CN102392047B - Bicistronic mRNA (messenger ribonucleic acid) expression vector suitable for cells of mammals and application thereof - Google Patents
Bicistronic mRNA (messenger ribonucleic acid) expression vector suitable for cells of mammals and application thereof Download PDFInfo
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Abstract
The invention discloses a bicistronic mRNA (messenger ribonucleic acid) expression vector which is suitable for cells of mammals and can realize high-efficient expression of exogenous genes and an application thereof. The bicistronic mRNA expression vector comprises connecting elements in the 5'-3' direction, namely (1) an open reading frame 1: containing the sequences of an early promoter/enhancer and an introne of cytomegalovirus, multiple clone sites and the sequence of a bovine growth hormone transcription terminator, which are sequentially arranged; (2) an open reading frame 2: containing the sequences of the early promoter/enhancer and the introne of the cytomegalovirus, the multiple clone sites and the sequences of the bovine growth hormone transcription terminator and a simian virus promoter, which are sequentially arranged; and (3) screening marker genes and the sequence of a simian virus 40 (SV40) terminator. The bicistronic mRNA expression vector has the high-efficient promoter, effective transcription termination signals, screening and gene amplification markers and two open reading frames, and can improve the expression efficiency of the exogenous genes and reduce the production cost.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of High Expression Vectors in Mammalian Cells.
Background technology
According to the type of host cell, gene expression system roughly can be divided into to protokaryon, yeast, plant, insect and mammalian cell expression system.With other system, compare, the advantage of mammalian cell expression system is to instruct the correct folding of protein, the multiple translation post-treatment functions such as complicated N-type glycosylation and O type glycosylation accurately are provided, thus expression product aspect molecular structure, physicochemical property and biological function close to natural higher organism protein molecule.From starting most with naked DNA direct transfection mammalian cell between so far more than 30 years, mammalian cell expression system has not only become the production platform of several genes engineering medicine, in the structure and function research of the discovery of new gene, protein, has also play a part very important.But zooblast exogenous protein expression efficiency is low, and cost is high, therefore improves zooblast exogenous protein expression efficiency, and reducing production costs is the work that current urgent need completes.
High-caliber mammalian cell expression vector should have following characteristics: efficient promotor is arranged, to start transcribing of goal gene; Effectively transcription termination signal, guarantee the correct termination of transcribing; Have screening and gene amplification sign, in convenient screening karyomit(e), high copy is integrated the cell of goal gene.At foreign gene, carry out in the expression process, select any expression vector to consider from many aspects, same expression vector, same promotor difference on effect in different cell types is larger, this existence with this transit cell record regulatory factor is relevant, need to constantly grope.
CHO (Chinese hamster ovary) cell is the engineering cell that current expression alien gene is the most often selected.CHO-dhfr wherein
-with CHO-GS be the main flow cell strain of mammalian cell expression, CHO-dhfr-system is the engineering cell strain (external and domestic a plurality of preservation mechanism on sale) of widespread use, it can make the copy number amplification of foreign gene under methotrexate (MTX) pressure, makes foreign protein obtain high-caliber expression.And this cell strain is easy to transfection, can carry out suspension culture, serum-free culture and high density fermentation.Suitable to industrial a large amount of Restruction albumen.CHO-GS system is a kind of efficient exogenous protein expression system of Lonza company exploitation.In mammalian cell, glutamine synthetase (Glutamine synthetase, GS) the synthetic glutamine (glutamine) of catalysis glutaminate (glutamate) and ammonium salt (ammonium), this reaction is unique source of glutamine in cell.But the cell of some vitro culture (as murine myeloma cell) is natural can not produce enough GS molecules, they need to add ectogenic glutamine in substratum just can maintain normal growth metabolism.Therefore, GS is considered to desirable transfection colony screening mark: under the condition without exogenous glutamine, the clone could be grown and form to the cell that only is integrated into external GS gene.Depress and can make CHO-GS cell high-efficient expression alien gene in adding of MSX, mode by the optimization expression carrier, can improve the expression of host cell to foreign gene, therefore, the efficient expression vector that builds a kind of mammalian cell that is applicable to, especially Chinese hamster ovary celI is significant in industrial production.
Summary of the invention
The object of the present invention is to provide a kind of two-cistron expression vector and application thereof that is applicable to mammalian cell the high efficient expression foreign protein of energy, to reduce the production cost of foreign protein.
One aspect of the present invention discloses a kind of two-cistron expression vector that is applicable to mammalian cell, and described carrier comprises the connect elements of following 5 '-3 ' direction:
(1) open reading frame 1: comprise cytomegalovirus (CMV) early promoter/enhanser of being arranged in order and intron sequences, multiple clone site, Trobest terminator sequence;
(2) open reading frame 2: comprise cytomegalovirus (CMV) early promoter/enhanser and intron sequences, multiple clone site, Trobest transcription terminator and the simian virus SV40 promoter sequence that are arranged in order;
(3) selection markers gene, simian virus SV40 terminator sequence.
Preferably, cytomegalovirus (CMV) early promoter/enhanser of described open reading frame 1 and open reading frame 2 and intron sequences are SEQ ID NO:1.
Preferably, the Trobest Transcription Termination subsequence of described open reading frame 1 is SEQ ID NO:2.
Preferably, the Trobest transcription terminator of described open reading frame 2 and simian virus SV40 promoter sequence are SEQID NO:3.
Preferably, described simian virus SV40 terminator sequence is SEQ ID NO:4.
Preferably, the multiple clone site of described open reading frame 1 comprises NheI restriction enzyme site and MluI restriction enzyme site; The multiple clone site of described open reading frame 2 comprises EcoRI restriction enzyme site and NotI restriction enzyme site.
Preferably, described selection markers gene is selected from Tetrahydrofolate dehydrogenase (DHFR) or glutamine synthetase (GS) gene.
Preferably, the described two-cistron expression vector that is applicable to mammalian cell is to be obtained by plasmid Pcmob3H transformation.
More excellent, the described two-cistron expression vector that is applicable to mammalian cell is between the SalI restriction enzyme site and NheI restriction enzyme site of plasmid Pcmob3H multiple clone site, from 5 '-3 ' direction, is inserted with described open reading frame 1, open reading frame 2 and selection markers gene and simian virus SV40 terminator sequence.
Preferably, described zooblast is Chinese hamster ovary (CHO) cell.
More excellent, described zooblast is selected from CHO-K1 or CHO-dhfr
-cell strain.
Second aspect present invention discloses a kind of application of two-cistron expression vector in mammalian cell expression albumen that is applicable to mammalian cell.
Preferably, described albumen is antibody, comprises a heavy chain and a light chain.
More excellent, described antibody is total man TNF alpha monoclonal antibodies, comprises the light chain of total man TNF alpha monoclonal antibodies and the heavy chain of total man TNF alpha monoclonal antibodies.
Optimum, the sequence of light chain of described total man TNF alpha monoclonal antibodies is SEQ ID NO:5; The sequence of heavy chain of described total man TNF alpha monoclonal antibodies is SEQ ID NO:6.
The present invention is applicable to the two-cistron expression vector of mammalian cell the high efficient expression foreign protein of energy, be respectively equipped with the polyclone restriction enzyme site in two open reading frame, can pass through double digestion, the method connected is inserted into place, enzyme point of contact, described polyclone position by the gene order of foreign protein: by the expression vector in the present invention through restriction enzymes double zyme cutting, electrophoresis, reclaim the satisfactory expression vector fragment of clip size, the gene of the foreign protein that the satisfactory expression vector fragment of size is processed with the restriction enzyme through identical is connected, obtain the efficient expression vector of corresponding albumen.
Third aspect present invention discloses a kind of two-cistron expression vector of the total man of being mounted with TNF alpha monoclonal antibodies gene, described carrier is for building on the basis at the two-cistron expression vector that is applicable to mammalian cell of the present invention, wherein, the multiple clone site of described open reading frame 1 is inserted with total man TNF alpha monoclonal antibodies light chain DNA sequence dna, and the multiple clone site of described open reading frame 2 is inserted with total man TNF alpha monoclonal antibodies heavy chain DNA sequence dna.
Preferably, the light chain DNA sequence dna of described total man TNF alpha monoclonal antibodies is SEQ ID NO:5, and the heavy chain DNA sequence dna of total man TNF alpha monoclonal antibodies is SEQ ID NO:6.
Preferably, the multiple clone site of described open reading frame 1 comprises NheI restriction enzyme site and MluI restriction enzyme site, and described total man TNF alpha monoclonal antibodies light chain DNA sequence dna is inserted between NheI restriction enzyme site and MluI restriction enzyme site; The multiple clone site of described open reading frame 2 comprises EcoRI restriction enzyme site and NotI restriction enzyme site, and described total man TNF alpha monoclonal antibodies heavy chain DNA sequence dna is inserted between EcoRI restriction enzyme site and NotI restriction enzyme site.
Beneficial effect of the present invention is: (1) has early promoter and the enhancer sequence of cytomegalovirus (CMV), can efficiently carry out transcribing of foreign gene; (2) contain one section intron sequences, can improve the stability of messenger RNA(mRNA) (mRNA), extend its transformation period; (3) contain Trobest polyadenylic acid transcription termination signal, protect the timely termination that positive mRNA transcribes; (4) contain Tetrahydrofolate dehydrogenase (DHFR), as screening and amplification sign, can screen the cell strain that obtains the stable integration goal gene, and make cell under the MTX selective pressure, improve the expression of goal gene.(5) light chain and heavy chain district are placed on to common transcript and expression in identical carrier, make heavy chain, light chain can guarantee the ratio expression of 1: 1, form whole immunoglobulin.Therefore, carrier of the present invention has efficient promotor, to start transcribing of goal gene; Effectively transcription termination signal, guarantee the correct termination of transcribing; Have screening and gene amplification sign, in convenient screening karyomit(e), high copy is integrated the cell of goal gene.The measurement result of the expression amount of antibody shows, with existing animal cell bicistronic expression vector, compare, the antibody expression amount of two-cistron expression vector of the present invention obtains significantly and promotes, and expression level has improved about 10-20 doubly, improved the preparation efficiency of animal derived antibody, reduced production costs.
The accompanying drawing explanation
Fig. 1: the structure of two-cistron expression vector
Fig. 2: the building process of pHAI-H plasmid
Fig. 3: the building process of pHAI-DH plasmid
Fig. 4: the building process of pHAI-N-DH plasmid
Embodiment
Following embodiment describes the present invention in detail, but its invention scope is not limited.
The amplification of embodiment 1pCMV-intron-L-BGH PA fragment
(1) pcr amplification of pCMV promotor and intron
Plasmid pCI-neo is eukaryon expression plasmid, take the pCI-neo plasmid as template, take cytomegalovirus (CMV) early promoter/enhanser and intron sequences (SEQ ID NO:1) is reference, design is carried out the PCR reaction containing the primer P1/P2 of SacI restriction enzyme site or NheI restriction enzyme site, the amplification two ends are with cytomegalovirus (CMV) early promoter/enhanser and the intron sequences of restriction enzyme site, and the PCR reaction conditions is as table 1.
Upstream primer (P1): 5 '-GCG
gAGCTCgGGCTATTGGCCATTGCATACGC-3 ' (underscore is the SacI restriction enzyme site) (SEQ ID NO:7)
Downstream primer (P2):
5 '-TGAACTGGGAGTGGACACCTGTGGAGAGAAAGGCAAG
cTAGCgC-3 ' (underscore is the NheI restriction enzyme site) (SEQ ID NO:8)
Set up following reaction system in 100 μ l thin wall centrifugal tubes:
Table 1PCR reaction conditions
The PCR product is carried out to agarose gel electrophoresis, and the size of PCR product conforms to expectation, the DNA fragmentation amplified is reclaimed to test kit with glue and reclaim.
(2) pcr amplification of the light chain of total man TNF alpha monoclonal antibodies
Get 5 milliliters of active rheumatoid arthritis patient peripheral bloods, separate white corpuscle with lymphocyte separation medium, cultivated, identify positive colony according to the ELISA result, a positive colony cell 5000-10000 obtained, separate total RNA with Trizol (GIBCO), by M-MLV ThermoScript II (Promega company product) to obtain cDNA.Take this cDNA as template, the sequence of light chain (SEQ ID NO:5) of antibody of take is reference, design is carried out the PCR reaction containing the primer P3/P4 of NheI restriction enzyme site or MluI restriction enzyme site, and the amplification two ends are with the light chain of the CMV antibody of restriction enzyme site, and the PCR reaction conditions is in Table 1.
Upstream primer (P3): 5 ' GC
gCTAGcTTGCCTTTCTCATCCCAGGTGTCCACTCCCAGTTCA-3 ' (underscore is the NheI restriction enzyme site) (SEQ ID NO:9)
Downstream primer (P4): 5 '-CTAGAAGGCACAGTCGCTAACACTCTCCCCTG
aCGCGTgC-3 ' (underscore is the MluI restriction enzyme site) (SEQ ID NO:10)
Set up following reaction system in 100 μ l thin wall centrifugal tubes:
The PCR product is carried out to agarose gel electrophoresis, and the size of PCR product conforms to the expectation base pair, by glue recovery test kit recovery for the DNA fragmentation amplified.
(3) pcr amplification of BGH PA transcription termination sequence
Plasmid pCI-neo (Promega) is eukaryon expression plasmid, what take is template, the BGH PA transcription termination sequence (SEQID NO:2) of take is reference, design is containing the primer P5/P6 in MluI restriction enzyme site or the XbaI enzyme cutting site performing PCR reaction of going forward side by side, the amplification two ends are with the BGH PA transcription termination sequence of restriction enzyme site, and the PCR reaction conditions is in Table 1.
Upstream primer (P5): 5 ' GC
aCGCGTcAGGGGAGAGTGTTAGCGACTGTGCCTTCTAG-3 ' (underscore is the MluI restriction enzyme site) (SEQ ID NO:11)
Downstream primer (P6): 5 ' ATAGAGCCCACCGCATCCCCAG
tCTAGAgC-3 ' (underscore is the XbaI enzyme cutting site) (SEQ ID NO:12)
Set up following reaction system in 100 μ l thin wall centrifugal tubes:
The PCR product is carried out to agarose gel electrophoresis, and the size of PCR product conforms to the base pair of expectation, the DNA fragmentation amplified is reclaimed to test kit with glue and reclaim.
(4) the overlapping extension PCR method splicing of pCMV-intron-L-BGH PA fragment
Take P1 and P6 as primer, pCMV-intron, Light, the BGH PA fragment of preparation are mixed as the full length sequence that is template amplification pCMV-intron-L-BGH PA, the PCR reaction conditions is in Table 1.
The PCR product is carried out to agarose gel electrophoresis, and the size of PCR product conforms to the expectation size, by glue recovery test kit recovery for the DNA fragmentation amplified.
(5) structure of recombinant plasmid pHAI-H
The purified product reclaimed is processed with sacI and XbalI double digestion, with same double digestion, process Pcmob3H be connected, the ligation system is as follows:
Reaction system cumulative volume 15 μ l, add each reactive material successively, mixes, and is placed in 16 ℃ of water-baths 16 hours.The transformed competence colibacillus bacillus coli DH 5 alpha, intestinal bacteria are inoculated in containing the LB liquid nutrient medium of penbritin (100 μ g/ml) and cultivate, filter out positive bacterium colony, amplification extracting plasmid order-checking, sequence is SEQ ID NO:24, order-checking is confirmed correct, the correct plasmid called after pHAI-H obtained.
The amplification of embodiment 2pCMV-H-BGHA-SV40 fragment
(1) pcr amplification of pCMV promotor and intron
Take the pCI-neo plasmid as masterplate, take cytomegalovirus (CMV) early promoter/enhanser and intron sequences (SEQ IDNO:1) is reference, design is carried out the PCR reaction containing the primer P7/P8 of xhoI restriction enzyme site or EcoRI restriction enzyme site, the amplification two ends are with cytomegalovirus (CMV) early promoter/enhanser and the intron sequences of restriction enzyme site, and the PCR reaction conditions is as table 1.
Upstream primer (P7): 5 '-GCG
cTCGAGgGGCTATTGGCCATTGCATACGC-3 ' (underscore is the xhoI restriction enzyme site) (SEQ ID NO:13)
Downstream primer (P8): 5 '-CTGGGAGTGGACATGGAGAGAAAGGCAAAGTGG
gAATTCcGC-3 ' (underscore is the EcoRI restriction enzyme site) (SEQ ID NO:14)
Set up following reaction system in 100 μ l thin wall centrifugal tubes:
The PCR product is carried out to agarose gel electrophoresis, and the size of PCR product conforms to the expectation size, by glue recovery test kit recovery for the DNA fragmentation amplified.
(2) pcr amplification of the heavy chain of total man TNF alpha monoclonal antibodies
Get 5 milliliters of active rheumatoid arthritis patient peripheral bloods, separate white corpuscle with lymphocyte separation medium, cultivated, identify positive colony according to the ELISA result, a positive colony cell 5000-10000 obtained, separate total RNA with Trizol (GIBCO), by M-MLV ThermoScript II (Promega company product) to obtain cDNA.Take this cDNA as template, the heavy chain of antibody sequence (SEQ ID NO:6) of take is reference, design is carried out the PCR reaction containing the primer P9/P10 of EcoRI restriction enzyme site or NotI restriction enzyme site, and the amplification two ends are with the heavy chain of the CMV antibody of restriction enzyme site, and the PCR reaction conditions is in Table 1.
Upstream primer (P9): 5 ' GCG
gAATTCcCACTTTGCCTTTCTCTCCATGTCCACTCCCAG-3 ' (underscore is the EcoRI restriction enzyme site) (SEQ ID NO:15)
Downstream primer (P10): 5 ' CTAGAAGGCACAGTCGTCATTTACCCGGAGACAGGG
gCGGCCGCcGC-3 ' (underscore is the NotI restriction enzyme site) (SEQ ID NO:16)
Set up following reaction system in 100 μ l thin wall centrifugal tubes:
The PCR product is carried out to agarose gel electrophoresis, and the PCR product conforms to the expectation size, by glue recovery test kit recovery for the DNA fragmentation amplified.
(3) amplification of Trobest (BGH PA) transcription terminator and simian virus SV40 promotor
Take BGH transcription terminator and simian virus SV40 initiating sequence (SEQ ID NO:3) is reference, design is containing the primer P11/P12 of NotI restriction enzyme site or SpeI restriction enzyme site, the pCI-neo of take carries out the PCR reaction as template, the amplification two ends are with Trobest (BGH) transcription terminator of restriction enzyme site and the amplification of simian virus SV40 promotor, and the PCR reaction conditions is in Table 1.
Upstream primer (P11): 5 '-GCG
gCGGCCGCcCCTGTCTCCGGGTAAATGACGACTGTGCCTTCTAG-3 ' (underscore is the NotI restriction enzyme site) (SEQ ID NO:17)
Downstream primer (P12): 5 ' GCTTTTTGCAAAAGCCTAGGC
aCTAGTgCG-3 ' (underscore is the SpeI restriction enzyme site) (SEQ ID NO:18)
Set up following reaction system in 100 μ l thin wall centrifugal tubes:
The PCR product is carried out to agarose gel electrophoresis, and the PCR product conforms to the size of expectation, the DNA fragmentation amplified is reclaimed to test kit with glue and reclaim.
(4) the overlapping extension PCR method splicing of pCMV-intron-H-BGH PA-SV40 fragment
Take P7 and P12 as primer, the glue of pCMV-intron, Heavy, BGH PA SV40 is reclaimed to product and mix, as the full-length gene order of template amplification pCMV-intron-H-BGH PA-SV40, the PCR reaction conditions is in Table 1.
The PCR reaction system is as follows:
The PCR product is carried out to agarose gel electrophoresis, and the PCR product conforms to the size of expectation, the DNA fragmentation amplified is reclaimed to test kit with glue and reclaim.
(5) structure of recombinant plasmid pHAI-DH
The PCR product of the pCMV-intron-H-BGH PA-SV40 of acquisition is connected with the pMD-19T carrier, and recombinant plasmid is cut to evaluation with the XhoI/SpeI enzyme, the positive plasmid pMD-pCMV-intron-H-BGH PA-SV40 built is carried out to DNA sequencing.Recombinant plasmid pMD-pCMV-intron-H-BGH PA-SV40, with the XhoI/SpeI double digestion, reclaims the pCMV-intron-H-BGHPA-SV40DNA fragment; The recombinant plasmid pHAI-H of embodiment 1 preparation is also processed with the XhoI/SpeI double digestion, pCMV-intron-H-BGH PA-SV40 gene after double digestion is processed is with after pHAI-H after the double digestion processing is connected, order-checking is confirmed, sequence is SEQ ID NO:25, sequencing result is correct, by the correct plasmid called after pHAI-DH obtained.
The ligation system is as follows:
The amplification of embodiment 3DHFR-SV40PA fragment
(1) amplification of DHFR fragment
The CHO-K1 cell extracting cell total rna that goes down to posterity and cultivate from routine, reverse transcription becomes cDNA as template, and the DHFR sequence (SEQ ID NO:23) of take is reference, and design primer P13/P14 carries out the PCR reaction, amplify the DHFR fragment of an end containing the SfiI restriction enzyme site, the PCR reaction conditions is in Table 1.
Upstream primer (P13): 5 ' GC
gGGCCATCGAGGCCgCCACCATGGTTCGACCATTG-3 ' (underscore is the SfiI restriction enzyme site) (SEQ ID NO:19)
Downstream primer (P14): 5 '-CATCAATGTATCTTATCAGCATCTTCCTGTTAGTC-3 ' (SEQ IDNO:20)
Set up following reaction system:
The PCR result is carried out agarose gel electrophoresis, and the size of PCR product conforms to the base pair size of expectation, the fragment amplified is reclaimed to test kit with DNA glue and reclaim.
(2) amplification of SV40PA fragment
Take plasmid pCI-neo as template, and the SV40PA sequence (SEQ ID NO:4) of take is reference, and design primer P15/P16, carry out the PCR reaction, and the end that increases is containing the SV40PA fragment sequence of NheI restriction enzyme site, and the PCR reaction conditions is in Table 1.
Upstream primer (P15): 5 '-GACTAACAGGAAGATGCTGATAAGATACATTGATG-3 ' (SEQ IDNO:21)
Downstream primer (P16): 5 ' GTTTATTGCAGCTTATAATGGTG
cTAGC-3 ' (underscore is the NheI restriction enzyme site) (SEQ ID NO:22)
Set up following reaction system in 100 μ l thin wall centrifugal tubes:
The PCR product is carried out to agarose gel electrophoresis, and the PCR product conforms to the size of expectation, the DNA fragmentation amplified is reclaimed to test kit with glue and reclaim.
(3) the overlapping extension PCR method splicing of DHFR-SV40PA fragment
It is template that the glue of DHFR and SV40PA of take respectively reclaims product, take P13 and P16 as primer, the full-length gene order of splicing DHFR-SV40PA, and the PCR reaction conditions is in Table 1.
The PCR reaction system is:
The PCR product is carried out to agarose gel electrophoresis, and the PCR product conforms to the size of expectation, the DNA fragmentation amplified is reclaimed to test kit with glue and reclaim.
(4) structure of two-cistron expression vector
The PCR product of the DHFR-SV40PA of acquisition is connected with the pMD-19T carrier, identifies that positive plasmid pMD-DHFR-SV40PA is checked order, result and expected sequence are in full accord.
Recombinant plasmid pMD-DHFR-SV40PA, with the SfiI/NheI double digestion, reclaims the pMD-DHFR-SV40PA fragment; Recombinant plasmid pHAI-DH, also with the SfiI/NheI double digestion, is attached thereto the pMD-DHFR-SV40PA gene, the order-checking confirmation, and sequence is SEQ ID NO:26, sequencing result is correct, the correct plasmid called after pHAI-N-DH obtained.
The ligation system is:
Embodiment 4pHAI-N-DH carrier transfection CHO cell is expressed antibody research
(1) recombinant expression plasmid pHAI-N-DH transfection CHO cell
Go down to posterity and cultivate the CHO-K1 cell by GMEM-S (Gibco-BRL) substratum routine, the GMEM-S substratum adds 10% foetal calf serum through overheated deactivation and dialysis treatment before using.When cell 80% merges, with 0.25% trypsin digestion and cell, passage is inoculated in the 35mm Tissue Culture Dish, carries out plasmid transfection next day.2 μ g plasmid pHAI-N-DH and 10 μ l lipofectamine Lipofectamine are not diluted containing the GMEM-S substratum of foetal calf serum with 100 μ l respectively, and remix to a pipe, mix gently, and room temperature is placed 40 minutes.Wash cell twice with the GMEM-S nutrient solution that does not add foetal calf serum therebetween, absorb nutrient solution.The GMEM-S nutrient solution that adds 800 μ l serum-frees in the room temperature placement transfection liquid of 40 minutes, mix gently, add again in the Tissue Culture Dish with the serum free medium washing, be placed in 37 ℃, the cell culture incubator of 5% carbonic acid gas, saturated humidity, add the GMEM-S nutrient solution of 1ml containing 20% foetal calf serum after 12 hours, continue to cultivate 48 hours.
(2) cell screening and expression product detect
After transfection 48 hours, by cell with 0.25% tryptic digestion, be inoculated in 5 90mm Tissue Culture Plates, add methotrexate (Methotrexate, MTX) (Sigma) to final concentration 25 μ mol/L, after 3-4 days, cell starts death, after two weeks, the cell of survival tends towards stability, and forms the colony (clone) formed by individual cells propagation, when the clone is of moderate size, select single clone, be inoculated in 24 orifice plates.Method: filter paper is cut into to approximately 2 * 2mm
2fragment, autoclaving, dry, and is soaked in 0.25% tryptic digestive juice.Nutrient solution in the culture plate of formation single cell clone is discarded, in the filter paper placement and single cell clone that will soak with 0.25% tryptic digestive juice with tweezers, after 3-5 minute, filter paper is taken out, be placed in 24 orifice plates, 60 single cell clones have been selected altogether, with the complete GMEM-S culture medium culturing cell containing 50 μ mol/L MTX, cultured cells supernatant, use indirect elisa method to measure TNF-alpha expression amount; At first will detect with TNF antigen and be diluted to applicable concentration with coated damping fluid, in 96 orifice plates, every hole adds the coating buffer of 100 μ l, 4 ℃ of coated spending the night of refrigerator.After being coated with, use washings continuous washing 5 times.Accurately take 0.5gBSA, be dissolved in 100ml PBS, mix, be 0.5%BSA.Every hole adds the confining liquid of 300 μ l, again puts into 4 ℃ of sealings and spends the night.Take out 96 orifice plates, wash continuously 5 times washing on the plate machine.The sample of serial dilution is added in enzyme plate successively, do feminine gender and positive control simultaneously, be put in 37 ℃ and hatch 1h.Take out 96 orifice plates, wash continuously 5 times washing on the plate machine.Every hole adds two anti-100 μ l, is placed in 37 ℃ and hatches 1h.Take out 96 orifice plates, wash continuously 5 times washing on the plate machine, every hole adds 100 μ lTMB nitrite ions, 37 ℃ of 10min.After developing the color, add the stop buffer (vitriol oil of 2M) of 50 μ l, measure the 0D value at the 450nm place
Result is as follows: the amount of expression that the TNF-Alpha antibodies of expression detected is: 1576.2ng/ml, and result shows, this expression vector can high efficient expression foreign protein.
Claims (14)
1. a two-cistron expression vector that is applicable to mammalian cell, described carrier comprises the connect elements of following 5 '-3 ' direction:
(1) open reading frame 1: comprise cytomegalovirus early promoter/enhanser of being arranged in order and intron sequences, multiple clone site, Trobest Transcription Termination subsequence;
(2) open reading frame 2: comprise the cytomegalovirus early promoter/enhanser and intron sequences, multiple clone site, Trobest transcription terminator and the simian virus SV40 promoter sequence that are arranged in order;
(3) selection markers gene, simian virus SV40 terminator sequence, described selection markers gene is selected from dihydrofolate reductase gene;
Cytomegalovirus early promoter/the enhanser of described open reading frame 1 and open reading frame 2 and intron sequences are SEQ ID NO:1; The described two-cistron expression vector that is applicable to mammalian cell is to be obtained by plasmid Pcmob3H transformation, and described mammalian cell is Chinese hamster ovary cell.
2. two-cistron expression vector as claimed in claim 1, is characterized in that, the Trobest Transcription Termination subsequence of described open reading frame 1 is SEQ ID NO:2.
3. two-cistron expression vector as claimed in claim 1, is characterized in that, the Trobest transcription terminator of described open reading frame 2 and simian virus SV40 promoter sequence are SEQ ID NO:3.
4. two-cistron expression vector as claimed in claim 1, is characterized in that, described simian virus SV40 terminator sequence is SEQ ID NO:4.
5. two-cistron expression vector as claimed in claim 1, is characterized in that, the multiple clone site of described open reading frame 1 comprises NheI restriction enzyme site and MluI restriction enzyme site; The multiple clone site of described open reading frame 2 comprises EcoRI restriction enzyme site and NotI restriction enzyme site.
6. two-cistron expression vector as claimed in claim 1, it is characterized in that, the described two-cistron expression vector that is applicable to mammalian cell is between the SalI restriction enzyme site and NheI restriction enzyme site of plasmid Pcmob3H multiple clone site, from 5 '-3 ' direction, inserts described open reading frame 1, open reading frame 2, selection markers gene and simian virus SV40 terminator sequence.
7. two-cistron expression vector as claimed in claim 1, is characterized in that, described Chinese hamster ovary cell is selected from CHO-K1 or CHO-dhfr
-cell strain.
8. be applicable to the application of two-cistron expression vector in mammalian cell expression albumen of mammalian cell as described in claim as arbitrary as claim 1-7.
9. application as claimed in claim 8, is characterized in that, described albumen is antibody, comprises a heavy chain and a light chain.
10. application as claimed in claim 9, is characterized in that, described antibody is total man TNF alpha monoclonal antibodies, comprises the light chain of total man TNF alpha monoclonal antibodies and the heavy chain of total man TNF alpha monoclonal antibodies.
11. application as claimed in claim 10, is characterized in that, the DNA sequence dna of the light chain of described total man TNF alpha monoclonal antibodies is SEQ ID NO:5, and the DNA sequence dna of the heavy chain of total man TNF alpha monoclonal antibodies is SEQ ID NO:6.
A 12. two-cistron expression vector that is mounted with total man TNF alpha monoclonal antibodies gene, described carrier for to build on the basis of the described two-cistron expression vector of the arbitrary claim of claim 1-7, wherein, the multiple clone site of described open reading frame 1 is inserted with total man TNF alpha monoclonal antibodies light chain DNA sequence dna, and the multiple clone site of described open reading frame 2 is inserted with total man TNF alpha monoclonal antibodies heavy chain DNA sequence dna.
13. two-cistron expression vector as claimed in claim 12, is characterized in that, the light chain DNA sequence dna of described total man TNF alpha monoclonal antibodies is SEQ ID NO:5, and the heavy chain DNA sequence dna of total man TNF alpha monoclonal antibodies is SEQ ID NO:6.
14. two-cistron expression vector as claimed in claim 12, it is characterized in that, the multiple clone site of described open reading frame 1 comprises NheI restriction enzyme site and MluI restriction enzyme site, and described total man TNF alpha monoclonal antibodies light chain DNA sequence dna is inserted between NheI restriction enzyme site and MluI restriction enzyme site; The multiple clone site of described open reading frame 2 comprises EcoRI restriction enzyme site and NotI restriction enzyme site, and described total man TNF alpha monoclonal antibodies heavy chain DNA sequence dna is inserted between EcoRI restriction enzyme site and NotI restriction enzyme site.
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| 基于FMDV IRES的双顺反子载体的构建及体外表达分析;郑海学;《中国生物工程杂志》;20071231;第27卷(第8期);29-33 * |
| 张国强.哺乳动物细胞高效表达系统研究进展.《生物技术通讯》.2005,第16卷(第1期),56-59. |
| 郑海学.基于FMDV IRES的双顺反子载体的构建及体外表达分析.《中国生物工程杂志》.2007,第27卷(第8期),29-33. |
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