CN102586190A - CHO (Chinese Hamster Ovary) cell strain of efficiently-expressed recombinant human nerve growth factor and construction method thereof - Google Patents
CHO (Chinese Hamster Ovary) cell strain of efficiently-expressed recombinant human nerve growth factor and construction method thereof Download PDFInfo
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Abstract
The intervention relates to a CHO (Chinese Hamster Ovary) cell strain of an efficiently-expressed recombinant human nerve growth factor and a construction method thereof and belongs to the field of biotechnology. The preservation number of the cell strain is CGMCC No. 4541, and the recombinant human nerve growth factor (rhNGF) with biological activity can be stably expressed. The invention also discloses a method for constructing the CHO cell strain; and through cell transfection, gene amplification and subcloning screening carried out on an amplified cell line, an efficiently-expressed monoclonal cell strain suitable for serum-free suspension culture is obtained. The invention simultaneously discloses a recombinant human nerve growth factor sequence secretly expressed by the CHO cell strain; and the secrete recombinant human nerve growth factor has good biological activity, so that PC12 multiplication can be kept outside a human body, PC12 cell differentiation is induced, and the chick embryo dorsal root ganglion nerve fiber growth is stimulated to lay a good foundation for the large-scale industrialization preparation of the recombinant human nerve growth factor.
Description
Technical field
The present invention relates to biological technical field, Chinese hamster ovary celI strain, its construction process and the application of RHNGF (rhNGF) that particularly can stability and high efficiency secreting, expressing biologically active.
Background technology
Progress along with medical science; Various craniocerebral trauma, cerebral apoplexy, Spinal injury, newborn infant's ischemia, hypoxic ischemic encephalopathy; The survival rate of cerebral palsy of children and alzheimer's disease improves constantly, but damaged and patient's poor prognosis of neural function makes medical circle face a difficult problem.Central nervous system injury and peripheral nerve injury become the principal disease that threatens human health at present.Therefore, adding cerebral nerve protects the exploitation of type medicine, impaired nervus centralis and the peripheral nerve of reparation to become the powerful impellent that promotes the fast development of this type pharmaceutical market.Wherein, NGFF (Nerve Growth Factor; NGF) become focus (Hefti F, Weiner WJ.Ann.Neurol, the Nerve growth factor and Alzheimer ' s disease.1986 of Recent study; 20 (3): 275-281.Scott SA, Crutcher KA.Nerve growth factor and Alzheimer ' s disease.Rev Neurosci.1994; 5 (3): 179-211.).
NGFF is to come to light the earliest; One of neurotrophic factor that research at present is the most thorough; Also be find up to now unique, and have the bioactive molecules of regulating nerve injury reparation function not only to maincenter and the nutritious factor effect of peripheroneural normal neurocyte.From the isolating NGFF of mouse submandibular gland is that molecular mass is that 140KD, settling ratio are the mixture of 7S, the composition of proportions of pressing α 2 β 2 γ 2 by α, β, 3 kinds of peptide chains of γ.Wherein, β subunit is the active zone of 7S NGF, and the biological activity of whole NGF relies on β subunit entirely, is also referred to as 2.5S NGF.Research shows; β subunit is made up of two identical peptide chains; Each peptide chain contains 118 amino-acid residues, is by combining to constitute dimer (Shooter EM.Early days of the nerve growth factor proteins.Annu Rev Neurosci.2001 through non covalent bond; 24:601-629.).Form two peptide chains of β subunit; Its conserved structure mainly comprises two pairs of antiparallel β chains; The β chain has constituted the long and flat shape of NGF, comprising 6 cysteine residues, forms 3 pairs of disulfide linkage; These disulfide linkage are very important to the biological activity of keeping NGF; In case destroy then active total loss (Whittemore SR, Seiger A.The expression, localization and functional significance of beta-nerve growth factor in the central nervous system.Brain Res.1987; 434 (4): 439-464.).
In recent years, the gene structure of 2.5S NGF is also illustrated, is positioned on first pair of chromosomal near-end shortwall, and the NGF cDNA of people, chicken, ox, cavy is cloned in succession.CDNA with mouse NGF is a probe, from people's DNA library, clones human ngf gene.Arrange the aminoacid sequence of can deriving from nucleotide base, find that the homology of the NGF aminoacid sequence of human and mouse has 90%, and the structure of mouse NGF and human body NGF also has similarity highly, biological effect does not have specificity between tangible kind yet.Domestic from eighties of last century promptly begun the eighties mouse NGF purifying and application research (Liu Chuan, Li Jinping, Hua Zhongwei, etc. the purifying and the activity identification of mouse submandibular gland 2.5S NGFF. institute of Military Medical Science Institute periodical, 1987,11:28～33; Shen Xinliang; Pan Xiaodong; Bai Minggang; Deng; The new purposes of NGFF in treatment organic solvent poisoning property peripheral neuropathy. Chinese patent: CN99127304.4), the injection mouse nerve growth factor of China's development is at present obtaining the kind biological product New Drug Certificate that State Food and Drug Administration (SFDA) issues.Wuhan Haite Bio-pharmaceutical Co., Ltd, Xiamen Beidazhilu Biological Engineering Co., Ltd had obtained the production certification that SFDA issues respectively in 2003, respectively with " Jin Lujie " and " grace is through multiple " listing.SFDA in 2006 has ratified SHUTAISHEN (Beijing) medicine company injection NGFF again and has produced, commodity " Soviet Union's peptide is given birth to " by name, and so far Chinese market has formed the situation of standing like the legs of a tripod.Yet; The inhuman source NGF in mouse and other source is different from people's NGF molecule after all on primary structure and space conformation; Although its purity can reach more than 98%, still can not get rid of long-term a large amount of the use and produce anti-mouse NGF antibody and the possibility that causes allergic reaction.Human body produces anti-mouse NGF antibody will cause its transformation period shortening in vivo, have influence on clinical therapeutic efficacy and patient's drug safety.
Therefore the recombinant human NGF that all adopted the expressing cho cell system expression of domestic and international many research institutions; Determination of activity is the result show; Property NGF compares with the mouse source, and recombinant human NGF has good biological activity (Jiang Jing, Yu Shuping; Jiang Guixiang, etc. the recombinant human beta nerve-growth factor that two step column chromatography purifying CHO express (β-rhNGF). Chinese biological engineering magazine .2008; 28 (10): 84～89.; Chen Yong Jiang Lin Yang Jun, etc. the biological activity of human's Construction of eukaryotic and expression product thereof. the Chinese biological goods are learned magazine .2005; 18 (6): 457-460.).But; Because China's upstream gene reorganization and cell strain structure and triage techniques lag behind American-European developed country level; The CHO engineering cell strain of domestic structure is not only also cultivated at the blood serum medium that has of continuing to use the interpolation Ox blood serum; And adherent Chinese hamster ovary celI also is difficult to realize high-density culture, must just can amplify through the suspension culture domestication and carry out industrialization production.On the other hand, the Recombinant Protein Expression level is several times lower than than the U.S. even tens times, does not reach the laboratory scale output of external 20-30g/ far away, has said nothing of realization extensive industrialization cheaply.For example, the homemade expressing cho cell product of China's approval listing only has two kinds: EPO and Hepatitis B virus vaccine.Although but the using dosage of these two kinds of products has only the Gamma Magnitude level; It produces the very original and primary state that also is in; Many producers even still with rolling bottle cultivating and producing (Hu Xianwen; Chen Huipeng, Tang Zhongming, etc. the present situation of bio-pharmaceuticals and future (one): history and reality market. Chinese biological engineering magazine .2004; 24 (12): 95-101.; Hu Xianwen, Chen Huipeng, Tang Zhongming, etc. the present situation of bio-pharmaceuticals and future (two): development trend and hope. Chinese biological engineering magazine .2005; 25 (1): 86-93.).
Since conventional Chinese hamster ovary celI need be under the situation that has Ox blood serum or analogous components to exist adherent growth, serum in the cell culture medium or protein component have very big influence to expression and the protein purification process in later stage of recombinant protein in cell.If carry out industrialization, also need at first carry out serum-free domestication, make cell adapted serum free medium and well-grown and continuous release produce recombinant protein to reduce cost and to make things convenient for the exploitation of downstream purification technology therein.Often cause the genetically engineered cell strain unstable in the cell non-serum domestication process, cause the protein production ability drop.Current mammalian cell serum-free, extensive, high-density, suspension culture have become one of most important gordian technique of field of biological pharmacy, and have promoted the very strong driving force that biotechnology industry develops with deeply becoming with rapid progress of its research.But; Existing recombinant human NGF research document is not seen relevant report; Do not point out us just can obtain a kind ofly both need not carrying out the serum-free acclimation shaking culture in forefathers' the document, can carry out the approach that the subclone screening obtains efficiently expressing recombinant human NGF cell strain again very easily through which kind of method.
Summary of the invention
The CHO DG44-NGF-118 cell strain that the purpose of this invention is to provide a kind of efficiently expressing recombinant human NGFF; This cell strain is at Chinese common micro-organisms culture presevation administrative center (China General Microbiological Culture Collection Center; CGMCC) preservation, its deposit number is: CGMCC No.4541.
Another object of the present invention is to obtain to have the method for CHO DG44-NGF-118 cell strain that stability and high efficiency is expressed the RHNGF (rhNGF) of biologically active through setting up screening; Solve the deficiency that the above-mentioned background technology exists, thereby set up a kind of technology platform of expressing and produce with complex construction and bioactive recombinant protein.Described technology platform is through utilizing serum-free suspension culture technology and methylcellulose gum semisolid medium subclone technology; Can omit the serum-free domestication process of prior art; Not only improve the subclone success ratio, but also greatly facilitate the downstream purification of recombinant protein.
Its host cell is the CHO DG44 of the domestication that suspends through serum-free.
Said Chinese hamster ovary celI strain has proteinic nucleotide sequence shown in the coding SEQ ID No2.
Said nucleotide sequence is shown in SEQ ID No.1.
The construction process of above-mentioned Chinese hamster ovary celI strain may further comprise the steps:
(1) nucleotide sequence of synthetic shown in SEQ ID No.1 is inserted into the MCS of pOptiVEC-polylinker, obtains recombinant plasmid,
(2) with plasmid change over to serum-free suspend domestication CHO DG44 in,
(3) mode that progressively the improves methotrexate concentration screening of pressurizeing obtains mixing clone cell,
(4) adopt the methylcellulose gum semisolid medium to carry out subclone and select the monoclonal cell strain, adopt the expression amount of ELISA method detection monoclonal cell strain, the Chinese hamster ovary celI strain of the RHNGF that screening obtains efficiently expressing.
Wherein nucleotide sequence is inserted between the Xba I and Not I restriction enzyme site of pOptiVEC-polylinker.
Said recombinant plasmid is pOptiVEC/NGF-118; Its preparation method comprises the steps: the nucleotide sequence of (A) synthetic shown in SEQID NO.1; And be connected on the pcDNA3.1 carrier and make up successful pcDNA3.1/NGF-118 recombinant plasmid; (B) use Xba I and Not I double digestion pcDNA3.1/NGF-118 recombinant plasmid and pOptiVEC-polylinker respectively; Ngf gene fragment after reclaiming enzyme then and cutting is connected with linearizing pOptiVEC carrier, and the transformed into escherichia coli competent cell extracts plasmid after the picking mono-clonal enlarged culturing; Identify positive recombinant with double digestion method and sequencing method, obtain the pOptiVEC/NGF-118 recombinant plasmid.
Said methotrexate concentration is 400nM.
Said step (3) pressurization screening is respectively 20nM according to concentration, 40nM then for being that 10nM selects substratum to begin the pressurization screening containing methotrexate concentration; 60nM; 80nM, 100nM, the gradient of 200nM and 400nM progressively improves the concentration of selecting methotrexate in the substratum.
The application of said Chinese hamster ovary celI strain in producing the growth factor of human nerve pharmaceutical grade protein.
In order to realize above-mentioned purpose of the present invention, the application provides following embodiment:
1. obtain growth factor of human nerve (Nerve growth factor through the UniProtKB database retrieval; NGF) ngf gene sequence; It is optimized design; Shown in SEQ ID NO.1, be inserted into the MCS of pOptiVEC-polylinker, form the pOptiVEC/NGF-118 recombinant plasmid.
2. the pOptiVEC/NGF recombinant plasmid is changed in the CHO DG44 cell, improve the screening of pressurizeing of MTX concentration gradually, progressively improve the RHNGF expression level.
3. pressurization is screened the mixing clone cell of the express recombinant growth factor of human nerve that obtains, utilize the methylcellulose gum semisolid medium to carry out the monoclonal cell that subclone will obtain the high expression level amount.
4. the quantitative ELISA method is measured the growth curve and the rhNGF expression amount variation tendency of the stable cell line of mono-clonalization.
5. with chick embryonic dorsal root ganglion method (Dorsal root ganglion, DRG) biological activity of qualitative detection RHNGF.
6. induce the biological activity of experiment qualitative detection RHNGF with P of Rats C12 cells in vitro.
The Chinese hamster ovary celI strain of the efficiently expressing recombinant human NGFF that the present invention set up, the expression amount of rhNGF is the highest in No. 5 monoclonal cell strain culture supernatant wherein, reaches 25.5mg/L, called after CHO DG44-NGF-118.CHO DG44-NGF-118 cell strain of the present invention, its host cell are the CHO-DHFR of the domestication defective strain host cell CHO DG44 that has adapted to the serum-free suspension culture, and it is with the advantage that existing bibliographical information method is compared:
In the existing document strain of employed CHO-DHFR deficient cells need be under the situation that has Ox blood serum or analogous components to exist adherent growth, serum in the cell culture medium or protein component have very big influence to expression and the protein purification process in later stage of recombinant protein in cell.If carry out industrialization, also need at first carry out serum-free domestication, make cell adapted serum free medium and well-grown and continuous release produce recombinant protein to reduce cost and to make things convenient for the exploitation of downstream purification technology therein.Often cause the genetically engineered cell strain unstable in the cell non-serum domestication process; Cause the protein production ability drop; And the present invention uses the CHO-DHFR host cell CHO DG44 of the adaptation serum free medium of having tamed can omit this step, helps improving the stability of genetically engineered Chinese hamster ovary celI strain.
In addition; The animal serum or other the animal derived and/or humanized's protein ingredient that use in the serum cultivation are arranged; Not only increase production cost, but also increased the viral pollution of chance, particularly Ke-Ya Shi sick (being usually said mad cow disease) that the external source invasive organism pollutes.The serum-free that uses among the present invention/no albumen synthetic medium does not contain any animal derived and albumen property composition; CHO-DHFR host cell CHO DG44 after the domestication has obtained good conformity therein; And cell growth is with the product expression level and have blood serum medium suitable; Its technical characterstic is and can in extensive bio-reactor, realizes high-density culture by sustenticular cell, and helps foam control.
Strain classification: the Chinese hamster ovary celI strain, its deposit number is: CGMCC No.4541
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center
Preservation date: on January 5th, 2011
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica
Description of drawings
Fig. 1: the A09025-1 plasmid enzyme restriction is identified collection of illustrative plates
Fig. 2: the pOptiVEC/NGF-118 plasmid enzyme restriction is identified collection of illustrative plates
The M:DM1000DNA molecular weight standard; 1.Xba I and Not I double digestion pOptiVEC/NGF-118 plasmid; 2.pOptiVEC/NGF-118 plasmid
Fig. 3: ELISA detects 24 monoclonal cell culture supernatant of institute's picking
Fig. 4: quantitative ELISA detects 3 monoclonal cell culture supernatant that increase in 6 orifice plates
Fig. 5: Western blotting method detects 3 monoclonal cell culture supernatant that increase in 6 orifice plates
M: low molecular weight protein molecular weight standard; 1,2,3,4 swimming lanes: be respectively mouse NGF reference substance, No. 5, No. 8 and No. 20 monoclonal cell strain culture supernatant.
Fig. 6: the growth curve of CHO DG44-NGF-118 cell strain and reorganization NGF titre variation tendency
Fig. 7: the biological activity of chick embryonic dorsal root ganglion method (DRG) qualitative detection RHNGF
A, B: chick embryonic dorsal root ganglion (DRG) was induced 0 hour; After chick embryonic dorsal root ganglion (DRG) is induced 48 hours.Wherein, A, C are that mouse NGF (25ng/ml) induces group; B, D are that recombinant human NGF (25ng/ml) induces group.
Fig. 8: the Analytical Chemical Experiment of P of Rats C12 cell induction detects the biological activity of RHNGF.
A, B, the C:PC-12 cell induction was cultivated 0 hour; D, E, the F:PC-12 cell induction was cultivated 72 hours; G, H, the I:PC-12 cell induction was cultivated 144 hours.Wherein, A, D, the negative control group of G, B, E, H are that mouse NGF (1 μ g/ml) induces group; C, F, I are that recombinant human NGF (1 μ g/ml) induces group.
The practical implementation method:
The more detailed experimental technique of the present invention can be referring to embodiment.Present embodiment is to be used for explaining, rather than limits the present invention with method in any form.
Employed cell strain, bacterial strain, plasmid and reagent are the commercially available prod in the embodiment of the invention.
Embodiment one: the pOptiVEC/NGF-118 construction of recombinant plasmid
1.NGF the acquisition of gene
The sequence of people NGF protein (P01138) and gene in the inquiry UniProtKB DB; Ngf gene is optimized design; Ngf gene nucleotide sequence after the optimization shown in SEQ ID No.1, trust money only intelligence Bioisystech Co., Ltd to carry out full gene synthetic and be connected on the pcDNA3.1 carrier (Invitrogen company), make up successful pcDNA3.1/NGF-118 recombinant plasmid; Be numbered A09025-1, enzyme is cut and is identified that collection of illustrative plates is as shown in Figure 1.
2.pOptiVEC/NGF-118 the acquisition of recombinant plasmid
According to conventional molecule clone technology operation; PcDNA3.1/NGF-118 recombinant plasmid and pOptiVEC-polylinker carrier (Invitrogen company) are used Xba I and Not I (Dalian Takara company) double digestion respectively; The NGF-118 gene fragment of using glue to reclaim after test kit (Beijing health is the century bio tech ltd) recovery enzyme is cut then is connected with linearizing pOptiVEC carrier; Link product transformed into escherichia coli competent cell Trans10 (Beijing Quanshijin Biotechnology Co., Ltd); Utilize the little extraction reagent kit of plasmid (Beijing health is the century bio tech ltd) to extract plasmid after the picking mono-clonal enlarged culturing; Through Xba I and Not I double digestion and insertion sequence mensuration evaluation positive recombinant, obtain the pOptiVEC/NGF-118 recombinant plasmid, the double digestion qualification result is as shown in Figure 2.
Embodiment two: the CHO DG44 cell strain that obtains express recombinant people NGF
CHO DG44 cell is the Chinese hamster ovary line of dhfr defective, available from Invitrogen company.The by specification routine goes down to posterity before the CHO DG44 cell transfecting, with 3 * 10
5Cell/ml density is inoculated in the aseptic triangular flask of 125ml that contains 30ml CD DG44 nutrient solution, makes the next day cell density can reach 5 * 10
5Viable cell/ml, the transfection step is pressed the FreeStyle of Invitrogen company
TMMAX transfection reagent specification sheets carries out.With 15 μ l FreeStyle
TMMAX transfection reagent and 9 μ gpOptiVEC/NGF-118 recombinant plasmid dnas join 1.2ml OptiCHO
TMIn the SFM serum free medium, soft mixing, incubated at room 10min.1.2ml plasmid-transfection reagent mixed solution slowly and is uniformly splashed in the Tissue Culture Flask, put CO
2The incubator shaking culture.Behind the transfection 24h, be replaced by and contain the CD OptiCHO that methotrexate concentration is 10nM
TMThe substratum screening of pressurizeing, treat that cell viability and density are recovered after, respectively according to 20nM; 40nM, 60nM, 80nM; 100nM, the concentration gradient of 200nM and 400nM progressively improves the amplification of pressurizeing of the concentration of selecting methotrexate in the substratum, and enlarged culturing is carried out subsequent experimental.When MTX concentration is brought up to 400nM, by 3 * 10
5The density of/ml is inoculated in the 125ml culturing bottle, cultivates the culture supernatant of getting the cell mixing strain after 3 days and carries out the quantitative ELISA detection, and the result shows that it is 3160.5ng/ml that RHNGF is expressed titre.In order to obtain the monoclonal cell of high expression level, carried out cloning according to embodiment three described methods to mixing clone cell, in the hope of obtaining the monoclonal cell strain of high expression level.
Embodiment three: methylcellulose gum semisolid medium subclone is selected the monoclonal cell strain
Adopt CloneMatrix semisolid medium (available from Genetix company) to carry out cloning, operate according to products instruction.In brief, will mix clone cell by 1 * 10
3The density of/ml is inoculated into 100ml and contains in the CloneMatrix substratum of MTX of 400nM, at 37 ℃, 5%CO
2Condition under cultivate.Cultivate after 14 days, picking independently disperses, well-grown monoclonal cell, changes 96 orifice plates over to and cultivates, and gets culture supernatant after 3 days and carries out the ELISA detection.According to the ELISA detected result, choose the OD value than higher mono-clonal porocyte, change 24 orifice plate enlarged culturing over to, carry out ELISA after 5 days and detect, the result is as shown in Figure 3.According to ELISA result, choose the OD value and change 6 orifice plate enlarged culturing over to, freeze-stored cell when treating that cell density is suitable than 6 higher mono-clonal holes 5,8,16,20,22 and 24.Collect the cells and supernatant in each mono-clonal hole respectively, (its rhNGF content DY256) detects in R&D company, presses the operation of test kit specification sheets to use the ELISA test kit.Because effectively increased in mono-clonal hole 16,22 and 24 in the enlarged culturing process, have only 3 monoclonal cells 5,8 and 20 to obtain increasing and having carried out the quantitative ELISA detection, the result is as shown in Figure 4.The expression amount of rhNGF is the highest in No. 5 monoclonal cell strain culture supernatant, reaches 1618ng/ml, called after CHO DG44-NGF-118.Get the culture supernatant of 3 monoclonal cell strains and carry out the Tricine-SDS-PAGE electrophoresis; Resolving gel concentration is 10%; (SIGMA company N6655) carries out Western blotting and detects, and the result is as shown in Figure 5 to utilize the anti-mouse NGF of rabbit polyclonal antibody behind the commentaries on classics film; A specific band is arranged about molecular weight 13KD, consistent with the expection molecular weight.
Embodiment four: the growth curve of stable cell line and rhNGF titre variation tendency
Be the cell density of detection CHO DG44-NGF-118 cell strain and the relation between the rhNGF titre, the CHODG44-NGF-118 cell strain is pressed 1.7 * 10
5The density of/ml is inoculated into the OptiCHO that does not contain MTX
TMIn the Medium substratum, at 37 ℃, 5%CO
2With in the 500ml Erlenmeyer flask, carry out shaking culture under the condition of 130rpm, every 24h sampling detects the expression amount of cell density and rhNGF.After the continuous monitoring 9 days; Is X-coordinate according to measuring the result to cultivate fate; Cell density is left ordinate zou, and NGF expresses titre and is cell density and the rhNGF titre changing trend diagram that right ordinate zou is drawn CHO DG44-NGF-118 cell strain cultured continuously, and is as shown in Figure 6.The result showed, increases gradually like the prolongation cell density along with incubation time behind the cell inoculation, reached peak 64 * 10 at the 6th day
6/ ml, the titre of rhNGF then continues to increase, and the rhNGF titre reaches mxm. 25.5mg/L during to the 9th day.This prompting serum-free suspension culture helps high-density culture, reaches the highest back at cell density and causes cell to begin death because of the consumption of nutritive ingredient and the accumulation of metabolic waste, and cell density descends but rhNGF expresses also and continuing increase.If in bio-reactor, adopt feeding culture and dissolved oxygen, pH value, pol and meta-bolites are regulated and control, can also obtain the more rhNGF expression level of maxicell culture density and Geng Gao.
Embodiment five: the biological activity of DRGs method (DRG) qualitative detection RHNGF
For detecting the expressed rhNGF biological activity of CHO DG44-NGF-118 cell strain; Get the culture supernatant that contains rhNGF and behind ultrafiltration and concentration, carry out concentration calibration with the quantitative ELISA method; Be canonical reference article article with commercially available injection mouse NGF (SHUTAISHEN pharmaceutcal corporation, Ltd) then; Chicken embryo DRG induction method according to classics carries out this experiment, and concrete operation method is following:
1. separation chick embryonic dorsal root ganglion: obscure glass is placed on the super clean bench horizontal plane, got the chicken embryo, remove the part Ovum crusta Gallus domesticus of air cavity face gently, observe the inner membrance that spreads all over fresh capillary vessel; Break the back and take out the chicken embryo, behind the disconnected neck chicken four limbs are sticked on the obscure glass, dissect, gill with pincet; The careful backbone that exposes is under it placement stereoscopic microscope, in the time of observation; Take out DRGs with the microdissection tweezers, put to (Poly-L-Lysine is in 24 orifice plates that PLL) encapsulated with poly-lysine; 2～3 neuroganglions in every hole add the DMEM basic medium of 1ml, place in 37 ℃, the incubator of 5%CO2 and cultivate 1h.
2.NGF induce chick embryonic dorsal root ganglion: will cultivate ganglionic DMEM substratum and be replaced by DMEM pastille substratum (NGF of different concns); Be divided into two groups of NGF sample to be measured and standard substance; NGF sample wherein to be measured and standard substance are done 2 times of doubling dilutions with the DMEM substratum; Each sample is done 5～6 extent of dilution (should be diluted to and negative findings occur), under inverted microscope, observes ganglionic state behind the cultivation 48h.As shown in Figure 7; NGF is the same with mouse; We expressed rhNGF has good biological activity, can induce chick embryonic dorsal root ganglion growth, can be observed fine and close under the inverted microscope and elongated a large amount of nerve fiber in DRGs, be radial to around growth.
Embodiment six: the biological activity of P of Rats C12 cell induction method qualitative detection RHNGF
NGF can induce rat have a liking for chromium neurocytoma PC12 differentiation and produce a large amount of radial nerve fiber aixs cylinders to around radiation growth and being coupled to each other.Therefore, we are standard substance with commercially available injection mouse NGF (SHUTAISHEN pharmaceutcal corporation, Ltd), adopt PC12 cell induction method to detect the biological activity of rhNGF, and concrete operation method is following:
1. the preparation of standard solution: (30 μ g/ bottles after BA >=15000AU) by specification redissolves, are diluted to 15 μ g/ml with basic culture solution to get mouse NGF standard substance.Configuration standard article solution in the 5ml centrifuge tube, weaker concn is 2 μ g/ml in advance, carries out 2 times of serial dilutions then, totally 5 extent of dilution are transferred to 24 orifice plates (encapsulating with poly-lysine in advance) with standard solution, corresponding 1 hole of each concentration, every hole 500 μ l.
2. the preparation of detected sample solution: detected sample is diluted to 2 μ g/ml with basic culture solution; Carry out 2 times of serial dilutions then; Totally 5 extent of dilution are transferred to 24 orifice plates (encapsulating with PLL in advance) with standard solution, corresponding 1 hole of each concentration; Every hole 500 μ l more than operate under the aseptic condition and carry out.More than operate under the aseptic condition and carry out.Alternative is selected 2 holes and is added negative control solution 500 μ l (the not substratum of drug), more than operates under the aseptic condition and carries out.
3. activation measurement: the PC12 cell is cultivated in 37 ℃, 5% carbonic acid gas with perfect medium, and the control cell concn is that every 1ml contains 1.0 * 10
5～5 * 10
5Individual cell.Go down to posterity the back after 24～36 hours, be used for Determination of biological activity.It is warm in advance to 37 ℃ to test used solution.Get capacity PC12 cell culture, centrifugal collection PC12 cell is resuspended in basic medium and is made into every 1ml and contains 2 * 10
4The cell suspension of individual cell is inoculated in the hole of containing NGF standard substance or sample in the 24 porocyte culture plates, every hole 500 μ l.Place 37 ℃, 5% carbonic acid gas is cultivated, and whenever changes fresh liquid medium at a distance from 3 days, keeps identical drug level.The state of observation of cell growth and photograph under inverted microscope.As shown in Figure 8; Inductive PC12 cell quantity does not increase to some extent; But do not see the nerve fiber growth, and the same with mouse NGF, rhNGF of the present invention has good biological activity; Can induce the growth of PC12 cytodifferentiation and nerve fiber, can be observed under the inverted microscope the elongated nerve fiber that is branch dress from the PC12 cell be radial to around grow and be coupled to each other.
The above only is preferred embodiment of the present invention, is not the present invention is done any pro forma restriction.Though the present invention with preferred embodiment openly as above, yet be not that any way limits the present invention.Any those skilled in the art; In technical scheme scope of the present invention; Allly utilizing above-mentioned disclosed technology contents to make a little change or modification, should be regarded as the equivalent embodiment of equivalent variations, is not break away from technical scheme content of the present invention in every case;, all still belong in the scope of technical scheme of the present invention any simple modification, equivalent variations and modification that above embodiment did according to technical spirit of the present invention.
Claims (10)
1. the CHO DG44-NGF-118 cell strain of efficiently expressing recombinant human NGFF, its deposit number is: CGMCC No.4541.
2. CHO DG44-NGF-118 cell strain according to claim 1, its host cell is the CHO DG44 of the domestication that suspends through serum-free.
3. Chinese hamster ovary celI strain according to claim 1, said Chinese hamster ovary celI strain have proteinic nucleotide sequence shown in the coding SEQ ID No.2.
4. Chinese hamster ovary celI strain according to claim 3, said nucleotide sequence is shown in SEQ ID No.1.
5. the construction process of the arbitrary described Chinese hamster ovary celI strain of claim 1-4 may further comprise the steps:
(1) nucleotide sequence of synthetic shown in SEQ ID No.1 is inserted into the MCS of pOptiVEC-polylinker, obtains recombinant plasmid,
(2) with plasmid change over to serum-free suspend domestication CHO DG44 in,
(3) mode that progressively the improves methotrexate concentration screening of pressurizeing obtains mixing clone cell,
(4) adopt the methylcellulose gum semisolid medium to carry out subclone and select the monoclonal cell strain, adopt the expression amount of ELISA method detection monoclonal cell strain, the Chinese hamster ovary celI strain of the RHNGF that screening obtains efficiently expressing.
6. construction process according to claim 5, wherein nucleotide sequence is inserted between the Xba I and Not I restriction enzyme site of pOptiVEC-polylinker.
7. construction process according to claim 6; Said recombinant plasmid is pOptiVEC/NGF-118; Its preparation method comprises the steps: the nucleotide sequence of (A) synthetic shown in SEQ ID No.1; And be connected on the pcDNA3.1 carrier and make up successful pcDNA3.1/NGF-118 recombinant plasmid, (B) use Xba I and Not I double digestion pcDNA3.1/NGF-118 recombinant plasmid and pOptiVEC-polylinker respectively, the ngf gene fragment after reclaiming enzyme then and cutting is connected with linearizing pOptiVEC carrier; The transformed into escherichia coli competent cell; Extract plasmid after the picking mono-clonal enlarged culturing, identify positive recombinant, obtain the pOptiVEC/NGF-118 recombinant plasmid with double digestion method and sequencing method.
8. construction process according to claim 5, the ultimate density of said methotrexate are 400nM.
9. construction process according to claim 8; Said step (3) pressurization screening is respectively 20nM according to concentration, 40nM then for being that 10nM selects substratum to begin the pressurization screening containing methotrexate concentration; 60nM; 80nM, 100nM, the gradient of 200nM and 400nM progressively improves the concentration of selecting methotrexate in the substratum.
10. the application of the arbitrary said Chinese hamster ovary celI strain of claim 1-4 in producing the growth factor of human nerve pharmaceutical grade protein.
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