CN1717486A - Expression vector, methods for the production of heterologous gene products and for the selection of recombinant cells producing high levels of such products - Google Patents

Abstract

Disclosed is an expression vector for eukaryotic cells comprising a gene that codes for a protein of interest in a functional bond with a hamster ubiquitin/S27a promoter, and a gene which codes for a fluorescent protein. Preferably, said expression vector also comprises an amplifiable selection marker gene. Also disclosed are host cells, preferably mammal cells, which are transfected with the expression vector, methods for producing heterologous gene products, and a method for selecting high-producing cells.

Description

The system of selection of the preparation method of expression vector, heterologous gene products and high yield reconstitution cell

Invention field

The present invention relates to the system of selection of high yield reconstitution cell, prepare the method for heterologous gene products and expression vector, and with host cell its transfection, that can in this method, use.

The background technology of invention

Mammalian cell is to be used for producing the complicated proteinic preferred host cell of biopharmacy, because its modification of carrying out after translation, not only also can be compatible with the mankind on pharmacokinetics on the function.At commercial relevant cell type is hybridoma, myelomatosis, CHO (Chinese hamster ovary) cell and BHK (young hamster kidney) cell.Under serum-free and nonprotein working condition, carry out the cultivation of host cell more and more.Its reason is that relevant therewith cost reduces, the interference on purification of recombinant proteins matter reduces, and reduces the possibility that imports cause of disease (for example PrPC, virus).Use Chinese hamster ovary celI and become more extensive as host cell, be because this cell adapted in not containing serum and proteinic substratum suspension growth, and also be considered as and accept as safe production cell for administrative authority.

In order to produce the stable mammal cell line of expressing the purpose heterologous gene, usually by transfection, but with heterologous gene together with selectable marker gene, for example neomycin phosphotransferase is introduced in the required clone together.Heterologous gene and selectable marker gene can meanwhile or by a single carrier be expressed together, or by the vector expression that separates separately of cotransfection.After transfection 2 to 3 days, cell moved to contain selective reagents, for example when using neomycin phosphotransferase gene in the substratum as G418, and under this selection condition, cultivate several weeks.The expression of the gene product that the cell of the tool resistance of separable appearance, and institute then needs.Because at random and nondirectional integration, obtain cell colony in the host cell gene group, described cell colony presents the complete different expression rates of heterologous gene.These also can be the cell of non-expression, though described cell is expressed selective marker, do not express goal gene.In order to identify the cell clone that presents the high expression of allos goal gene, therefore must check and test a large amount of clones, this causes high time, labour and cost cost.

Gene amplification is phenomenon very universal in animal cell culture, is used for producing the biopharmacy protein of reorganization.Gene amplification has significantly improved the originally low relatively productive rate of many mammalian cell strains.A kind of widely used amplification technique is to be the based gene amplification system with Tetrahydrofolate dehydrogenase (DHFR), described system usually uses in the Chinese hamster ovary of DHFR-defective (CHO) cell, utilize the appropriate carriers system at this, described carrier system encoding D HFR and target protein matter, the Chinese hamster ovary celI of transfection DHFR-defective, for example CHO-DUKX (ATCC CRL-9096) or CHO-DG44 (Urlaubet al 1983).In the substratum of no glycine, xanthoglobulin and thymidine, select transfectant then.Add methotrexate (MTX), a kind of inhibitor of Tetrahydrofolate dehydrogenase (Kaufmanet al 1982 through increasing ground gradually; US 4,656, and 134), the foundation of reaching the high yield clone that increases and therefore reach.Randomly assigne is also obeyed in the follow-up screening of the high yield cell that is obtained and based on chance, therefore this screening step is highly labour-intensive, and expends time in.

Various methods in order better and more promptly to monitor gene transformation and expression, to have developed.This at first comprises the operation report molecule, as paraxin-Transacetylase, Luci, beta-galactosidase enzymes, or uses the fused protein of the coding region of containing beta-galactosidase enzymes or Luci.The shortcoming of these corresponding reporter-gene assays is that cell must be fixed or cracking, and must hatch with material and cofactor that external source is added.Therefore, may further not cultivate the cell of having analyzed.A kind of novel method is the coexpression based on the intestinal bacteria enzyme beta-galactosidase, though allow that by the FACS device viable cell is chosen people such as (, 1988) Nolan needs hypotonic anticipating, and is loaded in the cell so that will produce fluorescent substance.This activity also was suppressed before the letter sorting based on FACS-certainly.

Through the introducing of the green fluorescent protein (GFP) that derives from Victoria jellyfish (Aequorea victoria), and develop the GFP mutant that as reporter molecules, make the evaluation of the cell of expression of heterologous genes become easier from it.The coexpression of GFP is allowed instant analysis in vivo, and based on its fluorescence, the selection transfectant does not need extra material or auxilliary-factor.In various publications, describe and use GFP to monitor transgenosis as reporter molecules.At United States Patent (USP) 5,491, in 084 and 6,146,826, people such as Chalfie have described the method for the cell of selecting to express target protein matter.This method comprises the dna molecular with the encoding sequence that contains target protein matter, and second dna molecular of coding GFP-gene, common transfectional cell.Select to express the cell of GFP then.Gubin etal (1997) has studied and has lacked under the selective growth condition, and GFP is expressed in the stability in the Chinese hamster ovary celI.Both to contain the plasmid transfection cell that GFP also contains neomycin phosphotransferase.Mosser etal (1997) uses the plasmid contain the bicistronic mRNA expression cassette, its coding GFP and target gene (also being called goal gene), but identify and select to express the cell of induced product.This target gene is under the control of adjustable promotor.Use viral IRES (internal ribosome entry site) element, reach the coupling that GFP and target gene are expressed, the encode bicistronic mRNA of GFP and target protein matter of result is expressed.But itself do not contain any selectable marker gene at this employed plasmid.Therefore, through second plasmid in cotransfection or follow-up transfection with its importing.Opposite, people such as Levenson (1998) use the retroviral vector that has the bicistronic mRNA expression cassette, wherein goal gene can be cloned into before the IRES sequence.The optional marker gene of selecting of sequence phase Gray code after the IRES sequence, this or invest to G418, tetracycline, hygromycin B, histidinol D or the drug-fast mark of phleomycin or GFP.

Described the carrier that contains the IRES element that derives from Picornaviridae already, but this IRES element is placed in (people such as Pelletier, 1988 between product gene and the selectable marker gene; People such as Jang, 1989; People such as Davies, 1992).

Also successfully GFP and resistant maker gene are merged.For example, people (1998) such as Bennett describes GFP/ Leo mycin (zeomycin) fused protein.Successfully use the selective marker of this dual-use function, identify and select the process mammalian cells transfected.People (1998) such as opposite Primig use the fused protein of GFP and neomycin phosphotransferase, carry out the research of its enhanser.

In people's such as Meng (2000) open and International Patent Application WO 01/04306, use a kind of expression system, goal gene is expressed, to select and to identify the cell of highly expressing recombinant protein with increased selectable marker gene DHFR that derives from single carrier and GFP gene.These three genes are blended in the transcription unit, or separately in two units.All three genes these spaces in a single expression vector and the contact of transcribing make the probability of its coamplification under selective pressure improve, and therefore identify and select the high yield clone.Best clone expresses target protein with the order of magnitude in maximum 3-4.5pg/ cells sky, and the DHFR selective marker of described best clone by using available reorganization selective reagents amplification chosen with FACS based on GFP and separated.Utilize attached cell and containing in the substratum of serum at this to experimentize, promptly utilize well-known stronger in fact and it is characterized in that cell and condition than the higher baseline productive rate.

Summary of the invention

Therefore, task of the present invention is the reconstitution cell development selective system for the productive rate with raising, and it satisfies following demand:

(1) shortens development high yield cell with the time that preparation biopharmacy protein is spent, reduce development cost simultaneously;

(2) select the high yield cell with less capacity cost with highoutput;

(3) use " it is strong to ferment " high yield cell, it for example demonstrates when increasing methotrexate concentration has lower infringement to growth;

(4) transfection, selection and cultivation adapt to the cell of suspension, preferably in the substratum that does not contain serum;

(5) reduce required gene amplification step.

The present invention further task provides expression vector and with the host cell of its transfection, it can be used in this clonal selection system, and uses this host cell to prepare the method for heterologous gene products.

According to an aspect of the present invention, expression vector by the gene (also being called " goal gene " below) of coding target protein matter, solved this task, described gene is connected with the proteinic gene function of coding fluorescence with hamster ubiquitin/S27a promotor.

Expression vector preferably also contains the selectable marker gene that can increase, for example Tetrahydrofolate dehydrogenase (DHFR) gene.Preferred expression vector also contains other regulatory element, for example enhanser that is connected with promotor on function.In addition, expression vector preferably also contains internal ribosome entry site (IRES), and its bicistronic mRNA of allowing proteinic gene of coding fluorescence and goal gene is expressed.

The present invention is also about carrier is carrier, and it has multiple clone site to replace goal gene, to mix one of these genes, that is to say a sequence area with a plurality of restriction enzyme cleavage sites.

Another aspect of the present invention is about utilizing a kind of host cell of mentioned expression vector transfection.These are eukaryotic host cell, the preferred mammal cell, rodent cells wherein, as hamster cell and especially Chinese hamster ovary celI or bhk cell for preferred especially.

Another aspect of the present invention is the method for preparing heterologous gene products, is wherein allowing under the condition of this gene product expression, cultivates the host cell with expression vector transfection according to the present invention, and separates this gene product from culture or substratum.

In particular specific embodiment of the present invention, with expression vector according to the present invention, and extraly with one or more carriers that have the gene of one or more other target protein matter of encoding, transfection, preferably cotransfection host cell.

In this relation, the invention provides and prepare the method for heterodimer protein to be used, wherein under the condition of allowing this heterodimer protein expression, cultivate the host cell of this type, described host cell has utilized the expression vector cotransfection of these proteinic different subunits of heterodimer of coding, and separates this heterodimer protein from culture or substratum.A special cases of these class methods is to produce antibody and subunit thereof.

Another aspect of the present invention is the method about the host cell of selecting expression target protein matter, wherein allowing under the condition that this target protein matter and fluorescence protein are expressed, cultivate host cell population, and identify and select to demonstrate cell or a plurality of cell of the high expression level rate of fluorescence protein with expression vector transfection according to the present invention.Preferably use fluorescence-activated cell sorting device (FACS) to select.

Surprising is, have been found that the system prepared in accordance with the present invention that uses, might be in the shortest time isolated cell aggregate, it expresses the average specificity productive rate that surpasses 15 pico-grams (single chain protein matter) or 10 pico-grams (humanized antibody) recombinant protein/cell sky, and does not need the step of gene amplification.Through single be the based gene amplification step with DHFR-, can make this specificity gain in yield to each more than cell pico-gram every days 30.The productivity that so reaches in cell aggregate is than disclosed optimum cell clone's taller 8 to 10 the factor of maximum yield so far.

Surprising is between the expression and fluorescence protein expression of target protein matter, splendid connection also to be arranged.This in addition be suitable for cotransfection, if as in the example of expressing antibodies two immunoglobulin chains its own vector expression of mat respectively, and in FACS chooses, depend on the coupling that itself and fluorescence protein are being transcribed, can only select the expression of a chain.However, the high expression level rate of fluorescence protein, for the growth of cell and vigor without any negative influence.In addition, compare with traditional gene amplification strategy that progressively carries out, also can reduce select the high yield cell development time at least half, the result has reduced development usefulness and cost significantly.

Description of drawings

Fig. 1 shows the comparison with the expression level that recombinant cell clone was obtained, wherein under the control of CMV promotor, or under the control of hamster ubiquitin/S27a promotor, and the expression of heterologous genes product.Two promotors connect cmv enhancer on function, pause sequence, and BGH poly A is identical in each situation.At CMV ¹Situation in, expression vector based on pcDNA3-(Invitrogen, Kalsruhe, DE), at CMV ²In, its for based on the expression vector of pBluescript-(Stratagene, La Jolla, CA, US), and in the situation of CHO, it is expression vector people such as (, 1998) Werner based on pAD-CMV-.By the expression of lysosomal enzyme, all expression vectors all contain the selective marker Tetrahydrofolate dehydrogenase (DHFR) that can increase, and through follow-up amplification step, utilize aminopterin (MTX) to increase expression of heterologous genes.For two chains of expressing antibodies (Ab), utilize second carrier that contains as the Xin Meisu-resistant gene of selective marker to carry out cotransfection.So that tiring of gained or specificity productive rate to be provided with respect to the expression based on the CMV promotor, being set is 1 (CMV ¹Be enzyme, CMV ²Be antibody).

Fig. 2 shows that be used for the schematic preparation " P/E " of in the CHO-DG44 cell carrier is carrier of express recombinant protein matter is the combination of cmv enhancer and hamster-ubiquitin/S27a promotor, only there be " P " to represent promoter element, and " T " abort signal for transcribing, its polyadenylation by the mRNA that process is transcribed is essential.Point out position and the direction that each transcription unit's internal transcription is initial with arrow.In order to clone heterologous gene, after promoter element, insert the sequence area (multiple clone site-mcs) of cleavage site with a plurality of retriction endonucleases.The selective marker Tetrahydrofolate dehydrogenase that can increase is abbreviated as " dhfr ", and the selective marker neomycin phosphotransferase is abbreviated as " neo ".The IRES element in encephalomyocarditis virus source is served as internal ribosome entry site in bicistronic mRNA transcription unit inside, and is allowed the translation of the green fluorescent protein matter " GFP " of back.

Fig. 3 shows the schematic preparation of eukaryote expression vector, and it is encoding human pharmacy protein respectively, and be used to transfection CHO-DG44 cell." P/E " is the combination of cmv enhancer and hamster-ubiquitin/S27a promotor, only has " P " to represent promoter element, and " T " abort signal for transcribing, its polyadenylation by the mRNA that process is transcribed is essential.Point out position and the direction that each transcription unit's internal transcription is initial with arrow.The selective marker Tetrahydrofolate dehydrogenase that can increase is abbreviated as " dhfr ", and the selective marker neomycin phosphotransferase is abbreviated as " neo ".The IRES element in encephalomyocarditis virus source is served as internal ribosome entry site in bicistronic mRNA transcription unit inside, and is allowed the translation of the green fluorescent protein matter " GFP " of back.Adhesion molecule (US5,412,216) in " sICAM " coding soluble cell, and " F19HC " and " F19LC " encode respectively weight or light chain (EP953 639) of humanized antibody F19.

Fig. 4 is presented at the connection between sICAM productive rate and the GFP fluorescence, adopts cell aggregate ZB1 as an example.This cell aggregation is available from the transfection that utilizes carrier pBIDG-sICAM, wherein by bicistronic mRNA transcription unit express therapeutic protein sICAM and GFP together.This aggregate is carried out the FACS letter sorting of successive based on GFP.In each separation step (selecting) afterwards, judge sICAMs concentration in the cell culture supernatant of aggregate, and calculate each cell specificity productive rate (pico-gram/cell * days) of every day through ELISA.Each material point is represented the mean value that at least three cultivations are gone down to posterity.Carry out altogether selecting for six times.

Fig. 5 shows through the FACS letter sorting based on GFP, separates the sICAM cell of high expression level, adopts cell aggregate ZB1 as an example.This cell aggregate is available from the transfection that utilizes carrier pBIDG-sICAM, wherein by bicistronic mRNA transcription unit express therapeutic protein sICAM and GFP together.The pair set body carries out the FACS letter sorting of successive based on GFP.After selecting at every turn, judge sICAMs concentration in the cell culture supernatant of aggregate through ELISA, and calculate each cell specificity productive rate (pico-gram/cell * days) of every day.At least three mean values that cultivation is gone down to posterity of each material point representative.Carry out altogether selecting for six times.

Fig. 6 shows through will be linked together based on selection and the MTX amplification step of GFP, the increase on the sICAM productive rate of being reached, and employing cell aggregate ZB1 is as an example.This cell aggregate available from the transfection that utilizes carrier pBIDG-sICAM, and carries out the FACS letter sorting of successive based on GFP.After the 4th time is selected or select for the 6th time,, carry out the gene amplification of DHFR mediation through in substratum, adding methotrexate (MTX) (5nM, 50nM, 500nM or 2 μ M MTX).Judge sICAMs concentration in the cell culture supernatant of aggregate through ELISA, and calculate each cell specificity productive rate (pico-gram/cell * days) of every day.At least three mean values that cultivation is gone down to posterity of each material point representative.

Fig. 7 is presented at after the methotrexate that adds different high dosages in the substratum, the survivability mode of cell aggregate.The cell aggregate ZB1 that obtains through the transfection that utilizes carrier pBIDG-sICAM (Fig. 3) is carried out the FACS letter sorting of successive based on GFP.After the 4th time is selected or select for the 6th time,, carry out the gene amplification of DHFR mediation through in substratum, adding methotrexate (MTX).During the selection phase,, judge cell number and survival and in many days, monitor and cultivate (dic) through with trypan blue staining.

Fig. 8 is presented at the connection between antibody production rate (monoclonal antibody F19) and the GFP fluorescence, adopts cell aggregate ZB1 as an example.This cell aggregate is available from the transfection that utilizes carrier combinations pBIDG-F19HC and pBIN-F19LC (Fig. 3).This aggregate is carried out the FACS letter sorting of successive based on GFP.After selecting at every turn, judge the concentration of antibody F19 in the cell culture supernatant of aggregate through ELISA, and calculate each cell specificity productive rate (pico-gram/cell * days) of every day.At least three mean values that cultivation is gone down to posterity of each material point representative.Carry out altogether selecting for six times.

Fig. 9 shows through the selection based on GFP, use FACS, and the monoclonal antibody F19 cell aggregation of separation high expression level adopts cell aggregate ZB1 as an example.To carry out the FACS letter sorting of successive through this cell aggregate that obtains with carrier pBIDG-F19HC and pBIN-F19LC (Fig. 3) transfection based on GFP.After selecting at every turn, judge the concentration of antibody F19 in the cell culture supernatant of aggregate through ELISA, and calculate each cell specificity productive rate (pico-gram/cell * days) of every day.At least three mean values that cultivation is gone down to posterity of each material point representative.

The specific descriptions of invention and preferred implementation

Expression vector according to the present invention contains the gene (" goal gene ") of coding target protein matter, and described gene is connected with hamster ubiquitin/S27a promotor and the proteinic gene of coding fluorescence on function.This expression vector preferably also contains the selectable marker gene that can increase.

Hamster-ubiquitin/S27a promotor

The ubiquitin of hamster/S27a promotor is strong homologous promoter, describes in WO 97/15664.This class promotor has one of preferred following at least feature: the sequence area, Sp1 binding site, poly-pyrimidine element, the shortage TATA box that are rich in GC.Particularly preferably be the promotor that has the Sp1 binding site but lack the TATA box.The promotor of preferred in addition constitutive activation, and particularly similarly have active promotor under the serum culture condition containing serum, low serum and do not contain.In another specific embodiment, it is an inducible promoter, particularly the activated promotor by removing serum.

Particularly advantageous embodiment is the promotor with the nucleotide sequence that is contained in Fig. 5 of WO 97/15664.Particularly preferred is the promotor that contains the position-161 that the derives from Fig. 5 sequence to-45 at this.

The promotor of using in the example of specification sheets of the present invention contains the dna molecular that has from the sequence of the position 1923 to 2406 of the SEQ ID NO:1 of the sequence table of appendix respectively.This sequence is equivalent to derive from the fragment-372 of Fig. 5 of WO 97/15664 to+111, and represents preferred promotor, and promptly preferred promoter should comprise this sequence area.Another suitable promoter fragment contains the sequence (being equivalent to-161 to+111 among Fig. 5 of WO 97/15664) of position 2134 to 2406.The promotor that only contains the sequence of position 2251 to 2406 no longer has function (be equivalent among Fig. 5 of WO 97/15664 position-45 to+111).Might 2143 begin to prolong promoter sequence from the position in 5 ' direction.

Also may use the function subfragment of complete hamster ubiquitin/S27a promoter sequence, and the functional mutants variant of complete sequence or its subfragment, it is modified through for example replacing, insert or deleting.Also corresponding subfragment, mutant or variant are called " through the promotor of modifying " below.

Through the promotor of modifying, described promotor preferably makes up with other regulatory elements, preferably have transcriptional activity, it is corresponding to-372 to+111 of Fig. 5 of the promoter fragment WO 97/15664 of the nucleotides sequence column position 1923 to 2406 that provides in SEQ ID NO:1).It is useful being found on the meaning of the present invention through the promotor of revising, if it has transcriptional activity in comparative reporter-gene assays, this activity is at least 50% of 1923 to 2406 fragments (372 to+111 fragment), preferably at least 80%, more preferably at least 90%, and more preferably at least 100%.Particularly preferably be promotor through modifying, its wild-type sequence SEQ ID NO:1 to hamster ubiquitin/S27a promotor has at least 80%, preferably at least 85%, preferably at least 90%, again preferably at least 95%, and particularly preferably be at least 97% lowest series homology, and in comparative reporter-gene assays, has corresponding promoter activity.

In corresponding comparative reporter-gene assays, the promoter fragment of desire test comprises is cloned into the reference sequences that is positioned at separately before the promoterless reporter gene, and this report genes encoding is Luci, secretion property alkaline phosphatase or egfp (GFP) for example.Subsequently through transfection, this construct (promoter sequence+reporter gene) is imported subject cell, for example in the CHO-DG44, and through the protein intensive amount of measurement report gene, judge the promoter fragment inductive reporter gene expression by separately.People such as for example Ausubel, Current Protocols in Molecular Biology, 1994, exemplary in the upgraded edition also have a corresponding test.

The promoter sequence of hamster ubiquitin/S27a promotor and promotor through modifying, it also can comprise for example 5 ' non-translational region or its fragment of therefrom selecting, and ubiquitin/S27a gene or its segmental coding region of selecting, can from understanding, use 1989 by those skilled in the art for example people such as Sambrook to the sequence of description the WO 97/15664; People such as Ausubel, the various standard methods of describing in 1994 obtain.For example, can begin, select suitable fragment from the sequence of among WO 97/15664, describing, and with the synthetic oligonucleotide probe that contains this fragments sequence of chemical mode.For example, this class probe be can use, for example ubiquitin/S27a gene or its 5 ' non-translational region or other fragments from the hamster genomic library, cloned through hybridization.Use above-mentioned reporter-gene assays, those skilled in the art do not cost very great strength just can identify the fragment with promoter activity, and is used for purpose of the present invention.The corresponding primer of genomic dna or genomic library be can derive from through the pcr amplification utilization, 5 ' non-translational region or its specific fragment obtained easily.Also can from bigger dna fragmentation, obtain the fragment of 5 ' non-translational region through restriction nuclease excision enzyme III digestion.Can also chemical mode synthetic this class dna molecular, or through connecting to produce the chemical mode synthetic fragment.

Can produce disappearance, insert and replace mutant through " site-specific sudden change nucleus formation " and/or " based on the sudden change generation technique of PCR ".In for example Lottspeich and Zorbas 1998 the 36.1st chapter and further, mention suitable method in the reference paper.

Derive from 5 ' non-translational region of hamster ubiquitin/S27a gene or derive from the intersection-hybridization of probe of the S27a part of hamster ubiquitin/S27a gene through utilization, also may be from other, in the preferably mammiferous corresponding homologous gene, identify and separate suitable promoter sequence.In for example Lottspeich and Zorbas 1998 the 23rd chapter, corresponding techniques has been described.For the purposes of the present invention, " homology " is such gene, if the nucleotide sequence of gene pair manifests at least 70% with the nucleotide sequence of this gene, preferably at least 80%, preferably at least 90%, again preferably at least 95%, and particularly preferably be at least 97% consistence, then this gene is " homologous ".

Goal gene

The goal gene that is contained in expression vector according to the present invention comprises the nucleotide sequence of any length of coding purpose product.Gene product or " purpose product " be protein, polypeptide, peptide or its fragment or derivative normally.Yet it can also be RNA or sense-rna.Goal gene can its total length, the form of shortening, exists as fusion gene or the gene that is labeled.It can be a genomic dna, or preferably cDNA or corresponding fragment or fusion.Goal gene can be natural gene order, maybe can be sudden change or otherwise modification.This class is modified codon optimization and the humanization effect that adapts to the host cell of determining that comprise.Goal gene, codified secretion property, cytoplasmic, the position combines in nuclear, with film or with cell surface bonded polypeptide.

Oligonucleotide, Nucleotide, polynucleotide and fragment thereof represented in " nucleotide sequence " or " nucleotide sequence " speech, and the DNA or the RNA in genome or synthetic source, and it exists with list or two strands, and can represent the coding or the noncoding strand of gene.Can use standard technique, generate or the sudden change nucleus formation of PCR mediation (, describing in 1989 or people such as Ausubel, 1994), come the modification of nucleic acids sequence for example people such as Sambrook as site-specific sudden change.

" coding " means the characteristic or the performance of the Nucleotide of particular sequence in nucleic acid, the gene in karyomit(e) or mPNA for example, in bioprocess,, be used for synthesizing other polymkeric substance and macromole, for example rRNA, tRNA, mRNA, other RNA molecule, cDNA or polypeptide as matrix.Therefore, if through the transcribing and follow-up translation of mRNA, in cell or other biological system, produce required protein, then dna encoding the protein.Can its nucleotide sequence is identical with the mRNA sequence, and normally also at sequence library, the coding strand that provides among EMBL or the GenBank for example, also have as the gene noncoding strand of transcribing matrix or cDNA both, be called and be used for coded product or protein.The nucleic acid of coded protein also comprises based on the degeneracy genetic code, have different IPs nucleotide sequence order, but the result produces the nucleic acid of proteinic same acid sequence.The nucleotide sequence of coded protein also can contain intron.

Thymus nucleic acid represented in " cDNA " speech, through reverse transcription, and prepared by mRNA or synthetic the 2nd DNA chain of other RNA that gene produces.If cDNA occurs with the form of double chain DNA molecule, it contains coding and noncoding strand.

The non-coding nucleotide sequence of any length represented in " intron " speech.Their natural appearing in many eukaryotic genes, and through the process that is called splicing, from the mRNA precursor of before having transcribed, remove.These need be 5 ' and 3 ' end accurately cut away intron, and correctly connect the mRNA end of gained, and produced the mRNA of ripe processing, having can be for the correct reading frame of synthetic protein successfully.Many splicing donor and acceptor splicing site sites that relate to this splicing, promptly directly the position is characterized in the sequence of exon-intron or intron-exon adjoiner.About introduction, referring to people such as Ohshima, 1987.

Target protein matter

Protein/polypeptide with biopharmacy importance comprises for example antibody, enzyme, cytokine, lymphokine, adhesion molecule, acceptor and derivative thereof or fragment, but is not limited thereto.Usually, all are as agonist or antagonist, and/or have treatment or a diagnostic uses polypeptide all be valuable.

" polypeptide " speech is used for aminoacid sequence or protein, and means the polymer of amino acid of any length.After this speech also is included in translation, through the protein of modifying as the reaction of glycosylation, phosphorylation, acetylizing or protein processing.Can be through for example replacing, lack or insert amino acid, and merge with other protein, still keep its biological activity simultaneously, come the structure of modified polypeptide.

The example of therapeutic protein is a Regular Insulin, rhIGF-1, human growth hormone (hGH) and other somatomedins, tissue plasminogen activator (tPA), erythropoietin (EPO), cytokine, white plain (IL) for example is situated between, as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, Interferon, rabbit (IFN)-α,-β,-γ,-ω or-τ, tumour necrosis factor (TNF) is as TNF-α, β or-γ, TRAIL, G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.Other example is mono-clonal, polyclone, polyspecific and single-chain antibody, and fragment, similarly is for example Fab, Fab ', F (ab ') ₂, Fc and Fc ' fragment, light (L) and weight (H) immunoglobulin chain, and constant, variable or hypervariable region, and Fv and Fd fragment people such as (, 1999) Chamov.This antibody can be the mankind or non-human source.Humanization and mosaic type antibody also are possible.

(Fab=Fab) be made up of the variable region of two chains, it maintains together through adjacent constant region the Fab fragment.They for example can as papoid, produce from traditional antibody or through dna clone through with protease treatment.Other antibody fragments are F (ab ') ₂Fragment, it can be through utilizing pepsic proteolytic digestion to produce.

Also may be through gene clone, the antibody fragment of preparation brachymemma, it only is made up of the variable region of heavy (VH) and light chain (VL).It is called as Fv fragment (fragment of variable fragment=variable part).Because in these Fv fragments, can not be via the halfcystine group covalent attachment of constant chain, make the Fv fragment stable through some other method usually.For this purpose, usually through about 10 to 30 amino acid, 15 amino acid whose small peptide fragments especially preferably link together the variable region of heavy and light chain.This produces the single polypeptide chain that wherein VH and VL link together through peptide linker.Also this antibody-like fragment is called strand Fv fragment (scFv).The example of scFv antibody is known and has been described, reference, people such as Huston for example, 1988.

In recent years, developed the strategy that various production polymer scFv derivatives.Be intended that and produce pharmacokinetic properties, open the recombinant antibodies that increases the bonded avidity with improvement.In order to reach the segmental multimerization effect of scFv (Multimerisierung), the form that can have the fused protein of multimerization structural domain is produced them.Poly effect structural domain can be CH3 district or the spirane structure (" coiled coil structure ") of for example IgG, as leucine-zipper territory.In another strategy, use the interaction between segmental VH of scFv and VL district to carry out multimerization effect (for example miniature bifunctional antibody (diabodies), miniature three function antibodies (tribodies) and miniature five-function antibody (pentabodies)).

Those skilled in the art use " miniature bifunctional antibody " speech, represent the homodimer scFv derivative of divalence.In the scFv molecule peptide linker is foreshortened to 5-10 amino acid, the result forms homodimer through making the VH/VL chain overlapping.Also can make miniature bifunctional antibody more stable through inserting disulphide bridges.Can find the example of miniature bifunctional antibody in 1994 the document people such as for example Perisic.

Those skilled in the art use " miniantibody " (minibody), represent the homodimer scFv derivative of divalence.It is made up of fused protein, and it contains the immunoglobulin (Ig) as the dimerization zone of action, preferably IgG, especially preferably the CH3 district of IgG1.This is connected with the scFv fragment with connector area through the hinge area that also belongs to IgG.By people such as Hu, 1996 have described the example of this class miniantibody.

Those skilled in the art use " miniature three function antibodies ", represent tervalent homotrimer scFv derivative (people such as Kortt, 1997).The direct fusion of VH-VL need not used joint sequence, causes trimerical formation.

Those skilled in the art are called the fragment of little antibody (mini antibody), it has two-, three-or quaternary structure, also be the segmental derivative of scFv.Through two-, three-or tetrameric " coiled coil " structure, reach multimerization effect (people such as Pack, 1993 and 1995; People such as Lovejoy, 1993).

The proteinic gene of coding fluorescence

Expression vector according to the present invention contains the proteinic gene of coding fluorescence, on function, is connected with goal gene, and under the control of hamster-ubiquitin/S27a promotor, the hamster-ubiquitin/S27a promotor of passing through modification or its homologue.

Fluorescence protein can be the fluorescence protein of for example green, blue-greenish colour, blueness, yellow or other colors.Specific example is the green fluorescent protein (GFP) available from Victoria jellyfish or kidney shape jellyfish (Renilla reniformis), and from wherein developing the mutant that, referring to people such as for example Bennet, 1998; People such as Chalfie, 1994; WO 01/04306 and the document of wherein mentioning.

In WO 00/34318, WO 00/34326, WO 00/34526 and WO 01/27150, described other fluorescence protein and their gene of encoding, be incorporated herein by reference.These fluorescence proteins are species Anthozoa (Anthozoa), for example the fluorophore of the abiotic luminous organism of Ma Hanuo coral (Anemonia majano), plumage coral (Clavularia sp.), button coral (Zoanthus sp.) I, button coral II, sea anemone (Discosomastriata), mushroom coral (Discosoma sp.) " redness ", mushroom coral " green ", mushroom coral " red-purple ", wrinkle Zhe sea anemone (Anemonia sulcata).

Except open-air type protein, fluorescence protein used according to the invention also comprise natural or with the mutant of genetic technique preparation and variant, its fragment, derivative or for example with other protein or the variant that merges of peptide.Employed sudden change for example can change this proteinic exciting or emmission spectrum, chromophoric formation, optical extinction coefficient or stability.In addition, can improve the expression in Mammals or other species through the effect of codon optimization.According to the present invention, but also can use the fluorescence protein that merges with selective marker, the selective marker that can increase preferably is as Tetrahydrofolate dehydrogenase (DHFR).

The fluorescence that fluorescence protein is emitted makes to detect this protein, for example through the flow cytometry with fluorescence activated cell sorter (FACS) or through fluorescent microscope.

Other regulatory elements

Can on function, hamster-ubiquitin/S27a promotor and other be regulated sequence association, so that increase/be adjusted in the transcriptional activity in the expression cassette.

For example, can on function, promotor be connected with enhancer sequence, so that increase transcriptional activity.For this, can use several copies of one or more enhanser and/or enhancer sequence, for example CMV or SV40 enhanser.

Therefore polynucleotide sequence represented in " enhanser " speech, and it acts on the activity of promotor with cis position, and stimulate on function and this promotor banded gene transcription.Unlike promotor, the influence of enhanser and position and orientation-independent, and therefore they can be placed on before or after the transcription unit, in intron, or or even in the coding region.Enhanser can be close to transcription unit, and from one section distance quite far away of promotor.Also may overlapping on physics and the function be arranged with promotor.Those skilled in the art know the many enhansers that derive from various sources and (and are deposited in the database, as GenBank, for example SV40 enhanser, cmv enhancer, polyomavirus enhanser, adenovirus enhanser), the form of can independent component or being cloned into the element in the polynucleotide sequence obtains (for example be deposited in ATCC, obtain from the commercial and industrial source).Many promoter sequences also contain enhancer sequence, as the CMV promotor of frequent use.People's cmv enhancer is one of the strongest enhanser of identifying so far.The example of a derivable enhanser is the metallothionein(MT) enhanser, can stimulate it through glucocorticosteroid or heavy metal.

Other possible modifications are the importing of for example a plurality of Sp1 binding sites.Promoter sequence also can with regulate sequence association, described adjusting sequence is allowed the control/adjusting of transcriptional activity.Therefore, promotor can be made can prevent/derivable.For example, can through with its with just-or the sequence of the binding site of negative transcription factor of regulating be connected, carry out.Transcription factor Spl mentioned above for example has positive influence to transcriptional activity.Another example is the binding site of activator AP-1, and it can act on positive and negatively and transcribe.Can control the activity of AP-1 through the factor of numerous species, for example somatomedin, cytokine and serum (people such as Faisst, 1992 and reference wherein).Also can change promoter sequence, increase and transcribe efficient, judge in reporter-gene assays then whether this has increased promoter activity through via one, two, three or the sudden change of more a plurality of bases (replace, insert or disappearance).

Basically, extra regulatory element comprises promotor, enhanser, termination and the polyadenylation signal beyond hamster-ubiquitin/S27a promotor, and other expression controlling elements.Knownly concerning various cell types induce and regulate sequence at composing type." transcription regulatory element " is usually included in promotor, transcription initiation and the termination site of gene order upstream to be expressed, and polyadenylation signal.

Polynucleotide sequence represented in " promotor " speech, and it is allowed and is controlled at transcribing of connected gene on the function or sequence.Promotor contains promising RNA polymerase in conjunction with the recognition sequence that provides, and the initiation site of transcribing (transcription initiation site).In order in some cell type or host cell, to express the sequence of wanting, must select the appropriate functional promotor.Those skilled in the art are familiar with deriving from the various promotors in various sources, are included in composing type, the derivable and promotor that can prevent.They are deposited in the database, GenBank for example, and can the discrete element, or the form of being cloned into the element in the polynucleotide sequence that derives from commerce or industrial source obtains.In inducible promoter, can react to signal and reduce or increase the activity of promotor.The example of an inducible promoter is tsiklomitsin (tet) promotor.This promotor contains tetracycline operator sequence (tetO), can induce it through the trans-activator matter (tTA) of tsiklomitsin-adjusting.When tsiklomitsin occurring, suppressed combining of tTA and tetO.The example of other inducible promoters be jun, fos, metallothionein(MT) and heat-shocked promotor (also referring to people such as Sambrook, 1989; People such as Gossen, 1994).Be particularly suitable in eukaryote camber expression promoter, terminal repetition district of length of SV40 early promoter, adenovirus major late promoter, mouse metallothionein(MT)-I promotor, Rous sarcoma virus and human cytomegalovirus's early promoter are for example arranged.The example of other allos mammalian promoters is Actin muscle, immunoglobulin (Ig) or heat-shocked promotor.

" transcription initiation site " speech means the nucleic acid in construct, and it is equivalent to and is impregnated in main transcript, i.e. first nucleic acid of mRNA precursor.Transcription initiation site can be overlapping with promoter sequence.

" transcription pausing site " speech means normally the nucleotide sequence at 3 ' end of goal gene or gene section to be transcribed, and it causes the termination of transcribing of RNA polymerase.

" polyadenylation signal " is a signal sequence, and the specific site at 3 of eukaryote mRNA ' end causes incision, and mixes about 100-200 adenine nucleotide (poly A tail) at the 3 ' end that is cut open after transcribing.Polyadenylation signal is included in the sequence A ATAAA of about 10-30 the Nucleotide upstream of cutting the site, and the position is in the sequence in downstream.Known various polyadenylation element is as tk polyadenylic acid, SV40 late period and early stage polyadenylic acid or BGH polyadenylic acid (at for example US5,122,458 in describe).

In a preferred specific embodiment of the present invention, each transcription unit all has promotor or promotor/enhancer element, goal gene and/or marker gene, and the transcription pausing element.In another preferred specific embodiment, transcription unit also contains translation and regulates unit.

" translation adjusting element " comprises translation initiation site (AUG), ends password and polyadenylic acid signal for each treats polypeptide expressed.For optimum expression, remove, add or change 5 of nucleotide sequence to be expressed '-and/or 3 '-non-translational region, may be wise, so as to get rid of any may unsuitable extra translation initiation password, or other may influence and transcribe or the sequence of the expression of expression level.In order to promote to express, the total binding site of rrna can be inserted the upstream that is right after in initiator codon separately.In order to produce secreted polypeptide, goal gene contains signal sequence usually, its coded signal precursor peptide, and the synthetic polypeptide is transported to and by the ER film.Signal sequence is common, but be not always the position and can after this protein being inserted the ER film, cut in the amino terminal of secreted protein through signal peptidase.Gene order usually but not necessarily contain its signal sequence.If the natural signals sequence does not occur, then can import allogenic signal sequence in known manner.Those skilled in the art will know that many signal sequences of this kind, and be deposited in the sequence library, as GenBank and EMBL.

The regulatory element of a particularly important according to the present invention is internal ribosome entry site (IRES).The IRES element comprise do not rely on upstream region of gene 5 '-sequence that on function, activates translation initiation of terminal methyl group guanosine-(guanosinium) cap (CAP structure), and in zooblast, allow translation two cistrons (open reading frame) from single transcript.This IRES element is the translation that is right after at the open reading frame in downstream, and independently ribosome entry site(RES) is provided.Opposite with the mRNA of bacterium, it can be polycistronic, and promptly how different polypeptide or the products of its codified translated one by one through mRNA, and the mRNA that great majority derive from zooblast is monocistronic, and only encode protein or product.In the situation of the polycistronic transcription thing in eukaryotic cells, translate, and will end codon by first and end, will from rrna, discharge transcript subsequently immediate translation initiation site from the upstream.Therefore, will only produce first polypeptide or the product of encoding at translate duration by mRNA.Opposite, polycistronic transcription thing with IRES element, this IRES is being connected with second or follow-up open reading frame in transcript on the function, allow the follow-up translation that framework is read in the open reading frame volume in its downstream in the position, and be able in eukaryotic cells, produce two or a plurality of by identical transcript encoded polypeptides or product.

The IRES element can have all lengths and various source, and can rise and be derived from for example encephalomyocarditis virus (EMCV) or other picornaviruss.Various IRES sequences and the purposes on the construction carrier thereof have been described in the literature, referring to people such as for example Pelletier, 1988; People such as Jang, 1989; People such as Davies, 1992; People such as Adam, 1991; People such as Morgan, 1992; People such as Sugimoto, 1994; People such as Ramesh, 1996; People such as Mosser, 1997.

The gene order of position in the downstream is connected with 3 of IRES element ' end on function, promptly selects spacing distance, so that do not influence or only influence expression of gene a little, or sufficient expression is arranged for the purpose of wanting.Can be through simple experiment, through changing at interval, and the operation report genetic testing, judge as function at interval and to express speed, judge that in IRES element and position the best that can Gong give full expression to is permitted distance between the initiator codon of the gene in its downstream.

Through described measurement, might obtain the optimum expression box, it expresses quite valuable for heterologous gene products.Therefore, measuring the expression cassette that is obtained through one or more this class, is further target of the present invention equally.

The selectable marker gene that can increase

According to preferred vector of the present invention, contain the selectable marker gene that can increase extraly, it allows the amplification of the marker gene that can increase, and the common amplification of the transcription unit that preferably is made up of the gene of hamster-ubiquitin/S27a gene, goal gene and fluorescence protein.About this point, in the presence of the appropriate selection agent, cultivate host cell with the corresponding expression vector transfection, feasible those host cells that only have many gene copies of the selectable marker gene that can increase at least can duplicate (being replicated).This is preferably in the presence of the selective agent of cumulative content, through culturing cell progressively and reach.

The selectable marker gene that can the increase eukaryotic cells required enzyme of under some culture condition, growing of encoding usually.The selectable marker gene codified Tetrahydrofolate dehydrogenase (DHFR) that for example, can increase.In this case, if cultivation is with the host cell of its transfection in the presence of selective agent methotrexate (MTX), this gene then increases.

Following table 1 provides the selectable marker gene that increases that other can be used according to the invention and the example of relevant selective agent, and at Kaufman, " Enzymology method " described in the summary of 185:537-566 (1990).

Table 1: the selectable marker gene that can increase

The selectable marker gene that can increase	The preservation number	Selective agent
			Tetrahydrofolate dehydrogenase	M19869 (hamster) E00236 (mouse)	Methotrexate (MTX)
Metallothionein(MT)	D10551 (hamster) M13003 (mankind) M11794 (rat)	Cadmium
			(carbamylphosphate synthetase: aspartic acid changes the carbamyl enzyme to CAD: dihydroorotase)	M23652 (hamster) D78586 (mankind)	N-phosphoric acid ethanoyl-L-aspartic acid
Adenosine deaminase	K02567 (mankind) M10319 (mouse)	Xyl-A-or adenosine, 2 ' deoxycoformycin
			AMP (adenylic acid (AMP))-desaminase	D12775 (mankind) J02811 (rat)	VITAMIN B4, azaserine, coformycin
The UMP-synthetic enzyme	J03626 (mankind)	The 6-azauridine, the pyrazoles furans
			IMP 5 '-desaturase	J04209 (hamster) J04208 (mankind) M33934 (mouse)	Mycophenolic acid
Xanthine-guanine-phosphoribosyltransferase	X00221 (intestinal bacteria)	Mycophenolic acid and limited xanthine
			The HGPRT enzyme of sudden change or the thymidine-kinases of sudden change	J00060 (hamster) M13542, K02581 (mankind)	Xanthoglobulin, aminopterin and thymidine (HAT)

	J00423, M68489 (mouse) M63983 (rat) M36160 (simplexvirus)
			Thymidylic acid-synthetic enzyme	D00596 (mankind) M13019 (mouse) L12138 (rat)	Floxuridine
P-glycoprotein 170 (MDR1)	AF016535 (mankind) J03398 (mouse)	Multiple medications, for example Zorubicin, vincristine(VCR), colchicine
			Ribonucleotide reductase	M124223, K02927 (mouse)	The aphid rhzomorph of dwelling
Glutamine-synthetic enzyme	AF150961 (hamster) U09114, M60803 (mouse) M29579 (rat)	Methionine sulfoxide amine (Methioninsulfoximin) (MS X)
			L-asparagine-synthetic enzyme	M27838 (hamster) M27396 (mankind) U38940 (mouse) U07202 (rat)	β-aspartyl hydroxamic acid, albizziine, 5 ' U-18496
Argininosuccinate synthetase	X01630 (mankind) M31690 (mouse) M26198 (ox)	The sword bean nitronic acid
			Ornithine-decarboxylase	M34158 (mankind) J03733 (mouse) M16982 (rat)	Alpha-difluoromethyl ornithine
The HMG-CoA-reductase enzyme	L00183, M12705 (hamster) M11058 (mankind)	Closely knit rhzomorph
			N-ethanoyl Glucoamino-transferring enzyme	M55621 (mankind)	Tunicamycin

Threonyl-tRNA-synthetic enzyme	M63180 (mankind)	Borrelidin
			Na +K +-ATP enzyme	J05096 (mankind) M14511 (rat)	Unabain

According to the present invention, as preferably the encode gene of polypeptide of the selectable marker gene that can increase with DHFR function, DHFR or for example from the fused protein of fluorescence protein and DHFR.DHFR is that the biosynthesizing of purine is necessary.The cell that lacks the DHFR gene can not be grown in purine-defective substratum.Therefore but DHFR is useful selective marker, in the cell of cultivating at the substratum of purine-containing not, is used for selecting and amplification gene.The selection medium that uses with the DHFR gene is methotrexate (MTX).Therefore, the present invention includes the method for recombinant host cell of preparation high yield, it contains the following step: (i) with the transfected host cell of encode at least one target protein matter, fluorescence protein and DHFR; (ii) cultivate this cell under the condition that range gene is expressed allowing, but and (iii) through at the selective agent of allowing at least one selectable marker gene that can increase of amplification, as culturing cell under the existence of methotrexate, the gene that amplification is integrated altogether.Preferably in the substratum that does not contain xanthoglobulin/thymidine, lacking under the serum, and adding the MTX of cumulative concentration, cultivating through cells transfected.In first amplification step, the concentration of MTX is 200nM at least preferably, and in preferred specific embodiment, is 500nM at least, and can progressively increases to 1 μ M.In the discrete situation, can use the concentration that surpasses 1 μ M.

Mammalian cell, preferably mouse myeloma and hamster cell are to use the preferred host cell of DHFR as the selective marker that can increase.Cell strain CHO-DUKX (ATCC CRL-9096) and CHO-DG44 people such as (, 1983) Urlaub are particularly preferred, because they are not because results of mutation itself has the DHFR activity.In order to have in their own active cell types of endogenous DHFR at other, the amplification effect that uses DHFR to rely on, may use the DHFR gene of sudden change in the process of transfection, its coding has reduction to the protein of the susceptibility of methotrexate (people such as Simonson, 1983; People such as Wigler, 1980; People such as Haber, 1982).

According to preparation of expression vectors of the present invention

In principle, the traditional method that can use always through those skilled in the art is for example described by people such as Sambrook (1989), prepares according to expression vector of the present invention.The functional component of carrier is wherein also described, for example suitable promotor (except hamster ubiquitin/S27a promotor), enhanser, termination and polyadenylation signal, antibiotics resistance gene, selective marker, replication orgin and splicing signal.Can use traditional cloning vector to prepare them, for example plasmid, phage, phagemid, clay or virus vector, as baculovirus, retrovirus, adenovirus, adeno associated virus and hsv, and synthetical karyomit(e)/microchromosome.Eukaryotic expression vector also contains the prokaryotic organism sequence usually, for example replication orgin and antibiotics resistance gene, and its allowable carrier duplicates in bacterium and selects.Known many eukaryote expression vectors that contains the multiple clone site that is used for importing polynucleotide sequence, and some can be available from various companies, as Stratagene, and La Jolla, CA, USA; Invitrogen, Carlsbad, CA, USA; Promega, Madison, WI, USA or BD Biosciences Clontech, Palo Alto, CA, USA.

One of in a manner familiar to those skilled in the art, with hamster-ubiquitin/S27a promotor, goal gene, the proteinic gene of coding fluorescence, the preferred selectable marker gene that can increase in addition, Tetrahydrofolate dehydrogenase for example, and optional extra regulatory element, as internal ribosome entry site (IRES), enhanser or polyadenylation signal, import in the expression vector.According to expression vector of the present invention, minimumly contain ubiquitin/S27a promotor, goal gene, and the proteinic gene of coding fluorescence.Expression vector preferably also contains the selectable marker gene that can increase.According to the present invention, also can use ubiquitin/S27a promotor, ubiquitin/S27a promotor of for example describing in the present invention through modifying through modifying.Particularly preferably be and wherein on function, the proteinic gene of ubiquitin promoter, goal gene and coding fluorescence linked together or be in the functional expression vector that is connected.

In the scope of this explanation, " functional connection " or " connecting on function " speech mean two or a plurality of nucleotide sequence or partial sequence, settle them to make it be carried out the function that it is wanted.For example, if promotor/enhanser can be with position forward, the transcribing of the gene order that control or regulate has connected, then it is connected with coding gene sequence on function.Usually, but not necessarily, the dna sequence dna that connects on function abuts against together, and if connect two coding gene sequences, or in the situation of secretory signal sequence, be in identical reading frame.Though the promotor that connects on function normally position not necessarily must be close with it in the upstream of coding gene sequence.Enhanser equally needn't be closely close, as long as it helps transcribing of gene order.For this purpose, they can be at the upstream and downstream of gene order, and are optional from its segment distance.The polyadenylation action site is connected with gene order on function, if it is placed in 3 of gene order ' end, makes and transcribes via encoding sequence to polyadenylation signal.Can connect according to traditional recombination method, for example through round pcr, through connection, or through splicing at suitable restricted cleavage site place.If there is not available suitable cleavage site, then can use synthetic oligonucleotide joint or conjugant in known manner.According to the present invention, preferably do not carry out connection on function via intron sequences.

In one of described specific embodiment, on function, ubiquitin/S27a promotor or the proteinic gene of its modified forms, goal gene and coding fluorescence are linked together.This for example means, and from identical ubiquitin/S27a promotor or its modified forms, expresses goal gene and the proteinic gene of coding fluorescence.In a particularly preferred specific embodiment, carry out connection on this function via the IRES element, and be able to from the synthetic bicistronic mRNA of two genes.According to expression vector of the present invention, can contain enhancer element extraly, it plays a role to one or more promotor on function.Particularly preferably be wherein ubiquitin/S27a promotor or its modified forms and enhancer element, for example the expression vector that connects of SV40 enhanser or cmv enhancer element.

Basically, the expression of gene in expression vector can begin to carry out from one or more transcription unit.Transcription unit is defined as the zone of containing one or more gene to be transcribed.Gene in transcription unit is connected to each other on function, makes all genes in this class unit, all transcribing under the control at identical promoters or promotor/enhanser.The result that this gene transcription connects is to transcribe more than one protein or product from a transcription unit, and expresses it by this.Each transcription unit can contain at this gene order that wherein contained transcribed and translate necessary regulatory element.Identical or different regulatory element can be contained in each transcription unit.The function that can use IRES element or intron to carry out the gene in transcription unit connects.

Expression vector can contain the single transcription unit of the selectable marker gene that is used for expressing the gene of goal gene, fluorescence protein and can increases.Perhaps, also can with these sequences in the gene two or a plurality of transcription unit in.At this, the various combinations of gene all are possible in transcription unit.In another specific embodiment of the present invention, can be through cotransfection, or in continuous transfection according to any order, with more than one, by one, two or the expression vector formed of a plurality of transcription unit introduce in the host cell.Can be chosen in any combination of each carrier adjusted element and gene, as long as guarantee giving full expression to of transcription unit.If need, can be with other regulatory element and gene, for example extra goal gene or selective marker are placed on the expression vector.

Therefore, can be according to expression vector of the present invention at one or in two transcription units that separate, the selectable marker gene that contains the proteinic gene of coding fluorescence and can increase.Each transcription unit can transcribe and express one or more gene product.If contain two genes in a transcription unit, it and preferably uses IRES to guarantee that the function of all components connects under the control of identical promotor or promotor/enhanser.If proteinic gene of coding fluorescence and the selectable marker gene that can increase are comprised in two transcription units that separate, then it can be under the control of identical or different promotor/enhanser.Yet, for selectable marker gene, preferably use its natural or more weak allogeneic promoter, SV40 early promoter for example, and preferably do not use enhanser.In scope of the present invention, the expression vector that has two transcription units that separate is preferred.Goal gene and the proteinic gene of coding fluorescence are contained in (bicistronic mRNA) transcription unit, and the selectable marker gene that can increase is then contained in another transcription unit.Preferably through coding polyadenylic acid signal, preferably the sequence of tk polyadenylic acid, BGH polyadenylic acid or SV40 polyadenylic acid limits each transcription unit at 3 ' end.

According to the present invention, preferably also have those all multiple clone site to replace the carrier of goal gene, described multiple clone site is allowed the recognition sequence via restriction endonuclease, clone's goal gene.From prior art, know the multiple recognition sequence of the restriction endonuclease of all kinds, and affiliated restriction endonuclease.Preferably use the sequence of forming by at least six Nucleotide as recognition sequence.Can be people such as for example Sambrook, find the table look-up of suitable recognition sequence in 1989.

Host cell

For with expression vector transfection according to the present invention, use the eukaryote host cell, mammalian cell preferably, and more particularly be rodent cells, as mouse, rat and hamster cell system.With the corresponding cell of expression vector transfection according to the present invention, the result produces and passes through conversion, passes through modification, reorganization or genetically modified cell in heredity, and it also is a target of the present invention.

Preferred within the scope of the invention host cell is a hamster cell, as BHK21, BHK TK ^-, CHO, CHO-K1, CHO-DUKX, CHO-DUKX B1 and CHO-DG44 cell, or the derivative/offspring of these clones.Particularly preferably be CHO-DG44, CHO-DUKX, CHO-K1 and BHK21 cell, particularly CHO-DG44 and CHO-DUKX cell.The myeloma cell of mouse is fit to too, preferably NS0 and Sp2/0 cell, and the derivative/offspring of these clones.

The hamster that can use according to the present invention and the example of mouse cell are provided in following table 2.Yet, also can use the derivative of these cells and offspring, other mammalian cell, include but not limited to the mankind, mouse, rat, ape and monkey, rodentine cell strain, or eukaryotic cells, include but not limited to yeast, insect and vegetable cell, as producing the proteinic host cell of biopharmacy.

Table 2: hamster and mouse production clone

Clone	Preserving number
		NS0	No. the 85110503rd, ECASS
Sp2/0-Ag14	ATCC CRL-1581
		BHK21	ATCC CCL-10
BHKTK -	No. the 85011423rd, ECACC
		HaK	ATCC CCL-15

(2254-62.2 BHK-21-derivative)	ATCC CRL-8544
		CHO	No. the 8505302nd, ECACC
CHO-K1	ATCC CCL-61
		CHO-DUKX(＝CHOduk -，CHO/dhfr -)	ATCC CRL-9096
CHO-DUKX B1	ATCC CRL-9010
		CHO-DG44	People such as Urlaub; Cell 33[2], 405-412,1983
CHO Pro-5	ATCC CRL-1781
		V79	ATCC CCC-93
B14AF28-G3	ATCC CCL-14
		CHL	No. the 87111906th, ECACC

Through traditional method (people such as Sambrook, 1989; People such as Ausubel, 1994), carry out with polynucleotide or according to the effect of one of expression vector of the present invention transfection eukaryote host cell.The proper method of transfection comprises transfection, protoplastis fusion, microinjection and the virus infection that transfection, coprecipitation of calcium phosphate, electroporation, the polycation (for example deae dextran) of for example liposome mediation mediate.According to the present invention, preferably carry out stable transfection, wherein construct is incorporated into the genome of host cell, or in artificial chromosome/microchromosome, or with stable manner, be included in the host cell with episomal form.Provide best transfection frequency, and the transfection method of the expression of heterologous genes in host cell separately is preferred at this.According to definition, be called " heterologous sequence " or " heterologous gene " with respect to host cell with being inserted into each sequence in the host cell or each gene.Even the gene that sequence that desire imports or desire import is identical with the endogenous sequence or the native gene of this host cell, also be suitable for.For example, being imported into the hamster actin gene in the hamster host cell, also is allogenic gene according to definition.

At heterodimer protein, for example in the reorganization of monoclonal antibody (mAb) preparation, can carry out the transfection of suitable host cell through two kinds of diverse ways in principle.The monoclonal antibody of this kind is made up of many subunits, weight and light chain.The gene of these subunits of coding can be placed on single plasmid independently or in the polycistronic transcription unit, use this plasmid transfection host cell then.This attempt in being integrated into the host cell gene group after, guarantee the expression on the stoichiometric calculation of gene.Yet in the situation of independent transcription unit, the mRNA of the different proteins of must guaranteeing by this to encode shows identical stability, and transcribes and translation efficiency.In second kind of situation, through single promotor, carry out expression of gene in the polycistronic transcription unit, and only form a transcript.Through using the IRES element, obtain second and follow-up cistron in correct effective inner translation initiation of gene.Yet, first cistron of expression speed ratio of these cistrons low, the translation initiation of the preinitiation complex that it relies on through so-called " cap ", more effective than the translation initiation that IRES relies in fact.In order to reach the real equimolar expression of cistron, can for example import element between extra cistron, it causes the expression speed (WO 94/05785) of homogeneous with the IRES element.

Other also preferably prepare the possible path of many heterologous proteins simultaneously according to the present invention, be cotransfection, wherein integrate the gene in different expression vectors respectively.This has the advantage that can adjust some ratio of gene and gene product mutually, is equilibrated at mRNA stability by this, and transcribe with translation efficiency on difference.In addition, this expression vector because their size less be more stable, and in clone and transfection, be easier to operate.

Therefore, in special specific embodiment of the present invention, has the carrier transfection of the gene of one or more other target protein matter of coding extraly with one or more, preferably cotransfection host cell.Be used for other carriers or a plurality of carrier of common transfection, under the control of identical promotor/enhanser combination, coding is other target protein matter or a plurality of protein for example, and at least one other selective marker, for example neomycin phosphotransferase.

According to the present invention, preferably under the condition that does not contain serum, set up adaptation and cultivate host cell, choose wantonly in the substratum that does not contain animal protein/peptide and cultivate.The example of the substratum that can buy, comprise Ham ' s F12 (Sigma, Deisenhofen, DE), RPMI-1640 (Sigma), (Dulbecco ' s) is through the Eagle ' s substratum (DMEM of improvement for Du Bei Keshi; Sigma) MEM (MEM; Sigma), (Iscove ' s) is through Du Bei Keshi substratum (IMDM of improvement for Ai Sikefu; Sigma), CD-CHO (Invitrogen.Carlsbad, CA, USA), CHO-S-SFMII (Invitrogen), the CHO-substratum (Sigma) that does not contain serum and nonprotein CHO-substratum (Sigma).Optional replenish in these substratum each, for example hormone and/or other somatomedin (for example Regular Insulin, siderophilin, Urogastron, rhIGF-1), salt (for example sodium-chlor, calcium, magnesium, phosphoric acid salt), buffered soln (for example HEPES), nucleosides (for example adenosine, thymidine), glutamine, glucose or other nutrition of equal value, microbiotic and/or trace element with all cpds.Though according to the present invention, the substratum that does not contain serum is preferred, also can use and cultivate host cell with an amount of serum blended substratum.In order to select to express the cell through genetic modification of one or more selectable marker gene, one or more selective agent is added in this substratum.

" selective agent " speech means and hinders host cell growth that lacks the selectable marker gene of being discussed and the material of surviving.For example, in order to select expressed antibiotics resistance gene,, preferably use microbiotic Geneticin (G418) as medium additives as the existence of neomycin phosphotransferase.If the selectable marker gene (referring to table 1) of employed gene for increasing, then selective agent also can be the material that brings out the amplification of selectable marker gene.For example, methotrexate is to select medium, and it is fit to be used for increasing the DHFR gene.In table 1, enumerate other and brought out the example of the selective agent of amplification.

" selectable marker gene " is through adding corresponding selective agent in substratum, allowing that selection specifically contains the gene of the cell of this gene.Say that more expressly antibiotics resistance gene can be used as the positively charged selective marker and is used.Have only cell, can grow in the presence of antibiotic corresponding with this gene transformation, and therefore selected.The cell of opposite unconverted can not be grown or survive under these selection conditions.Positivity, negativity and bifunctional selective marker are arranged.The positivity selective marker allows through the resistance with respect to selective agent, or through metabolism or catabolism defective in the compensation host cell, selects and enrichment process cell transformed.Opposite, can optionally remove the cell of the gene of having accepted selective marker through the negative selective marker.The example is the thymidine kinase gene of hsv, and its expression in cell is by acycloguanosine (acyclovir) and 9-[1,3-dihydroxy-2-third oxygen methyl] add guanine (gancyclovir) time, cause the removing of cell.But the selective marker of Shi Yonging in the present invention, comprise the selective marker that can increase, comprise the mutant modified with mode of inheritance and variant, fragment, function equivalent, derivative, homologue, and with the fusion of other protein or peptide, as long as this selective marker keeps its selectivity characteristic.This analog derivative shows sizable homology in being considered as selectively zone or structural domain in aminoacid sequence.Document description many selectable marker genes, comprise bifunctional (positivity/negative) mark (referring to, for example WO 92/08796 and WO94/28143).The example of the selective marker in being commonly used in eukaryotic cells, the gene that comprises aminoglycoside phosphotransferase (APH), hygromix phosphotransferase (HYG), Tetrahydrofolate dehydrogenase (DHFR), thymidine kinase (TK), glutamine synthetase, asparagine synthetase, and mediation is to the gene of the resistance of Xin Meisu (G418), tetracycline, histidinol D, bleomycin, phleomycin and Li Ouxin (zeocin).

Also may sort art (FACS) through fluorescence activated cell selects through cell transformed.About this point, for example can use beta-galactosidase enzymes, cell surface marker or the fluorescence protein (egfp (GFP) and derive from Victoria jellyfish and the variant of kidney shape jellyfish or other species, DsRed matter and send out the protein of other color fluorescence for example of bacterium, and derive from the variant of abiotic luminous organism (for example mushroom coral, wrinkle Zhe sea anemone, plumage coral, button coral), select through cell transformed.

In the present invention, preferably use the DHFR gene to select (reorganization) host cell of modifying with mode of inheritance, as the selectable marker gene that can increase.At the basic cell that uses the DHFR feminine gender, during as CHO-DG44 or CHO-DUKX, this mark is particularly suitable for being used for selecting and follow-up amplification, because these cells are not expressed endogenous DHFR, and therefore can not grow in the substratum of purine-containing not.As a result, can use the DHFR gene here, and in the substratum that does not contain xanthoglobulin/thymidine, select through cell transformed as dominant selectable marker.In order to obtain the gene amplification of DHFR mediation, use methotrexate (MXT).Through adding MTX, growth characteristics have been influenced fatefully.Along with the increase of MTX concentration and amplification step, observe the substantial deterioration of fermentation robustness of cell.Yet surprising is has been found that use according to clonal selection of the present invention system, and in the presence of high density MTX, the recombinant host cell that shows many a lot of strong behaviors increases (referring to Fig. 7).Therefore, can be in the presence of 500nM MTX, preferably in the presence of 1 μ M MTX, the host cell that fluorescence-activating cells letter sorting (FACS) is identified and selected has been used in cultivation and amplification, and the result has increased productive rate significantly.

Therefore, a kind of method of high yield host cell of selecting is regarded as especially according to the present invention, if with expression vector transfection according to the present invention, and the host cell of expressing goal gene, fluorescence protein and DHFR gene at least is selected through the FACS letter sorting, and at 500nM at least, the existence of 1 μ M MTX experience gene amplification step down preferably.

" fermentation robustness " speech means the growth characteristics of cell, for example keep certain growth velocity, robustness to " amplifying in proportion " (" Upscaling ") (large-size of bio-reactor), and in kind of a system keeps, reach high cell counting and vigor, so that when amplifying in proportion, meet the industry speed that goes down to posterity.

Express

Expressing a speech is about the transcribing and/or translating in host cell of heterologous gene sequence.Usually appearing at the amount of corresponding mRNA in the host cell, or the amount to be produced by the gene product of goal gene coding, judgement expression speed.For example, through the in situ hybridization of RNA blot hybridization, rnase-RNA-protection, cell RNA or through PCR method (people such as Sambrook, 1989; People such as Ausubel, 1994), judge through the amount of transcribing the mRNA that nucleotide sequence produced that selects.Also can judge by selected nucleotide sequence coded protein through the whole bag of tricks equally, for example ELISA, Western blotting, radioimmunoassay, immuno-precipitation, the proteinic biological activity of detection, or through proteinic immunostaining and carry out facs analysis (people such as Sambrook, 1989 subsequently; People such as Ausubel, 1994).

" high expression level (speed) ", " high expression level ", " increase and express " or " high yield " mean and continue and expression of q.s ground level or the synthetic heterologous sequence that is imported in the host cell gene of the therapeutic protein of for example encoding.If through cultivating according to cell of the present invention according to one of method of the present invention of describing in this article, and if this cell generation every day surpasses the gene product (5 pico-gram/sky/cell) that about 5 pico-grams are wanted at least, then occur increasing or high expression level, or high expression level or speed, or high yield.(increase or high expression level then also appear in 10 pico-gram/sky/cells, or high expression level or speed, or high yield if according to producing cell of the present invention every day the gene product that surpasses about 10 pico-grams at least and want.Particularly, then occur increasing or high expression level if generation every day is wanted gene product (15 pico-gram/sky/cell) above about 15 pico-grams at least according to cell of the present invention, or high expression level or speed, or high yield.Particularly if surpass at least according to producing cell of the present invention every day that about 20 pico-grams want, increase or high expression level then appear in gene product (20 pico-gram/sky/cell), or high expression level or speed or high yield.Particularly, then occur increasing or high expression level if produce the gene product of wanting above about 30 pico-grams at least (30 pico-gram/sky/cell) every day according to cell of the present invention, or high expression level or speed, or high yield.

Can reach the height on the meaning of the present invention or expression, high yield or high expression level or the speed of increase in every way.For example, via the coexpression of goal gene, might select and identify that expression of heterologous genes reaches the cell of high level with the selectable marker gene that can increase.The selective marker that can increase is not only allowed the host cell of selecting stable transfection, also allows the gene amplification of purpose heterologous gene.The additional copy of nucleic acid can be integrated in the genome of host cell, to extra artificial/microchromosome, or extremely with in the localized polynucleotide of episome form.The selection of this way with the recombinant host cell of FACS support can be mixed, this host is one or more fluorescence protein (for example GFP) or the cell surface marker that includes as extra selective marker.Obtain to increase the additive method of expressing, described method also can be used the combination of different methods, be for example to use (synthetical) transcription factor, handle cell with the natural or synthesising preparation that raises endogenous or allogeneic gene expression, improve mRNA or proteinic stability (transformation period), improve the initial of mRNA translation, through using plasmid episomal (to use virus sequence as replication orgin, SV40 for example, polyomavirus, adenovirus, the sequence of EBV or BPV) increases gene dosage, use to promote the sequence (people such as Hemann of amplification, 1994), or be the basis based on the in vitro amplification system of DNA catenation people such as (, 1996) Monaco.

According to of the present invention be that the coupling of goal gene and the proteinic gene of coding fluorescence is transcribed.The bicistronic mRNA of gained is expressed target protein matter and fluorescence protein.Coupling with this target protein matter and fluorescence protein is expressed as the basis, might be easily according to the present invention, through expressed fluorescence protein, for example, select and the recombinant host cell that separates high yield through using fluorescence activated cell sorter (FACS) to select.

Selection shows high vigor, and increases the recombinant host cell of the expression speed of the gene product of wanting, and is the process in a plurality of stages.At least at coding fluorescence protein, and with goal gene link coupled expression of gene, research is with expression vector transfection according to the present invention, or it is optional with other carriers host cell of cotransfection for example, so that identify and select to show the cell/cell colony of the highest fluorescence protein expression rate.Preferably only pick out and belong to 10% cell, and further cultivate with the highest fluorescence protein expression rate.In fact, this means picks out the brightest 10% cell that fluoresces, and further cultivates.Correspondingly, also can pick out in the cell mixture the brightest 5%, preferably the brightest 3% or or even the brightest 1% the cell that fluoresces, and duplicate it.In particularly preferred specific embodiment, only pick out the brightest 0.5% or the brightest 0.1% cell that fluoresces, and duplicate it.

For this purpose, with expression vector cells transfected according to the present invention, this substratum is chosen wantonly and is contained the specific selective agent of selective marker that can increase before cultivating in selecting substratum.Can use the concentration that progressively increases selective agent, apply the selective pressure that increases gradually.

Can be in the enterprising row filter step of cell aggregation, or use the cell aggregation/cell clone of letter sorting in advance.Can carry out one or more, preferably two or a plurality of, and particularly three or a plurality of step of selecting, and select between the step one, can cultivate and one specific period of replicating cell, for example about 2 weeks in the situation of set.

Optional can make host cell experience one or more gene amplification step, so that increase goal gene at least and the copy number of the selectable marker gene that can increase.Using the progressively process of amplification gene of methotrexate, is according to the description in No. the 5th, 179,017, United States Patent (USP) for example.According to the present invention, accessible high yield, the gene copy that is not limited to increase.Yet but be the high-level efficiency clone, the feature of the stability of increase and fermentation robustness.Therefore might reduce the number of required gene amplification step, and for example only carry out single gene amplification.

Therefore, the method according to selection cell of the present invention comprises the following steps:

(i) transform appropriate host cell with support according to the present invention at least, wherein preferred DNA with expression vector stably mixes in the host cell gene group, or in synthetical karyomit(e)/microchromosome;

(ii) allowing under the condition that goal gene and fluorescence protein are expressed, cultivating through cell transformed;

(iii) in the presence of at least one selective agent, cultivate this cell, and those cells that only can grow are duplicated in the presence of described selective agent;

(iv) from cell mixture, select the cell that shows the highest fluorescence protein expression rate, wherein select and detect cell through fluorescence activated cell sorter (FACS);

(v) cultivate the cell of picking out with the highest fluorescence protein expression rate.

The optional cell that will v) obtain according to step, repeating step ii)-v) one or repeatedly.In addition, also can choose wantonly to make through cell transformed and experience one or more gene amplification step, wherein it is cultivated the amplification that it causes the selectable marker gene that can increase in the presence of selective agent.Can utilize the cell of not selecting as yet, and also can utilize and select one or cell repeatedly already in advance, carry out this step.

The present invention is also about wherein duplicating the cell through corresponding letter sorting, and is used for preparing the method for purpose encoding gene product.Preferably in the substratum that does not contain serum, cultivate selected high yield cell, and preferably in the suspension culture base, allowing under the condition of destination gene expression.Preferably from cell culture medium,, obtain target protein matter with the form of secretion property gene product.Yet, by expressing the protein of no secretion signal, also can be from the cell lysis product isolated genes product.In order to obtain the product of pure homogeneous, it does not contain other recombinant protein and host cell proteins matter in fact, carries out traditional purification step.At first, usually, from substratum or the molten product of born of the same parents, remove cell and cell debris.Then can through for example on immune avidity and ion exchange column fractional separation, ethanol precipitation, at sephadex, silica gel or Zeo-karb, as anti-phase HPLC or the chromatography on the DEAE, make the cellular products of wanting not contain soluble protein, polypeptide and the nucleic acid of pollution.The method of the heterologous protein that purifying is expressed by reconstitution cell for well known by persons skilled in the art, and is described in the literature, for example people (1995) and Scopes (1988) such as Harris.

Below, through non-limiting specific embodiment, be explained in more detail the present invention.

Embodiment

Abbreviation

AP: alkaline phosphatase

Bp: base pair

CHO: Chinese hamster ovary

DHFR: Tetrahydrofolate dehydrogenase

ELISA: enzyme-linked immunosorbent assay

FACS: fluorescence activated cell sorter

FAP: fibroblast activation protein matter

GFP: green fluorescent protein

HBSS: Han Keshi (Hanks) balanced salt solution

HT: xanthoglobulin/thymidine

HRPO: horseradish peroxidase

IRES: internal ribosome entry site

Kb: kilobase

MAb: monoclonal antibody

MTX: methotrexate

PCR: polymerase chain reaction

SICAM: adhesion molecule in the soluble cell

Method

1. cell cultures and transfection

Under 37 ℃, at the environment and the 5%CO of humidity ₂In, in Tissue Culture Flask, with the form of suspension cell, be supplemented with the CHO-S-SFMII substratum that does not contain serum of xanthoglobulin and thymidine (Invitrogen GmbH, Karlsruhe, DE) in, for good and all culturing cell CHO-DG44/DHFR-/-(people such as Urlaub, 1983).(SchaerfeSystem DE), or through trypan blue staining, judges cell counting and viability, then with 1-3 * 10 to utilize the CASY1 cell counter ⁵The concentration inoculation of/milliliter, and once went down to posterity in every 2-3 days.

Use Lipofectamine Plus reagent (Invitrogen GmbH) transfection CHO-DG44.For each transfection mixture, explanation according to the producer, will be altogether 1 microgram plasmid DNA, 4 microlitre Lipofectamine and 6 microlitre Plus reagent mix be in the same place, and with the volume of 200 microlitres add in 0.8 milliliter of CHO-S-SFMII substratum that is supplemented with HT 6 * 10 ⁵In the CHO-DG44 cell of individual exponential growth.Under 37 ℃, after in the cell thermostat container, cultivating 3 hours, add 2 milliliters of CHO-S-SFMII substratum that are supplemented with HT.As for based on DNFR, select the CHO-DG44 cell of stable transfection, transfection after 2 days, cell is moved in the CHO-S-SFMII substratum that does not add xanthoglobulin and thymidine, every 3 to 4 days replacing substratum.In selection based on DHFR and neomycin phosphotransferase, expression vector contains DHFR and another expression vector contains in the case of cotransfection of Xin Meisu-phosphotransferase selective marker therein, also, G418 (Invitrogen) is added in the substratum with the concentration of 400 mcg/ml.

Through in not containing the CHO-S-SFMII substratum of HT, (DE) obtaining is the based gene amplification with DHFR-through the heterologous gene integrated for Sigma, Deisenhofen to add the selective agent MTX of concentration 5-2000nM.

2. expression vector

The desire analysis is expressed, and uses the eukaryote expression vector, and it be the basis with pAD-CMV carrier people such as (, 1998) Werner, and through the combination of cmv enhancer/hamster ubiquitin/S27a promotor, mediates the constitutive expression (WO 97/15664) of heterologous gene.And basic carrier pBID contains the DHFR minigene, its serve as the selective marker that can increase (referring to, for example EP 0 393 438), in carrier pBIN, through neomycin resistance gene displacement DHFR minigene (Fig. 2).For this purpose, (Stratagene, La Jolla USA), as 1640 base pair Bsu36I fragments, separate the selective marker neomycin phosphotransferase, comprise SV40 early promoter and TK-polyadenylation signal from commercially available plasmid pBK-CMV.After filling up the reaction of segmental end with the Klenow-DNA-polysaccharase, the Bsu36I/StuI fragment of this fragment with 3750 base pairs of carrier pBID is connected, the latter also utilizes the Klenow-DNA-polysaccharase to handle.

In the underlying carrier pBIDG of bicistronic mRNA (Fig. 2), from carrier pIRES2-EGFP (Clontech, Palo Alto, CA, USA) separate the IRES-GFP gene regions in, and under the control of cmv enhancer/promotor, introduce carrier pBID, and be retained in the multiple clone site between promoter region and the IRES element.Use following operation.In PCR sudden change nucleus formation, wherein plasmid pIRES2-EGFP through using mutagenic primer, will change sequence A TGCTT at the HindIII cleavage site AAGCTT in the IRES sequence on the one hand as template, and therefore get rid of it.On the other hand through with 5 of IRES sequence ' end complementary primer, insert the XbaI cleavage site, or through with 3 of GFP sequence ' end complementary primer, importing SpeI cleavage site.With the PCR fragment of XbaI and SpeI digestion gained, it contains complete IRES and GFP sequence, and is cloned in the single XbaI cleavage site of the multiple clone site 3 ' end at carrier pBID.

From pAD-sICAM, with the segmental form of HindIII/SalI, separate human sICAM gene people such as (, 1998) Werner, and be cloned in the corresponding cleavage site of carrier pBIDG, the result is carrier pBIDG-sICAM (Fig. 3).

In order to express mono-clonal humanization F19 antibody, from plasmid pGlD105F19HC (NAGENESEQ:AAZ32786), with the segmental form of 1.5kb NaeI/HindIII, separate heavy chain, and be cloned in the carrier pBIDG that digested with EcoRI (filling up with the Klenow-DNA-polysaccharase) and HindIII, the result produces carrier pBIDG-F19HC (Fig. 3).On the other hand, from plasmid pKN100F19LC (NAGENESEQ:AAZ32784),, separate light chain, and be cloned in the corresponding cleavage site of carrier pBIN, produce carrier pBIN-F19LC (Fig. 3) with the segmental form of 1.3kb HindIII/EcoRI.

3.FACS

Utilize Coulter Epics Altra device to carry out flow cytometry analysis and letter sorting.The FACS that helium-argon laser is installed has 488 millimicrons excitation wavelength.Fluorescence intensity is absorbed at the wavelength that conforms to fluorescence protein, and handles through the software Coulter Expo32 that encloses.Speed with 8000-10000 incident/second is selected.The cell of centrifugal suspension (with 180 * g 5 minutes), and in HBSS, be adjusted to 1-1.5 * 10 ⁷The cell concn of/milliliter.Select cell according to its fluorescence protein signal then.Cell is put into the test tube that contains substratum already, centrifugal, and be seeded in the corresponding culture vessel according to the number of the cell selected.

4.ELISA

SICAM in the CHO-DG44 cell conditioned medium of stable transfection tires by ELISA with quantitative (the Ausubel et al. of standard operation, 1994, upgraded edition), wherein use two SICAM monoclonal antibody specifics that in mouse, develop (for example at US 5,284,931 and US 5,475,091 in describe).One in two antibody is HRPO conjugated antibody.The SICAM albumen that uses purifying is as standard.

Through ELISA, according to standard program (people such as Ausubel, 1994, upgraded edition), use on the one hand the anti-IgG Fc fragment of goat (Dianova, Hamburg, DE), use AP-conjugated goat to resist human κ light chain antibody (Sigma) on the other hand, quantitatively the F19 monoclonal antibody in the CHO-DG44 of stable transfection cell conditioned medium liquid.Use purified F19 antibody as standard substance.

Through formula pico-gram/((C _t-C _o) t/In (C _t-C _o) calculating productive rate (pico-gram/cell/sky), wherein C _oAnd C _tBe respectively the cell counting when inoculation or results, and t is an incubation time.

Embodiment 1: compare CMV and hamster-ubiquitin/S27a-promoter activity

For the activity of comparison hamster-ubiquitin/S27a-promotor and the activity of the CMV promotor in being commonly used in the eukaryote expression vector, with various recombinant vectors transfection CHO-DG44 cells.Under the control of CMV-promotor, or under the control of hamster-ubiquitin/S27a promotor, the expression of heterologous genes product is a lysosomal enzyme on the one hand, is IgGl-antibody on the other hand.Two promotors all are connected with the CMV-enhanser on function.Use the abort signal of BGH polyadenylic acid as heterologous gene.The expression vector that contains the CMV promotor is with the pcDNA3 (" CMV through modifying ¹", Invitrogen) or pBluescript carrier (" CMV ²", Stratagene) be the basis, and the selective marker Tetrahydrofolate dehydrogenase of encoding and can increase extraly.On the other hand, the expression vector with hamster promotor is to be the basis with pAD-CVM carrier people such as (, 1998) Werner.For the weight and the light chain of expressing antibodies, carry out cotransfection with second carrier that contains as the neomycin resistance gene of selective marker.Yet, can also replace cmv enhancer by the SV40 enhanser.

Through after transfection, the restriction dilution in 96 well culture plates is selected in the substratum that does not contain HT and isolated cell clone (in the situation of cotransfection, adding 400 mcg/ml G418).Through from 5nM, increase methotrexate concentration step by step via 50nM, 500nM to 2 μ M, together with the dilution in each situation clone, make the cell clone that for recombinant protein, has maximum output, experience progressively be the based gene amplification with DHFR-.In each amplification stage, select about 20 to 30 clones with maximum output.

Generally speaking, find that the hamster promotor has higher efficient.In the expression of the expression of lysosomal enzyme and antibody among both, the productive rate that it obtained or tire, Billy is with wherein under the control of CMV promotor, expression of heterologous genes cell obtained that those are higher 2 to 5 times.Fig. 1 shows that for example in the amplification stage separately, optimum cell clone's relative potency and relative specificity productive rate will be set at 1 (CMV based on the expression of heterologous genes separately of CMV promotor ¹Be lysosomal enzyme, CMV ²Be antibody).

Embodiment 2: through the FACS letter sorting based on GFP, transport disengaging height is expressed the cell of sICAM

The soluble form sICAM of intercellular adhesion molecule ICAM1, it is the possible therapeutical agent of flu, because it and ICAM receptor competition combine with rhinoviral, and this mode can reduce, or even can prevent the interaction of rhinovirus and ICAM acceptor, this interaction enters in the cell for it and the prerequisite of follow-up infection (people such as Bella, 1999; People such as Marlin, 1990).

SICAM is selected as the example of expressing single chain protein matter (480 amino acid) in Chinese hamster ovary celI.For this reason, with pBIDG-sICAM transfection CHO-DG44 (Fig. 3).The extra expression of GFP in the pBIDG-sICAM cells transfected makes it may use selection strategy based on FACS-.Through bicistronic mRNA transcription unit, expression treatment protein sICAM and GFP jointly, and express DHFR through the transcription unit that separates.In 2 to 3 weeks after in not containing the CHO-S-SFMII substratum of HT, selecting for the first time, pick out 5% cell with the highest GFP fluorescence.After the cultivation in about 2 weeks, isolate 5% cell once again with the highest GFP fluorescence.This is selected continuously and carries out altogether 6 times.Can between sICAM productive rate and GFP fluorescence, show good association (Fig. 4).Only the selection of supporting through FACS does not need any MTX amplification step, isolates the cell aggregation (Fig. 5) that has up to the high specific productive rate in 16 pico-grams/cell/sky in the shortest time.Made up through the selection and the single follow-up MTX amplification step that with GFP are the basis, even might be increased productive rate to 30 pico-gram/cell/sky above (Fig. 6).After the 4th time that utilizes 500nM MTX is selected, utilize the amplification of set, and also after the 6th time that utilizes 2 μ M MTX is selected, utilize the amplification of set, all reach these productive rates.With usually opposite in the progressively amplification that the extremely low MTX concentration of 5-20nM MTX begins from scope, it must use higher MTX concentration to begin, and just can reach the effect of amplification.Therefore, add 5 or 50nM MTX in deriving from the cell of selecting for the 4th time, or add 500nM MTX in deriving from the cell of selecting for the 6th time, the result does not have to increase significantly (Fig. 6) on productive rate.Obviously the level of DHFR has been so high in the set of beginning, so that only can utilize the MTX of high dosage to reach the inhibition of DHFR completely.In addition, although the high initial dose of MTX, the survival of the cell aggregation of selecting in advance during selecting still improves a lot, promptly in the shorter time, obtaining the cell colony (Fig. 7) of high vigor than tradition gene amplification strategy progressively.

Embodiment 3: through the FACS letter sorting based on GFP, transport disengaging height is expressed the cell of monoclonal antibody F19

In cotransfection, with plasmid combination pBIDG-F19HC and pBIN-F19LC (Fig. 3) transfection CHO-DG44 cell.Expressed humanized antibody F19, direct anti-surface molecular FAP, it is by reactive matrix inoblast synthetic (also referring to patent EP 0 953 639).In employed carrier configuration, express by its two protein chain of the antibody of the vector expression of oneself separately, its also extraly in the transcription unit that separates, encoding D HFR or neomycin phosphotransferase selective marker.In addition, another selective marker GFP then is included in the carrier pBIDG-F19HC.Be connected through GFP and transcribing of heavy chain expression by the IRES element, with carrier pBIDG-F19HC/pBIN-F19LC cotransfection CHO-DG44, can be only through using continuous FACS letter sorting to select to have the cell of high GFP intensive amount, the cell of transport disengaging height expressing antibodies F19 at short notice.For this reason, preceding 2-to 3-week in adding the CHO-S-SFMII substratum that does not contain HT of 400 mcg/ml G418, select through the cells transfected set after, pick out 5% cell with the highest GFP fluorescence through FACS.Carry out this altogether and select maximum 6 times, between selecting, be the incubation period in about 2 weeks at every turn.Surprising is, presents good connection (Fig. 8) between F19 productive rate and GRP fluorescence, though two protein chains from their vector expression, and in the FACS letter sorting based on GFP, only may be selected the expression of heavy chain, because it is transcribed with GFP.Can make gain in yield to 10 pico-gram/cell/sky (Fig. 9), and through single follow-up MTX amplification step, since the cell aggregation of selecting for the 5th time,, 1000nM MTX selects further to increase to average 37 pico-grams/cell/sky in the substratum through being added to.Also can be connected, obtain comparative data through the SV40 enhanser that on function, makes the hamster promotor with replaced C MV enhanser.Simultaneously, compare with traditional progressively gene amplification strategy, conventional measures generally include four amplification stages can reduce select the high yield cell development time half to about 120 days, and on development usefulness and cost, be attended by significantly and reduce.

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Sequence table

＜110〉(the BOEHRINGER INGELHEIM PHARMA GmbH ﹠amp of Boehringer Ingelheim Pharma KG; Co.KG)

＜120〉system of selection of the preparation method of expression vector, heterologous gene products and high yield reconstitution cell

<130>Case1-1412

<140>

<141>

<160>1

<170>PatentIn Ver.2.1

<210>1

<211>2406

<212>DNA

＜213〉Chinese hamster (cricetulus griseus)

<300>

<310>PCT/EP/96/04631

<311>1996-10-24

<312>1997-05-01

<400>1

gatctccagg acagccatgg ctattacaca gagaaaccct gtctggaaaa acaaaaaatt 60

agtgtccatg tgtaaatgtg tggagtatgc ttgtcatgcc acatacagag gtagagggca 120

gtttatggga gtcagttcct attcttcctt tatgggggac ctggggactg aactcaggtc 180

atcaggcttg gcagaaagtg cattagctca cggagcctta tcattggcga aagctctctc 240

aagtagaaaa tcaatgtgtt tgctcatagt gcaatcatta tgtttcgaga ggggaagggt 300

acaatcgttg gggcatgtgt ggtcacatct gaatagcagt agctccctag gagaattcca 360

agttctttgg tggtgtatca atgcccttaa aggggtcaac aacttttttt ccctctgaca 420

aaactatctt cttatgtcct tgtccctcat atttgaagta ttttattctt tgcagtgttg 480

aatatcaatt ctagcacctc agacatgtta ggtaagtacc ctacaactca ggttaactaa 540

tttaatttaa ctaatttaac cccaacactt tttctttgtt tatccacatt tgtggagtgt 600

gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgc 660

gcgcgcgcgc gcgctcggat cattctacct tttgtttaaa aaatgttagt ccaggggtgg 720

ggtgcactgt gaaagtctga gggtaacttg ctggggtcag ttctttccac tataggacag 780

aactccaggt gtcaactctt tactgacaga accatccaaa tagccctatc taattttagt 840

tttttattta tttatttttt gtttttcgag acagggtttc tctgtggctt tggaggctgt 900

cctggaacta gctcttgtag accaggctgg tctcgaactc agagatccac ctgcctctgc 960

ctcctgagtg ctgggattaa aggcatgcgc caccaacgct tggctctacc taattttaaa 1020

agagattgtg tgtcacaagg gtgtcatgtc gccctgcaac cacccccccc ccaaaaaaaa 1080

aaaaaaaaaa acttcactga agctgaagca cgatgatttg gttactctgg ctggccaatg 1140

agctctaggg agtctcctgt caaacagaat ctcaacaggc gcagcagtct tttttaaagt 1200

ggggttacaa cacaggtttt tgcatatcag gcattttatc taagctattt cccagccaaa 1260

aatgtgtatt ttggaggcag cagagctaat agattaaaat gagggaagag cccacacagg 1320

ttattaggaa gataagcatc ttctttatat aaaacaaaac caaaccaaac tggaggaggt 1380

ctacctttag ggatggaaga aaagacattt agagggtgca atagaaaggg cactgagttt 1440

gtgaggtgga ggactgggag agggcgcaac cgctttaact gtcctgtttt gcctattttt 1500

tggggacagc acatgttcct atttttccca ggatgggcaa tctccacgtc caaacttgcg 1560

gtcgaggact acagtcattt tgcaggtttc cttactgtat ggcttttaaa acgtgcaaag 1620

gtgaccatta accgtttcac gctgggaggg cacgtgcggc tcagatgctt cctctgactg 1680

agggccagga gggggctaca cggaagaggc cacacccgca cttgggaaga ctcgatttgg 1740

gcttcagctg gctgagacgc cccagcaggc tcctcggcta caccttcagc cccgaatgcc 1800

ttccggccca taacccttcc cttctaggca tttccggcga ggacccaccc tcgcgccaaa 1860

cattcggccc catcccccgg tcctcacctg aatctctaac tctgactcca gagtttagag 1920

actataacca gatagcccgg atgtgtggaa ctgcatcttg ggacgagtag ttttagcaaa 1980

aagaaagcga cgaaaaacta caattcccag acagacttgt gttacctctc ttctcatgct 2040

aaacaagccc cctttaaagg aaagcccctc ttagtcgcat cgactgtgta agaaaggcgt 2100

ttgaaacatt ttaatgttgg gcacaccgtt tcgaggaccg aaatgagaaa gagcataggg 2160

aaacggagcg cccgagctag tctggcactg cgttagacag ccgcggtcgt tgcagcgggc 2220

aggcacttgc gtggacgcct aaggggcggg tctttcggcc gggaagcccc gttggtccgc 2280

gcggctcttc ctttccgatc cgccatccgt ggtgagtgtg tgctgcgggc tgccgctccg 2340

gcttggggct tcccgcgtcg ctctcaccct ggtcggcggc tctaatccgt ctcttttcga 2400

atgtag 2406

Claims (25)

1. expression vector, described expression vector comprise the gene of coding target protein matter, and described gene is connected with the proteinic gene function of coding fluorescence with hamster-ubiquitin/S27a-promotor.

2. according to the expression vector of claim 1, it is characterized in that described carrier contains the selectable marker gene that can increase.

3. according to the expression vector of claim 1 or 2, it is characterized in that described carrier comprises the one or more enhansers that are connected with described one or more promoter function.

4. according to the expression vector of claim 1-3 in each, it is characterized in that described carrier contains internal ribosome entry site (IRES) extraly, described internal ribosome entry site is allowed the bicistronic mRNA expression of the gene of the proteinic gene of coding fluorescence and coding target protein matter/product.

5. according to the expression vector of claim 1-4 in each, it is characterized in that proteinic gene of described coding fluorescence and the selectable marker gene that can increase are to be arranged in the transcription unit that one or two separates.

6. according to the expression vector of claim 1-5 in each, it is characterized in that described functional connection is not to connect by intron sequences.

7. according to the expression vector of claim 1-6 in each, it is characterized in that described selectable marker gene coding Tetrahydrofolate dehydrogenase (DHFR) that increases or the fused protein of coding fluorescence protein and DHFR.

8. according to the expression vector of claim 3-7 in each, it is characterized in that described enhanser is CMV-or SV40-enhanser.

9. according to the expression vector of claim 1-8 in each, it is characterized in that described carrier comprises at least one polyadenylation signal extraly.

10. according to the expression vector of claim 1-9 in each, it is characterized in that multiple clone site required when described carrier comprises the gene that mixes coding target protein matter is to replace the gene of coding target protein matter/product.

11. an eukaryotic host cell, described host cell have been used the expression vector transfection in each according to claim 1-10.

12., it is characterized in that described host cell is a mammalian cell, preferably Chinese hamster ovary celI according to the host cell of claim 11.

13. host cell according to claim 11 or 12, it is characterized in that described host cell is extraly with one or more carrier transfections that have gene, one or more other target protein matter/products of described genes encoding, and at least one other selective marker.

14. a method for preparing heterologous gene products is wherein being allowed the host cell of cultivating under the condition of described gene product expression according to claim 11-13, and separates described gene product from culture or substratum.

15. method for preparing the protein/product of different aggressiveness form, wherein under the condition of allowing the protein of described different aggressiveness form/product expression, cultivate according to the host cell of claim 13, and from culture or substratum, separating the protein/product of described different aggressiveness form, described host cell is a plurality of expression vector cotransfections of the different subunits of the protein/product of the different aggressiveness form of usefulness coding.

16. according to the method for claim 15, the protein that it is characterized in that described different aggressiveness form is antibody.

17., it is characterized in that described host cell is carried out one or more gene amplification steps according to each method among the claim 14-16.

18. according to the method for claim 17, it is characterized in that the described selective marker that increases is DHFR, and the amplification agent is a methotrexate.

19., it is characterized in that only described host cell being carried out a step of utilizing the methotrexate amplification gene according to the method for claim 18.

20., it is characterized in that described method is to carry out in serum free medium according to each method among the claim 14-19.

21., it is characterized in that described host cell cultivates in suspension culture according to each method among the claim 14-20.

22. method of screening the host cell of expressing target protein matter/product, wherein allowing under the condition of expressing target protein matter/product and fluorescence protein, cultivation is according to the colony of the host cell of claim 11-13 in each, and identifies and/or select to demonstrate one or more cells of the high expression level of fluorescence protein.

23., it is characterized in that described selection use fluorescence activated cell sorter (FACS) carries out according to the method for claim 22.

24., it is characterized in that described selecteed host cell is carried out one or more extra gene amplification steps according to the method for claim 22 or 23.

25., it is characterized in that the described selective marker that increases is DHFR, and described amplification agent is a methotrexate according to the method for claim 24.

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