8 μ g recombinant plasmid pFUSE-hIgG1e4-Fc2-Col17, mix with the incomplete substratum of 0.5ml DMEM.
3, get 20 μ l Lipofectamine2000, mix with the incomplete substratum of 0.5ml DMEM, incubated at room 5 minutes.
4, the liquid-phase system mixing that liquid step 2 obtained and step 3 obtain, incubated at room 20 minutes.
5, the liquid-phase system that step 4 obtains is added in the Tissue Culture Dish of step 1, in 37 DEG C containing 5%CO
2incubator in hatch 6 hours, abandon supernatant, add DMEM perfect medium and in 37 DEG C containing 5%CO
2incubator in cultivate 24 hours.
6, collect the cell that step 5 obtains, cultivate with after trysinization at the DMEM perfect medium containing 600 μ g/ml bleomycins, the DMEM perfect medium containing 600 μ g/ml bleomycins more renewed for every 2 days, in 37 DEG C containing 5%CO
2cultivate 14 days in incubator.
7, collect the cell that step 6 obtains, in containing the DMEM perfect medium of 400 μ g/ml bleomycins, contain 5%CO in 37 DEG C with after trysinization
2incubator in cultivate (passage afterwards all carries out according to the method for this step), be replaced by after cell covers with the incomplete substratum of DMEM and under the same conditions cultivate two days, collect supernatant liquor.
Three, Col17-IgG1Fc fusion rotein qualification
1, get step 27 supernatant liquors obtained, the centrifugal 10min of 12000rpm, gets supernatant.
2, get the supernatant that 20ml step 1 obtains, add in 50kD ultra-filtration centrifuge tube, 4 DEG C, the centrifugal 15min of 4000g, obtain 1mL protein concentrated solution, with the total protein concentration in NANODROP2000c spectrophotometric determination protein concentrated solution.
Total protein concentration in the protein concentrated solution that first re-treatment obtains is 0.8mg/ml.
Total protein concentration in the protein concentrated solution that second re-treatment obtains is 0.42mg/ml.
Total protein concentration in 3rd protein concentrated solution that re-treatment obtains is 0.35mg/ml.
Total protein concentration in 4th protein concentrated solution that re-treatment obtains is 0.26mg/ml.
Total protein concentration in 5th protein concentrated solution that re-treatment obtains is 0.37mg/ml.
The protein content contained in the supernatant liquor that first re-treatment obtains is the highest.
3, protein concentrated solution step 2 obtained carries out SDS-PAGE electrophoresis, electrophorogram is shown in that (swimming lane 1 and swimming lane 2 represent first restructuring and process the protein concentrated solution obtained Fig. 2, swimming lane 3 represents the protein concentrated solution that second re-treatment obtains, swimming lane 4 represents the protein concentrate liquid that the 5th re-treatment obtains, swimming lane 5 represents the protein concentrate liquid that the 3rd re-treatment obtains), all show about 70kD obviously band, the protein concentration in the protein concentrated solution that first re-treatment obtains is significantly higher than the protein concentrated solution that other four re-treatments obtain.
4, the protein concentrated solution that first re-treatment step 2 obtains is carried out western blot, the results are shown in Figure 3.Primary antibodie is anti-BP patient activity phase serum (the 1:10 dilution of diluting with the TTBS damping fluid containing 5% skim-milk, active period, standard was that ELISA method records anti-COL17NC16A antibody horizontal and is greater than 150U/mL), two resist the Goat anti human IgG(H+L for horseradish peroxidase mark) antibody time the results are shown in Figure 3 swimming lane 1.Primary antibodie is Goat anti human IgG(H+L) antibody, two resist for horseradish peroxidase mark rabbit anti goat igg antibody time the results are shown in Figure 3 swimming lane 2.Primary antibodie be horseradish peroxidase mark Goat anti human IgG(H+L) antibody, do not add two anti-time the results are shown in Figure 3 swimming lane 3.Using the serum of anti-COL17NC16A antibody positive as primary antibodie, HRP mark Goat anti human IgG(H+L) antibody be two resist, show clear band at about 70kDa.With Goat anti human IgG(H+L) antibody is that primary antibodie is hatched, is two anti-ly to hatch with the rabbit anti goat igg antibody of HRP mark, shows clear band at about 70kDa.Directly with the Goat anti human IgG(H+L of HRP mark) antibody incubation, have no band.
5, the band of about 70kD in recycling step 3, deliver to Beijing Hua Da protein research and development centre, MALDI-TOF/TOF mass spectrometry method is utilized to carry out mass spectrometric detection to albumen, the theoretical value of mass-spectrometric data and Col17-IgG1Fc fusion rotein is compared, Mascot scoring is 22 points (threshold value is 13 points), illustrates that aminoacid sequence overlap ratio has significance.
6, the band of about 70kD in recycling step 3, check order, N holds 15 amino-acid residues and Col17-IgG1Fc fusion rotein N to hold 15 amino-acid residues consistent.
Four, the repeated pruning of clone
By in first re-treatment, the cell that 6 of step one obtains as P0 for cell, by P0 for cell by step one 7 method continuous passage 5 times, successively called after P1 for cell, P2 for cell, P3 for cell, P4 for cell and P5 for cell.By P1 for cell to P5 for cell respectively by step one 7 method obtain supernatant liquor, then carry out 1 and 2 of step 2,0.78mg/ml, 0.81mg/ml, 0.80mg/ml, 0.81mg/ml, the 0.81mg/ml successively of the total protein concentration in protein concentrated solution.
Five, the preservation of clone
By in first re-treatment, the 293T clone BPKU of the cell strain called after COL17NC16A-IgG1Fc plasmid stabilisation transfection that 6 of step one obtains, be called for short clone BPKU, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on 06 25th, 2013 and (be called for short CGMCC, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), its deposit number is CGMCC No.7801.
The Isolation and Identification of embodiment 2, peripheral blood B cell
One, the separation of peripheral blood B cell
From the active period BP patient of 3 informed consents (not carrying out any treatment such as hormone or immunosuppressor, without immune deficiency relative disease medical history before gathering whole blood) and healthy volunteer's peripheral blood of 3 informed consents, be separated B cell respectively, method is as follows:
1, gather whole blood with anticoagulant heparin pipe, dilute with D-Hank's liquid equal-volume.
2, in 50ml centrifuge tube, add the human lymphocyte parting liquid that GE company of the 15ml Ficoll-Paque(U.S. produces), then slowly add the diluent that 30ml step 1 obtains at liquid level place, then 18 DEG C, the centrifugal 30min of 400g, now cell layering.
3, draw the plasma layer (i.e. the superiors) in centrifuge tube, be experiment serum; Tunica albuginea layer (namely from up to down counting the second layer) in gentle aspiration centrifuge tube, move into another centrifuge tube, add appropriate PBS buffer solution, then the centrifugal 10min of 2000rpm, collecting cell precipitates.
4, add 1ml1 × erythrocyte cracked liquid in the cell precipitation obtained to step 3, softly blow and beat, room temperature places 10min, the then centrifugal 10min of 2000rpm, and collecting cell precipitates.
5, with the cell precipitation that the resuspended step 4 of appropriate PBS damping fluid obtains, with blood cell counting plate counting, PBMC sum is.
6, the centrifugal 10min of cell suspension 2000rpm step 5 obtained, collecting cell precipitates, according to every 10
7the ratio of the corresponding 80 μ l magnetic bead sorting damping fluids of individual cell and 20 μ l CD19 magnetic beads adds magnetic bead sorting damping fluid and CD19 magnetic bead, and fully latter 4 DEG C of mixing hatches 15min, then according to every 10
7the ratio of individual cell corresponding 2ml magnetic bead sorting damping fluid adds magnetic bead sorting damping fluid to wash, the then centrifugal 10min of 300g, and collecting cell precipitates, resuspended with 500ul magnetic bead sorting damping fluid.
7, the cell suspension that step 6 obtains is splined on LS type cell sorting post, add 9ml magnetic bead sorting damping fluid, preserve effluent liquid (containing the cell beyond B cell in total PBMC in effluent liquid, by the cell called after sorting negative cells beyond B cell in total PBMC, by this effluent liquid called after sorting negative cells liquid) parallel cell counting, then in LS type cell sorting post, 5ml magnetic bead sorting damping fluid is added, retain together with magnetic bead at interior suspension (containing B cell in suspension, by its called after sorting positive cell liquid).
Two, the qualification of peripheral blood B cell
1, get the sorting positive cell liquid that step one obtains, being adjusted to PBS damping fluid is 2 × 10
5individual cell/ml.
2,2 μ l CD19-PE are added in the cell suspension obtained in 1ml step 2, after abundant concussion, lucifuge hatches 30min on ice, add the PBS buffer solution of 2ml precooling, 4 DEG C, the centrifugal 5min of 250g, collecting cell precipitates, add the 0.5ml1% paraformaldehyde aqueous solution, utilize the ratio of cells were tested by flow cytometry CD19 positive cell (i.e. B cell).
The purity that the cells were tested by flow cytometry of the sorting positive cell liquid of first BP patient the results are shown in Figure 4, CD19 positive cell reaches 92.58%.Be separated from first BP patient 30ml peripheral blood and obtain B cell 8 × 10
5individual, sorting negative cells 3 × 10
7individual.Be separated from second BP patient 30ml peripheral blood and obtain B cell 11 × 10
5individual, sorting negative cells 7.6 × 10
7individual.Be separated from the 3rd BP patient 30ml peripheral blood and obtain B cell 14 × 10
5individual, sorting negative cells 9.1 × 10
7individual.Be separated from first healthy volunteer 30ml peripheral blood and obtain B cell 10 × 10
5individual, sorting negative cells 6 × 10
7individual.Be separated from second healthy volunteer 30ml peripheral blood and obtain B cell 12 × 10
5individual, sorting negative cells .3 × 10
7individual.Be separated from the 3rd healthy volunteer 30ml peripheral blood and obtain B cell 10 × 10
5individual, sorting negative cells 5 × 10
7individual.
The external functional examination of embodiment 3, Col17-IgG1Fc fusion rotein
One, the mark function of fluorescin measures
Adopt APEX Antibody Labling Kit by specification to be marked by 2 protein concentrated solutions obtained of the step 3 of first of embodiment 1 re-treatment, obtain fluorescent marker protein liquid first (by the upper 488nm fluorescence dye of Col17-IgG1Fc fusion rotein mark).
Adopt APEX Antibody Labling Kit by specification to be marked by Rituximab, obtain fluorescent marker protein liquid second (by the upper 488nm fluorescence dye of Rituximab mark).
Carry out application of sample according to table 1, by DMEM perfect medium adjustment concentration, ensure that the final concentration of each composition in corresponding culture dish is consistent.When adding sorting positive cell liquid (also known as the B cell enchylema) that embodiment 2 prepares, its concentration in culture system is 4 × 10
5individual cell/ml.When adding the sorting negative cells that embodiment 2 prepares, its concentration in culture system is 2 × 10
6individual cell/ml.When adding the experiment serum that embodiment 2 prepares, its concentration in culture system is 50%(volume ratio).When adding fluorescent marker protein liquid first, the concentration of total protein is 4 μ g/ml.When adding fluorescent marker protein liquid second, the concentration of total protein is 4 μ g/ml.Utilize Laser Scanning Confocal Microscope system, at 37 DEG C, 5%CO
2dynamically cell fluorescence and metamorphosis is observed under culture condition.Continuous observation to 24 hour.
Table 1 is for the composition of each culture system first BP patient and first healthy volunteer
Second BP patient and second healthy volunteer, each culture system composition of the 3rd BP patient and the 3rd healthy volunteer is with table 1.
The result (cell cultures 24 hours) of first BP patient is shown in Fig. 5.The result of other two BP patients is consistent with Fig. 5.Fluorescently-labeled Rituximab makes nearly all B cell surface display fluorescence of BP patient and normal people.Fluorescently-labeled Col17-IgG1Fc fusion rotein makes minority B cell surface display fluorescence (i.e. COL17NC16A specific b cells) in BP patient's blood, and the equal unstressed configuration of Normal Human B Cells.Illustrate that fluorescently-labeled Col17-IgG1Fc fusion rotein possesses the effect of specific recognition COL17NC16A specific b cells.Observe to 24 hours, no matter have the existence of serum-free or sorting negative cells, the cell state of all fluorescent positive keeps good, has no obvious metamorphosis, illustrates that fluorescently-labeled Col17-IgG1Fc fusion rotein does not possess lethal effect.
Two, the killing ability of unstressed configuration labelled protein measures
Carry out application of sample according to table 2 respectively 0 hour and 2 hours (this time point cell state and fluorescent signal are stablized), by DMEM perfect medium adjustment concentration, ensure that the final concentration of each composition in corresponding culture dish is consistent.
0 little first time constantly application of sample: when adding sorting positive cell liquid (also known as the B cell enchylema) that embodiment 2 prepares, its concentration in culture system is 4 × 10
5individual cell/ml; When adding the sorting negative cells that embodiment 2 prepares, its concentration in culture system is 2 × 10
6individual cell/ml; When adding the experiment serum that embodiment 2 prepares, its concentration in culture system is 50%(volume ratio); When adding fluorescent marker protein liquid first, the concentration of total protein is 4 μ g/ml; When adding fluorescent marker protein liquid second, the concentration of total protein is 4 μ g/ml.
2 hours second time application of samples: add the step 3 of embodiment 12 protein concentrated solutions obtained or Rituximab also make the wherein contained concentration of total protein in culture system be 4 μ g/ml.
Utilize Laser Scanning Confocal Microscope system, at 37 DEG C, 5%CO
2dynamically cell fluorescence and metamorphosis is observed under culture condition.Continuous observation to 24 hour.
Table 2 is for the composition of each culture system first BP patient
Second BP patient and second healthy volunteer, each culture system composition of the 3rd BP patient and the 3rd healthy volunteer is with table 2.
The result (cell cultures 24 hours) of first BP patient is shown in Fig. 6.The result of other two BP patients is consistent with Fig. 5.Add unstressed configuration mark Rituximab or Col17-IgG1Fc fusion rotein after, all can be observed under serum or PBMC existent condition, corresponding fluorescencepositive cell surface shrinkage, form is irregular, lose intact cell form gradually, be decomposed into cell debris.Fluorescence negative cells state keeps good, has no obvious metamorphosis.Illustrate that Col17-IgG1Fc fusion rotein can specific killing COL17NC16A specific b cells, and to normal B cells without lethal effect.
Under serum existent condition, there is morphologic change in Col17-IgG1Fc fusion rotein about the 15min that fluorescencepositive cell is adding unstressed configuration mark, 4 hours later cell forms completely lose; Under PBMC existent condition, there is morphologic change in Col17-IgG1Fc fusion rotein 1 hours that fluorescencepositive cell is adding unstressed configuration mark, 18 hours later cell forms completely lose.Illustrate that the CDC effect of the Col17-IgG1Fc fusion rotein induction that unstressed configuration marks is early than ADCC effect.
Claims (10)
- The 293T clone BPKU of 1.COL17NC16A-IgG1Fc plasmid stabilisation transfection, its deposit number is CGMCC No.7801.
- 2. the application of clone described in claim 1 in preparation Col17-IgG1Fc fusion rotein; The protein of described Col17-IgG1Fc fusion rotein for being made up of the aminoacid sequence shown in sequence in sequence table 1.
- 3. prepare a method for Col17-IgG1Fc fusion rotein, comprise the steps: cultivate clone described in claim 1 and collect culture supernatant, be the solution containing described Col17-IgG1Fc fusion rotein; The protein of described Col17-IgG1Fc fusion rotein for being made up of the aminoacid sequence shown in sequence in sequence table 1.
- 4. the application of clone described in claim 1 in the medicine for the preparation for the treatment of bullous pemphigoid.
- 5. be used for the treatment of a medicine for bullous pemphigoid, the Col17-IgG1Fc fusion rotein that its activeconstituents prepares for method described in claim 3.
- 6.Col17-IgG1Fc the application of fusion rotein in the medicine for the preparation for the treatment of bullous pemphigoid; The protein of described Col17-IgG1Fc fusion rotein for being made up of the aminoacid sequence shown in sequence in sequence table 1.
- 7. be used for the treatment of a medicine for bullous pemphigoid, its activeconstituents is Col17-IgG1Fc fusion rotein; The protein of described Col17-IgG1Fc fusion rotein for being made up of the aminoacid sequence shown in sequence in sequence table 1.
- 8.Col17-IgG1Fc fusion rotein is the protein be made up of the aminoacid sequence shown in sequence in sequence table 1.
- 9. the gene of Col17-IgG1Fc fusion rotein described in coding claim 8.
- 10. gene as claimed in claim 9, is characterized in that: described gene is as shown in the sequence 2 of sequence table.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114656572A (en) * | 2022-02-25 | 2022-06-24 | 北京大学第一医院 | BP180 antibody rapid detection kit and preparation method thereof |
| CN117986389A (en) * | 2024-04-02 | 2024-05-07 | 百肽德医药生物科技(广东)有限公司 | Recombinant humanized XVII type collagen, preparation method and application |
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| CN117986389B (en) * | 2024-04-02 | 2024-06-07 | 百肽德医药生物科技(广东)有限公司 | Recombinant humanized XVII type collagen, preparation method and application |
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