CN106008722B - A kind of recombinant beta-hNGF-Fc fusion protein, Preparation method and use - Google Patents
A kind of recombinant beta-hNGF-Fc fusion protein, Preparation method and use Download PDFInfo
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- CN106008722B CN106008722B CN201610617323.4A CN201610617323A CN106008722B CN 106008722 B CN106008722 B CN 106008722B CN 201610617323 A CN201610617323 A CN 201610617323A CN 106008722 B CN106008722 B CN 106008722B
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/48—Nerve growth factor [NGF]
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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Abstract
The invention discloses a kind of recombinant beta-hNGF-Fc fusion proteins, Preparation method and use.Successively the amino acid sequence shown in people β NGF, short peptide linkers and human IgG Fc forms for it.Recombinant beta-hNGF-Fc the fusion protein on a molar basis, has the Bioactivity equal or higher with people NGF.The present invention also protects recon and cell strain containing the recombinant beta-hNGF-Fc fusion protein, has the purposes for peripheral neuropathy, the recovery of neurotrosis and the application of nerve growth factor long-actingization.
Description
Technical field
The present invention relates to restructuring biotechnology fields, especially design a kind of recombinant beta-hNGF-Fc fusion protein, preparation method
And purposes.
Background technique
Extensively, wherein β subunit is the active region of NGF for nerve growth factor (NGF) effect.β-the mNGF being commercialized at present
It (NOBEX), is obtained from the separation of adult male mice salivary gland and purifying.It is outer to be widely used in ophthalmology, nerve
The treatment of the diseases such as section, orthopaedics, Neurology, paediatrics, division of endocrinology's neuratorphy, neurodegeneration, wound reparation.Just because of note
It penetrates and is constantly increased with the demand of mouse nerve growth factor, NGF product now has the potential defect of two o'clock: 1, due to being source of mouse
Protein, with potential immunogenicity；2, a large amount of qualified male mice salivary gland is needed, this is needed at guarantee yield
The big bottleneck of one asked.And NGF is recombinantly expressed by prokaryotic expression system (such as E.coli), have and is repaired after can not carrying out protein translation
The shortcomings that decorations.
Usually said β-hNGF is 118 amino acid mature peptides.In human body, the expression of β-NGF has a following process: 1.
β-NGF coded sequence (723bp) is translated as prepro-hNGF (241aa), including signal peptide (1-18aa), leads peptide (19-
121aa), mature peptide (122-241aa)；2.In endoplasmic reticulum, signal peptide is hydrolyzed, forms pro-hNGF, including leads peptide and maturation
Peptide.Leading peptide end has 4 amino acid residue (118-121aa) Arg-Ser-Lys-Arg, is mammal premise Protein processing enzyme
Recognition site leads peptide and processes enzyme hydrolysis by precursor protein in golgiosome, formed hNGF (120aa)；3.hNGF is in 7S hNGF
The ArgAla of the middle lower excision C-terminal of γ subunit effect forms mature peptide (118aa).
The immunoglobulin of IgG class is protein abundant in human body.Its Half-life in vivo may be up to 21 days.There are many
The region IgGde Fc and other protein are combined into fusion protein to improve the report of half-life period.Prototype fusion protein passes through
The cysteine residues of IgG Fc hinge area are connected as dimer, are protein-based to be similar to IgG molecule.Fc fusion protein is in vivo
Show the comparable inside and outside foreign object dynamic characteristic of same kind human IgG.This method has been used for some clinically critically important
Cell factor (such as IL-2).
In NGF clinical application, there is the adverse reaction that injection site pain or injection survey lower limb pain, this and NGF
It is related to participate in pain nerve reaction.Therefore long-actingization NGF is developed, patient's administration number of times and frequency is reduced, is conducive to reduction of patient
Adverse reaction.In conclusion the preparation method of exploitation recombination human nerve growth factor β Fc fusion protein, has important face
Bed value.
Summary of the invention
The purpose of the present invention is to provide a kind of recombinant beta-hNGF- with people NGF equal or higher Bioactivity
Fc fusion protein.
To achieve the above object, the present invention provides a kind of recombinant beta-hNGF-Fc fusion protein, which is characterized in that described heavy
Successively the amino acid sequence shown in people β NGF, short peptide linkers and human IgG Fc forms group β-hNGF-Fc fusion protein.
Further, the short peptide linkers sequence is the sequence of not more than 20 amino acid；Preferably, the short peptide linkers contain
There is the sequence of at least two amino acid in glycine, serine, alanine and threonine；It is furthermore preferred that the short peptide linkers sequence
It is classified as lysine-threonine-Gly-Gly-Gly-Serine-Glycine-Gly-Gly-serine-figured silk fabrics
Sequence shown in propylhomoserin-glutamic acid.
Further, the human IgG Fc segment hinge area containing human IgG, the area CH2 and CH3.
Further, sequence shown in the fusion protein is as shown in SEQ ID NO:1；Preferably, the fusion protein institute is right
The nucleic acid sequence answered is as shown in SEQ ID NO:2.
Further, the recombinant beta-hNGF-Fc fusion protein on a molar basis, has equal or higher with people NGF
Bioactivity.
The present invention also provides a kind of recons, which is characterized in that contains the recombinant beta-hNGF-Fc fusion protein sequence.
The present invention also provides a kind of cell strains, which is characterized in that contains recon described in claim 6.
Further, the cell strain is mammalian cell strain；Preferably, the mammalian cell strain is Chinese hamster ovary celI
Derivative cell strain.
The present invention also provides a kind of recombinant beta-hNGF-Fc fusion protein preparation methods, which is characterized in that the method packet
It includes: the recombinant expression carrier of the building fusion protein of-hNGF-Fc containing β；
Recombinant expression carrier is transfected into mammalian cell, and screens the cell strain of high efficiency stable expression；
The cell strain of high efficiency stable expression obtained by fermented and cultured collects the β-hNGF- that culture solution and purifying cells secret out of
Fc fusion protein.
Further, the protein sequence of the β-hNGF-Fc is as shown in SEQ ID NO:1, it is preferred that corresponding β-
HNGF-Fc nucleic acid sequence is as shown in SEQ ID NO:2.
Further, the mammalian cell be Chinese hamster ovary celI, people NIH293 cell, COS cell or mdck cell and they
Derived cell system.
Recombinant beta-hNGF-Fc the fusion protein, the recon or the cell strain are used for peripheral neuropathy, nerve
The purposes of the recovery of damage and the application of nerve growth factor long-actingization.
HNGF-hFc fusion protein amino acid sequence is as shown in SEQ ID NO:1.Wherein 1-18aa is signal peptide, 19-
121aa is leader peptide, and 122-239aa is mature peptide, and 240-251aa is connection small peptide, and 252-478aa is human IgG Fc segment,
Wherein 252-270aa is hinge area, and 271-376aa is the area CH2, and 377-478aa is the area CH3.It is obtained through recombinant secretor expression
HNGF-hFc fusion protein structure is mature peptide-connection small peptide-hFc.Corresponding nucleic acid sequence as shown in SEQ ID NO:2,
Wherein 1-54bp is hNGF signal peptide sequence；55-363bp is that hNGF leads peptide sequence；364-717bp is hNGF maturation peptide sequence；
718-753bp is connection short peptide sequence, which is that lysine-threonine-Gly-Gly-Gly-serine-is sweet
Propylhomoserin-Gly-Gly-Serine-Valine-glutamic acid；754-1437bp is hIgG Fc sections of sequence.
People NGF is synthesized in the form of precursor in vivo, including signal peptide, leader peptide and mature peptide.Table of the leader peptide in NGF
Reach, fold, secrete in play important function.In leader peptide there are two conservative region be NGF expression, enzymatic hydrolysis formed mature peptide with
And necessary to secretion.Research has shown that leader peptide plays the role of Intramolecular chaperone in NGF folding process, helps
In the correct folding of NGF.Leader peptide includes potential N glycosylation site, after N is glycosylation modified, facilitates NGF and transports out endoplasm
Net.Thus compared with directly expressing maturation hNGF sequence, addition leader peptide sequences will recombinate in the present invention conducive to the external source of hNGF
Expression promotes albumen to carry out folding after correctly expressing, modification and secretion, forms the mature hNGF with biological activity.
The region Fc of human immunoglobulin(HIg) plays an important role in immune response.The effector function of IgG is mediated by Fc
Two kinds of mechanism realize: 1. combination cell surface Fc receptors (FcgRs), cause antibody-dependant cytotoxic effect (ADCC) way
Diameter is ingested pathogen by killing cell by phagocytosis or splitting action；2. and the part C1q of the first complement component C1 is tied
It closes, causes cytotoxicity (CDC) approach for depending on complement, to crack pathogen.In tetra- kinds of hypotypes of IgG, IgG1 and
IgG3 can be effectively combined the low an order of magnitude of the affinity ratio IgG1 or 3 of FcgR, IgG4 and FcgR, and IgG2 is not combined then
FcgR.Human IgG1 and 3 can effectively combine C1q, and activating complement cascade reaction, and IgG2 is very weak to the combination of complement,
IgG4 not activating complement cascade reaction.When hNGF-hFc is used for human body therapy, when in conjunction with receptor, it should be avoided stronger immune
Cell cracking caused by effector function.Therefore in the present invention, tetra- kinds of subtype sequences of IgG are compared, are devised a kind of from people
The mixing human IgG Fc sequence of IgG2 and IgG4, for weakening the effect of its immune effector.The sequence is mixed with the hinge of human IgG2
The area CH2 and CH3 region sequence of sequence and IgG4, and carried out codon optimization.
Wherein an embodiment shows that β-hNGF-Fc has vitro stability more stronger than hNGF to the present invention, it is prompted to have
The potentiality of long-actingization application.
The present invention provides a kind of preparation method for recombinating human nerve growth factor β Fc fusion protein (β-hNGF-Fc), special
Sign is, includes the following steps:
1) building of expression vector:
A variety of mammalian cell expression vectors can be used, the element being connected in expression vector comprising following operability:
Promoter, exogenous array insertion point, riddled basins.It preferably, include enhancer or interior between promoter and Insert Fragment
Regulate and control original part containing son etc., to enhance the expression of Insert Fragment.Preferably, the promoter be CMV promoter, PGK promoter,
The derivative promoter of any one and they in RSV promoter or SV40 promoter is marked for starting β-hNGF-Fc and screening
Remember the expression of gene.The riddled basins are dihyrofolate reductase (DHFR) gene, glutamine synthelase (GS) base
Cause and antibiotics resistance gene (neomycin resistance gene, puromycin resistance gene, blasticidin resistance gene or pluramycin
Resistant gene) in any one.
Artificial synthesized β-hNGF-Fc sequence, insertion expression vector obtain fusion protein expression vector.Inserted mode can be limited
Clone, the TA clone, dependent/non-dependent clone (Ligase IndependnetClone) of property restriction endonuclease mediation processed.
The fusion protein expression carrier of acquisition is transformed into E.coli TOP10, picking positive colony, PCR identification, matter
Granzyme cuts identification and sequencing identification positive colony.The positive colony that identification obtains freezes in 15% glycerol.
2) building of engineering cell strain；
A variety of mammal cell lines can be used.Preferably, the cell line is Chinese hamster Chinese hamster ovary celI, people NIH293
Cell, COS cell or mdck cell and their derived cell system.
Above-mentioned recombinant expression carrier is transfected into mammalian cell, a kind of preferred transfection method is fat transfection,
Other methods, including coprecipitation of calcium phosphate, electroporation, Protoplast fusion can be used.
After transfection, according to riddled basins, screens and stablize expression cell, and cultivated.Pass through limiting dilution method pair
Stablize expression cell to be subcloned, and each monoclonal expression quantity is measured by IgG Fc ELISA, it is higher to filter out expression quantity
Stablize expression monoclonal cell strain.
3) purifies and separates β-hNGF-Fc
Fermented and cultured high efficiency stable expression cell strain, a kind of preferred training mode are that fed-batch mode carries out feed-batch culture.
Terminate to cell viability lower than 70%.
Collect fermented and cultured product, and purifies and separates β-hNGF-Fc.A variety of chromatography methods may be incorporated for β-hNGF-Fc
Isolate and purify, including ion-exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration.It is preferable that with
Protein A affinitive layer purification separates β-hNGF-Fc.
Detailed description of the invention
Fig. 1 is pCHO1.0 Vector map figure；
Fig. 2 is β-hNGF-Fc positive colony PCR verifying electrophorogram；
Fig. 3 is β-hNGF-Fc positive colony plasmid digestion verification electrophorogram；
Fig. 4 is
Purifer100 tomographic system Protein A affinitive layer purification β-hNGF-Fc spectrogram；
Fig. 5 is SDS-PAGE analysis β-hNGF-Fc figure；
Fig. 6 is the dose-effect curve figure of β-hNGF-Fc fusion protein stimulation TF-1 cell Proliferation.
Fig. 7 is β-hNGF-Fc and hNGF external activity and time plot.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.Embodiment
In particular technique or condition person is not specified, described technology or conditions or according to the description of product according to the literature in the art
Book carries out.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.
The building of embodiment 1:pCHO1.0-hNGF-hFc expression vector
HNGF-hFc nucleic acid sequence translates resulting amino acid sequence such as SEQ ID NO:1 institute as shown in SEQ ID NO:2
Show.
SEQ ID NO:1 is as follows:
Wherein 1-18aa is signal peptide, and 19-121aa is leader peptide, and 122-239aa is mature peptide, and 240-251aa is connection
Small peptide, 252-478aa are Fc segment, and the hNGF-hFc fusion protein structure obtained through recombinant secretor expression is mature peptide-connection
Small peptide-hFc.
SEQ ID NO:2 is as follows:
Wherein 1-54bp is hNGF signal peptide sequence；55-363bp is that hNGF leads peptide sequence；364-717bp is hNGF mature
Peptide sequence；718-753bp is connection short peptide sequence, which is lysine-threonine-Gly-Gly-Gly-silk
Propylhomoserin-Gly-Gly-Gly-Serine-Valine-glutamic acid；754-1437bp is hIgG Fc sections of sequence.
By chemically synthesized method composition sequence SEQ ID NO:3, the upstream and downstream of sequence introduces the I digestion position Xba respectively
Point and I restriction enzyme site of BstZ17 and protection base, primer Kozak sequence between I restriction enzyme site of Xba and ATG homing sequence
GCCACC.Wherein, TCTAGA be I restriction enzyme site of Xba (
It is shown), agacccaagctggctagc is protection base
(_ _ _ _ _ shown).GTATAC be I restriction enzyme site of BstZ17 (
It is shown), ctagactcgagcggccgc be protection base (It is shown).
SEQ ID NO:3 is as follows:
Expression vector used in the present embodiment is pCHO1.0, and Vector map is as shown in Figure 1.By site 1, the load
Body-DHFR the gene expression frame of promoter containing PGK, for expressing DHFR gene；EF2/CMV mixed promoter-insetion sequence expression
Frame, for expressing the β-hNGF-Fc sequence of insertion；PGK promoter-puromycin resistance gene expression cassette, it is fast for expressing
Purine mycin resistant gene.Methotrexate (MTX) (MTX) and puromycin can be used to carry out Double Selection for cell clone containing the carrier.
Using restriction enzyme Xba I and I double digestion composition sequence of BstZ17, using restriction enzyme A vr II with
I double digestion plasmid pCHO1.0 of BstZ17, wherein Xba I and Avr II is isocaudarner.Then the digestion of ethanol precipitation recovery purifying produces
Object.Digestion products are attached reaction using T4DNA Ligase and obtain recombinant expression carrier pCHO1.0-hNGF-hFc.It will
Connection product is converted to E.coliTop10 (Novagen), and through bacterium solution PCR identification, (result is shown in that Fig. 2, M marker, 1 and 2 are inspection
The bacterium colony of survey has the obvious band of 1500bp or so, illustrates that 1 and 2 be the positive bacterium colony of PCR identification), plasmid enzyme restriction identification
(result is shown in Fig. 3, and M marker, 1 is to detect the digestion of plasmid as a result, there is the clear item of 1500bp or so as we can see from the figure
Band, illustrate detect plasmid enzyme restriction qualification result it is correct) and sequencing identify after, by correct pCHO1.0-hNGF-hFc grams of sequence
Grand -80 DEG C freeze in 15% glycerol, spare.
The building and screening of 2: β-hNGF-Fc recombinant cell strain of embodiment
Plasmid purification: the big extraction reagent kit extraction purification plasmid pCHO1.0-hNGF-hFc of plasmid is used, Bio-Spec- is used
The concentration and purity of Nano measurement plasmid.>=1.0 μ g/ μ l, A260/A280=1.7-1.9 are needed for its concentration of the plasmid of transfection.
If concentration is insufficient, using isopropanol precipitating plasmid and it is concentrated into debita spissitudo.
Chinese hamster ovary celI activation: being removed from liquid nitrogen the Chinese hamster ovary celI frozen, and removal protection liquid adds appropriate CD FortiCHO to train
Supporting base to cell density is 5 × 105, in 125ml shaking flask, 37 DEG C, cultivate under the conditions of 100r/min, 5%CO2.Cell density
To 2 × 106When, 5 × 10 are diluted to using appropriate culture medium5, until cell viability is up to 90% or more.
Transfection: the transfection same day, cell density are 1 × 106, vigor should be greater than 90%.Cell, transfection reagent, plasmid ratio
Example is shown in Table 1:
The configuration of 1 rotaring redyeing system of table
By upper table, prepare 15ml Chinese hamster ovary celI, take 1.5ml CD FortiCHO culture solution, 15 μ g plasmids are added
PCHO1.0-hNGF-hFc is mixed, and 45 μ l transfection reagents are added, and is mixed, is stored at room temperature 20min, then by transfection reaction mixing
Object is added into cell, mixes gently.Cell is placed in 37 DEG C, cultivates under the conditions of 100r/min, 5%CO2.The cell of arbitrary volume
It can be transfected, transfection reagent and DNA can also be mixed with other ratios.
Stablize the screening of expression cell: after transfection for 24 hours, cell density being diluted to 3 × 105, MTX is added to final concentration
100nM, continues to cultivate under the conditions of 100r/min, 5%CO2 by 37 DEG C.Cell density is to 5 × 105When, it is diluted to 3 × 105, until
Cell viability shows that cell has adapted to concentration selection pressure up to 80% or more.Then it is incremented by the concentration of MTX, is followed successively by
200nM,500nM,1000nM.It is subcloned by limiting dilution method, β-hNGF-Fc table is measured by anti-igg Fc ELISA
Up to amount, the horizontal high monoclonal of secreting, expressing is selected.
The recombinant expression of 3: β-hNGF-Fc of embodiment isolates and purifies and qualitative
Recombinant expression
β-hNGF-Fc the recombinant cell strain obtained in embodiment 2 is subjected to fed-batch cultivation.Seed cell culture is to density 2
×106, 90% or more vigor can inoculation fermentation, ferment initial density 5 × 105.Daily the measurement density of cell, vigor and
Glucose content in culture solution, and FeedC culture medium is supplemented, make the final concentration of 4g/L of glucose in culture solution.Cell viability≤
Fermentation ends when 70%.Culture solution is collected by centrifugation, for isolating and purifying.
Protein A affinity protein purification purifies β-hNGF-Fc
It usesPurifer100 tomographic system carries out affinity chromatography, and chromatographic column is HiTrapMabSelect 1mL.
Instrumentation is carried out by operating guidance, and A1 pump is Buffer A (50mM phosphate buffer, 100mMNaCl, pH7.0), B1 pump
For Buffer B (50mM acetate buffer, pH3.5).
Cleaning: with 0.1M NaOH, flow velocity 1ml/min, rinsing 5 column volumes (CV) of ProteinA gel column, then with ultrapure
Water cleans 5cv with flow velocity 1ml/min, removes the magazine residual on ProteinA gel.
Balance: Buffer A rinses 10CV with 1ml/min flow velocity, to maintain ProteinA gel environment to be suitble to β-hNGF-
The combination of Fc and ProteinA.
Loading: fermentation liquid after clarification filtration with the rate of 1ml/min by ProteinA gel, make β-hNGF-Fc with
The combination of ProteinA energy specificity.
Punching is flat: Buffer A is rinsed to 280nm absorption value with 1ml/min flow velocity lower than 0.01.
Elution: with 1ml/min flow velocity, Buffer B rinses ProteinA gel, collects eluting peak.
Neutralize: eluting peak collects sample 2mol/L Tris-Cl, pH9.0 tune pH to 5.6.
As a result see Fig. 4.As can be seen from the figure: after pH is reduced to 3.5, affording a single, sharp elution
Peak.SDS-PAGE analysis: SDS-PAGE tests and analyzes the β-hNGF-Fc that purifying obtains.Use 12% separation gel.The results show that
Under reducing condition, the albumen of purifying is migrated to about 40kDa, and no significant polymer and foreign protein exist.As a result see Fig. 5.M is
Marker, 1 is the albumen of purifying.
Embodiment 4: Bioactivity analysis
2015 editions " Chinese Pharmacopoeia " mouse nerve growth factor biological activity assavs of reference, the second method: TF-1 cell/
MTS colorimetric method.
TF-1 cell method be the granulocyte macrophage colony stimulating factor (GM-CSF) based on TF-l cell line highly according to
Lai Xing, using NGF in conjunction with the NGF high-affinity receptor TrkA of TF-1 cell surface after induce TF-1 cell Proliferation the characteristics of,
The new NGF activity measuring method of one kind of foundation.This method has become detection recombinant human nerve growth factor (rhNGF) at present
The standard method of WHO reference material.
TF-1 cell culture: TF-1 cell strain is cultivated in 37 DEG C, 5% carbon dioxide incubator with complete medium, control
Cell concentration processed is that every 1ml contains 1.0 × 105~5.0 × 105A cell is used for NGF Determination of biological activity in second day after passage.
Cell suspension preparation: the amount of taking fully cell culture fluid 1000rpm is centrifuged 5min, discards supernatant, and collects TF-1 cell.5ml base is added
Cell is resuspended in basal culture medium, discards supernatant after 1000rpm centrifugation 5min, basal medium is resuspended in after so washing 3 times and is made into
Every 1ml is containing about 6.0 × 104The cell suspension of a cell, be placed in 37 DEG C, it is spare under 5% carbon dioxide conditions.
Standard solution preparation: take 1 recombinant human nerve growth factor activity criteria product dispensed (purchased from United Kingdom National
Biologic criteria and Quality Control object research institute (NIBSC), article No. 93/556), 10 times are diluted with basal medium, until every 1ml contains
100U。
The preparation of inspection product solution: inspection product carry out pre-dilution with basal medium, and every step dilution is maximum to be no more than 10 times, until every
1ml is containing about 100U.
The preparation of inspection product gradient:
96 porocyte culture plates choose adjacent two column in addition to the 1st column and the 12nd column and arrange or examine product column as standard items；
It is arranged in addition to the hole A in the inspection product column and standard items of 96 porocyte culture plates, 100 μ l basal mediums are added in every hole；
Inspection product column or the every hole in the hole standard items column A are separately added into 150 μ l inspection product solution or standard solution；
It is about to inspection product solution from A row to H or standard solution carries out 3 times and is serially diluted, totally 8 dilution, each dilution
Degree does 2 multiple holes, and every hole stays 100 μ l solution respectively, discards redundant solution in hole；
Every hole being arranged to inspection product column and standard items, 100 μ l cell suspensions being added, 200 μ l sterile waters are added to the every hole of blank column；
96 porocyte culture plates are put into wet box, the stationary culture 66- in 37 DEG C, the incubator of 5% carbon dioxide
72h。
Inspection product column and standard items arrange after 20 μ l of MTS solution is added in every hole and put back to 96 orifice plates in wet box, in 37 DEG C, 5% 2
Stationary culture 3h in the incubator of carbonoxide.
96 porocyte culture plates are put into microplate reader, using 550nm as reference wavelength, absorbance is measured at wavelength 490nm,
Record measurement result.
Result judgement:
Test data usesPro 5Software Data Analysis Software is handled, and is incorporated into and is counted as the following formula
Calculate result:
Inspection product biological activity (U/ml)=Pr × (Ds × Es)/(Dr × Er)
In formula, Pr is standard items biological activity, U/ml；
Ds is inspection product pre-dilution multiple；
Dr is standard items pre-dilution multiple；
Es is the extension rate that inspection product are equivalent to standard items median effective dose；
Er is the extension rate of standard items median effective dose.
Inspection product specific activity (U/mg)=inspection product biological activity/protein content.
β-hNGF-Fc fusion protein stimulates effect curve such as Fig. 6 of TF-1 cell Proliferation dosage, and four parameters are as shown in table 2,
Fit equation is y=(A-D)/(1+ (x/C) ^B)+D.It can be seen from the figure that the agent of β-hNGF-Fc fusion protein and standard items
Graded effect curve is consistent, illustrates under this condition, the effect of β-hNGF-Fc stimulation TF-1 cell is close with hNGF.The curve accords with
Close four parametric fit equations.Calculation shows that it is 3.57 × 10 that the ratio of β-hNGF-Fc fusion protein is living5U/mg.It is changed by molal weight
It calculates, relative to β-hNGF than work > 1 × 106U/mg, and the ratio work of standard items are suitable.Activity experiment shows that β-hNGF-Fc has
With the comparable In vitro biological activity of hNGF.
2 dosage effect parameter of table
Embodiment 5: vitro stability analysis
Taking β-hNGF-Fc and hNGF respectively, (recombinant human nerve growth factor activity criteria product are (purchased from United Kingdom National biology
Standard and Quality Control object research institute (NIBSC), article No. 93/556), concentration is 0.05 μm of ol/ml, is dissolved in pH 7.0,50mM phosphate
In buffer, it is placed in 37 DEG C of heat preservations.Activity residual amount is measured by sampling weekly, with initial than living as 100% relative activity, experiment
Three repetitions are set.Change curve is as shown in fig. 7, wherein NGF activity standard items are hNGF.As seen from the figure, by 4 weeks,
The loss of activity of β-hNGF-Fc is slow compared with hNGF, hNGF activity residual 2.24%, and β-hNGF-Fc activity residual 27.38%.
The vitro stability of β-hNGF-Fc is significantly stronger than hNGF as the result is shown.Before the result prompts β-hNGF-Fc to have long-actingization application
Scape.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art are not departing from the principle of the present invention and objective
In the case where can make changes, modifications, alterations, and variations to the above described embodiments within the scope of the invention.
Claims (9)
1. a kind of recombinant beta-hNGF-Fc fusion protein, which is characterized in that the fusion protein sequence is as shown in SEQ ID NO:1.
2. a kind of gene of recombinant beta-hNGF-Fc fusion protein described in coding claim 1, which is characterized in that the gene
Nucleic acid sequence is as shown in SEQ ID NO:2.
3. a kind of recombinant vector, which is characterized in that the core containing recombinant beta-hNGF-Fc fusion protein described in coding claim 1
Acid sequence.
4. a kind of cell strain, which is characterized in that contain recombinant vector described in claim 3.
5. cell strain as claimed in claim 4, which is characterized in that the cell strain is mammalian cell strain.
6. cell strain as claimed in claim 5, which is characterized in that the mammalian cell strain is cell derived from Chinese hamster ovary celI
Strain.
7. a kind of recombinant beta-hNGF-Fc fusion protein preparation method, which is characterized in that the described method includes:
Construct the recombinant expression carrier of the fusion protein of-hNGF-Fc containing β；
Recombinant expression carrier is transfected into mammalian cell, and screens the cell strain of high efficiency stable expression；
The cell strain of high efficiency stable expression obtained by fermented and cultured is collected the β-hNGF-Fc that culture solution and purifying cells secret out of and is melted
Hop protein；The amino acid sequence of the β-hNGF-Fc fusion protein is as shown in SEQ ID NO:1；Corresponding β-hNGF-Fc melts
The nucleic acid sequence of hop protein is as shown in SEQ ID NO:2.
8. recombinant beta-hNGF-Fc fusion protein preparation method as claimed in claim 7, which is characterized in that the mammal is thin
Born of the same parents are Chinese hamster ovary celI, people NIH293 cell, COS cell or mdck cell and their derived cell system.
9. recombinant beta-hNGF-Fc fusion protein described in claim 1, recombinant vector described in claim 3 or claim 4-6 appoint
One cell strain is used to prepare the use of peripheral neuropathy, the recovery of neurotrosis and the long-acting chemical drug object of nerve growth factor
On the way.
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| CN114933657B (en) * | 2021-08-25 | 2024-02-02 | 上海交通大学医学院 | Nerve growth factor mutant recombinant protein and its application |
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| WO2023088351A1 (en) * | 2021-11-19 | 2023-05-25 | 舒泰神（北京）生物制药股份有限公司 | Use of nerve growth factor in preparation of drug for treating or improving diseases of reproductive system |
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