CN1693467A - System and method of mammalian cell strain for fast constructing target gene high expression - Google Patents
System and method of mammalian cell strain for fast constructing target gene high expression Download PDFInfo
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Abstract
A system and method for quickly configuring the high-expression target gene of mammal's cell strain features that the mutation and recombination are used to screen the site able to be amplified and with high transcription activity, and inserting marker in the site. Said method include such steps as using the recombinants to recognize and severing a pair of recombinant signal sequences and site-specific recombination.
Description
Technical field
The present invention relates to the high-transcriptional activity site of using mutant and recombinant technology screening amplification property, and insert the method for mark in site.The invention still further relates to the quick method that makes up and obtain the mammal cell line of target gene high expression of practical site specificity recombination mechanism, promptly use recombinase identification and cut a pair of recombination signal sequence (integrate labeled vector and targeted expression carrier and respectively have), finish the locus specificity reorganization, realize the stable high expression level of target gene.The invention still further relates to integration labeled vector and the targeted expression carrier in the inventive method and comprise the system of integrating labeled vector, targeted expression carrier, recombinase carrier and the mammalian cell strain that sets out.
Background technology
Develop rapidly along with genetic engineering technique, existing many recombinant proteins are used for clinical as medicine, in numerous recombinant protein expression system, though protokaryon and yeast expression system cost are low, but often can not correctly assemble, fold and glycosylation the mammalian proteins with biological function beyond expression of words.And this advantage of mammalian cell expression system exactly, recombinant protein the expression of these systems without exception relate to reconfiguring and distributing of genetic material.
Genetic material can be recombinated by several different independent mechanisms, comprises homologous recombination, locus specificity reorganization and non-homogeneous reorganization, unusual reorganization.Homologous recombination relates to the extremely similarly reorganization between the dna segment of those sequences, and homologous sequence is along its chain formation pairing, and interchain takes place to intersect and exchange then, and intersection can occur in any site in the homologous fragment.The length of recombination efficiency and homologous sequence, the same degree of sequence, and homology is directly proportional with the ratio of non-homogeneous DNA in the construct.In general need 2～10 kilobase right at least, or bigger.
The locus specificity reorganization relates to the exchange of genetic material at predetermined site, and in this reaction, recombinase protein is attached on the recombination signal sequence, forms a chain break, and then the exchange of DNA chain takes place.The locus specificity reorganization is made up of three parts, and recombinase protein, recombination signal sequence and cofactor, some system only are made up of preceding two components, and do not need cofactor, for example, and FRT/flp system, Loxp/Cre system.Recombination signal sequence is generally very short, is approximately tens bases, thus this DNA construct is operated easier than the big segment of homologous recombination.
Non-homogeneous reorganization and unusual reorganization, occur between the genetic material that does not have remarkable sequence homology, its point of crossing does not occur in locus specificity recombination sequence place, foreign DNA is incorporated into karyomit(e) in non-homogeneous site, cause chromosomal transposition or disappearance, finish reorganization by the concatemerization of end connection, double-strand break reparation, bridge fracture and transfection sequence.
For obtaining the mammal cell line of stable transfection, realize the expression of target gene high level persistence, the mode of non-homogeneous reorganization is adopted in a large number, and the generation of the mammal cell line of stable transfection generally includes following steps; There is the dna vector of target gene and drug resistance gene to import host cell the clone, in the presence of medicine, select to cultivate the cell of successfully integrating foreign DNA subsequently, in most application, target gene links with the selected marker that adjacent gene copy number is increased, and the gene of coding Tetrahydrofolate dehydrogenase (DHFR) is modal marker gene.Culturing cell can cause the output of DHFR to increase because of the amplification of DHFR gene in the presence of the competitive inhibitor ammonia first dish purine of DHFR.In the time of DHFR gene amplification, the dna sequence dna of its flanking region also can be amplified, the scope of amplification is tens kilobase, because target gene links to each other with the DHFR gene, so target gene has also obtained a large amount of amplifications, the increase of copy number causes the increase of protein output, thereby realizes a day mark high level gene expression.Although this method has been proved to be success, but the non-homology reorganization has significant limitation, for example do not have target, the position effect of being integrated is controlled, quantity is seldom on karyomit(e) in the site of be fit to integrating and having a very high transcriptional activity, often need from up to ten thousand transfectants, screen and stablize high expressing cell system, and the minimum time that needs half a year of process that obtains, the non-constant of controllability.
The problem of non-homogeneous reorganization can be recombinated by homologous recombination or locus specificity and be overcome, because these two kinds of recombination forms all have very strong target, U.S. Ai De company in 1998 uses homologous recombination to realize Recombinant Protein Expression fast, and its patent is to the method for mammalian cell specific site and used carrier (Chinese patent application number 98804988.0) with gene integration.Though the frequency of homologous recombination spontaneous generation reorganization in yeast and other fungal organism is very high, but in higher eucaryote, the probability of this type of reorganization is extremely low, and in mammalian cell, the frequency that homologous recombination takes place is that one of percentage of non-homologous recombination occurrence frequency is to five thousandths.In addition, the frequency that homologous recombination takes place just is subjected to the influence of homologous fragment length, generally needs 2～10 kilobase at least, or bigger, actual adopt generally all surpassed 10 kilobase, the operation of the DNA construct of this molecular weight is extremely bothered.What the present invention was different with it is to adopt non-homogeneous reorganization and two kinds of mechanism of locus specificity reorganization to realize efficiently expressing of recombinant protein.
Summary of the invention
One aspect of the present invention relates to a kind of integration labeled vector, and it comprises following element or sequence:
A. dominant selectable marker;
B. selection markers;
C. one or more amplification gene;
The genetic elements of D. in microorganism, breeding;
E. recombination signal sequence;
Wherein, selection markers and dominant selectable marker insert recombination signal sequence with fusion protein form expression between promotor and the fusion rotein, and initiator codon ATG is positioned at before the recombination signal sequence, is controlled by the promotor of a reduction jointly; And amplification gene also adopts the viral promotors of reduction.
In preferred embodiments, the reduction promotor of regulation and control expressing fusion protein is the viral promotors of reduction.The promotor of regulating and control described amplification gene and expressing fusion protein can be identical, also can be different.
The present invention relates to a kind of targeted expression carrier on the other hand, and it comprises following element or sequence:
A. dominant selectable marker;
The genetic elements of B. in microorganism, breeding;
C. recombination signal sequence;
D. target gene expression cassette;
E. the replication orgin of eukaryotic virus,
Wherein, the dominant selectable marker in this carrier is different from the dominant selectable marker in the integration labeled vector of the present invention, and this dominant selectable marker gene is not activated son and initiator codon; Recombination signal sequence inserts before the dominant selectable marker, and this recombination signal sequence is identical with recombination signal sequence in the integration labeled vector of the present invention.
In preferred embodiments, the target gene expression cassette in the described carrier comprises SP163 enhanser, the upright early gene promotor of CMV, endoplasmic reticulum signal peptide, target gene sequence and Trobest adenylic acid (AMP) tailing signal.
Further aspect of the present invention relates to the construction process of the cell strain that contains the high-transcriptional activity site that can increase in the genome, may further comprise the steps:
A. with integration labeled vector of the present invention in non-key position linearizing, be as the criterion with the normal function that does not influence all elements, use this linearized vector to transform the mammalian cell strain that sets out then by non-homogeneous recombination mechanism;
B. utilize transformant, obtain to integrate the clone that labeled vector is integrated in the higher transcriptional activity site of genome at the selective pressure screening steps A acquisition of dominant selectable marker;
C. the clone's that obtains of determination step B selection markers gene expression amount obtains the clone of screening-gene high expression level, promptly integrates the clone that labeled vector inserts high-transcriptional activity site place in the genome;
D. utilize clone, obtain the clone that amplification gene is amplified, promptly contain the cell strain of the high-transcriptional activity site that can increase in the genome at the selective pressure screening step C acquisition of amplification gene.
In this method of the present invention, the selective pressure among step B and the D can progressively increase.
Another aspect of the invention relates to the method for the Mammals recombinant cell strain that obtains target gene high expression, may further comprise the steps:
A. utilize the construction process of the cell strain that contains the high-transcriptional activity site that to increase in the genome of the present invention to obtain to contain in the genome cell strain of the high-transcriptional activity site that can increase;
B. the cell strain that obtains with superhelix form cotransfection steps A with targeted expression carrier of the present invention and recombinase expression vector, described recombinase expression vector transient expression site-specific recombinase, the locus specificity reorganization takes place in this recombinase catalysis targeted expression carrier and the integration labeled vector that is incorporated on the genome at the recombination signal sequence place;
C. utilize transfection clone, obtain the clone that target gene inserts the amplification gene flank, i.e. the Mammals recombinant cell strain of target gene high expression at the selective pressure screening step B acquisition of selective marker in the targeted expression carrier.
In preferred embodiments, the present invention obtains the further comprising the steps of D of method of the Mammals recombinant cell strain of target gene high expression: utilize the clone at the selective pressure screening step C acquisition of amplification gene, obtain the Mammals recombinant cell strain of target gene high expression.
In the embodiment that is more preferably, the further comprising the steps of E of method of the Mammals recombinant cell strain of acquisition target gene high expression of the present invention: the Mammals recombinant cell strain of the target gene high expression that step C or step D are obtained goes down to posterity.Described step e can be carried out under selective pressure.Described selective pressure is the selective pressure at amplification gene.This selective pressure can progressively increase.
The invention still further relates to the system of the high secretion of a kind of quick structure recombinant protein cell strain, this system comprises:
A. integration labeled vector of the present invention;
B. targeted expression carrier of the present invention;
C. recombinase expression vector, but the locus specificity reorganization takes place in integration labeled vector and targeted expression carrier that this recombinase expression vector transient expression site-specific recombinase, described recombinase can catalysis be incorporated on the mammalian cell strain genome that sets out at the recombination signal sequence place;
D. mammalian cell strain sets out.
The described mammalian cell strain that sets out can be selected from CHO-dhfr
-, COS, Hela, NIH3T3 and NS0 myeloma cell.
Description of drawings
Figure 1A integrates labeled vector pTGS-FRT-DHFR synoptic diagram.
SV40 promotor: the SV40 virus early promoter of reduction
ATG: initiator codon
Identification of FRT site:Flp recombinase and cleavage site
LacZ-Zeocin: fusion rotein (beta-galactosidase enzymes and phleomycin resistance albumen)
Bla: ammonia benzyl resistant gene
Dhfr: dihydrofolate reductase gene
PUC origin: intestinal bacteria replicon
Figure 1B FRT recombination signal sequence synoptic diagram.
Fig. 2 pWS3 carrier synoptic diagram.
Ori: intestinal bacteria replicon
Bla: ammonia benzyl resistant gene
Dhfr: Tetrahydrofolate dehydrogenase (DHFR) gene
PMT-1: metallothionein(MT) I promotor
VK: antibody chain variable region sequence
CK: antibody Kappa chain constant region sequence
VH: antibody heavy chain variable region sequence
CH1/Fc:IgG1 type antibody constant region sequence
Fig. 3 pCMV-VKH-SARS synoptic diagram.
ColEI ori: intestinal bacteria replicon
Bla: ammonia benzyl resistant gene
Sp163: enhanser
PCMV: the upright promotor early of human cytomegalic inclusion disease virus
VK: antibody chain variable region sequence
CK: antibody Kappa chain constant region sequence
VH: antibody heavy chain variable region sequence
CH1/hinge/CH2/CH3:IgG1 type antibody constant region sequence
Dhfr: Tetrahydrofolate dehydrogenase (DHFR) gene
PA: Trobest adenylic acid (AMP) tailing signal
SV40pA:SV40 virus polyadenylic acid tailing signal
Fig. 4 targeted expression carrier pTGS-site synoptic diagram.
Target gene is expressed box
Identification of FRT:Flp recombinase and cleavage site
Hgr: hygromycin phosphotransferase gene
Ori: intestinal bacteria replicon
Bla: ammonia benzyl resistant gene
Fig. 5 IgG1 type antibody expression box (pTGS-SARS1-site belongs to this type).
PCMV: the upright promotor early of human cytomegalic inclusion disease virus
VK: antibody chain variable region sequence
CK: antibody Kappa chain constant region sequence
VH: antibody heavy chain variable region sequence
CH1/hinge/CH2/CH3:IgG1 type antibody constant region sequence
PA: Trobest adenylic acid (AMP) tailing signal
Fig. 6 IgG1 type antibody expression box (pTGS-GL-site belongs to this type).
PCMV: the upright promotor early of human cytomegalic inclusion disease virus
Sp163: enhanser
VK: antibody chain variable region sequence
CK: antibody Kappa chain constant region sequence
VH: antibody heavy chain variable region sequence
CH1/hinge/CH2/CH3:IgG1 type antibody constant region sequence
PA: Trobest adenylic acid (AMP) tailing signal
Fig. 7 Fab antibody expression box (pTGS-MH-140 belongs to this type).
PCMV: the upright promotor early of human cytomegalic inclusion disease virus
VK: antibody chain variable region sequence
CK: antibody Kappa chain constant region sequence
VH: antibody heavy chain variable region sequence
CH1:IgG1 type antibody constant region sequence
PA: Trobest adenylic acid (AMP) tailing signal
Fig. 8 Western Blot hybridization.
A:Pierce company produces human IgG; B:SARS antibody; C:CD20 antibody
Fig. 9 flow cytometry (FACS) and indirect immunofluorescence be figure as a result.
Left figure: the right figure of negative control: CD20 antibody
The common transfection method of Figure 10 and the application's transfection method passage number and the comparison of cloning positive rate.
The anti-SARS antibody cell strain of Figure 11 stably excreting antibody accumulation secretion spirogram.
Anti-SARS antibody cell strain multiplication curve of Figure 12 stably excreting and survival rate.
The a cell strain multiplication curve b pressurization cell strain multiplication curve that do not pressurize
The c cell strain survival rate d pressurization cell strain survival rate of not pressurizeing
Figure 13 subclone cell strain multiplication curve and survival rate.
Figure 14 subclone cell strain antibody accumulation secretion discharge curve.
Table 1ELISA analyzes transient transfection targeted expression carrier antibody secretory volume
Betagalactosidase activity before and after the pressurization of table 2CHO-DZ engineering cell strain
The comparative result of the common transfection method of table 3 and the application's transfection method
Embodiment
The invention provides rapid screening and obtain the transcriptional activity site of mammalian cell genome amplification property, and insert the method for mark in this site.The present invention also provides the novel method that required foreign DNA is integrated into target site in the mammalian cell genome via the locus specificity reorganization.Use the high expression level that this method can repeat to realize the different target gene.
Particularly, at first with integrating labeled vector, mark is inserted the transcriptional activity site of mammalian genes group amplification property, then with targeted expression carrier and the two plasmid co-transfection mammalian cells of recombinase expression vector, make target gene under the effect of locus specificity recombination system, be integrated into genomic mark, by the amplification effect of amplification gene, increase gene copy number fast, realize gene high expression.
Integrate labeled vector
The application's integration labeled vector comprises following element or sequence.
The first, dominant selectable marker.This dominant selectable marker helps to separate and identifies that will integrate labeled vector by non-homogeneous recombination mechanism is inserted into genomic cell.The example of dominant selectable marker comprises the gene of the following material of encoding: India's different wall streptomycete phleomycin resistance (Sh ble), hygromix phosphotransferase (hph), neomycin resistance (neo), hypoxanthine phosphoribosyltransferase (HPRT), tetracycline (pac), dihydroorotase glutamine synthetase (GS), Histidine D (hisD), carbamyl phosphate synthetase (CAD), Tetrahydrofolate dehydrogenase (DHFR), aspartate transcarbamylase, xanthine-guanine phosphoribosyl transferase (gpt) and adenosine deaminase (ada).
The second, selection markers.Selection markers can make carrier-containing cellular segregation come out, and does not need to apply medicine or other selective pressure to them.The example of selection markers comprises the gene of Codocyte surface protein, fluorescin and enzyme.The cell that comprises carrier can utilize the fluorescently-labeled antibody of cell surface protein or is that the substrate of fluorescence-causing substance separates with the enzymatic conversion of suppressed by vector coding by flow cytometer (FACS), perhaps selects by the specific reaction of enzyme-to-substrate.
The 3rd, one or more amplification gene.The existence of amplification gene, cell had overcome the ability of relative medicine lethal effect, but rising along with drug level, the cell that contains less amplification gene copy can't be resisted the effect of medicine and death, the cell that only contains more amplification gene copy could be survived, and the formation colony, thereby pick out the cell of the integration labeled vector that comprises that copy number increases.The example of amplification label includes, but not limited to Tetrahydrofolate dehydrogenase (DHFR), adenosine deaminase (ada), dihydroorotase glutamine synthetase (GS) and carbamyl phosphate synthetase (CAD).Realize gene amplification, different system is to the requirement difference of host cell, for example common system, the clone that Tetrahydrofolate dehydrogenase (DHFR)/methotrexate (MTX) system need suddenly change is as host cell, and dihydroorotase glutamine synthetase (GS)/methionine(Met) (MSX) system does not need the clone of suddenling change.
The 4th, the genetic elements of in microorganism, breeding, for example replication origin of microorganism and antibiotics resistance mark.
The 5th, recombination signal sequence includes, but are not limited to FRT, Loxp, AttB/AttP.
Integrate in the labeled vector at this, regulating and controlling sequence comprises elements such as promotor and enhanser.Usually contain the binding site of transcription factor on the regulating and controlling sequence, described transcription factor for example TATA is conjugated protein, CAAT binding protein, SP-1, AP-1.The source of regulating and controlling sequence can be cellular genome or viral genome.The example of cell regulate and control sequence includes, but not limited to metallothionein(MT) I gene, immunoglobulin gene, casein I gene.The example of virus regulating and controlling sequence includes, but not limited to the controlling element of the upright early gene of cytomegalovirus (CMV), SV40 gene, adenovirus late gene, hsv gene.In preferred embodiments, regulating and controlling sequence is a viral promotors.
The application's favourable part is selection markers and dominant selectable marker with fusion protein form expression, the promotor that is controlled by a reduction jointly, reduce transcriptional activity and make translation impaired, like this under host cell is in minimum lethal concentration at the dominant selectable marker medicine, those are integrated labeled vector and insert the clone of site for low transcriptional activity site, just can not resist the effect of medicine and death, the clone who only is incorporated into the higher position of transcriptional activity could survive, pass through the mensuration of selection markers gene expression amount again, select the high clone of expression amount, these clones are for integrating the clone that labeled vector is inserted into the genome high-transcriptional activity site.
The application's favourable part is that amplification gene adopts the viral promotors of reduction, just can reach the gene copy number that very high concentration medicine effect just can reach like this under the drug effect of lower concentration very, not only fast but also save medicine.For example select the amplification of Tetrahydrofolate dehydrogenase (DHFR) gene with methotrexate, do not use under the situation of reduction promotor, the concentration of methotrexate need reach 1-100nM, and for initial drug level about 1000 times just can reach satisfied copy number.Because the use of drug level increases step by step, the process of medicament selection reaches half a year like this, even the longer time.Adopt the application's method, the concentration of methotrexate only need reach 0.01-1nM, and for initial drug level 10 times need the bimestrial time at most, and gene just can increase and reach satisfied copy number.
Though the application adopts non-homogeneous reorganization to realize integrating the integration of labeled vector, but favourable part is screening and obtains high-transcriptional activity site is very fast, only need to eliminate the integration of most low transcriptional activity site, and common non-homogeneous reorganization need be removed the integration of eliminating low transcriptional activity site from several thousand clones by medicine.
The favourable part of another of the application is to insert recombination signal sequence between promotor and fusion rotein, and initiator codon ATG is positioned at before the recombination signal sequence.
The targeted expression carrier
The application's targeted expression carrier comprises following element or sequence.
The first, dominant selectable marker.This dominant selectable marker is different from the dominant selectable marker of integrating in the labeled vector, can be a kind of in the following example, India's different wall streptomycete phleomycin resistance (Shble), hygromix phosphotransferase (hph), neomycin resistance (neo), hypoxanthine phosphoribosyltransferase (HPRT), tetracycline (pac), dihydroorotase glutamine synthetase (GS), Histidine D (hisD), carbamyl phosphate synthetase (CAD), Tetrahydrofolate dehydrogenase (DHFR), aspartate transcarbamylase, xanthine-guanine phosphoribosyl transferase (gpt) and adenosine deaminase (ada).
The second, the genetic elements of in microorganism, breeding, for example replication origin of microorganism and antibiotics resistance mark.
The 3rd, with the identical recombination signal sequence of integration labeled vector, include, but are not limited to FRT, Loxp, AttB/AttP.
The 4th, treat the target gene expression cassette of great expression.Described target gene includes, but are not limited to immunoglobulin gene, cell surface receptor, intracellular receptor, hormone, cytokine, somatomedin, transcription factor, enzyme etc.Regulating and controlling sequence in this expression cassette comprises elements such as promotor and enhanser.Usually contain the binding site of transcription factor on the regulating and controlling sequence, described transcription factor for example TATA is conjugated protein, CAAT binding protein, SP-1, AP-1.The source of regulating and controlling sequence can be cellular genome or viral genome.The example of cell regulate and control sequence includes, but not limited to metallothionein(MT) I gene, immunoglobulin gene, casein I gene.The example of virus regulating and controlling sequence includes, but not limited to the controlling element of the upright early gene of cytomegalovirus (CMV), SV40 gene, adenovirus late gene, hsv gene.In preferred embodiments, regulating and controlling sequence is a viral promotors.
The 5th, the replication orgin of eukaryotic virus.The existence of virus replication starting point makes the targeted expression carrier can be used as the episome short period of time to be present in the cell, the carrier copy number increases severely at short notice, instantaneous high expression level target gene, favourable part is can obtain a large amount of target gene albumen in three days, be used for downstream analysis, and estimate carrier after various transformations, the expression to target gene does not exert an influence.The example of useful virus replication starting point includes, but are not limited to SV40ori and EBV ori.
The application's favourable part is to insert recombination signal sequence before the dominant selectable marker on the targeted expression carrier, this dominant selectable marker gene is not activated son and initiator codon, have only when the targeted expression carrier and integrate correct locus specificity reorganization has taken place between the labeled vector, integrate the expression that promotor on the labeled vector and initiator codon can start dominant selectable marker like this, under the selective marker medicine pressure of targeted expression carrier, cell could be survived.So just finish the colony screening that the reorganization of correct site takes place fast.
According to the application's preferred embodiment, in the target gene expression cassette, promotor is the upright early gene promotor of CMV, and enhanser is SP163, and signal peptide is the endoplasmic reticulum signal peptide, and tailing signal is a Trobest adenylic acid (AMP) tailing signal.This kind expressed box, can realize the high expression level of target gene.
The recombinase expression vector
The recombinase expression vector is commercially available in invitrogen or promega company, and this carrier can instantaneous high expression level site-specific recombinase, and the recombinase expression vector comprises following element.
The first, the genetic elements of in microorganism, breeding, for example replication origin of microorganism and antibiotics resistance mark.
The second, recombinase gene is expressed box, comprises viral promotors, recombinase gene, adenylic acid (AMP) tailing signal.
Recombinase expression vector and targeted expression carrier cotransfection are behind host cell, recombinase carrier express recombinant enzyme, cause the targeted expression vector integration at genome recombination signal sequence place, in case the targeted expression vector integration advances genome, then no longer need recombinase, on the contrary, too much recombinase can cause the targeted expression carrier to cut down from genome.Just because of this, the recombinase expression vector that the application selects for use lacks the dominant selectable marker of targeted expression carrier, and therefore, along with going down to posterity of cell, this plasmid can be lost faster.
The structure that contains the cell strain of the high-transcriptional activity site that can increase
At first will integrate labeled vector in non-key position linearizing, be standard with the normal function that does not influence all elements, adopts non-homogeneous recombination mechanism, utilize means known in the art with the carrier transfered cell (as CHO-dhfr
-, COS, Hela, NIH 3T3, NS0 myeloma cell etc.), introduction method includes, but not limited to electroporation, coprecipitation of calcium phosphate, DEAE-dextran, liposome transfection.Integrating the promotor that has adopted two reductions on the labeled vector simultaneously, first is the promotor of selection markers and dominant selectable marker fusion rotein, and second is the attenuated virus promotor of amplification gene.Under the situation that selective pressure exists, use the reduction function of first promotor and can pick out the clone that higher transcriptional activity site is integrated fast.Owing to adopted this strategy, make and be filtered into power and reach about 1/10th.Then, the clone's that mensuration integration labeled vector inserts at random selection markers gene expression amount, the clone that the selection markers gene expression amount is high is and integrates the clone that labeled vector inserts high-transcriptional activity site place in the genome.
Though the acquisition of high-transcriptional activity site can realize the high expression level of foreign gene, expression amount does not reach satisfied degree, and one of effective ways of solution are that the copy number of realizing high-transcriptional activity site place gene increases severely, even reach thousands of copy numbers.But not every transformation cell lines can stand karyomit(e) specific site DNA to be duplicated in local recirculation, have only the part transformation cell lines can stand the rotaring redyeing gene that those zones produce thousands of copies, in addition, its capacity variance that allows gene copy number to increase of different transcriptional activity sites is very big, therefore, realize that foreign gene at this regional high expression level, is necessary at first to filter out the high-transcriptional activity site with high amplification property, reduce the blindness that transformation cell lines makes up.
The favourable part of the application be when rapid screening behind high-transcriptional activity site because the use of second reduction promotor, subsequently promptly can rapid screening to high-transcriptional activity site with amplification property, screen successful ratio greatly about about 1/40th.Integrating labeled vector is the cell strain that contains the high-transcriptional activity site of amplification with the cell that mark is inserted into the high-transcriptional activity site that genome can increase.
The structure of the recombinant mammalian cells strain of target gene high expression
After having obtained to contain the cell strain of the high-transcriptional activity site that can increase, then use targeted expression carrier and recombinase expression vector with this cell strain of superhelix form cotransfection, utilize the locus specificity recombination mechanism, adopt means known in the art with the carrier transfered cell, these methods comprise, but be not limited to electroporation, coprecipitation of calcium phosphate, DEAE-dextran, liposome transfection.
Use targeted expression carrier and recombinase expression vector separately, cell does not show the dominant selectable marker resistance on the targeted expression carrier, and like this, under the selective marker medicine pressure of targeted expression carrier, cell can not be survived.Have only when the targeted expression carrier and integrate correct locus specificity reorganization has taken place between the labeled vector, cell could be survived, and the clone of growth is the clone who is inserted into amplification property transcriptional activity site.By the insertion of targeted expression carrier, target gene has been realized the purpose adjacent with amplification gene.
Culturing cell in containing the selection substratum of one or more selective reagents.Each drug level that increases all can obtain some resistance colonies, can pick out single colony and identify amplification label and the copy number of goal gene, the analysis purposes expression of gene is picked out the highest single clone of destination gene expression level and is used for further amplification under higher drug level.
The application's favourable part only need to be seldom the screening that raises of the drug level of round, can realize that common transfectional cell ties up to the amplification copy number of the target gene that the highest drug level just can reach.The application's favourable part also is to have inserted recombination signal sequence in amplification property transcriptional activity site before targeted expression carrier transfectional cell.Therefore, use the application's method and system, the possibility that the amplification of target gene to be expressed and high expression level are realized is 100%, and common infection protocol has only ten thousand/about.
Below specify the application's technical scheme by specific examples.For the application's superior effect is described, in example, selected the expression difficulty to be higher than general proteic macromolecule antibody.
Application example 1
Integrate the structure example of labeled vector pTGS-FRT-DHFR and target labeled vector pTGS-SARS1-site or pTGS-CD20-site
Make up material and the method used
The employed biomaterial of this example comprises: COS7; 293T; Escherichia coliXL1-Blue (available from invitrogen); PFRT/lacZeo2, pOG44, pcDNA5/FRT, pcDNA3.1/hgr+ (available from invitrogen); PWS3, pWSD3, pTGS-VKH (Beijing Tian-Guang Biotechnology Co., Ltd. Is), pGEM-T easy (available from promega).Restriction enzyme is available from Acc65I, ApaLI, BglII, BssHII, EcoNI, MfeI, NaeI, PciI, PmlI, PmeI, SnaBI (available from New England Biolabs); ApaI, BamHI, BglII, EcoO65I, EcoRI, EcoRV, HindIII, NcoI, NdeI, PstI, PvuII, SacII, XbaI, XhoI (available from Takara company).T4DNA ligase enzyme, klenow fragment, T4DNA polysaccharase, alkaline phosphatase; Albumen dyes molecular weight standard (available from NewEngland Biolabs) in advance; Premix Taq (available from Takara company); Lipofextamine 2000, Platinum Pfx polysaccharase (available from invitrogen); Dna molecular amount standard DL2000, DLl5000 (available from Takara company); Gene ruler 1Kb dna ladder degree molecular weight standard; The dna fragmentation purifying reclaims test kit available from Qiagen; The plasmid extraction test kit is available from promega and vast Tyke.
This example electricity changes competent preparation, but design of primers, fragment recovery and the enzyme of purifying, DNA cut be connected, segmental benefit is flat and the equal reference molecule of dephosphorylation is cloned second edition (cold spring harbor laboratory).
Integrate labeled vector pTGS-FRT-DHFR (Fig. 1), wherein contain:
The first, one dominant selectable marker gene India different wall streptomycete phleomycin resistant gene (Shble), this gene protein are given cell and are resisted the antibiotic ability of Zeocin.
The second, one selection markers beta-galactosidase gene.
The 3rd, an amplification gene Tetrahydrofolate dehydrogenase (DHFR) gene, amplification gene adopts the SV40 viral promotors of reduction, this promotor is deleted one of them 72bp enhancer sequence (321～-250), 5～7 bit bases of another 72bp enhancer sequence are sported GTT (245～-243) by TGG, and this amplification gene is expressed box and is derived from the existing carrier pWS3 of our company.This amplification gene and promotor thereof can be positioned at any position on the carrier, only otherwise the function that influences other elements gets final product.
The 4th, the genetic elements of in microorganism, breeding, the bla gene of replication origin ColE1 and expression amicillin resistance.
The 5th, recombination signal sequence FRT.
In this carrier, by translating direction, be viral SV40 reduction promotor (lacking 98 base enhancer sequence (321～-224)) successively, initiator codon ATG, recombination signal sequence FRT, the selection markers beta-galactosidase gene, dominant selectable marker India different wall streptomycete phleomycin resistant gene.Selection markers and dominant selectable marker form fusion rotein, and first initiator codon of fusion rotein is the initiator codon ATG before the recombination signal sequence.The FRT recombination signal sequence is 34bp, comprises three 13bp inverted repeats, the core sequence of a 8bp, the cutting of chain be connected the both sides (Figure 1B) that occur in the 8bp sequence.
Targeted expression carrier pTGS-SARS1-site (Fig. 4, Fig. 5) and pTGS-CD20-site (Fig. 4, Fig. 6), wherein:
The first, contain and the identical recombination signal sequence FRT of integration labeled vector.
The second, contain the genetic elements of in microorganism, breeding, the bla gene of replication origin ColE1 and expression amicillin resistance.
The 3rd, contain a dominant selectable marker hygromix phosphotransferase (hph) gene, be recombination signal sequence FRT before this gene, whole box does not have promotor and initiator codon, but tailing signal is arranged.Its expression depends on the targeted expression carrier and is incorporated into correct reorganization between the genomic integration labeled vector.The locus specificity reorganization takes place in two identical FRT recombination signal sequences under the effect of recombinase (FLP), article two, before the result of chain exchange makes preceding promotor of selection markers beta-galactosidase gene and initiator codon directly be placed on hygromix phosphotransferase (hph) gene, form complete expression frame, give the ability that cell is resisted Totomycin.
The 4th, target gene is expressed box and is respectively the expression box of antibody gene light chain and the heavy chain expression box of IgG1 type antibody.The expression box of antibody gene light chain is the SP163 enhanser by translating direction successively, and CMV founds early gene promotor, endoplasmic reticulum signal peptide, variable region of light chain, constant region of light chain, Trobest adenylic acid (AMP) tailing signal.The heavy chain expression box of IgG1 type antibody is the SP163 enhanser by translating direction successively, and CMV founds early gene promotor, endoplasmic reticulum signal peptide, variable region of heavy chain, CH, Trobest adenylic acid (AMP) tailing signal.
The 5th, comprise the complete replication orgin SV40ori of eukaryotic virus.Owing to contain the SV40 replication orgin, this carrier all can be realized instantaneous high expression level at 293T among COS7 and the Chinese hamster ovary cell CHO.Table 1ELISA analyzes the transient transfection result of targeted expression carrier, and transient transfection adopts common liposome transfection method.
Targeted expression carrier pTGS-SARS1-site inserts and derives from the antibody gene that screens in people's target storehouse, and expressing antibodies is a people source SARS antibody.This carrier not only has very high transient expression amount in the COS7 cell, can reach the transient expression amount of μ g/mL in the CHO-dhfr-cell equally.Data are the ELISA result of three transient transfections in the table.
Targeted expression carrier pTGS-GL-site, the chimeric IgG1 antibody-anti-CD20 antibodies of expressing human-mouse, this antibody sequence is the open antibody sequence ZH7 of NCBI, and this carrier has very high transient expression amount in the 293T cell, and data are the ELISA result of three transient transfections in the table.
Targeted expression carrier pTGS-MH-140-site, antibody gene derives from hybridoma p140, expressing human-mouse chimeric Fab antibody.This carrier has higher transient expression amount in the 293T cell, data are the ELISA result of three transient transfections in the table.
The elisa assay method:
Goat-anti human Fab or IgG antibody are diluted with coating buffer, 100 μ L/ holes bag is by affine 96 orifice plates of ELISA, 4 ℃ are spent the night, with 37 ℃ of sealings of 10% calf serum in 200 μ L/ holes 30min～1 hour, PBST washes plate three times, standard substance and testing sample are added to 96 orifice plates with 100 μ L/ holes, room temperature 2 hours, or 4 ℃ spent the night.PBST washes plate three times, adds horseradish peroxidase mark goat anti-human antibody Fc, Fab or IgG100 μ L/ hole, and room temperature is placed 40min, and PBST washes plate four times, with the colour developing of OPD Color Appearance System, surveys the OD492nm absorbancy.Data in the selected linearity range are made curve with standard substance absorbancy (OD492) to the logarithm (lnC) of standard substance concentration, are X-coordinate with concentration, OD492 is an ordinate zou, do broken line graph, get typical curve OD492=alnC+b, according to typical curve calculation sample concentration.
Table 1ELISA analyzes the transient transfection result of targeted expression carrier
| Anti-CD20 antibodies | ??350μg/mL(293T) |
| Anti-SARS antibody | ??80μg/mL(COS7) |
| ??60μg/mL(CHO-dhfr-) | |
| Anti-gpIIb/IIIaFab antibody | ??60μg/mL(293T) |
The screening in application example two amplification property transcriptional activity sites
PstI endonuclease single endonuclease digestion is integrated labeled vector pTGS-FRT-DHFR, Qiagen glue reclaims test kit and reclaims, reclaiming back concentration is 100ng/mL, adopts BioRad electric shock instrument, and 200ng linearized vector electricity changes the Chinese hamster ovary cell CHO-dhfr of dihydrofolate reductase gene defective type
-, the electric shock cell density is 3 * 10
5Cell/mL, DMEM electricity changes damping fluid, final plasmid concentration is 1 μ g/mL, changes the mixed solution of plasmid and cell over to electric shock glass, 160 volts of Square wave patterns, 15 milliseconds of electric shocks once, transitional cell is cultivated to substratum immediately, changes liquid after 12 hours; Divide bottle after 48 hours, make cell melt the sheet rate less than 20%, the substratum of this moment changes into selects substratum (containing 100 μ g/mL Zeocin microbiotic), and 100 μ g/mL Zeocin antibiotic concentrations are the minimum drug fatality concentration of host cell.The clone appearred in about 30 days.Choose 400 mono-clonals respectively to 96 orifice plates, test monoclonal betagalactosidase activity (Bradford standard measure protein commonly used on the betagalactosidase activity detection kit (kinds of goods catalogue K1455-01) of use invitrogen company and the pharmacopeia).400 clones all express beta-galactosidase enzymes, have clone's enzyme work of 1/4th to reach 100 Miller units.These 100 clones are carried out subclone, the betagalactosidase activity of continuous 20 generations test subclone, 10% subclone has the characteristic of stable high expression level beta-galactosidase enzymes as a result.Select 100 mono-clonals with 4 * 10
-7The M methotrexate, the selection culture medium culturing cell of no xanthoglobulin and thymidine, this process is a cell pressure process, in case cell changes the selection substratum over to, mass mortality appears in cell very soon, has only dihydrofolate reductase gene to be inserted in can to increase the cell in site just can resist the effect of medicine.New clone occurs in succession after 18 days, and trysinization is cloned, and tests these clones' betagalactosidase activity, and the result has 12% clone to show enzyme increase alive, shows that the high-transcriptional activity site that these clones are labeled has the property of amplification.To be 12 strains integrated the clone of labeled vector, these cell strain called afters CHO-DZ engineering cell strain in the transcriptional activity site of amplification property with table 2.
Betagalactosidase activity before and after the pressurization of table 2CHO-DZ engineering cell strain
| Mono-clonal number | Betagalactosidase activity (milliunits) before the pressurization | Pressurization back betagalactosidase activity (milliunits) | Enzyme is lived increases multiple |
| ??M54-4 | ??121 | ??400 | ??3.3 |
| ??W37-13 | ??73 | ??114 | ??1.56 |
| ??M24-1 | ??260 | ??346 | ??1.33 |
| ??W62-14 | ??71 | ??101 | ??1.42 |
| ??W2-15 | ??113 | ??150 | ??1.32 |
| ??M42-21 | ??92 | ??169 | ??1.84 |
| ??W92-8 | ??50 | ??91 | ??1.82 |
| ??M53-24 | ??66 | ??100 | ??1.51 |
| ??M24-3 | ??44 | ??79 | ??1.80 |
| ??W21-2 | ??105 | ??134 | ??1.28 |
| ??M42-1 | ??71 | ??212 | ??2.99 |
| ??W92-2 | ??62 | ??89 | ??1.44 |
The structure of application example 3 high secretion SARS human antibody stably transfected cell lines
Liposome transfection CHO-DZ engineering cell, 1 * 10
5Individual cell, 100ng targeted expression carrier pTGS-SARS1-site and 1 μ g FLP recombinase expression vector pOG44 cotransfection, pOG44 is commercially available in invitrogen, pOG44 transient expression site-specific recombinase, this enzyme acts on the FRT site on CHO-DZ engineering cell strain and the targeted expression carrier, by the cutting and the exchange of two site chains, with targeted expression carrier site-directed integration to karyomit(e).Transfection is rare biography after 48 hours, makes cell melt the sheet rate less than 20%, and the substratum of this moment changes new selection substratum (containing 250 μ g/mL Totomycin, no Zeocin microbiotic) into, and 250 μ g/mL Totomycin concentration are the minimum drug fatality concentration of CHO-DZ engineering cell strain.
Simultaneously, the method that adopts common stably transfected cell line to make up, estimate the efficient that makes up clone, (antibody gene is identical with pTGS-SARS1-site for 350ng pCMV-VKH-SARS, the genetic expression box is also identical) transfection CHO-dhfr-cell, transfection conditions is identical with the application's locus specificity recombination method.100 clonal analysis antibody-secretings of picking amount, positive rate and clone's stability at random after the transfection.
Single FRT site is arranged on the targeted expression carrier, and transfection can not given the cell hygromycin resistance separately.Targeted expression carrier and FLP recombinase expression vector cotransfection CHO-DZ engineering cell, the reorganization of FRT/FLP locus specificity takes place, the cell expressing hygromycin resistance, with this as selection markers, visible significantly hygromycin resistance clonal growth about 25 days after the transfection, ELISA measures the antibody-secreting amount, and trysinization is gone down to posterity, in 17 generations of METHOD FOR CONTINUOUS DETERMINATION, positive rate is 100%.The clone's positive rate that obtains with this method is 100%, and the expressing quantity between each clone substantially all is in the identical scope, and these clones are carried out the secretory volume that entire pressurisation can improve recombinant protein in a large number.
In case the targeted expression vector integration is in genome, then no longer need recombinase, the lasting meeting of recombinase causes the reduction of integration efficiency, recombinase expression plasmid express recombinant enzyme in mammalian cell among the present invention, do not express the resistance screening mark, therefore can be degraded gradually.
The cell strain that obtains behind the pTGS-SAR1-site site-directed integration is pressurizeed, and first week adopted 4 * 10
-7The pressurization of M methotrexate, second week was continued pressurization, and methotrexate concentration increases to 1 * 10
-6M, the 3rd week was used dimethyl sulfoxide (DMSO) (DMSO) freeze-stored cell, the 5th all recovery cells, recover test cell multiplication performance and protein secretion two days later.Uninflated sample went down to posterity 2 months, with the cell of pressurization back recovery, with 1.5 * 10
5Cell/mL 2mL altogether inoculates 6 orifice plates, receives supernatant in per 24 hours, and elisa assay protein secretion, platform are expected the sum and the mortality ratio of basket dyeing calculating cell.Figure 11 is the anti-SARS antibody cell strain of a stably excreting antibody accumulation secretion discharge curve, and Figure 12 is anti-SARS antibody cell strain multiplication curve of stably excreting and survival rate.Figure 11 and 12 data are the mean value of three experiments.The table with test results clear-cells can double in 24 hours, and the pressurization cell reached the quick secretory phase at the 5th day, and its output reaches more than the 160 μ g/mL.Cell to the week of recovering carries out subclone, selects the subclone of high expression level, with 1.5 * 10
5Cell/mL 2mL altogether inoculates 6 orifice plates, received supernatant in per 24 hours, the elisa assay protein secretion, platform is expected the sum and the mortality ratio of basket dyeing calculating cell, test cell multiplication performance and protein secretion, Figure 13 shows subclone cell strain multiplication curve and survival rate, and Figure 14 is a subclone cell strain antibody accumulation secretion discharge curve.Antibody-secreting the most vigorous one day was the 6th day, 1.68 * 10
6Individual total cellular score, antibody-secreting amount are 203.1111ug/mL, and therefore, output is 203.1111ug/mL * 2mL/1.68=241.8ug/24h/1 * 10
6Individual cell.7 days antibody-secreting total amounts are 564.5556ug/mL, and promptly 1.129mg/7 days/6 orifice plates are cultivated, and its expression amount has reached commercial standard (CS).
The method that adopts common stable cell lines to make up, along with going down to posterity of cell, positive rate constantly descends, and expression amount also constantly descends.Table 3 is comparative results of common transfection method and the application's transfection method, and Fig. 9 is common transfection method and the application's transfection method passage number and the comparison of cloning positive rate.
The comparative result of the common transfection method of table 3 and the application's transfection method
| The application's transfection method | Common transfection method |
| Cell: CHO-DZ engineering cell strain | Cell: CHO-dhfr |
| Carrier: targeted expression carrier pTGS-SARS 1-site | Carrier: antibody expression vector pCMV-VKH-SARS |
| Algebraically: 17 | Algebraically: 15 |
| Positive rate: 100% | Positive rate: reduce to 50% of the 15th generation by 78% of the 1st generation |
| Expression amount: 5 ~ 10 μ g/mL | Expression amount: 2 μ g/mL ~ 0.1 μ g/mL |
The structure of application example 4 high secretion CD20 chimeric antibody stably transfected cell lines
Transfection method and application example 3 are identical, and this method is equally applicable to the structure of other antibody high yielding cell sarain.This example targeted expression carrier adopts pTGS-GL-site.After treating the hygromycin resistance clonal growth, trysinization and collection clone are gone down to posterity, and the antibody-secreting amount before the pressurization is 25ug/24 hour/1 * 10
6Individual cell.At present to the amplification of pressurizeing of this height secretion anti-CD20 antibodies cell strain.
To using the antibody employing SDS-PAGE electrophoretic separation that example 3 and 4 is expressed, the human IgG that standard substance select for use Pierce company to produce, sample is SARS IgG1 type antibody and CD20IgG1 type antibody, is transferred to nitrocellulose filter after the electrophoretic separation, 4 ℃, 100V, transferase 45 0min.The taking-up film, 5% skimmed milk TBST room temperature sealing 1h washes film three times, adds horseradish peroxidase mark goat anti-human antibody IgG (H+L) room temperature reaction 1h, washs ECL system (Pierce) colour developing, X sensitive film exposure 30～60s, development, photographic fixing three times.Western Blot result shows that the molecular weight of albumen of expression equates with human IgG, and Fig. 7 is Western Blot figure as a result.
The CD20IgG1 type antibody of using example 4 is carried out indirect immunofluorescence and stream type cell analyzer (FACS) analysis, and the Daudi cell in the vegetative period of taking the logarithm, 2%FBS-PBS are washed 3 times, every pipe packing 2 * 10
5Individual cell is got respective sample 50uL and Daudi cell and is hatched 20min for 4 ℃, and 2%FBS-PBS washes 3 times, adds FITC-goat anti-human igg Fc 50uL, 4 ℃ of lucifuge reaction 20min.2%FBS-PBS washes 4 times, inverted fluorescence microscope observation of cell film surface fluorescence, and 1% Paraformaldehyde 96 is fixed, stream type cell analyzer (FACS) analysis of cells surface average fluorescent strength and positive rate.Fig. 8 is flow cytometry (FACS) and indirect immunofluorescence figure as a result, and it is very strong in conjunction with active to show that the anti-CD20 antibodies of expressing has.
After the instruction of those skilled in the art in understanding present specification, the scope that is appreciated that the application fully is not limited to the specific examples in the embodiment of the present application, and the application's scope is defined by the claims.
Claims (35)
1. integrate labeled vector for one kind, comprise following element or sequence:
A. dominant selectable marker;
B. selection markers;
C. one or more amplification gene;
The genetic elements of D. in microorganism, breeding;
E. recombination signal sequence;
Wherein, selection markers and dominant selectable marker insert recombination signal sequence with fusion protein form expression between promotor and the fusion rotein, and initiator codon ATG is positioned at before the recombination signal sequence, is controlled by the promotor of a reduction jointly; And amplification gene also adopts the viral promotors of reduction.
2. the carrier of claim 1, the promotor of wherein regulating and control expressing fusion protein are the viral promotors of reduction.
3. the carrier of claim 1, wherein regulating and control fusion rotein is identical attenuated virus promotor with the amplification gene expression promoter.
4. the carrier of claim 1, wherein dominant selectable marker is selected from the gene of the following material of coding: India's different wall streptomycete phleomycin resistance, hygromix phosphotransferase, neomycin resistance, hypoxanthine phosphoribosyltransferase, tetracycline, dihydroorotase glutamine synthetase, Histidine D, carbamyl phosphate synthetase, Tetrahydrofolate dehydrogenase, aspartate transcarbamylase, xanthine-guanine phosphoribosyl transferase and adenosine deaminase.
5. the carrier of claim 1, wherein selection markers is the gene of Codocyte surface protein, fluorescin and enzyme.
6. the carrier of claim 1, wherein amplification gene is selected from Tetrahydrofolate dehydrogenase, adenosine deaminase, dihydroorotase glutamine synthetase and carbamyl phosphate synthetase.
7. the carrier of claim 1, the genetic elements of wherein breeding in microorganism comprises replication origin and the antibiotics resistance mark of microorganism.
8. the carrier of claim 1, wherein recombination signal sequence is selected from FRT, Loxp and AttB/AttP.
9. the carrier of claim 1, wherein contain:
A. the different wall streptomycete of dominant selectable marker gene India phleomycin resistant gene;
B. selection markers beta-galactosidase gene;
C. amplification gene dihydrofolate reductase gene, the SV40 viral promotors that this gene is weakened is regulated and control, deleted-321～-250 72bp enhancer sequence in this promotor, 5～7 bit bases of-245～-243 72bp enhancer sequence have been sported GTT by TGG;
D. replication origin ColE1 and express the bla gene of amicillin resistance;
E. recombination signal sequence FRT;
Wherein, in carrier,, be viral SV40 reduction promotor successively by translating direction, initiator codon ATG, recombination signal sequence FRT, selection markers beta-galactosidase gene, dominant selectable marker India different wall streptomycete phleomycin resistant gene; Wherein lacked-321～-224 98bp enhancer sequence in this SV40 reduction promotor.
10. the carrier of claim 1, it is pTGS-FRT-DHFR.
11. a targeted expression carrier comprises following element or sequence:
A. dominant selectable marker;
The genetic elements of B. in microorganism, breeding;
C. recombination signal sequence;
D. target gene expression cassette;
E. the replication orgin of eukaryotic virus,
Wherein, the dominant selectable marker in this carrier is different from the dominant selectable marker in the integration labeled vector of claim 1, and this dominant selectable marker gene is not activated son and initiator codon; Recombination signal sequence inserts before the dominant selectable marker, and the recombination signal sequence in the integration labeled vector of this recombination signal sequence and claim 1 is identical.
12. the carrier of claim 11, wherein dominant selectable marker is selected from the following proteinic gene of coding: India's different wall streptomycete phleomycin resistance, hygromix phosphotransferase, neomycin resistance, hypoxanthine phosphoribosyltransferase, tetracycline, dihydroorotase glutamine synthetase, Histidine D, carbamyl phosphate synthetase, Tetrahydrofolate dehydrogenase, aspartate transcarbamylase, xanthine-guanine phosphoribosyl transferase and adenosine deaminase.
13. the carrier of claim 11, the genetic elements of wherein breeding in microorganism are replication origin and the antibiotics resistance mark of microorganism.
14. the carrier of claim 11, wherein recombination signal sequence is selected from FRT, Loxp and AttB/AttP.
15. the carrier of claim 11 wherein comprises the SP163 enhanser in the target gene expression cassette, CMV founds the early gene promotor, endoplasmic reticulum signal peptide, target gene sequence and Trobest adenylic acid (AMP) tailing signal.
16. the carrier of claim 15, wherein the target gene coding is selected from following albumen: immunoglobulin (Ig), cell surface receptor, intracellular receptor, hormone, cytokine, somatomedin, transcription factor, enzyme.
17. the carrier of claim 11, wherein the eukaryotic virus replication orgin is SV40 ori or EBV ori.
18. the carrier of claim 11 wherein contains:
A. dominant selectable marker hygromycin phosphotransferase gene;
B. replication orgin ColE1 and express the bla gene of amicillin resistance;
C. recombination signal sequence FRT;
The D.SP163 enhanser, CMV founds the early gene promotor, endoplasmic reticulum signal peptide, antibody chain variable region, constant region of light chain and Trobest adenylic acid (AMP) tailing signal;
E. eukaryotic virus replication orgin SV40 ori.
19. the carrier of claim 11 wherein contains:
A. dominant selectable marker hygromycin phosphotransferase gene;
B. replication orgin ColE1 and express the bla gene of amicillin resistance;
C. recombination signal sequence FRT;
The D.SP163 enhanser, CMV founds the early gene promotor, endoplasmic reticulum signal peptide, antibody heavy chain variable region, CH and Trobest adenylic acid (AMP) tailing signal;
E. eukaryotic virus replication orgin SV40 ori.
20. the carrier of claim 11, it is pTGS-SARS1-site, pTGS-GL-site or pTGS-MH-140-site.
21. contain the construction process of the cell strain of the high-transcriptional activity site that can increase in the genome, may further comprise the steps:
A. with the integration labeled vector of one of claim 1-10 in non-key position linearizing, be as the criterion with the normal function that does not influence all elements, use this linearized vector to transform the mammalian cell strain that sets out then by non-homogeneous recombination mechanism;
B. utilize transformant, obtain to integrate the clone that labeled vector is integrated in the higher transcriptional activity site of genome at the selective pressure screening steps A acquisition of dominant selectable marker;
C. the clone's that obtains of determination step B selection markers gene expression amount obtains the clone of screening-gene high expression level, promptly integrates the clone that labeled vector inserts high-transcriptional activity site place in the genome;
D. utilize clone, obtain the clone that amplification gene is amplified, promptly contain the cell strain of the high-transcriptional activity site that can increase in the genome at the selective pressure screening step C acquisition of amplification gene.
22. the method for claim 21, wherein the selective pressure among step B and the D progressively increases.
23. the method for claim 21, wherein the transform mode in the steps A is electroporation, coprecipitation of calcium phosphate, DEAE-dextran or liposome transfection.
24. the method for one of claim 21-23, wherein integrating labeled vector is the carrier of claim 10, and the mammalian cell strain that sets out is CHO-dhfr
-
25. obtain the method for the Mammals recombinant cell strain of target gene high expression, may further comprise the steps:
A. utilize the method for one of claim 21-24 to obtain to contain in the genome cell strain of the high-transcriptional activity site that can increase;
B. use the targeted expression carrier of one of claim 11-20 and the cell strain that the recombinase expression vector obtains with superhelix form cotransfection steps A, described recombinase expression vector transient expression site-specific recombinase, the locus specificity reorganization takes place in this recombinase catalysis targeted expression carrier and the integration labeled vector that is incorporated on the genome at the recombination signal sequence place;
C. utilize transfection clone, obtain the clone that target gene inserts the amplification gene flank, i.e. the Mammals recombinant cell strain of target gene high expression at the selective pressure screening step B acquisition of selective marker in the targeted expression carrier.
26. the method for claim 25, further comprising the steps of D: utilize clone, obtain the Mammals recombinant cell strain of target gene high expression at the selective pressure screening step C acquisition of amplification gene.
27. the method for claim 26, further comprising the steps of E: the Mammals recombinant cell strain of the target gene high expression that step C or step D are obtained goes down to posterity.
28. the method for claim 27, wherein step e is carried out under selective pressure.
29. the method for claim 28, wherein selective pressure is the selective pressure at amplification gene.
30. the method for claim 25, wherein the mode of cotransfection is electroporation, coprecipitation of calcium phosphate, DEAE-dextran or liposome transfection among the step B.
31. the method for one of claim 25-30, wherein selective pressure progressively increases.
32. the method for claim 31, wherein integrating labeled vector is the carrier of claim 10, and the targeted expression carrier is the carrier of claim 20, and recombinase carrier is pOG44, and the mammalian cell strain that sets out is CHO-dhfr
-
33. a system that makes up high secretion recombinant protein cell strain fast, this system comprises:
A. the integration labeled vector of one of claim 1-10;
B. the targeted expression carrier of one of claim 11-20;
C. recombinase expression vector, but the locus specificity reorganization takes place in integration labeled vector and targeted expression carrier that this recombinase expression vector transient expression site-specific recombinase, described recombinase can catalysis be incorporated on the mammalian cell strain genome that sets out at the recombination signal sequence place;
D. mammalian cell strain sets out.
34. the system of claim 33, the mammalian cell strain that wherein sets out is selected from CHO-dhfr
-, COS, Hela, NIH 3T3 and NS0 myeloma cell.
35. the system of claim 33, wherein said integration labeled vector are the carriers of claim 10, the target labeled vector is the carrier of claim 20, and the recombinase expression vector is pOG44, and the mammalian cell strain that sets out is CHO-dhfr
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| CN107384932B (en) * | 2016-08-31 | 2020-10-20 | 北京天广实生物技术股份有限公司 | Anti-human CD20 humanized monoclonal antibody MIL62, preparation method and application thereof |
| US11970541B2 (en) | 2016-08-31 | 2024-04-30 | Beijing Mabworks Biotech Co. Ltd. | Anti-CD20 antibody |
| CN107058346A (en) * | 2017-05-03 | 2017-08-18 | 李军涛 | A kind of hepatitis B virus surface antigen gene and expression vector and cell line |
| CN110982790A (en) * | 2019-12-26 | 2020-04-10 | 海珂分子（北京）科技有限责任公司 | Cell strain for protein display and expression and preparation method and application thereof |
| CN110982790B (en) * | 2019-12-26 | 2022-07-19 | 海珂分子（北京）科技有限责任公司 | Cell strain for protein display and expression and preparation method and application thereof |
| CN112175993A (en) * | 2020-09-02 | 2021-01-05 | 海珂分子（北京）科技有限责任公司 | Construction method and application of single-copy cell strain for stably and highly expressed protein |
| CN112175993B (en) * | 2020-09-02 | 2023-02-28 | 海珂分子（北京）科技有限责任公司 | Construction method and application of single-copy cell strain for stably and highly expressed protein |
| CN112538499A (en) * | 2020-12-22 | 2021-03-23 | 北京鼎成肽源生物技术有限公司 | Recombinant plasmid vector, antibody display cell line and application thereof |
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