CN103131674A - Chinese hamster ovary (CHO) cell line capable of expressing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, creation method and application thereof and cell base built by CHO cell line - Google Patents
Chinese hamster ovary (CHO) cell line capable of expressing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, creation method and application thereof and cell base built by CHO cell line Download PDFInfo
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Abstract
The invention relates to a Chinese hamster ovary (CHO) cell line capable of showing Rta albumen of Elzatein-Barn (EB) virus stably and efficiently, a creation method and application thereof and a cell base built by the CHO cell line, and belongs to the technical field of biology. Preservation number of the recombination cell line CHO/RTA is CGMCC6955, the CHO cell base expressing the Rta albumen of the EB virus can be used for practical production and formed by the recombination cell line CHO/RTA. The invention further provides a method for creating the CHO cell line. Dihydrofolate reductase defect type Chinese hamster ovary cells (CHO/dhfr-) serve as host cells, and the cell line capable of expressing the Rta albumen of the EB virus stably and efficiently is obtained after screening and amplification of the host cells.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of stability and high efficiency is expressed the Chinese hamster ovary celI strain, its establishment method, application of Epstein-Barr virus Rta albumen and by the cell bank of its foundation.
Background technology
Epstein-Barr virus is a kind of gamma herpes viruses, and there is this virus in the whole world near adult's infection of 95%.Ebv infection can be divided into latent infection phase and cracking replicative phase two states, and after primary infection, this virus can be set up lifelong latent infection in host.Epstein-Barr virus enters the cracking replication status by latent state and infects, and is relevant with the generation of series of human malignant tumour.In IARC (IARC) criteria for classification to carcinogen in 1999, Epstein-Barr virus has clearly been classified as one of carcinogenic factor of the mankind.
The Rta albumen of BRLF1 genetic expression is that Epstein-Barr virus enters the essential active element of cracking replication status.In all tumours relevant to Epstein-Barr virus, the closest with the relation of nasopharyngeal carcinoma (Nasopharyngeal Carcinoma, NPC).Nasopharyngeal carcinoma is the south east asia kinds of tumor, with the Chinese three large tumours of esophagus cancer, liver cancer and title, and occurred frequently in south China number province, hold pride of place in the various malignant tumours in south.
In application number is 200610113403 national patent, announced with the method for RT-PCR main EBV lysis genes has been studied at the expression in the middle of Nasopharyngeal Biopsy Tissues (comprising tumor tissues and healthy tissues), discovery expression of BZLF1, BALF2 and BCLF in NPC sample and control sample all can detect, and the expression of BRLF1 only can detect in the NPC sample, and fails in healthy tissues to detect.The result shows all can occur, but the expression of BRLF1 gene only to occur the reactivation of Epstein-Barr virus in tissues of nasopharyngeal carcinoma and healthy tissues in Biopsies from Patients with Nasopharyngeal Carcinoma, this result shows the vital role of BRLF1 gene in the nasopharyngeal carcinoma genesis.
Development along with science and technology, gene recombination technology more and more has been widely used in medical field, be in 200610113403 patents at application number, announced with the base sequence of 514～1068bp in Epstein-Barr virus cracking state early gene BRLF1 and the base sequence of 1153～1603bp and be reconstituted in the pGEX-5X-3 expression vector and carry out expressing protein in intestinal bacteria, and with albumen coated elisa plate after purifying, set up the ELISA test kit that is used for nasopharyngeal carcinoma examination, early diagnosis and therapy effect prediction.Yet, the limitation that the existence of prokaryotic cell prokaryocyte heterologous gene expression system self is difficult to overcome, so its application and popularization are extremely restricted.The eukaryotic cell heterologous gene expression system is widely used in expressing the biological activity protein that needs posttranslational modification, particularly mammalian expression system has post transcriptional modificaiton function accurately, system compares with yeast expression system with procaryotic cell expression, the albumen of expressing near the native protein molecule, is the first-selected system that present recombinant protein is produced aspect molecular structure, physicochemical property and biological function.
In different mammalian cell expression systems; Chinese hamster ovary cell (CHO) exogenous protein expression system uses through practice for many years; become the mammal cell line of the most successful expression foreign protein; its advantage is that foreign gene can be stably integrated in cell chromosome; and be easy to mass-producing and cultivate, these two characteristics are that exogenous protein large-scale preparation has been created condition.Wherein Tetrahydrofolate dehydrogenase (dhfr) defective type Chinese hamster ovary celI lacks the endogenous Tetrahydrofolate dehydrogenase, in the time of containing the expression vector cotransfection of dihydrofolate reductase gene and goal gene, Tetrahydrofolate dehydrogenase (dhfr) gene and goal gene are integrated in host chromosome same site usually, under the condition that competitive inhibitor-aminomethyl petrin (MTX) exists of dhfr, thereby the increase of dhfr gene copy number drives the purpose that reaches raising destination gene expression amount.
At present, there is no in prior art by with pCDNA3.1(+)-Chinese hamster ovary celI of EBV-Rta and pSV2-dhfr cotransfection DHFR absence type, after the screening amplification, the report of the Recombinant CHO cell line of efficient, stably express EBV-Rta.
Summary of the invention
According to demand and the deficiency in above-mentioned field, the invention provides a kind of Recombinant CHO cell line CHO/RTA of high efficiency stable expression Epstein-Barr virus Rta albumen, its preserving number is: CGMCC6955.
Cell strain CHO/RTA of the present invention is mainly to utilize Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO/dhfr) as host cell, is stablized after screening amplification, the cell strain of high efficient expression Epstein-Barr virus Rta albumen.
The expression vector of cell strain CHO/RTA of the present invention is not limited to specific expression vector, as long as it can be recombinated with described DNA fragmentation, forms suitable expression plasmid and gets final product;
Preferably, the expression vector of cell strain CHO/RTA of the present invention is the eukaryotic cell heterologous gene expression system, and it can be recombinated with described DNA fragmentation, is easy to synthesize, can effectively be secreted in nutrient solution, and can form the expression plasmid that suitable expression has activated protein.
The recombinant C HO/RTA cell strain of the present invention by filtering out, and then set up the Chinese hamster ovary celI storehouse that can be used for the expression Epstein-Barr virus Rta albumen of actual production.
Epstein-Barr virus albumen Rta is characterized in that, by its preserving number is: the Recombinant CHO cell line CHO/RTA secretion of CGMCC6955 gets.
The purposes of Epstein-Barr virus albumen Rta.
Described purposes refers in based on the detection reaction of immune response principle as antigen.
Described purposes refer to based on the detection kit of immune response principle as envelope antigen.
The construction process of recombinant C HO/RTA cell strain of the present invention comprises the following steps:
(1) will use TAP(CellSiganaling Technology, Inc., Danvers, MA, USA) stimulation B95-8 cell strain (ATCC Number:CRL-10624
TM) in the EBV genome that infects carry out RT-PCR(TaKaRa, TAK RR019A) amplification, obtain BRLF1 full length sequence cDNA, it is cloned into carrier for expression of eukaryon pCDNA3.1(+) in (American I nvitrogen company), build mammalian cell expression vector pCDNA3.1(+)-EBV-Rta.
(2) with positively charged ion plasmalogen Lipofectamine2000(American I nvitrogen company) with pCDNA3.1(+)-EBV-Rta and pSV2-dhfr(American Type Culture Collection, VMA0061) cotransfection CHO/dhfr
-
(3) use the G418(Gbico product) (concentration range be 0.1～1.0mg/ml) and xanthoglobulin (Gbico product) (concentration range is 0.01～0.2mmol/L), (concentration range is 0.001～0.03mmol/L) to thymus pyrimidine (Gbico product), the selection Screening of Media of glycine (concentration range 10-50mg/L) is integrated the stable transfected cells strain of EBV BRLF1 and dhfr gene.
(4) progressively increase progressively the mode of aminomethyl petrin (MTX) (Sigma product) concentration (0.01～1000 μ mol/L), the copy number of amplifying target genes.
(5) with the EBV-Rta in WesternBlot detection recombinaant CHO cell substratum.
(6) be separated to mono-clonal by limiting dilution assay from mix the clone, utilize the expression amount of EBV-Rta in ELISA method detection mono-clonal substratum, screen the cell strain CHO/RTA of high efficient expression EBV-Rta.
(7) above CHO/RTA cell strain in culturing process secreting, expressing EBV-Rta in substratum.
The application in the Chinese hamster ovary celI storehouse of Epstein-Barr virus Rta albumen is expressed in above-mentioned Chinese hamster ovary celI strain in actual production.
The present invention is by utilizing Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO/dhfr
-) as host cell, selection can with the restructuring of described DNA fragmentation, forms the carrier of suitable expression plasmid, stablized after screening is increased, the cell strain of high efficient expression Epstein-Barr virus Rta albumen.
Preferably, the expression vector of cell strain CHO/RTA of the present invention is the eukaryotic cell heterologous gene expression system, and it can be recombinated with described DNA fragmentation, is easy to synthesize, can effectively be secreted in nutrient solution, and can form the expression plasmid that suitable expression has activated protein.
The recombinant C HO/RTA cell strain of the present invention by filtering out, and then set up the Chinese hamster ovary celI storehouse that can be used for the expression Epstein-Barr virus Rta albumen of actual production.
The present invention has filtered out cell strain stable, high efficient expression Epstein-Barr virus Rta albumen, the process that has greatly advanced nasopharyngeal carcinoma examination, diagnosis and result for the treatment of to predict.
In early transfection of the inventive method and screening positive clone step, the concentration range of described G418 is 0.1～1.0mg/ml; Described hypoxanthic concentration range is 0.01～0.2mmol/L; The concentration range of described thymus pyrimidine is 0.001～0.03mmol/L, and the concentration range of described glycine is 10-50mg/L, can obtain the stable transfected cells strain, in this concentration range, is conducive to the cell attachment growth.
Although escherichia expression system can extract and solve many problems on quantity and consuming cost from the technology fussy degree, as a kind of prokaryotic expression system, can not overcome all the time the problem that self exists, the antigen expressed quality is unsatisfactory.Its reason is mainly, and prokaryotic expression system lacks after protein translation the function that further processing is modified, and recombinant protein can not correctly be folded and exists with the form of inclusion body.The Epstein-Barr virus albumen that recombinant protein not only produces with human body B cell two, on tertiary structure has larger difference, only can simulate the antigenic determinant that is formed by the adjacent amino acid residue in parent protein, it is linear epitope, a little less than causing the antigen-antibody binding ability, for detection of in patients with nasopharyngeal carcinoma during corresponding antibodies, easily undetected.And the sex change dissolving of inclusion body and renaturation etc., the loaded down with trivial details and complexity of process has also improved production cost.In addition, in the crowd, persons quite a lot infected intestinal bacteria, had Chinese People's Anti-Japanese Military and Political College's enterobacteria antibody in body, and therefore cross reaction easily occurs, and Interference Detection causes result false positive to occur.In order to improve the quality of antigen, the Epstein-Barr virus Rta albumen that the Recombinant CHO cell line of setting up by present method is expressed has more similar physico-chemical property and biological activity to natural Epstein-Barr virus Rta albumen, expression product efficient and cheap is for the preparation of antigen provides sufficient, stable protein source.
Strain classification: stable transfection contains the CHO/dhfr-cell strain of Epstein-Barr virus BRLF full length gene gene, and its deposit number is: CGMCCNo.6955
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation date: 2012.12.06
Preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica
Description of drawings
Fig. 1 is recombinant plasmid pCDNA3.1(+)-the EBV-Rta collection of illustrative plates
Fig. 2 IgG/Rta monoclonal antibody Western Blot detects EBV Rta recombinant protein
Wherein, recombinant C HO/RTA secretory protein after the 1-purifying; 2-empty carrier plasmid pCDNA3.1(+) secretory protein after transfection CHO/dhfr cell; M-albumen Marker
The typical curve that Fig. 3 BCA-100 quantification of protein kit measurement test kit standard protein measured value is made
Fig. 4 is test kit calibration object of the present invention matched curve
Fig. 5 is normal human blood Rta antibody concentration
Fig. 6 is Patients With Npc Rta anti-body contg
Fig. 7 is the variation of Rta antibody serum concentration mean value before and after six routine radiotherapy in patients with nasopharyngeal carcinomas
Fig. 8 is the variation before and after each patients serum Rta anti-body contg treatment of diagram
Embodiment
The more detailed experimental technique of the present invention can be referring to embodiment.The present embodiment is for explanation, rather than limits the present invention with method in any form.
The bacterial strain that uses in the embodiment of the present invention, plasmid and reagent are the commercially available prod.
The source of reagent of the present invention and medicine is itemized as follows:
Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO/dhfr
-) be purchased from American Type Culture Collection(ATCC) company's product (SCO610);
10% foetal calf serum is purchased from Hangzhou folium ilicis chinensis biotechnology company limited;
Recombinant protein Rta and mouse IgG/Rta monoclonal antibody is purchased from chamicon company;
Carrier for expression of eukaryon pCDNA3.1(+) be purchased from American I nvitrogen company;
Positively charged ion plasmalogen Lipofectamine2000 is purchased from American I nvitrogen company;
G418, xanthoglobulin and thymus pyrimidine, glycine are the Gbico product;
Aminomethyl petrin (MTX) is purchased from Sigma company;
BCA-100 quantification of protein test kit derives from Beijing and matches the biology of speeding, product article No. 300001-B.
The TAP full name is Tobacco Acid Pyrophosphatase, is purchased from Cell Siganaling Technology, Inc., Danvers, MA, USA.
Embodiment 1.CHO/RTA cell strain is cultivated preparation
(1) cell cultures
Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO/dhfr
-) be American Type Culture Collection(ATCC) company's product (SCO610), the complete culture solution of this cell must be added 0.1mmol/L xanthoglobulin and 0.016mmol/L thymus pyrimidine, 10% foetal calf serum (Hangzhou folium ilicis chinensis biotechnology company limited), 37 ℃ of cell culture incubators are cultivated.
(2) structure of eukaryon expression plasmid
To be that in the CN200610113403 Chinese patent, disclosed method acquisition BRLF1 full length sequence cDNA is cloned into carrier for expression of eukaryon pCDNA3.1(+ with application number) in, build mammalian cell expression vector pCDNA3.1(+)-EBV-Rta.
(3) transfection and screening positive clone
With the plasmid pCDNA3.1(+ that builds)-EBV-Rta and pSV2-dhfr mix according to mol ratio 5:1, with positively charged ion plasmalogen Lipofectamine2000(Invitrogen product) cotransfection CHO/dhfr
-Cell is used empty carrier plasmid pCDNA3.1(+ simultaneously) and pSV2-dhfr cotransfection CHO/dhfr
-Cell in contrast.Be the G418(Gbico product of 0.7mg/ml with concentration) and concentration be the stable transfected cells strain that the selection Screening of Media of the xanthoglobulin of 0.15mmol/L, thymus pyrimidine that concentration is 0.02mmol/L and the concentration glycine that is 30mg/L is integrated EBV BRLF1 and dhfr gene.
(4) pressurization of aminomethyl petrin (MTX) is cultivated
All positive colony cells of above-mentioned formation are inoculated into mixed culture in Tissue Culture Dish after with trysinization, after cell density is 80-90%, ratio according to 1:100-1:4000 is inoculated in new Tissue Culture Dish, after cell covers with, add aminomethyl petrin (MTX) (Sigma), concentration is from 10
-8Mol/L to 10
-4Mol/L, finally obtaining can be 10
-6The mixing clone strain of the recombinaant CHO cell of aminomethyl petrin (MTX) normal growth of mol/L, called after CHO/RTA.
(5) recombinant protein obtains and purifying
with the preserving number of above acquisition be: the CHO/RTA cell strain of CGMCC6955 is inoculated in Tissue Culture Dish, the complete culture solution of this cell must be added 0.1mmol/L xanthoglobulin and 0.016mmol/L thymus pyrimidine, 10% foetal calf serum (Hangzhou folium ilicis chinensis biotechnology company limited), reach the density of 90% left and right until cell after, use the serum free medium that does not add aminomethyl petrin (MTX) instead, collecting cell nutrient solution after 1-2 days, adopt the albumen in trichloroacetic acid precipitation method extraction cell culture fluid, use again the target protein in the streptavidin sepharose high performance purifying cells supernatant liquor of GE company.
(6) Immunological Identification (Western Blot method)
After recombinant protein Rta after purifying was carried out the SDS-PAGE electrophoresis, electrotransfer carried out immunoblotting assay to pvdf membrane.Result shows: recombinant protein Rta and mouse IgG/Rta monoclonal antibody (chamicon company product), be positive, and be about specific band of 66KDa place's appearance at molecular weight.
(7) detection of Rta expressing quantity
(biology of speeding is matched in Beijing according to BCA-100 quantification of protein test kit, product article No. 300001-B) process specifications is tested, be ordinate according to the equal OD value of standard protein institute's lining, the protein concn in each hole (mg/ml) is abscissa production standard albumen curve, get regression equation through linear regression fit, OD value substitution with sample calculates protein concentration.
Calculate formula and the coefficient of determination R of upper figure Plays curve with the EXCEL table
2For: y=0.2539x+0.0152, R
2=0.992R
2Close to 1, this typical curve is effective, can be used for inferring the concentration of tested albumen.
Detectable level sees Table 1:
After table 1 purifying, the concentration of restructuring Rta protein is calculated
By above typical curve, the Rta albumen that the recombinant C HO/RTA cell strain that filters out is expressed carries out quantitative analysis, and after purifying, the concentration of restructuring Rta protein reaches 2.35mg/ml.
Step 1: preparation enzyme plate
With coated damping fluid (pH value be 9.6,0.1mol/L carbonate buffer solution), above Epstein-Barr virus Rta albumen is carried out the 1:10000 dilution, join in the enzyme plate hole every hole 100 μ l, 37 ℃ of reactions 2 hours, after getting rid of coating buffer, pat dry, every hole adds the confining liquid (containing final concentration is 2% bovine serum albumin phosphate buffered saline buffer) of 200 μ l, and 37 ℃ were reacted 2 hours, get rid of confining liquid, pat dry, drying is preserved with the aluminium foil bag vacuum packaging.
Step 2: reagent preparation:
1) working concentration enzyme solution preparation
HRP mark goat anti-human igg needs through affinitive layer purification, and species specificity is strong, and purity is greater than 95%, and tiring is not less than 1:2000.Select commercialization enzyme connection thing diluent, company produces by the biopanda diagnostic reagent.
2) preparation of calibration object
HIV antibody, HCV antibody and HBsAg are all negative after testing, detect EBV Rta-IgG with test kit of the present invention positive, are corresponding unit concentration (200U/ml, 100U/ml, 50U/ml, 25U/ml, 12.5U/ml, 0U/ml) through dilution.Step 3: test kit sensitivity, precision, accuracy and preservation period detect
1) test kit precision test:
The test kit that is prepared different batches (001 batch, 002 batch, 003 batch) by the preparation method of embodiment 3 test kits, therefrom respectively extract 3 test kits, test, measure the concentration value of 100U/ml, 25U/ml calibration object solution, each sample repeats 10 times, calculates respectively the variation coefficient (CV%).
Table 1 different concns sample withinrun precision certification test result
Table 2 different concns sample betweenrun precision certification test result
In sum, this test kit is criticized interpolation, difference between batch all less than 10%, meets the regulation that test kit precision should be less than or equal to international external diagnosis reagent case precision≤15% fully.
2) test kit accuracy test:
The 100U/ml sample that detects concentration known with the reagent of different batches is respectively made concentration after 2 times, 4 times dilutions with the calibration object I, do recovery experiment, test each time diluting rear sample duplicate detection 3 times, comparative measurement value and theoretical sign value are obtained the rate of recovery (%), according to the accuracy in detection of result with examination development reagent.
The accuracy certification test result of table 3 test kit
| Lot number | 001 | 002 | 003 |
| The rate of recovery | 95.10% | 99.00% | 93.00% |
As shown in table 3, the test kit rate of recovery satisfies the test kit rate of recovery in 100% ± 10% requirement between 93%-99%.
3) test kit lowest detectable limit (sensitivity)
With test kit testing calibration product I of the present invention 20 times, the mean value of measurement result adds that 2 times of standard deviations are as the lowest detectable limit of test kit.Show that through test the lowest detection of test kit is limited to 10U/ml.
4) test kit linear relationship
Reagent with different batches detects enterprise's self-control calibration object respectively, to be used for the investigating linear relationship that this test kit can guarantee the sensing range of certain accuracy, the results are shown in Table 4
Table 4 linearity range test-results
| Lot number | 001 | 002 | 003 |
| Dose response curve relation conefficient (r) | 0.9982 | 0.9970 | 0.9986 |
5) test kit preservation period test
The test kit preservation condition is 2-8 ℃, and through the mensuration of 12 months, the linear relationship of test kit, precision, accuracy, quality control product practical measurement value were all within normal range.Consideration has improper preservation condition and occurs in transportation and use procedure, and test kit was placed 9 days under the condition of 37 ℃ of preservations, carries out accelerated test, and result shows that this test kit indices meets the requirements fully.Can draw test kit from above result can preserve 12 months at least at 2-8 ℃.
6) the test kit clinical trial is estimated
The nasopharyngeal cancer patient of not treating nasopharyngeal cancer patient and 6 routine treatment by stages of 30 routine normal people's samples and 20 examples being made a definite diagnosis with the test kit of the present invention preparation carries out Parallel testing, and trace routine and result judgement are carried out in strict accordance with the using method that embodiment 1 provides.Detected result is as shown in table 5,6,7.
6.1. detection reference pH-value determination pH: by 30 routine health volunteer's serum are detected, obtain the benchmark numerical value distribution situation (see figure 2) of normal human blood Rta antibody concentration.Normal people Rta antibody concentration mean value and standard deviation are: 10.32 ± 6.85U/ml.The data of this experiment are recorded in table 5.
Table 5 normal people Epstein-Barr virus Rta-IgG content detection result
6.2. through clinical definite but the 20 routine nasopharyngeal cancer patients of not yet receiving treatment through showing that with this quantification kit Epstein-Barr virus Rta-IgG content is except an exception, 19 routine serum Rta anti-body contg significance ground rising (see figure 3)s, average content is up to fruit 101.57 ± 42.22U/ml.This experimental test data sees Table 6.
Table 6 is not treated nasopharyngeal cancer patient Epstein-Barr virus Rta-IgG content detection result
6.3.Rta the clinical illustration of antibody detection by quantitative monitoring treatment of nasopharyngeal carcinoma effect
6 routine treatment by stages Nasopharyngeal Carcinoma Patients are followed the trail of Epstein-Barr virus Rta-IgG content detection find, through the increase (from one-period to four cycle) for the treatment of cycle, patient's Epstein-Barr virus Rta-IgG content is obvious reduction trend, as shown in Figure 4; These detection data of testing each example are as shown in Fig. 5 and table 7.
Table 7 Staging System for Nasopharyngeal Carcinoma treatment patient Epstein-Barr virus Rta-IgG content detection result
As shown in table 5, table 6, table 7, higher than clinical appraisal value 30U/ml, all the other are all lower than the clinical appraisal value except an example for 30 routine normal people's Epstein-Barr virus Rta-IgG content, and specificity is 96.67%(29/30); 20 examples are not treated in nasopharyngeal cancer patient 19 routine Epstein-Barr virus Rta-IgG content all higher than clinical appraisal value, susceptibility 95%(19/20); 6 routine treatment by stages Nasopharyngeal Carcinoma Patients are followed the trail of Epstein-Barr virus Rta-IgG content detection find, along with the increase for the treatment of cycle and treatment intensity, patient's Epstein-Barr virus Rta-IgG content is obvious reduction trend.Above results suggest, this test kit has very important meaning for the monitoring of nasopharyngeal cancer patient result for the treatment of.
The above is only preferred embodiment of the present invention, is not the present invention is done any pro forma restriction.Although the present invention with preferred embodiment openly as above, yet be not that any mode limits the present invention.Any those skilled in the art, in technical scheme scope of the present invention, allly utilize above-mentioned disclosed technology contents to make a little change or modification, should be considered as the equivalent embodiment of equivalent variations, in every case be not break away from the technical solution of the present invention content,, all still belong in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment does according to technical spirit of the present invention.
Claims (10)
1. the Recombinant CHO cell line CHO/RTA of high efficiency stable expression Epstein-Barr virus Rta albumen, its deposit number is: CGMCC6955.
2. Recombinant CHO cell line CHO/RTA according to claim 1, it is characterized in that, utilize Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO/dhfr-) as host cell, stablized after screening amplification, the cell strain of high efficient expression Epstein-Barr virus Rta albumen.
3. arbitrary described Recombinant CHO cell line CHO/RTA according to claim 2, its expression vector be can with described DNA fragmentation restructuring, form the bio-carrier of suitable expression plasmid.
4. Recombinant CHO cell line CHO/RTA according to claim 3, described expression vector is the eukaryotic cell heterologous gene expression system.
5. the application in the Chinese hamster ovary celI storehouse of Epstein-Barr virus Rta albumen is expressed in the arbitrary described Chinese hamster ovary celI strain of claim 1～4 in actual production.
6. the construction process of the arbitrary described Chinese hamster ovary celI strain of claim 1～4, comprise the steps:
(1) the EBV genome is carried out the RT-PCR amplification, get BRLF1 full length sequence cDNA; BRLF1 full length sequence cDNA is cloned into carrier for expression of eukaryon pCDNA3.1(+) in, build mammalian cell expression vector pCDNA3.1(+)-EBV-Rta;
(2) use positively charged ion plasmalogen Lipofectamine2000 with pCDNA3.1(+)-EBV-Rta and pSV2-dhfr cotransfection CHO/dhfr
-
(3) with G418 and xanthoglobulin, thymus pyrimidine, the selection Screening of Media integration EBV BRLF1 of glycine and the stable transfected cells strain of dhfr gene;
(4) progressively increase progressively the mode of aminomethyl petrin concentration, the copy number of amplifying target genes;
(5) with the EBV-Rta in WesternBlot detection recombinaant CHO cell substratum;
(6) be separated to mono-clonal by limiting dilution assay from mix the clone, utilize the expression amount of EBV-Rta in ELISA method detection mono-clonal substratum, screen the cell strain CHO/RTA of high efficient expression EBV-Rta.
7. construction process according to claim 6, is characterized in that, described EBV genome is B95-8 cell strain (the ATCC Number:CRL-10624 that stimulated with TAP
TM) the interior gained that infects.
8. construction process according to claim 7, is characterized in that, the concentration of described G418 is 0.1～1.0mg/ml; Described hypoxanthic concentration is 0.01～0.2mmol/L; The concentration of described thymus pyrimidine is 0.001～0.03mmol/L, and the concentration of described glycine is 10～50mg/L.
9. construction process according to claim 8, is characterized in that, the concentration of described aminomethyl petrin is 0.01～1000 μ mol/L.
10. a Chinese hamster ovary celI storehouse that is used for expressing Epstein-Barr virus Rta albumen, is characterized in that, set up by the arbitrary described recombinant C HO/RTA cell strain of claim 1～4 to form.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105044355A (en) * | 2015-07-08 | 2015-11-11 | 同昕生物技术（北京）有限公司 | Chemiluminescence kit for detecting EB virus Rta/IgG antibody and use thereof |
| CN110903357A (en) * | 2019-12-16 | 2020-03-24 | 东方海洋(北京)医学研究院有限公司 | Rta protein dominant epitope antigen and application thereof |
| CN111304243A (en) * | 2018-12-12 | 2020-06-19 | 普莱柯生物工程股份有限公司 | Construction method of CHO cell strain for efficiently expressing foreign protein, constructed CHO cell strain and application thereof |
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| CN102703502A (en) * | 2012-02-27 | 2012-10-03 | 同昕生物技术（北京）有限公司 | Preparation method for replication and transcription activator (Rta) protein and application of Rta protein to nasopharynx cancer detection reagent |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105044355A (en) * | 2015-07-08 | 2015-11-11 | 同昕生物技术（北京）有限公司 | Chemiluminescence kit for detecting EB virus Rta/IgG antibody and use thereof |
| CN105044355B (en) * | 2015-07-08 | 2016-10-19 | 同昕生物技术（北京）有限公司 | For detecting chemical luminescence reagent kit and the application thereof of Epstein-Barr virus Rta/IgG antibody |
| CN111304243A (en) * | 2018-12-12 | 2020-06-19 | 普莱柯生物工程股份有限公司 | Construction method of CHO cell strain for efficiently expressing foreign protein, constructed CHO cell strain and application thereof |
| CN110903357A (en) * | 2019-12-16 | 2020-03-24 | 东方海洋(北京)医学研究院有限公司 | Rta protein dominant epitope antigen and application thereof |
| CN110903357B (en) * | 2019-12-16 | 2021-06-18 | 东方海洋(北京)医学研究院有限公司 | Rta protein dominant epitope antigen and application thereof |
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| Publication number | Publication date |
|---|---|
| CN103131674B (en) | 2015-01-28 |
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