CN106497881A - Stability and high efficiency expression recombined human hyaluronidase CHO K1SP cell lines and its construction method - Google Patents
Stability and high efficiency expression recombined human hyaluronidase CHO K1SP cell lines and its construction method Download PDFInfo
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- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2474—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
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- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01035—Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
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Abstract
The present invention provides the CHO K1SP cell lines and its construction method of stability and high efficiency expression recombined human hyaluronidase.Including following step：Step A：Artificial synthesized nucleotide sequence as shown in SEQ ID N0.2, is inserted into the MCS of pGenHT1.0 carriers, obtains recombinant plasmid；Step B：The CHO K1SP cells that plasmid is proceeded to serum free suspension domestication；Step C：Pressurization screening is carried out in CD CHO+MSX screening and culturing mediums, obtains mixing clone cell；Step D：Monoclonal cell strain is selected, screening obtains the CHO K1SP cell lines of the recombined human hyaluronidase of high efficient expression.Serum-free used in the present invention/do not contain any animal derived and Proteinaceous composition without albumen synthetic media, and cell growth and Product Expression level are with to have blood serum medium suitable, its technical characterstic is that energy sertoli cell realizes High Density Cultivation in Large Scale Biology reactor, and is conducive to foam control.
Description
Technical field
The invention belongs to technical field of bioengineering, is related to the CHO- that a kind of stability and high efficiency expresses recombined human hyaluronidase
K1SP cell lines.
Background technology
Hyaluronic acid (HA) is principally found in the connective tissue of mammal, skin, cartilage and in synovia.Hyalomitome
Acid is also the main constituents of eye vitreous.In connective tissue, the water of the aquation related to hyaluronic acid is produced
Space between tissue, so as to being that cell movement and propagation create favourable environment.Hyaluronic acid is being had with cell movement
Key effect is played in the biological phenomena of pass, and these cell movements include Rapid development (rapid development), again
Life, reparation, embryo's generation, embryonic development, wound healing, Angiogenesiss and tumour occur.
Hyaluronidase is the hydrolase of a class specific for hydrolysis hyaluronic acid, is widely present in eucaryote and protokaryon life
In thing, hyaluronic acid is hydrolyzed mainly, is a kind of important physiological activator.Many important biologies are participated in animal body
Process, such as cell division, intercellular connection, the activity of reproduction cell, the transfection of DNA, embryonic development, wounded tissue
Repair, and normal cell and tumor cell proliferation.Many pathological change process are usually associated with hyaluronidase and hyalomitome
The change of acid, implies that they may play an important role.Simultaneously hyaluronidase can also change in body some medicines and
The distribution situation of physiological activator.
Hyaluronidase has three major types:1. mammalian-type hyaluronidase (EC 3.2.1.35), which is interior-N- second
Acylamino- hexoside enzyme, using tetrose and hexose as main end-product.They have hydrolysis and transglycosylase activity, can drop
Solution hyaluronic acid and chondroitin sulfate (CS), especially C4-S and C6-S.2. bacterial hyaluronidase (EC 4.2.99.1) degraded
Hyaluronic acid, and the CS and DS that degrades to some extent.They are interior-P-N- acetyl hexosaminidases, and they are eliminated anti-by P
Should play a role, main generation disaccharides end-product.3. from the hyaluronidase of leech, other parasites and shellfish
(EC 3.2.1.36), which is interiorGlycuronidase, produces tetrose and hexose end-product by hydrolyzing P 1-3 keys.Mammal
Hyaluronidase can be further divided into two groups:Neutral active (neutral active) and acidity activity (acidactive)
Enzyme.There are six kinds of hyaluronidase sample genes in human genome:HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and PH20/
SPAM1.HYALPl is pseudogene, and HYAL3 is not shown with enzymatic activity to any of substrate.HYAL4 is cartilage
Plain enzyme (chondroitinase), lacks activity to hyaluronic acid.HYALl be prototype acidity organized enzyme, acid reactive transparent matter
Sour enzyme, such as HYALl and HYAL2 lack catalysis activity in neutral pH.For example, more than pH4.5's HYALl lives without catalysis in vitro
Property (Frost et al Anal Biochemistry, 1997).HYAL2 is acid organized enzyme, and there is low-down ratio in vitro
Activity.PH20/SPAM1 is prototype neutral active enzyme, and it can be catalyzed transglycosylation, therefore, during hydrolysis HA
Hexose, disaccharides and eight carbon sugar can be formed.
Hyaluronic acid hydrolysis enzyme is clinically of many uses as a kind of pharmacological active substance, and the field of application includes medicine
Penetrating agent, cancer, diabetes, antibacterials, infant's medication and first aid fluid-supplement therapy.Hyaluronidase conventional use be for
The infiltration for improving other injectable drugs and the tissue permeability for increasing other drugs and promotion diffusion disperse.Modal should
With the rapid osmotic (such as dentistry) for being ophthalmologic operation and local anesthetic, make when cannot find blood vessel in child vein drop
With.Transparent bright matter acid enzyme is that malignant tissue's glycosaminoglycan content after hyaluronic acid ferment treatment increases in the mechanism of action of anti-tumor aspect
Plus, be conducive to tumour cell to combine more cancer therapy drugs.As it can strengthen anti-cancer ability of the adriamycin to breast cancer, reduce
Recurrence rate of carcinoma of urinary bladder etc..In clinical practice application, hypodermic injection is coordinated to replace intravenous drip increase with monoclonal antibody
The bioavilability of antibody drug；On treating diabetes market, hypodermic injection is combined with injection of insulin agent, can promote pancreas islet
The absorption of element, reduces the usage amount of insulin, reduces hypoglycemic incidence.
The commercially available hyaluronidase polyphyly of China is extracted from animal (ox, sheep) testis tissue at present, and purity is low, foreign protein
Content is higher, and immunogenicity is stronger.The hyaluronidase that animal tissue extracts occurs obvious bad reaction in clinical practice,
Such as there is serious inflammatory reaction and corneal injury in early stage hyaluronidase after injected into anterior chambers, may be the hyalomitome of injection
Sour enzyme impurities cause.As hyaluronidase is in the development of ophthalmic applications, its anaphylactoid report gradually increases, seriously
Limit hyaluronidase market at home.Low in order to solve domestic hyaluronidase purity, immunogenicity is not suitable for by force people
Defect, we build people source hyaluronidase expression plasmid, by Chinese hamster ovary celI in serum-free by gene recombination technology
Culture medium in expression and purification obtain hyaluronidase, without any animal derived components, without potential pathogenic risk, be difficult to produce
Raw immunity and inflammatory reaction, and its production scale will substitute the transparent of animal tissue's extraction not by organizing raw material sources to be limited
Matter acid enzyme, meets domestic great market needs, brings huge economic benefit.
Content of the invention
It is an object of the invention to provide a kind of CHO-K1SP cell lines of efficiently expressing recombinant human hyaluronidase, the present invention
Another purpose be by set up screening obtain have stability and high efficiency expression have biologically active recombined human hyaluronidase
The method of CHO-K1SP cell lines, sets up a kind of skill of expression and production with labyrinth and the recombinant protein of biologically active
Art platform.Described technology platform is by using serum free suspension culture technique, it is convenient to omit the serum-free domestication of prior art
Process, not only improves subclone success rate, but also greatly facilitates the downstream purification of recombinant protein.Its host cell be through
Cross the CHO-K1SP of serum free suspension domestication.
The construction method of above-mentioned Chinese hamster ovary celI strain, comprises the following steps:
(I) artificial synthesized nucleotide sequence as shown in SEQ ID NO.2, is inserted into the polyclonal of pGenHT1.0 carriers
Site, obtains recombinant plasmid,
(2) plasmid is proceeded in the CHO k1sp of serum free suspension domestication
(3) pressurization screening is carried out in CD-CHO+msx screening and culturing mediums, obtain mixing clone cell,
(4) monoclonal cell strain is selected using limiting dilution assay, is detected using Dotblot methods and screening positive clone, sieve
Choosing obtains the Chinese hamster ovary celI strain of the recombined human hyaluronidase of high efficient expression.With DNS methods, nephelometry and ELISA method detection cell line
The activity of the hyaluronidase of culture supernatant.
A kind of CHO-K1SP cell lines, can efficiently expressing recombinant human hyaluronidase.
Present invention also offers a kind of construction method of CHO-K1SP cell lines, including following step：
Step A：Artificial synthesized nucleotide sequence as shown in SEQ ID NO.2, is inserted into many grams of pGenHT1.0 carriers
Grand site, obtains recombinant plasmid；
Step B：Plasmid is proceeded in the CHO k1sp of serum free suspension domestication；
Step C：Pressurization screening is carried out in CD-CHO+msx screening and culturing mediums, obtains mixing clone cell,
Step D：Monoclonal cell strain is selected, screening obtains the Chinese hamster ovary celI strain of the recombined human hyaluronidase of high efficient expression.
Preferably, step A includes following step：
Step A1:The acquisition of rHuPH20 genes：People's hyaluronidase (rHuPH20) in inquiry UniProtKB databases
The sequence of gene, chooses the extracellular regions of rHuPH20, and amino acid sequence is carried out to its nucleotide sequence as shown in SEQ ID N0.1
Codon optimization, then carry out full genome and synthesize and be connected on puc57 carriers, build puc57/rHuPH20 recombinant plasmids；
Step A2:The acquisition of pGenHT1.0/rHuPH20 recombinant plasmids：Based on the method carrier construction of homologous recombination, profit
RHuPH20 fragments are expanded with puc57/rHuPH20 recombinant plasmids PCR, with Hind III and ECOR I double digestions by pGenHT1.0
The PCR primer of rHuPH20, and the pGenHT1.0 carriers through Hind III and ECOR I double digestions is added in aseptic EP pipes
Fragment, T4DNA polymerases and T4 buffer solutions, after reaction, by product transformed competence colibacillus bacillus coli DH 5 in LB (Amp+)
Flat board coating culture, screening positive clone are transferred in LB (Amp+) nutrient solution, are tried using a small amount of rapid extraction after incubated overnight
Agent box carries out plasmid extraction, enters performing PCR identification.
Step A3:The preparation of recombinant plasmid：Using extract pGenHT1.0/rHuPH20 recombinant plasmids, through centrifugation after, thoroughly
After analysing, precipitate, dry and dissolving, save backup.
Preferably, step D includes following step：
Step D1：Stable transfection:To routinely pass on before CHO-K1SP cell transfectings, after inoculation, transfection, then transfection is tried
Agent and pGenHT1.0/rHuPH20 recombinant plasmid dnas are added to OptiCHOTMIn SFM serum free mediums, soft mixing, incubation,
Again the plasmid for obtaining-transfection reagent mixed liquor slowly and is uniformly added dropwise in Tissue Culture Flask, shaken cultivation；After transfection,
By in passage to Tissue Culture Dish, change into after being further continued for passing on and screened containing screening and culturing medium, treat cell viability and close
After degree recovers, cloning has been carried out to mixing clone cell, to obtain the monoclonal cell strain of high expression；
Step D2：Select monoclonal：By counting to suspension cell, cell is carried out a series of dilution paving orifice plate
On, then cultivated, taking culture supernatant carries out detection selection monoclonal hole cell, continues Amplification Culture, treat that cell density is suitable
When freeze-stored cell.
The CHO-K1SP cell lines of the efficiently expressing recombinant human hyaluronidase set up by the present invention, No. 5 Dan Ke therein
RHuPH20 expression highests in grand cell line culture supernatant, reach 1500U/ml.The CHO K1-rHuPH20 born of the same parents of the present invention
Strain, its host cell is the CHO K1-rHuPH20 of domestication for having adapted to serum free suspension culture, itself and existing document report side
The advantage that method is compared is:
The Chinese hamster ovary celI strain of the expression recombinant protein used in existing document is needed with the presence of cow's serum or analogous components
In the case of adherent growth, expression of the serum or protein component in cell culture medium to recombinant protein in cell and after
The protein purification procedures of phase have a great impact.Therefore, if industrialization is tamed firstly the need of the serum-free for carrying out cell line, but
Domestication Cheng Zhonghui causes genetically engineered cell strain unstable, causes expressing quantity to reduce, and what present invention use had been tamed
Adapt to the CHO-GS host cell CHO K1SP of serum free medium, it is convenient to omit this step, be conducive to improving genetic engineering CHO
The stability of cell line.Additionally, the animal blood serum having used in serum free culture system or other animal derived and/or humanized's albumen into
Point, production cost is not only increased, and also add the chance that external source invasive organism pollutes, particularly Ke-Ya Shi diseases
The pollution of (i.e. usually said rabid ox disease) virus.Serum-free used in the present invention/without albumen synthetic media is without any
Animal derived and Proteinaceous composition, and cell growth and Product Expression level, with to have blood serum medium suitable, its technical characterstic exists
High Density Cultivation is realized in Large Scale Biology reactor in energy sertoli cell, and is conducive to foam control.
The CHO--k1sp systems of the present invention are belonging to GS systems, compared to DHFR- gene delections CHO- that tradition is used
The gene magnification screening system of DG44 host cells, GS systems have the obvious time excellent in cell line screening and growth course
Gesture, because GS systems can form high producer cell line by initial transfectant, is not required to by improving constantly many of selective reagent concentration
Take turns selection to realize gene magnification.And CHO-K1SP is than the CHIO-GS of gene defect, CHO-DG44G is easier to cultivate and strong
Strong, glutamine need not be added in culture medium, glutamine decomposition can be avoided to cause, and ammonia level in cultivating system is high to ask
Topic, reduces the difficulty of technology controlling and process, is conducive to improving cell fermentation density and extends the cells survival time.
Description of the drawings
Fig. 1 is the electrophoresis pattern of pGenHT1.0/rHuPH20 recombinant plasmids, and wherein 1 is that pGenHT1.0/rHuPH20 is carried
Body, 2 is MLUI the and Sal I double digestions of pGenHT1.0/rHuPH20 carriers, and 3 is the Hind of pGenHT1.0/rHuPH20 carriers
III and EcorR I double digestions.
Fig. 2 is the secreting, expressing of recombined human hyaluronidase in SDS-PAGE methods detection positive colony cells and supernatant.
Fig. 3 is the secretion table of recombined human hyaluronidase in Western blot methods detection positive colony cells and supernatant
Reach.
Fig. 4 is the Dot blot screening figures of the cell line for expressing recombined human hyaluronidase.
Specific embodiment
Below in conjunction with the accompanying drawings, the preferably embodiment of the present invention is described in further detail：
The structure of 1 pGenHT1.0/rHuPH20 recombinant plasmids of embodiment
The acquisition of rHuPH20 genes：
In inquiry UniProtKB databases, the sequence of the gene of people's hyaluronidase (rHuPH20), chooses rHuPH20 born of the same parents
Outer region, amino acid sequence carry out codon optimization as shown in SEQ ID N0.1 to its nucleotide sequence, entrust Nanjing gold this
Auspicious Bioisystech Co., Ltd carries out full genome and synthesizes and be connected on puc57 carriers (genscript companies), successfully constructs
Puc57/rHuPH20 recombinant plasmids.
The acquisition of pGenHT1.0/rHuPH20 recombinant plasmids
As shown in figure 1, the method carrier construction based on homologous recombination, is expanded using puc57/rHuPH20 recombinant plasmids PCR
RHuPH20 fragments, pGenHT1.0 is added in aseptic EP pipes 4ul rHuPH20's with Hind III and ECOR I double digestions
The pGenHT1.0 carrier segments of PCR primer and 1ug through Hind III and ECOR I double digestions, 0.5ul T4DNA polymerases,
4ulT4 buffer solutions, plus distilled water complements to 20ul, in room temperature reaction 30min.By product transformed competence colibacillus Escherichia coli
The coating culture of DH5, LB (Amp+) flat board, screening positive clone are transferred in LB (Amp+) nutrient solution, using little after incubated overnight
Amount rapid extraction kit carries out plasmid extraction, enters performing PCR identification.Nanjing Jin Sirui Bioisystech Co., Ltd is entrusted to restructuring
Plasmid carries out sequencing confirmation, final acquisition .pGenHT1.0/rHuPH20 recombinant plasmids.
A large amount of preparations of 1.3 recombinant plasmids
PGenHT1.0/rHuPH20 recombinant plasmids are extracted in a large number using alkaline lysis, through cesium chloride density gradient centrifugation after,
Supercoil state plasmid is reclaimed, through dialysis, precipitation, after drying and dissolving, is saved backup in -20 DEG C.
Embodiment 2 obtains the CHO-K1SP cell lines of expression recombined human hyaluronidase rHuPH20
2.1 stable transfection
Routinely pass on before CHO--k1sp cell transfectings, with 3XIO5Cell/ml density is inoculated in containing 30ml nutrient solutions
The aseptic triangular flasks of 125ml in, enable next day cell density to reach 5XIO5Living cells/ml, it is public that transfection procedure presses Invitrogen
The FreeStyle of departmentTMMAX transfection reagents specification is carried out.By 15 μ l FreeStyleTMMAX transfection reagents and 9 μ g
PGenHT1.0/rHuPH20 recombinant plasmid dnas are added to 1.2ml OptiCHOTMIn SFM serum free mediums, soft mixing,
Incubated at room lOmin.1.2ml plasmids-transfection reagent mixed liquor slowly and is uniformly added dropwise in Tissue Culture Flask, CO2 is put
Incubator shaken cultivation.After transfection 24h, according to 1:10 ratio passes on 24h by passage to 150mm Tissue Culture Dish
Change the screening and culturing medium containing 25uMmsx afterwards into be screened, maintain pressure screening and culturing surrounding, treat that cell viability and density are extensive
After multiple, cloning is carried out to mixing clone cell using limiting dilution assay, to obtaining the monoclonal cell strain of high expression.
2.2 select monoclonal using limiting dilution assay
By counting to suspension cell, by cell carry out a series of be diluted to 5/ml, then with 200 μ I's of every hole
Cell suspension spreads 96 orifice plates, in 37 DEG C, volumetric concentration 5%CO2Under conditions of cultivate two weeks, taking culture supernatant carries out dot blot
Detection.According to testing result, the high monoclonal hole cell of expression is chosen, proceed to 24 orifice plate Amplification Cultures, culture turned after 3-7 days
Enter 6 orifice plate Amplification Cultures, return again to T25 and 125ml shaking flask Amplification Cultures, freeze-stored cell when cell density is suitable.
2.3Dotblot methods select positive monoclonal
Dotblot methods are to collect cell culture supernatant as sample, put film, using pvdf membrane (need to steep methyl alcohol), press
According to sampfe order point film, 5ul/ points, ghost nutrient solution are carried out mark as negative control with ball pen, film are placed in 37 DEG C
Dry, about 10-15min；Closing, prepares 5% skimmed milk power with TBST, and room temperature shaker closes 1h；TBST is washed 1-2 time；One resists
(TBST preparations) is incubated 1h；TBST is washed three times, each 10min；Two anti-(TBST preparations) incubation 1h；TBST is washed three times, per
Secondary 10min；ECL develops, and as a result as shown in figure 4, screening 30 positive monoclonals altogether, selects 10 high Dan Ke of expression
Grand be amplified culture, freeze-stored cell is used as CHO-K1SP-rHuPH20 engineering cell strains.
Embodiment 3 identifies expression of the rHuPH20 in the cell culture of CHO-K1SP-rHuPH20 engineering cell strains
The culture supernatant that have selected the positive colony of former of expression carries out SDS-PAGE and Western blot detections.
Method is：Cell culture supernatant is collected, the Loading buffer of respective amount is added, is boiled 5min；SDS-PAGE electrophoresis；Turn
Film, after electrophoresis is complete, glue is carefully peeled, and is installed with filter paper, fiber mat, nitrocellulose membrane in the correct order, -20 DEG C be soaked in turn
Move buffer solution 40min；Electrophoretic blotting box is installed, rotor, ice chest is put into, is connected power supply, 100V constant pressures turn on magnetic stirring apparatus
Move lh；Transfer device is removed after the completion of transfer, film is taken off, positive and negative (film is front with the one side that sticker) has been marked, has been put into
Ponceaux dye liquor dyes 5-10min, takes out, and deionized water washes away loose colour, observes transfer effect, and Ponceaux dyeing can be done can not
Do；Confining liquid is added, 4 DEG C overnight, or room temperature lh；TBST washes film 1-2 time；Resisted with TBST dilutions one, be placed in room on horizontal shaker
Temperature incubation lh；TBST is washed three times, each 10min；Resisted with TBST dilutions two, be placed in incubated at room lh on horizontal shaker；TBST
Washing three times, each 10min, ECL develop the color.SDS-PAGE is shown in Fig. 2, and 10 plants of engineering cell strains sun occur near 60KD
Property band, consistent with predicting the outcome.Western blot results are as shown in figure 3,10 plants of engineering cell strains occur near 60KD
Positive band, consistent with predicting the outcome, the molecular weight of glycosylated rHuPH20 is 66KD.Result of the test shows successfully structure table
CHO-K1SP engineering cell strains up to rHuPH20.
The biologically active of rHuPH20 in the detection culture supernatant of embodiment 4
DNS is reduced to amino-compound by the reduced sugar in haase degraded ha products in alkaline solution, shows in boiling water bath
Color time 5min, determines the absorbance A 540 at 540nm immediately after cooling.The HA solution of 0.5ml 0.5% and 0.5ml sample liquids
Mixing, 37 water-bath 30min are boiled 5min and stop reaction, and centrifugation albuminate takes supernatant 0.4ml, plus 0.8ml DNS
Solution.DNS is reduced to amino-compound by the reduced sugar in HAase degraded HA products, boils 5min and is allowed to fully colour developing, cooling
Determine immediately afterwards.Blank group replaces sample liquid with phosphate buffer.
After 10 plants of CHO-K1SP-rHuPH20 engineering cell strains culture one week, nutrient solution supernatant is extracted respectively, dilute 50 times
The activity that HAase is determined with DNS methods, the results are shown in Table 1.CHO-K1SP cells are not detected by the work of HAase as negative control
Property.As a result as shown in table 1, the enzyme activity expression of 10 plants of CHO-K1SP-rHuPH20 engineering cell strains is all higher than 1000U/ml, most
Up to more than 1500U/ml.
The dynamic observation and stability analysis of 5 CHO-K1SP-rHuPH20 engineering cell strains of embodiment secretion rHuPH20
The dynamic that 10 plants of CHO-K1SP-rHuPH20 engineering cell strains secrete rHuPH20 is observed, such as Fig. 3 institutes
Show, after cell line passed on for 60 generations in the case where MSX pressure conditions are removed, remain to stably excreting rHuPH20, show through screening the height for obtaining
The CHO-K1SP cell lines of effect expression rHuPH20 are more stable, are worth with commercialization.
Table 1
| Cell line ID | Hyaluronidase activity (unit/ml) |
| 28 | 1090 |
| 133 | 1500 |
| 79 | 1100 |
| 140 | 1405 |
| 25 | 1510 |
| 65 | 1385 |
| 4 | 1112 |
| 5 | 1315 |
| 71 | 1275 |
| 18 | 1145 |
SEQ ID No1：RHuPH20 amino acid sequences
MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLD
MSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVID
WEEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPD
CYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPV
FAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCS
QVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDA
VDVCIADGVCIDAFLKPPMETEEPQIFY
SEQ ID NO2：Nucleotide sequence after rHuPH20 optimizations
ATGGGCGTGCTGAAGTTCAAGCACATCTTCTTCCGGTCCTTCGTGAAGTCCTCCGGCGTGTCCCAGATC
GTGTTTACCTTCCTGCTGATCCCCTGCTGCCTGACCCTGAACTTTCGGGCCCCTCCCGTGATCCCCAACGTGCCATT
TCTGTGGGCCTGGAACGCCCCCTCCGAGTTCTGTCTGGGCAAGTTCGACGAGCCCCTGGATATGTCCCTGTTCTCCT
TCATCGGCTCCCCCCGGATCAATGCTACCGGCCAGGGCGTGACCATCTTCTACGTGGACCGGCTGGGCTACTACCCC
TACATCGACTCCATCACCGGCGTGACCGTGAACGGCGGCATCCCCCAGAAGATCTCCCTGCAGGACCACCTGGACAA
GGCCAAGAAGGACATCACCTTCTACATGCCCGTGGACAACCTGGGCATGGCCGTGATCGACTGGGAGGAATGGCGGC
CTACCTGGGCCAGAAACTGGAAGCCCAAGGACGTGTACAAGAACCGGTCCATCGAGCTGGTGCAGCAGCAGAACGTG
CAGCTGTCTCTGACCGAGGCCACCGAGAAGGCTAAGCAGGAATTCGAGAAGGCCGGCAAGGACTTCCTGGTGGAAAC
CATCAAGCTGGGCAAGCTGCTGCGGCCCAATCACCTGTGGGGCTACTATCTGTTCCCCGACTGCTACAACCACCACT
ACAAGAAGCCCGGCTACAACGGCTCCTGCTTCAACGTGGAAATCAAGCGGAACGACGACCTGTCCTGGCTGTGGAAC
GAGTCCACCGCCCTGTACCCCTCCATCTACCTGAACACCCAGCAGAGCCCTGTGGCCGCCACACTGTACGTGCGGAA
CAGAGTGCGCGAGGCCATCAGAGTGTCCAAGATCCCCGACGCCAAGTCCCCCCTGCCTGTGTTCGCCTACACCCGGA
TCGTGTTCACCGACCAGGTGCTGAAATTCCTGAGCCAGGATGAGCTGGTGTATACCTTCGGCGAGACAGTGGCCCTG
GGCGCCTCTGGAATCGTGATCTGGGGCACCCTGTCCATCATGCGGTCCATGAAGTCCTGCCTGCTGCTGGACAACTA
CATGGAAACAATCCTGAACCCTTACATCATCAACGTGACCCTGGCCGCCAAGATGTGTTCTCAGGTGCTGTGCCAGG
AACAGGGCGTGTGCATCCGGAAGAACTGGAACTCCTCCGACTACCTGCACCTGAACCCCGACAACTTCGCCATTCAG
CTGGAAAAGGGCGGCAAGTTCACCGTGCGGGGCAAGCCCACACTGGAAGATCTGGAACAGTTCTCCGAGAAGTTCTA
CTGCTCCTGCTACTCCACCCTGAGCTGCAAAGAAAAGGCCGACGTGAAGGACACCGACGCCGTGGACGTGTGTATCG
CCGACGGCGTGTGTATTGACGCCTTCCTGAAGCCCCCCATGGAAACCGAGGAACCTCAGATCTTCTAC
Above content is further description made for the present invention with reference to specific preferred embodiment, it is impossible to assert
The present invention be embodied as be confined to these explanations.For general technical staff of the technical field of the invention,
On the premise of without departing from present inventive concept, some simple deduction or replace can also be made, should all be considered as belonging to the present invention's
Protection domain.
Claims (6)
1. a kind of CHO-K1SP cell lines, it is characterised in that can efficiently expressing recombinant human hyaluronidase.
2. the construction method of CHO-K1SP cell lines as claimed in claim 1, it is characterised in that including following step：
Step A：Artificial synthesized nucleotide sequence as shown in SEQ ID N0.1, is inserted into the polyclonal position of pGenHT1.0 carriers
Point, obtains recombinant plasmid；
Step B：Plasmid is proceeded in the CHO-K1SP of serum free suspension domestication；
Step C：Pressurization screening is carried out in CD-CHO+msx screening and culturing mediums, obtains mixing clone cell；
Step D：Monoclonal cell strain is selected, screening obtains the Chinese hamster ovary celI strain of the recombined human hyaluronidase of high efficient expression.
3. method as claimed in claim 2, it is characterised in that step A includes following step：
Step A1:The acquisition of rHuPH20 genes：The gene of people's hyaluronidase (rHuPH20) in inquiry UniProtKB databases
Sequence, choose the extracellular regions of rHuPH20, amino acid sequence carries out password to its nucleotide sequence as shown in SEQ ID N0.1
Son optimizes, then carries out full genome and synthesize and be connected on puc57 carriers, builds puc57/rHuPH20 recombinant plasmids；
Step A2:The acquisition of pGenHT1.0/rHuPH20 recombinant plasmids：Based on the method carrier construction of homologous recombination, utilize
Puc57/rHuPH20 recombinant plasmids PCR expands rHuPH20 fragments, pGenHT1.0 is existed with Hind III and ECOR I double digestions
The PCR primer of rHuPH20, and the pGenHT1.0 carrier-pellets through Hind III and ECOR I double digestions is added in aseptic EP pipes
Section, T4DNA polymerases and T4 buffer solutions are after reaction, flat in LB (Amp+) by product transformed competence colibacillus bacillus coli DH 5
Plate coating culture, screening positive clone is transferred in LB (Amp+) nutrient solution, using a small amount of rapid extraction reagent after incubated overnight
Box carries out plasmid extraction, enters performing PCR identification；
Step A3:The preparation of recombinant plasmid：Using pGenHT1.0/rHuPH20 recombinant plasmids are extracted, through centrifugation after, dialysis, heavy
After forming sediment, dry and dissolving, save backup.
4. method as claimed in claim 2, it is characterised in that step D includes following step：
Step D1：Stable transfection:To routinely pass on before CHO-K1SP cell transfectings, after inoculation, transfection, then by transfection reagent and
PGenHT1.0/rHuPH20 recombinant plasmid dnas are added in OptiCHO SFM serum free mediums, soft mixing, incubation, then will
The plasmid for obtaining-transfection reagent mixed liquor is slow and is uniformly added dropwise in Tissue Culture Flask, shaken cultivation；After transfection, will be thin
Born of the same parents are passaged in Tissue Culture Dish, change into and screened containing screening and culturing medium after being further continued for passing on, and treat that cell viability and density are extensive
After multiple, cloning has been carried out to mixing clone cell, to obtain the monoclonal cell strain of high expression；
Step D2：Select monoclonal：By counting to suspension cell, cell is carried out on a series of dilution paving orifice plate,
Cultivated again, taking culture supernatant carries out detection selection monoclonal hole cell, continues Amplification Culture, freezes when cell density is suitable
Deposit cell.
5. method as claimed in claim 2, it is characterised in that rHuPH20 amino acid sequences are：
MGVLKFKHIFFRSFVKSSGVSQIVFTFLLIPCCLTLNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIG
SPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDWEEWRPTW
ARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKK
PGYNGSCFNVEIKRNDDLSWLWNESTALYPSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVF
TDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAAKMCSQVLCQEQG
VCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADG
VCIDAFLKPPMETEEPQIFY.
6. method as claimed in claim 2, it is characterised in that the nucleotides sequence list of SEQ ID NO2 is：
ATGGGCGTGCTGAAGTTCAAGCACATCTTCTTCCGGTCCTTCGTGAAGTCCTCCGGCGTGTCCCAGATCGTGTTTAC
CTTCCTGCTGATCCCCTGCTGCCTGACCCTGAACTTTCGGGCCCCTCCCGTGATCCCCAACGTGCCATTTCTGTGGG
CCTGGAACGCCCCCTCCGAGTTCTGTCTGGGCAAGTTCGACGAGCCCCTGGATATGTCCCTGTTCTCCTTCATCGGC
TCCCCCCGGATCAATGCTACCGGCCAGGGCGTGACCATCTTCTACGTGGACCGGCTGGGCTACTACCCCTACATCGA
CTCCATCACCGGCGTGACCGTGAACGGCGGCATCCCCCAGAAGATCTCCCTGCAGGACCACCTGGACAAGGCCAAGA
AGGACATCACCTTCTACATGCCCGTGGACAACCTGGGCATGGCCGTGATCGACTGGGAGGAATGGCGGCCTACCTGG
GCCAGAAACTGGAAGCCCAAGGACGTGTACAAGAACCGGTCCATCGAGCTGGTGCAGCAGCAGAACGTGCAGCTGTC
TCTGACCGAGGCCACCGAGAAGGCTAAGCAGGAATTCGAGAAGGCCGGCAAGGACTTCCTGGTGGAAACCATCAAGC
TGGGCAAGCTGCTGCGGCCCAATCACCTGTGGGGCTACTATCTGTTCCCCGACTGCTACAACCACCACTACAAGAAG
CCCGGCTACAACGGCTCCTGCTTCAACGTGGAAATCAAGCGGAACGACGACCTGTCCTGGCTGTGGAACGAGTCCAC
CGCCCTGTACCCCTCCATCTACCTGAACACCCAGCAGAGCCCTGTGGCCGCCACACTGTACGTGCGGAACAGAGTGC
GCGAGGCCATCAGAGTGTCCAAGATCCCCGACGCCAAGTCCCCCCTGCCTGTGTTCGCCTACACCCGGATCGTGTTC
ACCGACCAGGTGCTGAAATTCCTGAGCCAGGATGAGCTGGTGTATACCTTCGGCGAGACAGTGGCCCTGGGCGCCTC
TGGAATCGTGATCTGGGGCACCCTGTCCATCATGCGGTCCATGAAGTCCTGCCTGCTGCTGGACAACTACATGGAAA
CAATCCTGAACCCTTACATCATCAACGTGACCCTGGCCGCCAAGATGTGTTCTCAGGTGCTGTGCCAGGAACAGGGC
GTGTGCATCCGGAAGAACTGGAACTCCTCCGACTACCTGCACCTGAACCCCGACAACTTCGCCATTCAGCTGGAAAA
GGGCGGCAAGTTCACCGTGCGGGGCAAGCCCACACTGGAAGATCTGGAACAGTTCTCCGAGAAGTTCTACTGCTCCT
GCTACTCCACCCTGAGCTGCAAAGAAAAGGCCGACGTGAAGGACACCGACGCCGTGGACGTGTGTATCGCCGACGGC
GTGTGTATTGACGCCTTCCTGAAGCCCCCCATGGAAACCGAGGAACCTCAGATCTTCTAC.
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