CN108220244A - Chinese hamster ovary celI strain, screening technique and its production technology of a kind of antibody gene containing recombinant human epidermal growth factor acceptor - Google Patents

Abstract

The invention discloses Chinese hamster ovary celI strain, screening technique and its production technologies of a kind of antibody gene containing recombinant human epidermal growth factor acceptor.The present invention provides a kind of recombinant human epidermal growth factor acceptor antibody genes, and the eukaryotic expression vector transfection for carrying recombinant human epidermal growth factor acceptor antibody gene is entered into CHO K1 cells, it repeatedly screens to obtain the cell of high expression recombinant human epidermal growth factor acceptor antibody, and establish the production technology of cell strain using flow cytometer.Recombinant protein expressed by the Chinese hamster ovary celI strain of the antibody gene provided by the invention containing recombinant human epidermal growth factor acceptor can close the EGFR ligand-binding sites point of a variety of cancer cell surfaces, so as to weaken or block the signal transduction in EGFR downstreams, reach the growth and invasion for inhibiting tumour, promote the purpose of Apoptosis.There is very big application value in terms of cancer caused by treatment EGFR overexpressions, treatment of cancer caused by being overexpressed for EGFR is worth with major application.

Description

Chinese hamster ovary celI strain, the screening of a kind of antibody gene containing recombinant human epidermal growth factor acceptor Method and its production technology

Technical field

The present invention relates to " the Chinese hamster ovary celI strain of antibody gene containing recombinant human epidermal growth factor acceptor a kind of, screening technique and Its production technology " belongs to biotechnology.

Background technology

In recent years, malignant tumour endangers human health as a kind of stubborn disease always.At present, the treatment master of cancer Will be still based on radiation and chemotherapy, but the damage that chemicotherapy is brought to human body is often unrepairable, such as radioactivity liver, lung Fibrosis etc..EGF-R ELISA (epidermal growth factor receptor, EGFR) is a kind of film surface Receptor with tyrosine kinase activity, can carry out cell signal transmission, and existing research confirms that EGFR is over-expressed and certain A little tumours, such as non-small cell lung cancer (NSCLC), colorectal cancer, breast cancer are closely related.In cancer patient, by artificial After intervention reduces EGFR, the state of an illness of cancer patient can be obviously improved.

The gene of encoding EGFR is located at 1st area of galianconism, 2 band of No. 7 chromosome, is under the jurisdiction of epidermal growth factor Sub (erbB) family, there are four members altogether for the family：ErbB1 (EGFR), ErbB2, ErbB3, ErbB4, EGFR is many such as head It is over-expressed in the solid tumor mass such as neck cancer, Skin Squamous Cell Carcinoma, the cancer of the esophagus, colorectal cancer and organ.After EGF combinations EGFR, promote EGFR monomers is made to carry out dimerization effect and form dimer, the dimerization of receptor causes the tyrosine protein kinase of intracellular region to be lived Property activation, then, signal transduction to nucleus by transcriptional control intragentic to core, plays corresponding biological action.Phase It closes the study found that EGF approach mainly includes RAS/RAF/MEK/ERK and PI3K/AKT accesses, the two accesses are multiple as one Miscellaneous network and run.The overexpression and activation of EGFR leads to several signal activation of intracellular, and cell Proliferation is not only made to reach high Tide, and it is also most important to the progression of other cancers such as Apoptosis, cell migration, angiogenesis.Epidermal growth factor The abnormal signal conduction of sub- receptor (EGFR) plays an important role in the development and maintenance of malignant phenotype, and therefore, this receptor is A kind of rational anti-tumor target.

At present, medicine used in being clinically directed to the tumour caused by EGFR unconventionality expressions is mainly small molecule Tyrosine kinase antagonist and monoclonal antibody.Micromolecular compound tyrosine kinase antagonist is mainly Gefitinib and Ai Luo For Buddhist nun.At present, approved listing has 20 kinds for treating cancer antibody drug, wherein, using EGFR as the therapeutic list of target spot Clonal antibody is mainly tri- kinds of Cetuximab, Panitumumab and Nimotuzumab, these EGFR monoclonal antibodies can seal The binding site of the corresponding ligand of EGFR extracellular regions is closed, inhibits its distinctive tyrosine kinase activity, slows down because of the signal path The speed of worsening of caused cancer.In the anti-hEGFR antibody of FDA approval listings, wherein, Cetuximab is fitted into list for people mouse Clonal antibody, although specific can be combined with EGFR, the preferable development for inhibiting cancer, in the process of human body application In often show stronger immunogenicity, be also easy to produce the reaction of people anti-mouse；Panitumumab is 100% humanized's monoclonal Antibody uses transgcnic mouse techniques to prepare, and by balb/c mouse cells secreting, expressing and modification, the modification of mouse source can cause sugar The shortcomings of baseization weakens, hypersensitivity, half-life short；Nimotuzumab be it is a kind of by humanization modified and acquisition The complementary of mouse IgG 2a is determined that domain (CDR) is transplanted in the antibody structure of people in transformation process by IgG1 type monoclonal antibodies, Mouse source antibody component is greatly reduced, however in the building process of expression vector, the light and heavy chain gene of antibody is connected to respectively Different carrier and be allowed to express respectively, the expression ratio that this method frequently can lead to light and heavy chain is in non-balanced state, together When reduce antibody packaging efficiency, decline the yield of the antibody with complete structure.In the present invention, the connection of antibody light and heavy chain Into same expression vector peedual, the coexpression of antibody light and heavy chain is realized.

Full length antibody molecule is four to be formed by connecting by two complete heavy chains and two complete light chains by disulfide bond Glycoprotein polyprotein precursor can just make it have high-affinity and with resisting during biological expression by the stringent processing modification of organelle Therefore former specific binding capacity, produces it just more stringent from the requirement on quality and quantity, the selection of expression system is also extremely It closes important.Prokaryotes are free of numerous organelles of Protein processing modification, and therefore, escherichia expression system may be only used for giving birth to Produce it is small, simple in structure, do not need to glycosylated antibody fragment such as Fab, Fab', scFv etc.；Lower eukaryotes expression system System, such as yeast and filamentous fungi, available for the production of full length antibody, but its type of glycosylation has differences compared with the mankind, such as Yeast glycosylation type is poly-malt carbohydrate type, and the type glycoprotein antibody half life is shorter, physiological activity is limited or even to people Body is toxic.Closest to human body be mammalian expression systems in protein modified system, the system can to albumen into Row appropriate folding, assembling and posttranslational modification, occupy an leading position in clinical practice, at present, Chinese hamster ovary cell (Chinese hamster ovary cells, CHO cells) is optimal eukaryotic expression host, passes through the system expression Albumen on configuration and conformation closer to native protein, be the preferred system of recombinant glycosylated protein production.

At present, obtaining many in the antibody drug of FDA approvals is produced by Chinese hamster ovary celI, and CHO-K1 cells are can to suspend Glutamine synthelase (GS) deficient cell of growth, therefore, the Chinese hamster ovary celI after transfection can be sieved with coamplification gene Choosing adds in GS genes as selection markers in expression vector upstream, while utilizes GS mortifier methionine sulfoxides (L- Methionine sulfoxide, MSX) pressurization screening is carried out, it is gradually increased with MSX concentration in culture medium, GS genes drive Foreign gene together connected in series is transcribed jointly, makes the transcriptional efficiency of target gene increase, so as to fulfill foreign gene High efficient expression.Since GS selection markers intuitively can not monitor and sort positive cell, the present invention is in the downstream of light chain Enhanced green fluorescence protein (EGFP) gene is introduced, EGFP is the mutant of GFP, has higher fluorescence intensity, in 488nm Green fluorescence can be generated under wavelength light excitation, so as to be detected and sort by flow cytometer.

Since conventional Chinese hamster ovary celI needs adherent growth, cell culture in the presence of having cow's serum or analogous components Serum or protein component in base have expression of the recombinant protein in cell and the protein purification procedures in later stage very big It influences.If carry out industrialization, it is also necessary to carry out serum-free domestication first, make cell adapted serum free medium and give birth to wherein Long good and continuous release generates recombinant protein to reduce cost and facilitate the exploitation of downstream purification technique.

Serum free suspension acclimatization technology is widely used in genetic engineering pharmaceutical.It is on the one hand to obtain and disclosure satisfy that life Produce the serum-free cell strain of requirement；On the other hand, it is to obtain the host cell of serum free suspension growth, is subsequent cell strain Exploitation lay the foundation, common host cell, such as Chinese hamster ovary celI, myeloma cell.Engineering cell acclimation method is adopted in the present invention With the domestication process in two stages, the first step adapts to serum-free culturing conditions culture, and second step makes cell adapted suspension.

Invention content

The present invention provides one kind to stablize, the suspension CHO-K1 monoclonal cell strains of high efficient expression anti-hEGFR, It can be used for the industrialization production of hEGFR antibody, obtained anti-hEGFR can close the corresponding ligand of EGFR extracellular regions Binding site inhibits its distinctive tyrosine kinase activity, slows down the speed of worsening of the cancer caused by the signal path.

Stabilization provided by the invention, the suspension CHO-K1 monoclonal cell strains of high efficient expression anti-hEGFR, by being free of The peedual-IRES-EGFP-hEGFR plasmid transfections of toxin enter CHO-K1 cell strains, and pass through methionine sulfoxide and pressurize It is repeatedly screened with flow cytometer, the domestication that suspends after selected by flow cytometry apoptosis monoclonal obtains.

The present invention also provides stabilization, the suspension CHO-K1 monoclonal cell strains of high efficient expression anti-hEGFR, which suspend, cultivates Production technology, i.e., 37 DEG C, 5%CO2,150rpm cultivate 8 days, added 10% culture body respectively at the 3rd day, the 5th day and the 7th day The long-pending F-68OptiCHO serum free mediums of Pluronic containing 1g/L, collect supernatant after culture, obtain anti- hEGFR。

Description of the drawings

Fig. 1 is the front and rear CHO-K1 cells of transfection

Fig. 2 is through MSX pressurization CHO-K1 cells after transfecting

Fig. 3 is the screening of CHO-K1 positive cells after transfection

Fig. 4 is detected for monoclonal cell positive rate

Fig. 5 is that the Dot Blot of monoclonal cell are detected

Fig. 6 is the expression suspension cell of anti-EGFR recombinant proteins and attached cell form

Fig. 7 is the culture expression anti-hEGFR CHO-K1 monoclonal cell strains SDS-PAGE analyses that suspend

Fig. 8 is anti-hEGFR purification results

Fig. 9 is anti-hEGFR and the elisa assay of antigen binding

Figure 10 is influences of the anti-hEGFR to the A431 cell cycles

Figure 11 is inhibiting effect of the anti-hEGFR to A431 cell invasions

Figure 12 influences MMP2, TIMP2, MMP9, TIMP1 gene mRNA expression in A431 cells for anti-hEGFR

Specific embodiment

Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method is conventional method unless otherwise specified.Test material used in following embodiments is certainly unless otherwise specified What routine biochemistry reagent shop was commercially available.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result makes even Mean value.CHO-K1 cells：This laboratory preserves.Fetal calf serum, IMDM culture mediums, DMEM culture mediums, serum-free MEM culture mediums, OptiCHO serum free mediums：GIBCO companies.L-Glutamine, methionine sulfoxide:Sigma companies.

Embodiment 1 obtains anti-hEGFR CHO-K1 cell strains

CHO-K1 cells are the Chinese hamster ovary lines of glutamine synthelase (GS) defect, are recovered from cell bank One CHO-K1 cell strain is passed on containing DMEM culture medium of 10% fetal calf serum containing glutamine twice, when cell is in During exponential phase, Tissue Culture Flask is inoculated in 1 × 10^6cells/ml cell densities, in 37 DEG C, 5%CO2 incubators Culture is until degree of converging is 70~80%, and with trypsin digestion, increase serum terminates digestion, and 1000rpm, 5min are used after centrifugation It is primary that PBS washes cell, and cell with serum-free MEM culture mediums (GIBCO companies MEM transfections serum free medium) is diluted and is counted Number, makes its concentration up to 1 × 10^7cells/mL.It takes after purification and without endotoxic peedual-IRES-EGFP-hEGFR matter 20 μ g of grain DNA are added in 400 μ L cell suspensions, and electricity is carried out under 250V, 1 000 Ω, 1 000 μ F parameters and is turned.It will be thin after transfection Dysuria with lower abdominal colic enters culture of recovering in the complete medium containing 10% fetal calf serum.Electricity is replaced after turning 24-30h containing 50 μM of GS mortifiers Methionine sulfoxide (L-Methionine sulfoxide, MSX), 10% fetal calf serum IMDM culture mediums of no glutamine into Row pressurization culture.Cellular morphology is as shown in Figure 1 before and after transfection.Anti-hEGFR CHO-K1 are thin after MSX pressurization cultures after transfection Born of the same parents' strain flow cytometer inspection positive rate is as shown in Figure 2.

The screening of embodiment 2, stabilization, high efficient expression anti-hEGFR CHO-K1 cell strains

The present invention introduces enhanced green fluorescence protein (EGFP) gene in the downstream of light chain, is excited in 488nm wavelength lights Under can generate green fluorescence, can be detected and sort by flow cytometer.It is used after transfection containing 50 μM of MSX, no glutamine 10% fetal calf serum IMDM culture mediums carry out pressurization culture eliminate the CHO-K1 for not being transferred to and transiently transfecting cells, wait to stablize The clone of transfection carries out the sterile sorting of flow cytometer after covering with Tissue Culture Flask.

The step of sorting is：Will be cells trypsinised, increase serum terminates digestion, 1000rpm, 5min, after centrifugation Cell is washed with PBS twice, and cell is resuspended in 500 μ L PBS, and the similary blanc cell for handling untransfected compares.Flow cytometer is known The higher cell sorting of the positive rate that does not go out is to containing 50 μM of MSX, and the six of 10% fetal calf serum IMDM culture mediums of no glutamine It in orifice plate, 37 DEG C, cultivates and observes in 5%CO2 incubators, after cell length to about 5~10 × 10^6cells, carry out next round Screening.

After the completion of four-wheel screening, the mixing clone group of more than 90% positive rate is obtained, expands culture, freeze-stored cell is established Cell bank.The results are shown in Figure 3 for four-wheel screening flow cytometer inspection anti-hEGFR CHO-K1 cell strains positive rate.

Embodiment 3 is stablized, the acquisition of high efficient expression anti-hEGFR CHO-K1 monoclonal cell strains

With 96 orifice plate monoclonal method for separating stable, high efficient expression list is filtered out from the mixing clone group that four sieves obtain Clonal cell line.

Monoclonal method for separating is as follows：First 96 orifice plates are added in per hole 200 μ L, 37 DEG C preheat in advance sterile contain 50 μM MSX, 10% fetal calf serum IMDM culture mediums of no glutamine, lateral opening addition PBS.Cell processing step such as embodiment 2 sorts 96 orifice plates are placed in 37 DEG C after the completion of screening, cultivated in 5%CO2 constant incubators, fluorescence microscope is utilized after 1~2 week by step Cell growth status is observed, and replaces culture medium, when monoclonal cell is proliferated to 1 more than 000cells, reaches the culture of 24 holes Plate expands culture successively according to the increase of cell Proliferation quantity, using flow cytometer under the conditions of 488nm exciting lights, detection The fluorescence intensity of each monoclonal cell strain.Flow cytometer screens to obtain anti-hEGFR monoclonal cell strains inspection positive rate such as Shown in Fig. 4.Highest 5 cell strains of flow cytometer detection value is selected to carry out the detection of Dot blot methods.Dot blot testing results are such as Shown in Fig. 5.Choose fluorescence intensity not only strong, Dot blot values but also high cell strain as anti-hEGFR expression cell strains, Amplification cultivation, freeze-stored cell.Establish Cell bank.

Embodiment 4 is stablized, and high efficient expression anti-hEGFR CHO-K1 monoclonal cell strains, which suspend, tames

It recovers from cell bank one and recombinates anti-hEGFR expression cell strains, with 50 μM of MSX, without glutamine 10% fetal calf serum IMDM culture mediums pass on twice, and when cell is in exponential phase, with trypsin digestion, increase serum is whole It only digests, 1000rpm, 5min, centrifuges, remove supernatant, add in (the GIBCO companies CHO serum-frees training of OptiCHO serum free mediums Support base), suspension is made in cell, is inoculated into Tissue Culture Flask with 5~6 × 10^5cells/ml, when cell density reaches 2~ 1 is pressed during 3 × 10^6cells/ml：5 cells pass on, and cultivate two weeks, make cell adapted to grow in serum free medium.Then Supernatant is removed in 1000rpm, 5min, centrifugation, adds in 1g/L Pluronic F-68OptiCHO serum free mediums (GIBCO companies CHO serum free mediums), 5~6 × 10^5cells/ml cell suspensions are made, are inoculated into sterile flasks, shaking speed is 150rpm, when cell density reaches 2~3 × 10^6cells/ml, by 1：3 passages, when passing on 5~6 times, when cell is in serum-free The middle speed of growth containing the speed of growth in serum with approaching, freeze-stored cell.Establish Cell bank.Cellular morphology compares before and after domestication See Fig. 6.

Embodiment 5 is stablized, and high efficient expression anti-hEGFR CHO-K1 monoclonal cell strains, which suspend, cultivates

It recovers one and is recombinated in anti-hEGFR CHO-K1 cell strains to sterile flasks from cell bank, with containing 1g/ LPluronic F-68OptiCHO serum free mediums are passed on twice, when cell is in exponential phase, with 3~5 × 10^ 5cells/ml cell densities be inoculated with, 37 DEG C, 5%CO2,150rpm cultivate 8 days, added respectively at the 3rd day, the 5th day and the 7th day The F-68OptiCHO serum free mediums of Pluronic containing 1g/L of 10% volume of culture, supernatant is collected after culture.To obtain the final product To anti-hEGFR.Micro supernatant is taken during culture daily, is analyzed through irreducibility SDS-PAGE (Fig. 7) as it can be seen that with culture The increase of time, anti-hEGFR expression quantity are continuously increased, and can reach 0.4mg/ml.

Embodiment 6, anti-hEGFR purifying

It is (public purchased from GE using 100 protein chromatography systems of AKTA Purifier after collecting cells and supernatant about 400mL Department) carry out antibody purification.With the phosphate buffer of PH 7.0 balance proteinA pillars (being purchased from GE companies), loading before loading It is then that the albumen of elution is saturating overnight in PBS afterwards with the citric acid-citrate buffer solution antibody elution albumen of PH 3.0 Analysis.Albumen after purification is analyzed (Fig. 8) with SDS-PAGE.

Embodiment 7, the detection of the biological activity of Anti-hEGFR

(1) ELISA detects the affinity of anti-hEGFR

Coated elisa plate after hEGFR antigen proteins are diluted to 20 μ g/mL with coating buffer, 4 DEG C overnight.It is separately added into two times The anti-hEGFR (3 μ g/mL of initial concentration) of gradient dilution, is not added with antibody as negative control, adds Cetuximab (3 μ of concentration G/mL) as positive control, each sample sets 3 multiple holes.The goat anti-human antibody of HRP labels is added in as secondary antibody, is used A values under microplate reader detection 450nm wavelength.The results are shown in Figure 9, with the reduction of hEGFR full length antibody concentration, light absorption value It decreases, in 0.047 μ g/mL concentration, OD450 values reach minimum, but still are 4.5 times of negative control.The result shows that Antibody concentration dependence is presented in the effect of hEGFR full length antibodies and hEGFR, and antibody titer is higher.

(2) the PI dyeing detection cell cycle

Through conventional pancreatin digestion process Control, anti-hEGFR and Cetuximab group A431 cells, with 70% ethyl alcohol After fixation and after PI is dyed, flow cytomery is carried out, the cell cycle fits software ModFit analysis results such as Figure 10 institutes Show：The cell proportion of G1/G0 phases is 62.43% in A431 cells without albumen processing, and anti-hEGFR groups and The cell proportion of Cetuximab group G1/G0 phases is respectively 77.96% and 79.15%.Anti-hEGFR groups, Cetuximab The cell proportion of group G1/G0 phases is substantially less than blank control group, significant difference (p<0.01)；And anti-hEGFR groups with Cetuximab groups are compared, and difference is not notable.

(3) influence of the full length antibody to A431 cell invasions

1) Boyden methods detection Anti-hEGFR is to the inhibiting effect of A431 cell invasions

In the experiment of Boyden vitro invasions, anti-hEGFR, Cetuximab are detected carefully after acting on A431 cells 48h respectively Born of the same parents penetrate the ability of Matrigel artificial basement membranes, and as a result (Figure 11) shows that it is thin that anti-hEGFR can significantly reduce A431 The theca cell number of wearing of born of the same parents (wears theca cell number to be reduced to 27.3 ± 3.05 by 34.0 ± 2.26 of blank control group, p< 0.01) indifference, and compared with Cetuximab groups (wear theca cell number for 26.3 ± 2.54).The result shows that cancer cell can be with The ability of vitro invasion is moved and has, anti-hEGFR can reduce A431 cells by being combined with cancer cell surfaces EGFR Invade transfer ability.

2) Anti-hEGFR adjusts the expression of MMP2, MMP9 and TIMP2, TIMP1 in A431 cells

MMPs and TIMPs is expressed in Various Tissues and organ, is primarily involved in the metabolism of extracellular matrix, MMPs has destructive effects to extracellular mechanism, and TIMPs can then inhibit the activity of MMPs, as MMP2 and MMP9 is thin in tumour Serve during born of the same parents' infiltration, transfer key.MMP2/TIMP2, MMP9/TIMP1Real-time PCR results (Figure 12) As it can be seen that the amount of MMP2 and MMP9 expression is higher than Cetuximab groups, but compared with blank control in anti-hEGFR groups, anti- In hEGFR groups, MMP2 and MMP9 expression quantity are 0.718756 and 0.0403273 times of blank control group respectively, and difference is up to notable Level (p<0.05)；MMP2 inhibitor TIMP2, TIMP1 expressions corresponding with MMP9 are：Anti-hEGFR groups TIMP2, TIMP1 expression quantity is respectively 4.67576 times and 757.155 times of blank control group, and respectively Cetuximab groups 1.93281 times and 2.28798 times, difference reaches the pole level of signifiance (p<0.01).It can illustrate, anti-hEGFR can pass through tune Ganglion cell's epimatrix destructive enzymes factor maintains the stabilization of extracellular matrix, so as to inhibiting the invasion of tumour cell.

Sequence table

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20 25 30

ata cac tgg tat cag caa aga aca aat ggt tct cca agg ctt ctc ata 144

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

aag tat gct tct gag tct atc tct ggg atc cct tcc agg ttt agt ggc 192

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

agt gga tca ggg aca gat ttt act ctt agc atc aac agt gtg gag tct 240

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

gaa gat att gca gat tat tac tgt caa caa aat aat aac tgg cca acc 288

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

acg ttc ggt gct ggc acc aag ctg gag ctg aag cga act gtg gct gca 336

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

cca tct gtc ttc atc ttc cct cca tct gat gag cag ttg aaa tct gga 384

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 432

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

aaa gtg cag tgg aag gtg gat aac gcc ctc caa tcc ggt aac tcc cag 480

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 528

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 576

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

gcc tgc gaa gtc acc cat cag ggc ctg agc tct ccc gtc aca aag agc 624

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

ttc aac agg gga gag tgt tga 645

Phe Asn Arg Gly Glu Cys

210

<210> 5

<211> 214

<212> PRT

<213> Artifical sequence

<400> 5

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 6

<211> 1350

<212> DNA

<213> Artifical sequence

<220>

<221> CDS

<222> (1)..(1350)

<400> 6

cag gtg cag ctg aag cag tca gga cct ggc ctc gtg cag ccc tca cag 48

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

agc ctg tcc atc acc tgc aca gtc tct ggt ttc tca tta act aac tat 96

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

ggt gta cac tgg gtt cgc cag tct cca gga aag ggt ctg gag tgg ctg 144

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

gga gtg ata tgg agt ggt gga aac aca gac tat aat aca cct ttc aca 192

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

tcc aga ctg agc atc aac aag gac aat tcc aag agc caa gtt ttc ttt 240

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

aaa atg aac agt ctg caa tct aat gac aca gcc ata tat tac tgt gcc 288

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

aga gcc ctc acc tac tat gat tac gag ttt gct tac tgg ggc caa ggg 336

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

act ctg gtc act gtc tct gca gcc tcc acc aag ggc cca tcc gtc ttc 384

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

ccc ctg gca ccc tcc tcc aag agc acc tct ggg ggc aca gcc gcc ctg 432

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

ggc tgc ctg gtc aag gac tac ttc ccc gaa cct gtg acg gtg tcc tgg 480

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

aac tca ggc gcc ctg acc agc ggc gtg cac acc ttc ccg gct gtc ctc 528

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

cag tcc tca gga ctc tac tcc ctc agc agc gtg gtg acc gtg ccc tcc 576

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

agc agc ttg ggc acc cag acc tac atc tgc aac gtg aat cac aag ccc 624

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

agc aac acc aag gtg gac aag cgc gtt gag ccc aaa tct tgt gac aaa 672

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

act cac aca tgc cca cct tgc cca gca cct gaa ctc ctg ggg gga cct 720

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

tca gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc atg atc tcc 768

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa gac 816

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat aat 864

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

gcc aag aca aag cct cgg gag gag cag tac aac agc acg tac cgg gtg 912

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag gag 960

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag aaa 1008

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

acc atc tcc aaa gcc aaa ggg cag ccc cga gaa cca cag gtg tac acc 1056

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

ctg ccc cca tcc cgg gag gag atg acc aag aac cag gtc agc ctg acc 1104

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

tgc ctg gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag 1152

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

agc aat ggg cag ccc gag aac aac tac aag acc acg cct ccc gtg ctg 1200

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

gac tcc gac ggc tcc ttc ttc ctc tat agc aag ctc acc gtg gac aag 1248

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag 1296

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

gct ctg cac aac cac tac acg cag aag agc ctc tcc ctg tct ccc ggt 1344

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

aaa tga 1350

Lys

<210> 7

<211> 457

<212> PRT

<213> Artifical sequence

<400> 7

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Leu Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe

65 70 75 80

Lys Met Glu Thr Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Glu Thr Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Glu Thr Thr Lys

355 360 365

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

370 375 380

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

385 390 395 400

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

405 410 415

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

420 425 430

Cys Ser Val Met Glu Thr His Glu Ala Leu His Asn His Tyr Thr Gln

435 440 445

Lys Ser Leu Ser Leu Ser Pro Gly Lys

450 455

Claims (9)

1. one kind contains recombinant human epidermal growth factor acceptor antibody gene (plasmid Peedual-IRES-EGFP-hEGFR) CHO suspension cell strains.

2. Chinese hamster ovary celI strain according to claim 1, host cell CHO-K1.

3. plasmid Peedual-IRES-EGFP-hEGFR according to claim 1, it includes IRES (internal ribosome into Angle of striking) sequence, EGFP (enhanced green fluorescence protein) sequences and EGFR (recombinant human epidermal growth factor acceptor antibody) sequence Row.

4. plasmid according to claim 3, wherein EGFP coding gene sequences are SEQ ID NO:Shown in 1, amino acid sequence It is classified as SEQ ID NO:Shown in 2；IRES EGFP fragment-encoding genes sequence is SEQ ID NO:Shown in 3.EGFR includes LC and HC Segment, wherein LC coding gene sequences are SEQ ID NO:Shown in 4, amino acid sequence is SEQ ID NO:Shown in 5；HC encodes base Because sequence is SEQ ID NO:Shown in 6, amino acid sequence is SEQ ID NO:Shown in 7.

5. according to the construction method of any Chinese hamster ovary celI strains of claim 1-4, include the following steps：

(1) extraction recombinant plasmid Peedual-IRES-EGFP-hEGFR；

(2) plasmid is transferred in CHO-K1.It takes after purification and without endotoxic peedual-IRES-EGFP-hEGFR plasmids 20 μ g of DNA are added in and electricity turn are carried out in 400 μ L cell suspensions, and electricity turns parameter as 250V, 1 000 Ω, 1 000 μ F；

(3) pressurization screening is carried out using GS mortifier methionine sulfoxide (L-Methionine sulfoxide, MSX), utilized Repeatedly screening obtains mixing clone cell to flow cytometer；

(4) monoclonal cell strain, flow cytomery positive rate, SDS-PAGE detection lists are obtained using selected by flow cytometry apoptosis The expression quantity of clonal cell line, Dot blot detections, screening obtain the Chinese hamster ovary celI of high efficient expression Urogastrone R monoclonal antibodies Strain.

6. construction method according to claim 5 obtains the CHO containing recombinant human epidermal growth factor acceptor antibody gene Cell strain.

7. construction method according to claim 5, the ultimate density of the methionine sulfoxide is 50 μM.

8. according to application of any Chinese hamster ovary celI strains of claim 1-5 in Urogastrone R monoclonal antibody drugs are produced.

9. according to Chinese hamster ovary celI strain described in claim 1-7 in the method for producing Urogastrone R monoclonal antibodies, it is characterised in that Culture 8 days, respectively at the 3rd day, the 5th day and the 7th day F- of Pluronic containing 1g/L for adding 10% volume of culture of culture 68OptiCHO serum free mediums.