CN102409060A - GS-DHFR dual genetic screening expression vector, structuring method and application thereof - Google Patents
GS-DHFR dual genetic screening expression vector, structuring method and application thereof Download PDFInfo
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Abstract
The invention discloses a GS-DHFR dual genetic screening expression vector, a structuring method and an application thereof, belonging to the biological technical field. The expression vector mainly sequentially contains multiple cloning sites, a DHFR sequence and a GS sequence. The invention further discloses a structuring method and an application of above expression vector; through the vector, molecular cloning technique can be used for fast structuring GS-DHFR dual genetic screening expression vector carrying the target gene, after transfecting of mammalian cell, the GS-DHFR dual genetic screening and system amplification are performed, so as to realize high-level expression of the target gene in the mammalian cell.
Description
Technical field
The present invention relates to biological technical field, the dual-gene screening expression vector of particularly a kind of GS-DHFR and construction process and application.
Background technology
The amplification of foreign gene in mammalian cell is to improve one of Critical policies of exogenous gene expression level.In general, the whole carrier in the expression system is made up of two independences and the expression unit that connects together: exogenous gene expression unit and amplification gene are expressed the unit, and amplification gene often also is a selective marker.Up to now; Kind of gene amplification selective system surplus having set up ten in the world; Tetrahydrofolate dehydrogenase (Dihyrofolate reductase wherein; DHFR) the gene amplification system is the most frequently used gene amplification selective system, and glutamine synthetase (Glutamine synthetase, GS) amplification system is the more effective amplification expression system of newly-developed.
Tetrahydrofolate dehydrogenase can be by folacin methotrexate (Methotrexate; MTX) suppress; With contain goal gene and the recombinant plasmid transformed CHO DHFR-cell of the DHFR gene that links to each other, the MTX that utilizes concentration to improve constantly selects the clone of anti-MTX, wherein DHFR gene and the goal gene that is attached thereto increase together; Thereby make goal gene high level expression (Looney JE, Hamlin JL.Mol cell Biol.1987; 7 (2): 569-577).Glutamine synthetase (GS) system is a kind of gene amplification system of new development in recent years.When glutamine synthetase provides energy in the ATP hydrolysis, utilize intracellular ammonia and Stimulina, under the culture condition that lacks the extracellular Stimulina; The inhibition of adding glutamine synthetase (Methionine sulphoximine, MSX), the goal gene amplification that can make the GS gene and be attached thereto; Reach the purpose (Bebbington CR, Renner G, the Thomson S that improve the destination gene expression level; Et al, Biotechnology.1992; 10 (2): 169-175.).
There are some researches show: GS system expression level height (Liu Wenjun, Yang Furong, Ruan Li, etc., viral journal, 1997; 13 (2): 103-109), but during the cell long-period cultured continuously, upgrowth situation is not good, and DHFR system expression level is low than the GS system, but the cell well-grown (Liu Wenjun, Yang Furong, Ruan Li, etc., hi-tech communication, 1997; 7 (2): 44-49).So, set up one and can efficiently express goal gene, can keep the screening amplification system of good cellular form again; The genetically engineered cell that has productive value for acquisition is highly significant; But do not see at present the report of this respect, there are some researches show, exogenous origin gene integrator goes in the genome of host cell to form integron (Integron) in prokaryotic system; Gene expression dose is relevant with order in the position of inserting the site with expression cassette; Because its inhibition separately of GS system and DHFR system is different, at present more what see is that two systems are building up in the different carriers then cotransformation respectively and in host cell, select the acquisition high expressing cell to be through pressurization; And how to make up to bring into play the advantage of the two simultaneously and play collaborative effect, not see report at present for the carrier of DE system.
Summary of the invention
The purpose of this invention is to provide the dual-gene screening expression vector of a kind of GS-DHFR and construction process and application.This carrier can utilize a large amount of amplifications of single plasmid realization foreign gene and efficiently express, and has simplified operation steps, has improved transfection efficiency, and has remedied the deficiency of DHFR and GS single-gene screening system.
The dual-gene screening expression vector of GS-DHFR; The dual-gene screening expression vector of GS-DHFR; Contain the CMV promotor successively; Foreign gene inserts the dihydrofolate reductase gene of site, the control of CMV promotor and the glutamine synthetase gene of SV40 promotor control, and the glutamine synthetase gene downstream are the SV40polyA signal sequence.
Said dihydrofolate reductase gene sequence and foreign gene insert also has the IRES sequence between the site.
It is MCS that said foreign gene inserts the site, has the nucleotide sequence shown in the Seq ID No.1.
The construction process of the dual-gene screening expression vector of above-mentioned GS-DHFR; Skeleton carrier is pOpti VEC; The nucleotide sequence of said MCS is inserted into the IRES sequence upper reaches of said pOpti VEC; Said glutamine synthetase gene is inserted in the Pvu II site of pOpti VEC, and the said glutamine synthetase gene upper reaches have the SV40 promotor, and downstream are the SV40polyA signal sequence.Sequence is shown in Seq ID No.5.
The application of the dual-gene screening expression vector of above-mentioned GS-DHFR is characterized in that inserting the site at said foreign gene inserts the purpose foreign gene, changes host cell then over to and obtains transformant; The inhibition that in the substratum that lacks Stimulina, adds folacin methotrexate and glutamine synthetase pressurizes screening to obtain high expressing cell system to transformant; High expressing cell system to obtaining carries out enlarged culturing, last separation and Extraction purpose expression of exogenous gene product.
Said host cell is a mammalian cell.
Said mammalian cell is a CHO DG44 cell.
Said high expressing cell system is respectively under the condition of 75uM and 200nM pressurization and screens acquisition for the concentration of the inhibition of folacin methotrexate in the substratum and glutamine synthetase.
Said purpose foreign gene is the human nerve growth factor gene, and its nucleotide sequence is shown in Seq ID No.2.
The present invention provides a kind of dual-gene screening expression vector, and screening-gene is dihydrofolate reductase gene (DHFR) and glutamine synthetase gene (GS), and purpose obtains to efficiently express goal gene, can keep the screening amplification system of good cellular form again.The contriver finds through a large amount of experiments; When the element on the carrier and Position Design thereof are following: " contain successively preceding dihydrofolate reductase gene expression cassette and after the glutamine synthetase gene expression cassette, foreign gene inserts the site and is in the dihydrofolate reductase gene expression cassette, the promotor of dihydrofolate reductase gene adopts CMV; the expression cassette of glutamine synthetase gene uses the SV40 promotor "; This carrier can get in host cell can efficiently express goal gene, can keep the screening amplification system of good cellular form again, in the carrier design of the present invention; Because the CMV promotor is strong than the SV40 promotor; Express the purpose that reaches enhancing foreign gene amplification efficiency through the reduction selectable marker gene, the expression of reduction selectable marker gene is when using the selective pressure identical with conventional expression vector; The coamplification gene copy number is higher, and corresponding expression of exogenous gene level also is improved.Promptly play carrier construction and cotransformation host respectively with two gene amplification systems than it; Under the situation that will obtain identical exogenous gene expression level; Carrier of the present invention institute cell transformed is applied the MSX selective pressure; Can practice thrift required reagent cost, promptly the building mode of double gene expression vector of the present invention makes and has reached collaborative effect between two gene amplification systems, rather than only the extraneous suppressor factor that gives of dependence applies amplification pressure.From expression of exogenous gene efficient also having been obtained outstanding effect, shown in embodiment 3, under the rejection condition with 75uM-MSX-200nM-MTX in substratum, the concentration of recombinant human NGF reaches 1.535mg/L in the culture supernatant of transformant; Except that the speed of growth slightly reduces; Microscopy finds that the transgenic DG 44 cell refractive powers of the dual-gene screening of GS-DHFR are good; The sparkling and crystal-clear bright form of cell is good; Each passage cell vigor is all more than 95%, and the carrier that the present invention's structure be described has been realized realizing high expression level, good passage cell form and the high viability that goes down to posterity simultaneously at single plasmid.Has advantages of simple operation when in addition, carrier of the present invention is used for transformed host cell.
Carrier of the present invention preferably inserted internal ribosome entry site sequence (IRES) before the DHFR gene, to strengthen the expression of exogenous gene in the expression cassette.
The foreign gene of the expression vector that the present invention makes up inserts the site can select different MCS nucleotide sequences according to the sequence characteristic of external source goal gene, and the present invention preferably nucleotides sequence shown in Seq ID No.1 classifies as and inserts the site as foreign gene before being inserted into DHFR.
The present invention also further provides the application of expression vector of the present invention, is mainly used in to transform the host, and the exogenous gene high-efficient expressed transgenic cell line of screening under amplification pressure is to obtain the exogenous gene expression product through the transgenic cell line that screens.
Because the screening-gene DHFR of the carrier that the present invention makes up and GS are mammalian cell screening-genes commonly used, therefore, the application of carrier of the present invention mainly refers to be transformed into goes down to posterity and expression alien gene with making the cell high-efficient rate in the mammalian cell.In a preferred embodiment of the invention; The expression vector called after pDG2.0 that makes up; Adopting CHO DG44 clone is host cell expression foreign gene NGFF gene; Experimental data like embodiment 3 shows; After adopting pDG2.0/NGF recombinant plasmid transfection DG 44 cells of the dual-gene screening system of GS-DHFR, under close MTX concentration suppressed, the expression level of recombinant human NGF was apparently higher than DG 44 cells of the pOptiVEC/NGF recombinant plasmid transfection of adopting DHFR single-gene screening system; Even DHFR single-gene screening system is when MTX concentration reaches 250nM, low about 10 times of the expression level of the dual-gene screening system of GS-DHFR when recombinant human NGF expression level also pressurizes than 75uM-MSX-200nM-MTX.
Description of drawings:
The enzyme of Fig. 1 .pOptiVEC-polylinker carrier is cut the checking collection of illustrative plates
1.DM15000DNA Marker; 2.pOptiVEC-polylinker carrier EcoR I and Pvu II double digestion
The PCR of GS gene checking collection of illustrative plates in Fig. 2 .pDG2.0 carrier
1.DM15000DNA Marker; 2.pDG2.0 the GS gene of pcr amplification in the carrier; 3.GS gene masculine contrast
Fig. 3 .pDG2.0 carrier structure synoptic diagram
Fig. 4 .pDG/NGF double digestion checking collection of illustrative plates
1.DM15000DNA Marker; 2.pDG/NGF carrier Xba I and Not I double digestion
Fig. 5. quantitative ELISA detects the typical curve of recombinant human NGF expression level
Fig. 6 .Western blot identifies recombinant human NGF expression map
1. the CHO DG44 cells and supernatant that pressurization is screened after the transfection; 2. mouse NGF sample; 3.ProteinRuler I LMWP standard (12kDa-80kDa)
The practical implementation method:
The more detailed implementation method of the present invention is referring to embodiment, and present embodiment is to be used for explaining, to explain rather than limit by any way the present invention.
Embodiment 1: the structure of carrier for expression of eukaryon pDG2.0 and evaluation
The definite polyclone enzyme of step 1. is cut site sequence
At first the annealing of a pair of complementary primer is formed the double chain DNA molecule of a 46bp, then through Japan spin company TA clone test kit add a base A at 3 ' end, sequence is following:
5 '-GAATTCAGTC GCGAGACGCG TCGTACGACC CGGGTGATCA CTCGAG-3 ' contains 7 restriction endonuclease sites commonly used respectively: EcoR I, Nru I, Mlu I, BsiW I, Xma I, Bcl I and Xho I from left to right.
Connect: get 1.5ml sterilization Eppendorf pipe and add above-mentioned double chain DNA molecule 6ul and pOptiVEC-TOPO plasmid (Invitrogen company); Contain internal ribosome entry site sequence IRES (Internal ribosome entry site on this carrier; IRES) and DHFR gene order 2ul; 5 * T4 connects damping fluid 2ul, and T4 ligase enzyme 1ul was hatched 2 hours for 22 ℃.
Transform: the aseptic 100ul of getting competent cell Trans10 (the full formula in Beijing King Company) places ice bath; Adding link product 10ul mixes; In ice bath, placed 30 minutes, and transferred in 42 ℃ of water-baths 90 seconds immediately, transfer to then in the ice bath and placed 2 minutes; Every then pipe adds the SOC nutrient solution of 0.5ml, 37 ℃ of shaking table 180rpm jogs 45 minutes.Get 200ul and transform thalline, evenly coat on the LB plate culture medium that contains 100mg/ml ammonia benzyl, after Bechtop dries up, put into 37 ℃ of incubators and be inverted overnight cultures.
Choose bacterium: picking colony is 5 in flat board, is inoculated in (5ml/ pipe) in the LB substratum that contains the 100mg/ml penbritin 37 ℃ of shaking table overnight cultures respectively.
Carrier is identified: with the plasmid that extracts, carry out double digestion checking (like Fig. 1) through EcoR I on the polyclone restriction enzyme site and the Pvu II on the pOptiVEC carrier, and the positive colony that checking obtains is carried out sequencing.Sequence results proof polyclone restriction enzyme site inserts the pOptiVEC carrier correctly, forms the pOptiVEC-polylinker carrier of middle transition.Add that after the single endonuclease digestion site on the pOptiVEC carrier, pOptiVEC-polylinker carrier MCS contains 12 restriction endonuclease sites commonly used from left to right respectively: Xba I, BamH I, EcoR I, Nru I, Mlu I, BsiW I, Xma I, BclI and Xho I, BamH I, Not I and Hpa I.
The clone of step 3.GS gene
With the pEE12.4 plasmid is template; With GS gene upstream and downstream Auele Specific Primer; (its nucleotide sequence of upstream primer is shown in Seq IDNo.3; Its nucleotide sequence of downstream primer is shown in Seq ID No.4) amplification GS gene, GS upstream region of gene SV40 promotor and SV40polyA site, GS gene downstream, 5 ' end of primer all adds Sma I restriction enzyme site.The pcr amplification system is: PfuMasterMix (the full formula in Beijing King Company) 25ul, and Primer1, each 1ul of Primer2, pEE12.4 template 1ul adds water to 50ul.The pcr amplification condition: 97 ℃ 1 minute, 61 ℃ 1 minute, 72 ℃ 2 minutes 30 seconds, totally 30 circulations.
Step 4.GS gene order is inserted the pOptiVEC-polylinker carrier
Single endonuclease digestion: get the GS gene fragment and the pOptiVEC-polylinker carrier of above-mentioned pcr amplification, the pOptiVEC-polylinker carrier is carried out single endonuclease digestion, the GS gene of pcr amplification is carried out single endonuclease digestion with Sma I with Pvu II.Enzyme is cut system: 10 * damping fluid 5ul, and carrier 10ul, restriction enzyme 2ul, moisturizing is to 50ul, 37 ℃ of water-baths 5 hours.Enzyme is cut after product and is cut glue recovery purpose fragment.(restriction enzyme Pvu II and Sma I purchase the Bioisystech Co., Ltd in NEB).
Dephosphorylation: the pOptiVEC-polylinker carrier after enzyme cut carries out dephosphorylation to be handled.Reaction system is: enzyme is cut product 30ul, SEAP 1ul, 10 * damping fluid 3.5ul, 37 ℃ of water-baths 5 hours.Enzyme is cut after product and is cut glue recovery purpose fragment.
Connect, it is said to transform and choose bacterium step such as step 2.
Step 5. carrier is identified: get 1ul incubated overnight bacterium, adopt the PCR method to carry out preliminary evaluation, reaction system is identical with GS gene clone condition with condition.Choose positive colony according to 1% agarose gel electrophoresis result, carry out sequencing.Accurate being inserted in the pOptiVEC-polylinker carrier of sequencing result proof GS gene order, the dual-gene screening expression vector establishment success of GS-DHFR promptly of the present invention, called after pDG2.0, its nucleotide sequence is shown in Seq ID No.5.
Embodiment two: the mammalian cell strain (is example with CHO DG44 cell) that obtains express recombinant people NGF
1.NGF the acquisition of gene
The sequence (Genbank ID:NM_002506.2) of people NGF protein (UniProtKB/Swiss-Prot ID:P01138) and gene obtains people's NGF gal4 amino acid complete sequence and wild-type nucleotide sequence in the inquiry UniProtKB DB.Then under the prerequisite that does not change over acquaintance NGF protein amino acid sequence; Utilize Vector NTI software to being optimized design; Modify the encoding sequence of indivedual codons, solve the problem that the inner restriction enzyme site of password bias and wild type gene too much influences vector construction.People's ngf gene coding nucleotide sequence after optimization is modified is shown in Seq ID No.2.Sequence after this is optimized, one side has kept 6 nucleotide sequence AGCGTA before initiator codon ATG, make it meet the Kozak sequence and require and help mRNA to translate and protein expression; On the other hand, before 3 ' end terminator codon TGA, deleted 6 nucleotide sequence AGAGCC, the mature human NGF protein amino acid sequence that causes thus expressing lacks 2 amino acid than wild-type people NGF protein amino acid sequence at carboxyl terminal.After ngf gene is optimized design; Insert Xba I site at 5 ' end; 3 ' end inserts Not I site, trust money only intelligence Bioisystech Co., Ltd to carry out full gene synthetic and be connected on the pcDNA3.1 carrier (Invitrogen company), make up successful pcDNA3.1/NGF recombinant plasmid.
2.pDG/NGF the acquisition of recombinant plasmid
PcDNA3.1/NGF recombinant plasmid and pDG2.0 carrier are used Xba I and Not I double digestion respectively, and the ngf gene fragment after enzyme is cut is connected with linearizing pDG2.0 carrier.As contrast; Ngf gene fragment after enzyme cut is inserted between the identical restriction enzyme site of pOptiVEC-polylinker carrier simultaneously; Connect after the product difference transformed into escherichia coli Trans 10; Identify with double digestion method and PCR sequencing PCR, obtain pDG/NGF recombinant plasmid and pOptiVEC/NGF recombinant plasmid.Concrete operations are all said with embodiment one.
3. recombinant plasmid pDG2.0/NGF and pOptiVEC/NGF recombinant plasmid transfection CHO DG44 cell
CHO DG44 cell is the Chinese hamster ovary line of DHFR defective type, available from Invitrogen company.The by specification routine goes down to posterity before the CHODG44 cell transfecting, with 3 * 10
5Cell/ml density is inoculated in the aseptic triangular flask of 125ml that contains 30ml CD DG44medium (Invitrogen company), makes the next day cell density can reach 5 * 10
5Viable cell/ml, the transfection step is pressed the FreeStyle of Invitrogen company
TMMAX Reagent specification sheets carries out.With 15ul FreeStyle
TMMAX Reagent and 9ug recombinant plasmid dna join 1.2ml OptiCHO
TMAmong the SFM (Invitrogen company), soft mixing, incubated at room 10min.1.2ml plasmid-transfection reagent mixed solution slowly and is uniformly splashed in the Tissue Culture Flask, put CO
2The incubator shaking culture.Behind the transfection 48h, do not contain the CD OptiCHO of Stimulina and HT (inferior sulphur purine and thymidine) with the DG 44 passages entering of pDG2.0/NGF recombinant plasmid transfection
TMCarry out preliminary screening among the Medium, treat that cell viability and density are recovered after, progressively improve the amplification of pressurizeing of MTX and MSX concentration respectively, enlarged culturing is carried out subsequent experimental; DG 44 passages with the transfection of pOptiVEC/NGF recombinant plasmid get into the CD OptiCHO that contains Stimulina, do not contain HT
TMCarry out preliminary screening among the Medium, treat that cell viability and density are recovered after, progressively improve the amplification of pressurizeing of MTX concentration, enlarged culturing is carried out subsequent experimental.
Embodiment three: the detection of people NGF in the recombinant C HO DG44 cells and supernatant
1. the acquisition of cells and supernatant
Behind pDG2.0/NGF recombinant plasmid transfection DG 44 cells, the CHO DG44 cell after MTX and the MSX amplification is pressed 2 * 10
5/ ml density has been inoculated in 30ml CD OptiCHO
TMCultivate in the aseptic triangle culturing bottle of the 125ml of Medium, after 4 days that cell culture fluid is centrifugal, discard cell precipitation, the harvested cell culture supernatant is for use.
Behind DG 44 cells with the transfection of pOptiVEC/NGF recombinant plasmid, the CHO DG44 cell after the MTX amplification is pressed 2 * 10
5/ ml density has been inoculated in 30ml CD OptiCHO
TMCultivate in the aseptic triangle culturing bottle of the 125ml of Medium, after 4 days that cell culture fluid is centrifugal, discard cell precipitation, the harvested cell culture supernatant is for use.
2. quantitative ELISA detects recombinant human NGF expression in the cells and supernatant
According to " growth factor of human nerve (NGF) ELISA test kit specification sheets " method of R&D company mensuration to people NGF expression amount in the cells and supernatant (sample 1) of DG 44 cells under 75uM-MSX-200nM-MTX pressurization concentration of pDG2.0/NGF transfection.Take identical test kit and working method, the expression of recombinant human NGF in the culture supernatant (sample 2) of DG 44 cells of the pOptiVEC/NGF recombinant plasmid transfection under the MTX pressurization concentration of detection 250nM, the concrete operations step is following:
1) .NGF standard solution preparation: add 1ml zero(ppm) water mixing before using, be made into the solution of 20ng/ml.If standard pipe 8 pipes, the first pipe mark-on article diluents (the PBS pH 7.2 that contains 1%BSA), 900 μ l, second to eight pipe adds sample diluting liquid 500ul.The NGF standard solution 100 μ l that in first pipe, add 20ng/ml with sample injector sucking-off 500 μ l, move to second pipe behind the mixing.Do the multiple dilution so repeatedly, from the 7th pipe, inhale and abandon 500 μ l, the 8th pipe is seen table 1 and table 2 for blank.
Table 1: quantitative ELISA detects result's (sample 1) that recombinant human NGF expresses
Table 2: quantitative ELISA detects result's (sample 2) that recombinant human NGF expresses
2). sample and washings dilution: with 1: 1000 diluting cells culture supernatant of sample diluting liquid (the PBS pH7.2 that contains 1%BSA); It is subsequent use that concentrated cleaning solution is diluted to working fluid with double distilled water at 1: 20.
3). application of sample: standard substance and testing sample 100 μ l are joined respectively in the reacting holes different on the enzyme plate, make in the Sptting plate reacting hole and hatch 120min in the rearmounted 37 ℃ of incubators of the abundant mixing of solution.
4). wash plate: with enzyme plate thorough washing 4-6 time, seal is done on filter paper with washings (PBS that contains 0.05%Tween 20, pH 7.2).
5). add first antibody working fluid (the anti-human's polyclonal antibody of biotinylated goat, working concentration 50ng/ml) 100 μ l in every hole, the rearmounted 37 ℃ of 60min of mixing wash plate, the same step 4) of method.
6). every hole adds horseradish peroxidase (HRP)-streptavidin binding substances working fluid 100 μ l.Wash plate, the same step 4) of method after Sptting plate put 37 ℃ of 30min.
7). every hole adds tmb substrate working fluid 100 μ l, puts to add 100 μ l stop buffer mixings after 15min are reacted in 37 ℃ of dark places, surveys light absorption value at the 450nm place with ELIASA in the 30min.
8). interpretation of result: the concentration value with standard substance is an X-coordinate; With the corresponding OD value of each concentration value is that ordinate zou utilizes GraphPad Prism software to carry out linear regression analysis to make typical curve and draw regression equation; Bring the OD value of testing sample into regression equation, income value multiply by extension rate (* 1000) and draws the NGF concentration in the cells and supernatant.The quantitative ELISA determination data shown in table one, typical curve such as Fig. 5, when MSX and two kinds of drug concentrations of MTX were respectively 75uM and 200nM, the concentration of recombinant human NGF was 1.535mg/L in the cells and supernatant.
What contrast with it is that with DG 44 cells of pOptiVEC/NGF recombinant plasmid transfection, the recombinant human NGF expression amount when MTX pressurization concentration is 250nM in the cells and supernatant is 0.1498mg/L (typical curve does not show).The result shows; After adopting pDG2.0/NGF recombinant plasmid transfection DG 44 cells of the dual-gene screening system of GS-DHFR; The expression level of recombinant human NGF is apparently higher than DG 44 cells of the pOptiVEC/NGF recombinant plasmid transfection of adopting DHFR single-gene screening system under close MTX concentration; Even DHFR single-gene screening system is when MTX concentration reaches 250nM, low about 10 times of the expression level of the dual-gene screening system of GS-DHFR of recombinant human NGF expression level during also than 75uM-MSX and 200nM-MTX pressurization.And except that the speed of growth slightly reduced, microscopy found that the transgenic DG 44 cell refractive powers of the dual-gene screening of GS-DHFR are good, and the sparkling and crystal-clear bright form of cell is good, and each passage cell vigor is all more than 95%.
3.Western people NGF expresses in the Blot qualitative detection cells and supernatant
The working method of Western Blot is following:
1). preparation SDS-PAGE glue: according to the spacer gel 3ml of " the molecular biology experiment guide third edition " method preparation 4%; 10% separation gel 8ml injects separation gel earlier, treats that separation gel solidifies back (0.5-1 hour); Inject spacer gel, treat that spacer gel solidifies back (0.5-1 hour) and carries out electrophoresis.
2). go up the appearance electrophoresis: get cells and supernatant (1.535ug/ml) and each 24ul of mouse NGF standard substance (15ug/ml) and 12ul under the 75uM-MSX-200nM-MTX pressurization concentration respectively; 5 * SDS the sample-loading buffer that adds 6ul and 3ul boiled 5 minutes; Centrifugal 2 minutes of 12000rpm gets supernatant and carries out protein electrophoresis.
3). change film: the gel behind the electrophoresis is immersed in 10-20min in 1 * transfering buffering liquid; Get with gel PVDF of the same size (pvdf) film (5 * 8cm), be immersed in the methanol solution behind the 5min; Filter paper is immersed in 5-10min in 1 * transfering buffering liquid; Make progress from the bottom of half-dried translator (SemiDry), spread filter paper, pvdf membrane, gel, filter paper successively, and in the shop, avoid the bubble between layer and the layer to produce, complete cathode electrode on the bonnet, open power supply, voltage 20V, 1 hour transfer printing time.
4) .Western blot reaction: the pvdf membrane after will shifting takes out, and immerses Tris-HCl buffer salt solution (TBS, the 20mmol/LTris of 1%BSA; 150mmol/LNaCl pH7.4) in the sealing, 4 ℃ spend the night after, discard confining liquid; With the TBS washed twice of vibrating, each 10min; Pvdf membrane is immersed the anti-mouse NGF of rabbit polyclonal antibody, and (N6655 in the anti-diluent (1: 2000) SIGMA), reacts 2h on the shaking table, reclaim one and resist, with TBST (the TBS pH7.4 that contains 1%Tween 20) vibration washing three times, each 10min; With pvdf membrane immerse AP-goat-anti rabbit two anti-(A3687 SIGMA) in the diluent (1: 10000), reacts 1h on the shaking table, reclaim two anti-, with TBST vibration washing five times, each 10min.
5). pvdf membrane is immersed in freshly prepared BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate salt/chlorination nitro blue tetrazolium) the colour developing liquid (the BCIP/NBT chromogenic substrate is the Promega Company products); After treating that band is clear; Outwell colour developing liquid, add the zero(ppm) water color development stopping.Western blot detected result is as shown in Figure 6, and there is a specific band at the place at the expection molecular weight.
The above only is preferred embodiment of the present invention, is not the present invention is done any pro forma restriction.Though the present invention with preferred embodiment openly as above, yet be not that any way limits the present invention.Any those skilled in the art; In technical scheme scope of the present invention; Allly utilizing above-mentioned disclosed technology contents to make a little change or modification, should be regarded as the equivalent embodiment of equivalent variations, is not break away from technical scheme content of the present invention in every case;, all still belong in the scope of technical scheme of the present invention any simple modification, equivalent variations and modification that above embodiment did according to technical spirit of the present invention.
Claims (9)
1.GS-DHFR dual-gene screening expression vector; Contain the CMV promotor successively; Foreign gene inserts the dihydrofolate reductase gene of site, the control of CMV promotor and the glutamine synthetase gene of SV40 promotor control, and the glutamine synthetase gene downstream are the SV40polyA signal sequence.
2. the dual-gene screening expression vector of GS-DHFR according to claim 1, said dihydrofolate reductase gene sequence and foreign gene insert also has the IRES sequence between the site.
3. the dual-gene screening expression vector of GS-DHFR according to claim 2, it is MCS that said foreign gene inserts the site, has the nucleotide sequence shown in the Seq ID No.1.
4. the construction process that carries is expressed in the dual-gene screening of the arbitrary described GS-DHFR of claim 1～3; Skeleton carrier is pOpti VEC; The nucleotide sequence in said foreign gene insertion site is inserted into the IRES sequence upper reaches of said pOpti VEC; Said glutamine synthetase gene is inserted in the Pvu II site of pOpti VEC, and the said glutamine synthetase gene upper reaches have the SV40 promotor, and downstream are the SV40polyA signal sequence.
5. the application of the dual-gene screening expression vector of the arbitrary said GS-DHFR of claim 1～3 the steps include: that inserting the site at said foreign gene inserts the purpose foreign gene, changes host cell then over to and obtains transformant; The inhibition that in the substratum that lacks Stimulina, adds folacin methotrexate and glutamine synthetase pressurizes screening to obtain high expressing cell system to transformant; High expressing cell system to obtaining carries out enlarged culturing, last separation and Extraction purpose expression of exogenous gene product.
6. application according to claim 5, said host cell are mammalian cell.
7. application according to claim 6, said mammalian cell are CHO DG44 cell.
8. application according to claim 5, said high expressing cell system is respectively under the condition of 75uM and 200nM pressurization and screens acquisition for the concentration of the inhibition of folacin methotrexate in the substratum and glutamine synthetase.
9. application according to claim 5, said purpose foreign gene is the human nerve growth factor gene, its nucleotide sequence is shown in SeqID No.2.
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| WO2013161958A1 (en) * | 2012-04-27 | 2013-10-31 | 日本ケミカルリサーチ株式会社 | Novel expression vector |
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| WO2014079309A1 (en) * | 2012-11-22 | 2014-05-30 | 苏州康宁杰瑞生物科技有限公司 | Gs-dhfrmut double gene screening expression vector, preparation method and use thereof |
| CN103468742B (en) * | 2012-11-22 | 2015-04-29 | 苏州康宁杰瑞生物科技有限公司 | GS-DHFRmut double-gene screening expression vector, and preparation method and application thereof |
| CN103865947A (en) * | 2012-12-14 | 2014-06-18 | 兰州生物制品研究所有限责任公司 | Eukaryotic expression vector containing attenuated SV40 promoter/dihydrofolate reductase expression element, and construction method thereof |
| CN104195173A (en) * | 2014-09-02 | 2014-12-10 | 北京比洋生物技术有限公司 | Glutamine synthetase expression vector with two expression cassettes |
| CN104946687A (en) * | 2015-06-09 | 2015-09-30 | 北京东方百泰生物科技有限公司 | Mammal dual gene highly efficient screening expression vectors and construction method thereof |
| CN104946687B (en) * | 2015-06-09 | 2018-09-14 | 北京东方百泰生物科技有限公司 | The dual-gene high frequency zone expression vector of mammal and its construction method |
| WO2018032464A1 (en) * | 2016-08-18 | 2018-02-22 | 广东东阳光药业有限公司 | High-throughput cell line screening method for cho-dhfr expression system |
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